JPH08337600A - Production of glucagon - Google Patents

Production of glucagon

Info

Publication number
JPH08337600A
JPH08337600A JP7146255A JP14625595A JPH08337600A JP H08337600 A JPH08337600 A JP H08337600A JP 7146255 A JP7146255 A JP 7146255A JP 14625595 A JP14625595 A JP 14625595A JP H08337600 A JPH08337600 A JP H08337600A
Authority
JP
Japan
Prior art keywords
gln
asp
ser
thr
phe
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
JP7146255A
Other languages
Japanese (ja)
Inventor
Kazuhisa Kashimoto
和久 樫本
Kazuya Hamanaka
和也 浜中
Yumiko Nagano
由美子 長野
Hikari Takahata
ひかり 高畠
Kinya Tomizaki
欣也 富崎
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ito Ham KK
Itoham Foods Inc
Original Assignee
Ito Ham KK
Itoham Foods Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ito Ham KK, Itoham Foods Inc filed Critical Ito Ham KK
Priority to JP7146255A priority Critical patent/JPH08337600A/en
Publication of JPH08337600A publication Critical patent/JPH08337600A/en
Pending legal-status Critical Current

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Classifications

    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Landscapes

  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

PURPOSE: To industrially advantageously obtain glucagon for medicine, etc., by eliminating an amino-protecting group by bonding amino group-protecting threonine to an insoluble resin bondable with carboxyl group, bonding amino acid in turn and eliminating peptide from the resin. CONSTITUTION: A threonine residue in 29th position is introduced into an insoluble resin (e.g. benzhydrylamine resin) having a functional group bondable with a carboxyl group by bonding a carboxyl group of amino group-protecting threonine, a protecting group of the threonine residue is removed and resultant free amino group is subjected to peptide bonding with amino group-protecting amino acid in turn by according to an amino acid sequence of glucagon expressed by the formula to synthesize a peptide chain in extending each one by one from 28th positioned asparagine residue to first positioned histidine residue and resultant peptide is treated with anhydrous hydrogen fluoride to be separated from the resin, then the protecting group is removed to afford the objective glucagon useful for an action of increasing a blood sugar value or testing of growth hormone secreting function, etc.

Description

【発明の詳細な説明】Detailed Description of the Invention

【０００１】[0001]

【産業上の利用分野】本発明は、医薬として有用なペプ
チドであるグルカゴンを化学合成手法を用いて効率的に
製造する方法に関する。TECHNICAL FIELD The present invention relates to a method for efficiently producing glucagon, which is a peptide useful as a medicine, by using a chemical synthesis method.

【０００２】[0002]

【従来の技術】グルカゴンは、膵臓のランゲルハンス島
Ａ細胞から分泌されるペプチドホルモンの一つであり、
インスリンとともに炭水化物の代謝に重要な役割を果た
している。インスリンが糖代謝を亢進させて血糖値を低
下させるの対し、グルカゴンは血中にブドウ糖を放出さ
せ、血糖値を上昇させる作用を有する。そのためグルカ
ゴンは成長ホルモン分泌機能検査、インスリノ−マ（島
細胞腫）の診断、肝糖原検査、低血糖時の救急処置、消
化管のＸ線及び内視鏡検査の前処置などに有用である。Glucagon is one of the peptide hormones secreted from the Langerhans islet A cells of the pancreas,
Together with insulin, it plays an important role in carbohydrate metabolism. Insulin promotes glucose metabolism and lowers blood glucose level, whereas glucagon has the action of releasing glucose into the blood and raising blood glucose level. Therefore, glucagon is useful for growth hormone secretory function test, diagnosis of insulinoma (islet cell tumor), hepatic glycogen test, emergency treatment for hypoglycemia, pretreatment for X-ray of digestive tract and endoscopy, etc. .

【０００３】グルカゴンのアミノ酸配列は公知であり、
Ｎ末端ヒスチジンに始まり、Ｃ末端スレオニンに終わる
29個のアミノ酸残基からなる単鎖ペプチドである。グル
カゴンは、ヒト、ウシ、及びブタにおいてアミノ酸配列
が同一であるため、工業的には、ウシ又はブタからイン
スリンを抽出する際の副産物として単離・精製すること
により得られており（以下、このような抽出法によって
得られたグルカゴンを抽出グルカゴンという。）、既に
製剤化されて市販されている。The amino acid sequence of glucagon is known,
Starts at the N-terminal histidine and ends at the C-terminal threonine
It is a single-chain peptide consisting of 29 amino acid residues. Since glucagon has the same amino acid sequence in human, bovine, and pig, it has been industrially obtained by isolating and purifying it as a by-product when extracting insulin from bovine or porcine (hereinafter, this Glucagon obtained by such an extraction method is referred to as extracted glucagon.), Which has already been formulated and is commercially available.

【０００４】しかしながら、医薬用インスリンの生産が
抽出法による生産から遺伝子組換えによる生産に変更さ
れたため、抽出グルカゴンを単独で生産するのは効率が
悪く、コストがかかるというのが現状である。そこで、
例えば、特開平３−２５４６９２号公報、特開平６−２
９２５９４号公報等において、遺伝子組換え技術を用い
たグルカゴン（以下、組換えグルカゴンという。）の生
産も検討されているが、未だ実用化には至っていない。
また、組換えグルカゴンは微生物を用いて生産されるた
め、安全性について細心の注意を払う必要があり、生産
後の処理が煩雑で、不経済であること等から工業的生産
においては十分満足できるものではない。However, since the production of medicinal insulin has been changed from the production by the extraction method to the production by gene recombination, it is currently inefficient and costly to produce the extracted glucagon alone. Therefore,
For example, JP-A-3-254692 and JP-A-6-2
The production of glucagon (hereinafter, referred to as recombinant glucagon) using gene recombination technology is also examined in Japanese Patent Publication No. 92594, but it has not been put to practical use yet.
In addition, since recombinant glucagon is produced using microorganisms, it is necessary to pay close attention to safety, and post-production processing is complicated and uneconomical. Not a thing.

【０００５】また、グルカゴンの合成については、M.Fu
jinoら（Synthesis of the Nonacosapeputide correspo
nding to Mammalian Glucagon, Chem. Pharm. Bull., 2
6(2), 539-548(1978)）や、N.A.Abrahamら（A NEW PHAS
E STRATRGY FOR THE SYNTHESIS OF MAMMALIAN GLUCAGO
N, Tetrahedron Letters, 32.5, 577-580(1991) ）が報
告しているが、大量に合成された報告はない。Regarding the synthesis of glucagon, M.Fu
jino et al. (Synthesis of the Nonacosapeputide correspo
nding to Mammalian Glucagon, Chem. Pharm. Bull., 2
6 (2), 539-548 (1978)) and NA Abraham et al. (A NEW PHAS
E STRATRGY FOR THE SYNTHESIS OF MAMMALIAN GLUCAGO
N, Tetrahedron Letters, 32.5, 577-580 (1991)), but no large amount of synthetic reports.

【０００６】[0006]

【発明が解決しようとする課題】本発明の課題は、化学
合成手法により、グルカゴンを効率よく大量に、しかも
安価に製造する方法を提供することにある。SUMMARY OF THE INVENTION An object of the present invention is to provide a method for efficiently producing glucagon in large quantities at low cost by a chemical synthesis method.

【０００７】[0007]

【課題を解決するための手段】上記課題を解決すべく鋭
意研究を重ねた結果、工業的生産に適したグルカゴンの
製造方法を見出し、本発明を完成した。本発明は、下記
式(1) ： H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- (1) Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH で表されるグルカゴンの製造方法であって、カルボキシ
ル基と結合可能な官能基を有する不溶性樹脂に、アミノ
基保護スレオニンのカルボキシル基を結合させて前記樹
脂に29位のスレオニン残基を導入する工程、該スレオニ
ン残基の保護基を除去して得られた遊離のアミノ基に、
上記式(1) のアミノ酸配列にしたがってアミノ基保護ア
ミノ酸を順次ペプチド結合させて28位のアスパラギン残
基から１位のヒスチジン残基まで一つずつ延長する工
程、及び得られたペプチドを前記樹脂から脱離させる工
程を含む製造方法（方法１）を提供する。As a result of intensive research to solve the above problems, a method for producing glucagon suitable for industrial production was found, and the present invention was completed. The present invention provides the following formula (1): H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala- Gln- (1) Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH is a method for producing glucagon represented by an insoluble resin having a functional group capable of binding to a carboxyl group, A step of introducing a threonine residue at position 29 into the resin by binding a carboxyl group of an amino group-protected threonine, to a free amino group obtained by removing the protecting group of the threonine residue,
According to the amino acid sequence of the above formula (1), amino-protected amino acids are sequentially peptide-bonded to extend one by one from the asparagine residue at the 28th position to the histidine residue at the 1st position, A manufacturing method (method 1) including a step of desorbing is provided.

【０００８】また、本発明は、上記式(1) で表されるグ
ルカゴンの製造方法であって、前記グルカゴンを構成す
る１又は複数のフラグメント（ここで、フラグメントを
構成するアミノ酸残基の総数は２〜28個である。）であ
って末端アミノ基を保護したものを合成する工程、カル
ボキシル基と結合可能な官能基を有する不溶性樹脂に、
アミノ基保護スレオニン又は29位のスレオニン残基を含
む末端アミノ基保護フラグメントのカルボキシル基を結
合させる工程、前記樹脂に結合したスレオニン残基又は
フラグメントの末端アミノ基の保護基を除去して得られ
た遊離のアミノ基に、上記式(1) のアミノ酸配列にした
がって末端アミノ基保護フラグメント及び／又は末端ア
ミノ基保護アミノ酸を順次ペプチド結合させて１位のヒ
スチジン残基まで延長する工程、及び得られたペプチド
を前記樹脂から脱離させる工程を含む製造方法（方法
２）を提供する。The present invention also provides a method for producing the glucagon represented by the above formula (1), wherein one or a plurality of fragments constituting the glucagon (wherein the total number of amino acid residues constituting the fragment is 2 to 28), in which a terminal amino group is protected, a step of synthesizing an insoluble resin having a functional group capable of binding to a carboxyl group,
A step of binding a carboxyl group of a terminal amino group-protected fragment containing an amino group-protected threonine or threonine residue at position 29, obtained by removing the terminal amino group-protecting group of the resin-bound threonine residue or fragment A step of sequentially peptide-bonding a terminal amino group-protected fragment and / or a terminal amino group-protected amino acid to the free amino group according to the amino acid sequence of the above formula (1) to extend to the histidine residue at position 1 Provided is a production method (method 2) including a step of releasing a peptide from the resin.

【０００９】更に、本発明は、上記式(1) で表されるグ
ルカゴンの製造方法であって、１位から12位のアミノ酸
残基に相当する下記一般式(7) ： Ｒ1-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-OH (7) （式中、Ｒ1は末端アミノ基の保護基である。）で表さ
れるペプチドと、13位から29位のアミノ酸残基に相当す
る下記式(8) ： Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr- OH (8) で表されるペプチドとを、トリプシン又はトリプシン様
酵素の存在下で反応させることを特徴とする製造方法
（方法３）を提供する。Further, the present invention is a method for producing the glucagon represented by the above formula (1), which is represented by the following general formula (7): R 1 -His- corresponding to the amino acid residues at the 1st to 12th positions. A peptide represented by Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-OH (7) (in the formula, R 1 is a protective group for the terminal amino group); The following formula (8) corresponding to the amino acid residues at positions 13 to 29: Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn- Provided is a production method (method 3), which comprises reacting the peptide represented by Thr-OH (8) with trypsin or a trypsin-like enzyme.

【００１０】更に、本発明は、上記式(1) で表されるグ
ルカゴンの製造方法であって、１位から18位のアミノ酸
残基に相当する下記一般式(9) ： Ｒ1-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-OH (9) （式中、Ｒ1は末端アミノ基の保護基である。）で表さ
れるペプチドと、19位から29位のアミノ酸残基に相当す
る下記式(10)： H-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH (10) で表されるペプチドとを、トリプシン又はトリプシン様
酵素の存在下で反応させることを特徴とする製造方法
（方法４）を提供する。Further, the present invention is a method for producing the glucagon represented by the above formula (1), which is represented by the following general formula (9) corresponding to the amino acid residues at positions 1 to 18: R 1 -His- Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-OH (9) (wherein R 1 is a protection of the terminal amino group. Represented by the following formula (10) corresponding to the amino acid residues at positions 19 to 29: H-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met- There is provided a production method (method 4), which comprises reacting with a peptide represented by Asn-Thr-OH (10) in the presence of trypsin or a trypsin-like enzyme.

【００１１】更に、本発明は、上記式(1) で表されるグ
ルカゴンの製造方法であって、１位から18位のアミノ酸
残基に相当する下記一般式(11)： Ｒ1-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys(Ｒ6)-Tyr-Leu-Asp-Ser-Arg-Arg-OH (11) （式中、Ｒ1は末端アミノ基の保護基であり、Ｒ6はリジ
ンの側鎖のアミノ基の保護基である。）で表されるペプ
チドと、上記式(10)で表されるペプチドとを、トリプシ
ン又はトリプシン様酵素の存在下で反応させることを特
徴とする製造方法（方法５）を提供する。Further, the present invention is a method for producing the glucagon represented by the above formula (1), which is represented by the following general formula (11) corresponding to the amino acid residues at positions 1 to 18: R 1 -His- Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys (R 6 ) -Tyr-Leu-Asp-Ser-Arg-Arg-OH (11) (wherein R 1 is a terminal a protecting group for an amino group, R 6 is a protecting group for the amino group of the side chain of lysine. and peptide represented by), and a peptide represented by the formula (10), trypsin or trypsin-like enzymes The present invention provides a production method (method 5), which comprises reacting in the presence of

【００１２】更に、本発明は、上記式(1) で表されるグ
ルカゴンの製造方法であって、前記グルカゴンを少なく
とも２個のフラグメントに分けて各フラグメントを液相
中で合成し、次いで液相中で各フラグメントを反応させ
てカップリングすることを特徴とする製造方法（方法
６）を提供する。本明細書において、アミノ酸、ペプチ
ド、保護基、溶媒その他に関して略号で表示する場合、
国際純正及び応用化学連合（ＩＵＰＡＣ）、国際生化学
連合（ＩＢＵ）の規定或いは該当分野における慣用記号
に従うものとする。ただしアミノ酸等に関し光学異性体
がありうる場合は、特に明示しなければＬ体を示すもの
とする。以下、その例を示す。Further, the present invention is a method for producing the glucagon represented by the above formula (1), wherein the glucagon is divided into at least two fragments and each fragment is synthesized in a liquid phase, and then the liquid phase is synthesized. There is provided a production method (method 6), which comprises reacting and coupling each fragment therein. In the present specification, when an abbreviation is used for amino acids, peptides, protecting groups, solvents, etc.,
The rules of the International Union of Pure and Applied Chemistry (IUPAC), the International Union of Biochemistry (IBU), or the conventional symbols in the relevant fields shall be followed. However, when an amino acid and the like may have optical isomers, the L form is shown unless otherwise specified. The example is shown below.

【００１３】 Ｔｙｒ：チロシン残基 Ｇｌｙ：グリシ
ン残基 Ｓｅｒ：セリン残基 Ａｒｇ：アルギ
ニン残基 Ａｓｐ：アスパラギン酸残基 Ｌｙｓ：リジン
残基 Ｌｅｕ：ロイシン残基 Ｔｈｒ：スレオ
ニン残基 Ｐｈｅ：フェニルアラニン残基 Ｇｌｎ：グルタ
ミン残基 Ｖａｌ：バリン残基 Ａｓｎ：アスパ
ラギン残基 Ｈｉｓ：ヒスチジン残基 Ａｌａ：アラニ
ン残基 Ｔｒｐ：トリプトファン残基 Ｍｅｔ：メチオ
ニン残基 Ａｏｃ ：t-アミルオキシカルボニル基 Ｂｏｃ ：t-ブトキシカルボニル基 Ｂｏｍ ：ベンジルオキシメチル基 Ｆｍｏｃ ：9-フルオレニルメチルオキシカルボニル基 Ｂｚｌ ：ベンジル基 Ｚ ：ベンジルオキシカルボニル基 Ｔｏｓ ：トシル基 ＯＭｅ ：メチルエステル ＯＢｚｌ ：ベンジルエステル ＯｃＨｅｘ：シクロヘキシルエステル ＯＢｕｔ ： t-ブチルエステル Ｂｕｔ ： t-ブチル基 ＯＮＰ ： p-ニトロフェニルエステル ＯＳｕ ： N-ヒドロキシコハク酸イミドエステル ＴＦＡ ：トリフルオロ酢酸 ＴＨＦ ：テトラヒドロフラン ＤＭＦ ：ジメチルホルムアミド ＤＣＣ ：ジシクロヘキシルカルボジイミド ＤＣＭ ：ジクロロメタン ＷＳＣ ： N-エチル-N’-ジメチルアミノプロピル-
カルボジイミド ＨＯＳｕ ： N-ヒドロキシコハク酸イミド ＨＯＢｔ ： 1-ヒドロキシベンゾトリアゾ-ル ＭｅＯＨ ：メタノール ＥｔＯＨ ：エタノール ＡｃＯＨ ：酢酸 ＤＩＥＡ ：ジイソプロピルエチルアミン方法１ 方法１は、上記式(1) で表されるグルカゴンを、いわゆ
るステップワイズ伸長法にて固相合成するものである。
より詳細には、メリフィ−ルド（ Merrifield.R.B.）の
方法［Solid phase peptide synthesis, J.Amer.Chem.S
oc., 85, 2149-2159 (1963) ］に従って行うことができ
る。まず、カルボキシル基と結合可能な官能基を有する
不溶性樹脂に、アミノ基保護スレオニンのカルボキシル
基を結合させて前記樹脂に29位のスレオニン残基を導入
する。次いで該スレオニン残基のアミノ基の保護基を除
去して得られた遊離のアミノ基に、上記式(1) で表され
るアミノ酸配列に従って、カルボキシル基を通常の方法
で活性化させたアミノ基保護アミノ酸を順次ペプチド結
合させてはそのアミノ基の保護基の除去するという操作
を繰り返して28位のアスパラギン残基から１位のヒスチ
ジン残基まで一つずつ延長する。続いて得られたペプチ
ドを前記樹脂から脱離させる。また、脱離させたペプチ
ドのＮ末端のアミノ基の保護基を除去し、側鎖に官能基
を有するアミノ酸残基の該官能基が保護されている場合
にはその保護基を除去することにより、グルカゴンを製
造することができる。Tyr: Tyrosine residue Gly: Glycine residue Ser: Serine residue Arg: Arginine residue Asp: Aspartic acid residue Lys: Lysine residue Leu: Leucine residue Thr: Threonine residue Phe: Phenylalanine residue Gln : Glutamine residue Val: Valine residue Asn: Asparagine residue His: Histidine residue Ala: Alanine residue Trp: Tryptophan residue Met: Methionine residue Aoc: t-amyloxycarbonyl group Boc: t-butoxycarbonyl group Bom : Benzyloxymethyl group Fmoc: 9-fluorenylmethyloxycarbonyl group Bzl: benzyl group Z: benzyloxycarbonyl group Tos: tosyl group OMe: methyl ester OBzl: benzyl ester OcHex: cyclohexyl ester OBT: t-butyl ester But: t-butyl group ONP: p-nitrophenyl ester OSu: N-hydroxysuccinimide ester TFA: trifluoroacetic acid THF: tetrahydrofuran DMF: dimethylformamide DCC: dicyclohexylcarbodiimide DCM: dichloromethane WSC: N-ethyl-N'-dimethylaminopropyl-
Carbodiimide HOSu: N-Hydroxysuccinimide HOBt: 1-Hydroxybenzotriazol MeOH: Methanol EtOH: Ethanol AcOH: Acetic acid DIEA: Diisopropylethylamine Method 1 Method 1 uses the glucagon represented by the above formula (1), Solid-phase synthesis is carried out by the so-called stepwise extension method.
More specifically, the method of Merrifield (RB) [Solid phase peptide synthesis, J.Amer.Chem.S]
oc., 85, 2149-2159 (1963)]. First, the carboxyl group of amino group-protected threonine is bound to an insoluble resin having a functional group capable of binding to a carboxyl group, and the threonine residue at position 29 is introduced into the resin. Then, the free amino group obtained by removing the protecting group of the amino group of the threonine residue is added to the amino group obtained by activating the carboxyl group by a usual method according to the amino acid sequence represented by the above formula (1). The procedure of sequentially peptide-bonding protected amino acids and removing the protecting group of the amino group is repeated to extend from the asparagine residue at position 28 to the histidine residue at position 1 one by one. The resulting peptide is subsequently released from the resin. In addition, by removing the protecting group of the amino group at the N-terminal of the eliminated peptide and removing the protecting group when the functional group of an amino acid residue having a functional group in the side chain is protected. , Can produce glucagon.

【００１４】方法１の固相合成は、溶媒の存在下に行
う。溶媒としては、ペプチド結合形成に使用し得ること
が知られている各種のもの、例えば、無水又は含水のジ
メチルホルムアミド（ＤＭＦ）、ジメチルスルホキシド
（ＤＭＳＯ）、ピリジン、クロロホルム、ジオキサン、
ジクロロメタン（ＤＣＭ）、テトラヒドロフラン（ＴＨ
Ｆ）、酢酸エチル、Ｎ−メチルピロリドン、ヘキサメチ
ルリン酸トリアミド（ＨＭＰＡ）等を、１種単独で、あ
るいは２種以上の混合溶媒として用いることができる。The solid phase synthesis of Method 1 is carried out in the presence of a solvent. As the solvent, various solvents known to be usable for peptide bond formation, for example, anhydrous or hydrous dimethylformamide (DMF), dimethylsulfoxide (DMSO), pyridine, chloroform, dioxane,
Dichloromethane (DCM), tetrahydrofuran (TH
F), ethyl acetate, N-methylpyrrolidone, hexamethylphosphoric triamide (HMPA) and the like can be used alone or as a mixed solvent of two or more kinds.

【００１５】また、方法１において用いる不溶性樹脂
は、カルボキシル基と結合可能な官能基を有する不溶性
樹脂であればいずれのものも使用できる。例えば、クロ
ルメチル樹脂、オキシメチル樹脂、アミノメチル樹脂、
ベンズヒドリルアミン樹脂（ＢＨＡ樹脂）、p-メチルベ
ンズヒドリルアミン樹脂（ＭＢＨＡ樹脂）、４−アミノ
メチルフェノキシメチル樹脂、４−ヒドロキシメチルフ
ェノキシメチル樹脂、４−オキシメチルフェニルアセタ
ミドメチル樹脂（ＰＡＭ樹脂）等が挙げられるが、それ
らの中でもアミノ基を有する樹脂が好ましい。ここで、
不溶性樹脂とは、上記の溶媒に不溶性の樹脂をいう。ま
た、不溶性樹脂の主鎖は、例えば、スチレンとジビニル
ベンゼンのコポリマー等から構成される。樹脂へのアミ
ノ基保護アミノ酸の結合、及び合成されたグルカゴンの
樹脂からの脱離は、従来公知の方法で行うことができ
る。尚、グルカゴンを樹脂から脱離させる場合、該グル
カゴンの末端アミノ基の保護基の脱離に用いる試薬と同
じものを使用すると効率的である。したがって、例え
ば、末端アミノ基の保護基としてＦｍｏｃを使用する場
合、該保護基の脱離は、トリフルオロ酢酸等で行うこと
から、樹脂としては４−ヒドロキシメチルフェノキシメ
チル樹脂等を用いるのが好ましい。また、例えば、末端
アミノ基の保護基としてＢｏｃを使用する場合、該保護
基の脱離は、フッ化水素等で行うことから、樹脂として
は４−オキシメチルフェニルアセタミドメチル樹脂等を
用いるのが好ましい。The insoluble resin used in Method 1 may be any insoluble resin having a functional group capable of binding to a carboxyl group. For example, chloromethyl resin, oxymethyl resin, aminomethyl resin,
Benzhydrylamine resin (BHA resin), p-methylbenzhydrylamine resin (MBHA resin), 4-aminomethylphenoxymethyl resin, 4-hydroxymethylphenoxymethyl resin, 4-oxymethylphenylacetamidemethyl resin (PAM resin) ) And the like, but among them, a resin having an amino group is preferable. here,
The insoluble resin means a resin insoluble in the above solvent. The main chain of the insoluble resin is composed of, for example, a copolymer of styrene and divinylbenzene. The amino group-protected amino acid can be bound to the resin and the synthesized glucagon can be released from the resin by a conventionally known method. In addition, when removing glucagon from the resin, it is efficient to use the same reagent used for removing the protective group of the terminal amino group of the glucagon. Therefore, for example, when Fmoc is used as the protective group for the terminal amino group, the protective group is removed with trifluoroacetic acid or the like, and therefore it is preferable to use 4-hydroxymethylphenoxymethyl resin or the like as the resin. . Further, for example, when Boc is used as a protective group for the terminal amino group, the protective group is removed with hydrogen fluoride or the like, and therefore 4-oxymethylphenylacetamidomethyl resin or the like is used as the resin. Is preferred.

【００１６】方法１においては、17〜25位のアミノ酸残
基に対応する各アミノ酸のカップリングの反応性が他の
位置と比較して劣っている。したがって、実際にアミノ
酸残基を延長させる場合、収率を上げるためにはかかる
部分の各アミノ酸のカップリング反応を何度か繰り返し
て行う必要がある。しかしながら、不溶性樹脂として、
カルボキシル基と結合可能な官能基の含有量が、好まし
くは 0.5mmol以下、更に好ましくは 0.1〜0.4mmol のも
のを使用すると、かかる部分の反応性を向上させること
ができるので、カップリング反応を繰り返さなくても収
率を上げることができる。また、官能基の含有量を 0.5
mmol以下とすることにより、グルカゴン生成の主反応に
関与しない官能基の量を低く抑えることができることか
ら副反応が生じにくくなり、より純度の高いペプチドを
得ることができる。したがって、その後の精製操作が容
易になる。また、反応時間も短縮することができる。更
に、グルカゴンの合成に用いる各種アミノ酸又はフラグ
メント、各種試薬、溶媒の量も低く抑えることができる
ので、効率的であり、生産性が向上する。方法２ 方法２は、上記式(1) で表されるグルカゴンを、いわゆ
るステップワイズ伸長法とフラグメント縮合法とを組み
合わせた方法、即ち、１又は複数の末端アミノ基保護フ
ラグメントと、１又は複数のアミノ基保護アミノ酸（フ
ラグメントではないもの）とを用いてグルカゴンを固相
合成する方法である。ここで、フラグメントとは、アミ
ノ酸残基を少なくとも２個含むものをいい、この方法２
においては、フラグメントを少なくとも１個用いること
から、フラグメントを構成するアミノ酸残基の総数は最
低２個である。また、アミノ基保護アミノ酸（フラグメ
ントではないもの）を少なくとも１個用いることから、
１又は複数のフラグメントを構成するアミノ酸残基の総
数は最高28個である。まず、上記式(1) で表されるグル
カゴンを構成する１又は複数のフラグメントであって、
末端アミノ基を保護したものを合成する。次いで、アミ
ノ基保護スレオニンのカルボキシル基、又は合成した末
端アミノ基保護フラグメントの中に、29位のスレオニン
残基を含む末端アミノ基保護フラグメントがある場合に
は該フラグメントのカルボキシル基を、カルボキシル基
と結合可能な官能基を有する不溶性樹脂に結合させる。
続いて、前記樹脂に結合したスレオニン残基又はフラグ
メントの末端アミノ基の保護基を除去して得られた遊離
のアミノ基に、上記式(1) のアミノ酸配列にしたがって
末端アミノ基保護フラグメント及び／又は末端アミノ基
保護アミノ酸を順次ペプチド結合させて１位のヒスチジ
ン残基まで延長する。次に、得られたペプチドを前記樹
脂から脱離させる。また、脱離させたペプチドのＮ末端
のアミノ基の保護基を除去し、側鎖に官能基を有するア
ミノ酸残基の該官能基が保護されている場合にはその保
護基を除去することにより、グルカゴンを製造すること
ができる。In the method 1, the reactivity of coupling of each amino acid corresponding to the amino acid residues at positions 17 to 25 is inferior to other positions. Therefore, when actually extending the amino acid residue, it is necessary to repeat the coupling reaction of each amino acid in such a portion several times in order to increase the yield. However, as an insoluble resin,
When the content of the functional group capable of binding to the carboxyl group is preferably 0.5 mmol or less, more preferably 0.1 to 0.4 mmol, the reactivity of such a portion can be improved, and thus the coupling reaction is repeated. Even without it, the yield can be increased. In addition, the functional group content should be 0.5
When the amount is less than or equal to mmol, the amount of the functional group that is not involved in the main reaction of glucagon formation can be suppressed to a low level, so that side reactions are less likely to occur and a peptide with higher purity can be obtained. Therefore, the subsequent purification operation becomes easy. Also, the reaction time can be shortened. Furthermore, the amounts of various amino acids or fragments, various reagents, and solvents used in the synthesis of glucagon can be kept low, which is efficient and improves productivity. Method 2 Method 2 is a method in which glucagon represented by the above formula (1) is combined with a so-called stepwise extension method and a fragment condensation method, that is, one or more terminal amino group-protected fragments and one or more This is a method for solid-phase synthesis of glucagon using an amino group-protected amino acid (not a fragment). Here, the fragment means a fragment containing at least two amino acid residues.
In, since at least one fragment is used, the total number of amino acid residues constituting the fragment is at least two. In addition, since at least one amino group-protected amino acid (which is not a fragment) is used,
The total number of amino acid residues constituting one or more fragments is 28 at maximum. First, one or more fragments constituting the glucagon represented by the above formula (1),
A product in which the terminal amino group is protected is synthesized. Then, if the carboxyl group of the amino group-protected threonine, or the synthesized terminal amino group-protected fragment has a terminal amino group-protected fragment containing the threonine residue at the 29-position, the carboxyl group of the fragment is replaced with the carboxyl group. It is bonded to an insoluble resin having a bondable functional group.
Then, the free amino group obtained by removing the protecting group of the terminal amino group of the threonine residue or fragment bound to the resin is added to the terminal amino group-protecting fragment and / or Alternatively, a terminal amino group-protected amino acid is sequentially peptide-bonded and extended to the histidine residue at the 1-position. Next, the obtained peptide is released from the resin. In addition, by removing the protecting group of the amino group at the N-terminal of the eliminated peptide and removing the protecting group when the functional group of an amino acid residue having a functional group in the side chain is protected. , Can produce glucagon.

【００１７】この方法２は、溶媒の存在下で行い、溶媒
としては、方法１で例示したものと同様のものが挙げら
れる。また、不溶性樹脂としても、方法１で例示したも
のと同様のものが挙げられる。また、方法２において、
29位のスレオニン残基を含む末端アミノ基保護フラグメ
ントを不溶性樹脂に結合させる場合、29位のスレオニン
残基のカルボキシル基を結合させてもよく、また、下記
一般式(2) ： Ｒ1-Ｘ-Asp-Thr( Ｒ3)-Ｒ2 (2) （式中、Ｒ1は末端アミノ基の保護基であり、Ｒ2は末端
カルボキシル基の保護基であり、Ｒ3はスレオニン残基
の側鎖の水酸基の保護基であり、Ｘは化学結合、Met、
又はLeu-Met である。）で表されるペプチドのアスパラ
ギン酸残基の側鎖のカルボキシル基を不溶性樹脂に結合
させてもよい。上記一般式(2) で表されるペプチドを用
いる場合、不溶性樹脂としては、最終的に得られたグル
カゴンを該樹脂から脱離させた時に樹脂への結合に関与
する上記アスパラギン酸のカルボキシル基がアミド化さ
れて、アスパラギン残基となるような樹脂を用いる。そ
のような不溶性樹脂としては、例えば、ベンズヒドリル
アミン樹脂、メチルベンズヒドリルアミン樹脂等が挙げ
られ、比較的安価なものを使用することができる。This method 2 is carried out in the presence of a solvent, and examples of the solvent include the same as those exemplified in the method 1. Further, as the insoluble resin, those similar to those exemplified in Method 1 can be mentioned. Also, in method 2,
When the terminal amino group-protected fragment containing the threonine residue at the 29-position is bound to an insoluble resin, the carboxyl group at the threonine residue at the 29-position may be bound, and the following general formula (2): R 1 -X -Asp-Thr (R 3 ) -R 2 (2) (wherein R 1 is a terminal amino group protecting group, R 2 is a terminal carboxyl group protecting group, and R 3 is a threonine residue side) X is a protective group for the hydroxyl group of the chain, X is a chemical bond, Met,
Or Leu-Met. The side chain carboxyl group of the aspartic acid residue of the peptide represented by () may be bound to the insoluble resin. When using the peptide represented by the general formula (2), as the insoluble resin, the carboxyl group of the aspartic acid involved in binding to the resin when the glucagon finally obtained is released from the resin, A resin that is amidated to an asparagine residue is used. Examples of such an insoluble resin include benzhydrylamine resin and methylbenzhydrylamine resin, and relatively inexpensive resins can be used.

【００１８】また、この方法２において、フラグメント
として19位から25位のアミノ酸残基に相当する下記一般
式(3) ： Ｒ1-Ala-Gln-Asp(Ｒ4)-Phe-Val-Gln-Trp-OH (3) （式中、Ｒ1は前記のとおりであり、Ｒ4はアスパラギン
酸残基の側鎖のカルボキシル基の保護基である。）で表
されるヘプタペプチドを用いるのが好ましい。上記方法
１で述べたように、17〜25位の各アミノ酸のカップリン
グの反応性は比較的低いが、上記一般式(3)で表される
ヘプタペプチドを用いることにより、カップリングを繰
り返さなくても収率を上げることができ、効率的であ
る。しかも副反応も低減することができるのでより高純
度のペプチドを得ることができることから、その後の精
製操作が容易になる。したがって、精製の回数や時間を
減らすことができ、精製に用いる溶媒の量も低減するこ
とができ、生産性が向上する。また、カップリングを繰
り返さなくてよいことから、アミノ基保護アミノ酸、反
応試薬、溶媒等の使用量を低減することができ、経済的
にも有利である。In this method 2, the following general formula (3) corresponding to the amino acid residues at positions 19 to 25 as a fragment: R 1 -Ala-Gln-Asp (R 4 ) -Phe-Val-Gln- It is preferable to use a heptapeptide represented by Trp-OH (3) (wherein R 1 is as described above and R 4 is a protecting group for a carboxyl group of the side chain of an aspartic acid residue). . As described above in Method 1, although the reactivity of coupling each amino acid at the 17th to 25th positions is relatively low, by using the heptapeptide represented by the above general formula (3), the coupling can be repeated. However, the yield can be increased and it is efficient. Moreover, side reactions can be reduced, and a peptide with higher purity can be obtained, which facilitates the subsequent purification operation. Therefore, the number and time of purification can be reduced, the amount of solvent used for purification can be reduced, and productivity is improved. Further, since the coupling does not have to be repeated, the amount of amino group-protected amino acid, reaction reagent, solvent, etc. can be reduced, which is economically advantageous.

【００１９】更に、方法２において、フラグメントとし
て、17位から25位のアミノ酸残基に相当する下記一般式
(4) ： Ｒ1-Arg(Ｒ5)-Arg(Ｒ5)-Ala-Gln-Asp(Ｒ4)-Phe-Val-Gln-Trp-OH (4) （式中、Ｒ1及びＲ4は前記のとおりであり、Ｒ5はアル
ギニン残基の側鎖のグアニジノ基の保護基である。）で
表されるノナペプチドを用いるのも、上記一般式(3) で
表されるヘプタペプチドを用いた場合と同様の理由で好
ましい。Further, in the method 2, as a fragment, the following general formula corresponding to the amino acid residues at positions 17 to 25 is given.
(4): R 1 -Arg (R 5 ) -Arg (R 5 ) -Ala-Gln-Asp (R 4 ) -Phe-Val-Gln-Trp-OH (4) (wherein R 1 and R 4 Is as described above, and R 5 is a protecting group for the guanidino group of the side chain of the arginine residue.) Is also used for the heptapeptide represented by the above general formula (3). It is preferable for the same reason as described above.

【００２０】更に、方法２において、フラグメントとし
て、19位から24位のアミノ酸残基に相当する下記一般式
(5) ： Ｒ1-Ala-Gln-Asp(Ｒ4)-Phe-Val-Gln-OH (5) （式中、Ｒ1及びＲ4は前記のとおりである。）で表され
るヘキサペプチドを用いるのも、上記一般式(3) で表さ
れるヘプタペプチドを用いた場合と同様の理由で好まし
い。また、かかるヘキサペプチドにはトリプトファン残
基が含まれていないことから、その合成が容易である。Further, in the method 2, as a fragment, the following general formula corresponding to the amino acid residues at positions 19 to 24 is given.
(5): Hexapeptide represented by R 1 -Ala-Gln-Asp (R 4 ) -Phe-Val-Gln-OH (5) (wherein R 1 and R 4 are as described above). It is also preferable to use for the same reason as when using the heptapeptide represented by the general formula (3). Moreover, since such a hexapeptide does not contain a tryptophan residue, its synthesis is easy.

【００２１】更に、方法２において、フラグメントとし
て、17位から24位のアミノ酸残基に相当する下記一般式
(6) ： Ｒ1-Arg(Ｒ5)-Arg(Ｒ5)-Ala-Gln-Asp( Ｒ4)-Phe-Val-Gln-OH (6) （式中、Ｒ1、Ｒ4及びＲ5は前記のとおりである。）で
表されるオクタペプチドを用いるのも、上記一般式(5)
で表されるヘキサペプチドを用いた場合と同様の理由で
好ましい。方法３ 方法３は、１位から12位のアミノ酸残基に相当する上記
一般式(7) で表されるペプチドと、13位から29位のアミ
ノ酸残基に相当する上記式(8) で表されるペプチドと
を、トリプシン又はトリプシン様酵素の存在下で反応さ
せるものである。Further, in the method 2, as a fragment, the following general formula corresponding to the amino acid residues at positions 17 to 24 is given:
(6): R 1 -Arg (R 5 ) -Arg (R 5 ) -Ala-Gln-Asp (R 4 ) -Phe-Val-Gln-OH (6) (wherein R 1 , R 4 and R 5 is as described above.) It is also possible to use the octapeptide represented by the general formula (5)
It is preferable for the same reason as the case of using the hexapeptide represented by. Method 3 Method 3 is represented by the peptide represented by the general formula (7) corresponding to amino acid residues 1 to 12 and the formula (8) corresponding to amino acid residues 13 to 29. The resulting peptide is reacted in the presence of trypsin or a trypsin-like enzyme.

【００２２】トリプシンは、タンパク質分解酵素の一つ
である。タンパク質分解酵素は、主としてペプチド結合
の開裂に使用されてきたが、その逆反応であるペプチド
結合の生成反応にも関与し得ることは古くから知られて
いる。しかし、そのようなタンパク質分解酵素を用いた
ペプチド結合生成反応は主にオリゴペプチド等に使用さ
れ、ほとんど直鎖ペプチドに限られている。Trypsin is one of the proteolytic enzymes. Proteolytic enzymes have been mainly used for the cleavage of peptide bonds, but it has long been known that they can be involved in the reverse reaction, that is, peptide bond formation reaction. However, such a peptide bond-forming reaction using a proteolytic enzyme is mainly used for oligopeptides and the like, and is mostly limited to linear peptides.

【００２３】本発明で用いるトリプシンは、国際生化学
連合(I.U.B) 酵素委員会に酵素番号EC.3.4.21.4 として
登録されており、ウシ膵から得られる酵素である。トリ
プシンは、フナコシ社等から市販されている。この反応
は、通常、pH４〜10、好ましくはpH５〜８の緩衝液を含
む媒質中で行われる。また、反応は、通常、０〜50℃、
好ましくは20〜40℃の範囲で行う。The trypsin used in the present invention is an enzyme obtained from bovine pancreas, which is registered in the International Union of Biochemistry (IUB) Enzyme Committee as Enzyme No. EC.3.4.21.4. Trypsin is commercially available from Funakoshi and the like. This reaction is usually carried out in a medium containing a buffer having a pH of 4 to 10, preferably pH 5 to 8. The reaction is usually 0 to 50 ° C,
It is preferably carried out in the range of 20 to 40 ° C.

【００２４】緩衝液としては、pH値が上記範囲内のもの
であればその種類は特に限定されることなくいずれのも
のも使用することができる。例えば、トリス塩酸緩衝
液、マツクイルベイン緩衝液、リン酸緩衝液、酢酸アン
モニウム緩衝液、アトキンス＆パンチン氏緩衝液、ベロ
ナール緩衝液等が挙げられる。上記のような緩衝液を反
応媒質として使用する場合、該緩衝液は、通常、水混和
性有機溶媒と混合して使用される。その水混和性有機溶
媒としては、例えば、ジメチルホルムアミド（ＤＭ
Ｆ）、ジメチルスルホキシド（ＤＭＳＯ）、ジメチルイ
ミダゾリジノン（ＤＭＩ）、ヘキサメチルホスホリルト
リアミド（ＨＭＰＡ）、メタノール（ＭｅＯＨ）、エタ
ノール（ＥｔＯＨ）等が挙げられ、中でも、特にジメチ
ルホルムアミド、メタノール、及びエタノールが好まし
い。これらの有機溶媒は１種単独で、又は２種以上の組
み合わせで使用することができる。上記緩衝液と水混和
性有機溶媒の混合割合は、緩衝液対有機溶媒の容積比
で、一般的には２：８乃至８：２の範囲であり、好まし
くは５：７乃至７：５の範囲である。The buffer solution is not particularly limited in its kind as long as it has a pH value within the above range, and any one can be used. For example, a Tris-hydrochloric acid buffer solution, a pineapple buffer solution, a phosphate buffer solution, an ammonium acetate buffer solution, an Atkins &Pantin's buffer solution, a veronal buffer solution and the like can be mentioned. When the above buffer solution is used as a reaction medium, the buffer solution is usually used by mixing with a water-miscible organic solvent. Examples of the water-miscible organic solvent include dimethylformamide (DM
F), dimethylsulfoxide (DMSO), dimethylimidazolidinone (DMI), hexamethylphosphoryltriamide (HMPA), methanol (MeOH), ethanol (EtOH), and the like, among which dimethylformamide, methanol, and ethanol are particularly preferable. Is preferred. These organic solvents can be used alone or in combination of two or more. The mixing ratio of the buffer solution and the water-miscible organic solvent is a volume ratio of the buffer solution to the organic solvent, and is generally in the range of 2: 8 to 8: 2, preferably 5: 7 to 7: 5. It is a range.

【００２５】トリプシン又はトリプシン様酵素の使用量
は、特に制限はなく、反応条件に応じて適宜変えること
ができる。また、反応は、トリプシン又はトリプシン様
酵素を、一般的な方法、例えば、担体結合法、架橋法、
包括法、その他の方法により固定化した固定化酵素を利
用して行うことができる。担体結合法において用いる担
体としては、例えば、セルロース、デキストラン、アガ
ロース等の多糖類の誘導体、ポリアクリルアミドゲル、
多孔性ガラス等が挙げられる。架橋法において用いる架
橋試薬としては、例えば、グルタールアルデヒド、ビス
ジアゾベンジジン、N,N-ポリメチレンビスヨードアセト
アミド、N,N-エチレンビスマレインイミド等が挙げられ
る。包括法で用いる素材としては、例えば、ポリアクリ
ルアミドゲル、ポリアクリルアルコールゲル、デンプ
ン、コンニャク粉、ナイロン、ポリウレア、ポリスチレ
ン、エチルセルロース、コロジオン、硝酸セルロース等
が挙げられる。但し、固定化法は何らこれらに限定され
るものではない。The amount of trypsin or trypsin-like enzyme used is not particularly limited and can be appropriately changed depending on the reaction conditions. In addition, the reaction is carried out using trypsin or a trypsin-like enzyme by a general method, for example, a carrier binding method, a crosslinking method,
It can be carried out using an immobilized enzyme immobilized by the encapsulation method or another method. The carrier used in the carrier binding method, for example, cellulose, dextran, derivatives of polysaccharides such as agarose, polyacrylamide gel,
Porous glass etc. are mentioned. Examples of the cross-linking reagent used in the cross-linking method include glutaraldehyde, bisdiazobenzidine, N, N-polymethylenebisiodoacetamide, N, N-ethylenebismaleinimide and the like. Examples of materials used in the encapsulation method include polyacrylamide gel, polyacrylic alcohol gel, starch, konjak powder, nylon, polyurea, polystyrene, ethyl cellulose, collodion, and cellulose nitrate. However, the immobilization method is not limited to these.

【００２６】上記一般式(8) で表されるペプチドとして
は、方法１〜方法２によりグルカゴンを製造する際の中
間体を用いることができる。また、上記一般式(7) で表
されるペプチドは、例えば、方法１に準じて、ステップ
ワイズ伸長法により固相合成することができる。この方
法３によれば、予め精製した上記一般式(7) で表される
ペプチドフラグメント及び上記一般式(8) で表されるペ
プチドフラグメントを使用することから、酵素反応によ
りグルカゴンを合成した後の精製操作が容易になる。即
ち、精製の回数を少なくすることができるので、精製に
使用する溶媒の量を低減することができ、時間も短縮す
ることができる。その上、精製によるロスも低減するこ
とができ、収率を上げることができる。また、酵素反応
を利用することから、反応が特異的で、ラセミ化などの
副反応が生じにくい。As the peptide represented by the above general formula (8), an intermediate for producing glucagon by methods 1 and 2 can be used. Further, the peptide represented by the above general formula (7) can be subjected to solid phase synthesis by the stepwise extension method according to Method 1, for example. According to this method 3, since the peptide fragment represented by the above-mentioned general formula (7) and the peptide fragment represented by the above-mentioned general formula (8) are used in advance, glucagon after synthesis by an enzymatic reaction is used. Purification operation becomes easy. That is, since the number of times of purification can be reduced, the amount of solvent used for purification can be reduced, and the time can also be shortened. In addition, the loss due to purification can be reduced and the yield can be increased. In addition, since an enzymatic reaction is used, the reaction is specific and side reactions such as racemization are unlikely to occur.

【００２７】この方法３においては、未反応物とともに
副生成物を酵素分解し、回収して再利用することができ
る。方法４ 方法４は、１位から18位のアミノ酸残基に相当する上記
一般式(9) で表されるペプチドと、19位から29位のアミ
ノ酸残基に相当する上記式(10)で表されるペプチドと
を、上記方法３と同様にトリプシン又はトリプシン様酵
素の存在下で反応させるものである。上記一般式(10)で
表されるペプチドとしては、方法１によりグルカゴンを
製造する際の中間体を用いることができる。また、上記
一般式(9)で表されるペプチドは、例えば、方法１に準
じて、ステップワイズ伸長法により固相合成することが
できる。この方法４は、上記方法３と同様の利点があ
る。方法５ 方法５は、１位から18位のアミノ酸残基に相当する上記
一般式(11)で表されるペプチドと、19位から29位のアミ
ノ酸残基に相当する上記式(10)で表されるペプチドと
を、上記方法３と同様にトリプシン又はトリプシン様酵
素の存在下で反応させるものである。上記一般式(11)で
表されるペプチドは、例えば、方法１に準じて、ステッ
プワイズ伸長法により固相合成することができる。この
方法の利点は、上記方法３と同様である。また、トリプ
シンによる分解反応を受けなくなるので、より高い収率
が得られるというメリットがある。方法６ 方法６は、グルカゴンを少なくとも２個のフラグメント
に分けて各フラグメントを液相中で合成し、次いで液相
中で各フラグメントを反応させてカップリングする、即
ち液相合成にてグルカゴンを製造するものである。この
方法６で用いる溶媒としては、方法１で例示したものと
同様のものが挙げられる。この方法６は、不溶性樹脂が
不要であり、アミノ基保護アミノ酸や、反応試薬、溶媒
等の使用量を低減することができる。また、製造設備の
規模を拡大することができるので、大量製造も可能であ
る。In this method 3, by-products as well as unreacted substances can be enzymatically decomposed, recovered and reused. Method 4 Method 4 is represented by the peptide represented by the above general formula (9) corresponding to amino acid residues 1 to 18 and the above formula (10) corresponding to amino acid residues 19 to 29. The obtained peptide is reacted in the presence of trypsin or a trypsin-like enzyme in the same manner as in Method 3 above. As the peptide represented by the general formula (10), an intermediate for producing glucagon by Method 1 can be used. Further, the peptide represented by the above general formula (9) can be subjected to solid-phase synthesis by the stepwise extension method according to Method 1, for example. This method 4 has the same advantages as the above method 3. Method 5 Method 5 is represented by the peptide represented by the general formula (11) corresponding to amino acid residues 1 to 18 and the formula (10) corresponding to amino acid residues 19 to 29. The obtained peptide is reacted in the presence of trypsin or a trypsin-like enzyme in the same manner as in Method 3 above. The peptide represented by the above general formula (11) can be subjected to solid phase synthesis by the stepwise extension method according to Method 1, for example. The advantage of this method is the same as that of the above method 3. Further, there is an advantage that a higher yield can be obtained because the decomposition reaction by trypsin is not performed. Method 6 Method 6 divides glucagon into at least two fragments and synthesizes each fragment in the liquid phase, and then reacts and couples each fragment in the liquid phase, that is, produces glucagon by liquid phase synthesis. To do. As the solvent used in this method 6, the same solvents as those exemplified in method 1 can be mentioned. This method 6 does not require an insoluble resin and can reduce the amounts of amino group-protected amino acids, reaction reagents, solvents and the like used. Further, since the scale of manufacturing equipment can be expanded, mass production is also possible.

【００２８】上記の本発明のグルカゴンの製造方法１〜
６においては、全て、アミノ酸のペプチド結合に関与す
るアミノ基への保護基の結合及び該保護基の脱離、並び
にアミノ酸のペプチド結合に関与するカルボキシル基の
活性化が必要である。また、必要に応じてアミノ酸の側
鎖の官能基にも保護基を結合する。これらの操作は、公
知の方法に従って行うことができる。例えば、「ザ．ペ
プチド(The Peptides)」第1巻(1966年)[Schreder and L
uhke著、Academic Press ,NewYork,U.S.A.] 、あるいは
「ペプチド合成」［泉屋ら著、丸善株式会社(1975年)］
に記載された方法に従い、例えば、アジド法、酸クロラ
イド法、酸無水物法、混合酸無水物法、ＤＣＣ法、活性
エステル法（P-ニトロフエニルエステル法、Ｎ−ヒドロ
キシコハク酸イミドエステル法、シアノメチルエステル
法等）、ウッドワ−ド試薬Ｋを用いる方法、カルボイミ
ダゾ−ル法、酸化還元法、ＤＣＣ−アデイテイブ（ＨＯ
ＮＢ、ＨＯＢｔ、ＨＯＳｕ）法等に従って行うことがで
き、固相合成及び液相合成のいずれにも適用できる。The above-mentioned glucagon production method 1 of the present invention
In 6 all, it is necessary to attach a protecting group to the amino group involved in the peptide bond of the amino acid and to remove the protecting group, and to activate the carboxyl group involved in the peptide bond of the amino acid. In addition, a protective group is also attached to the functional group of the side chain of the amino acid, if necessary. These operations can be performed according to known methods. For example, "The Peptides" Volume 1 (1966) [Schreder and L
uhke, Academic Press, NewYork, USA], or "peptide synthesis" [Izumiya et al., Maruzen Co., Ltd. (1975)]
In accordance with the method described in, for example, the azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method (P-nitrophenyl ester method, N-hydroxysuccinimide ester method) , Cyanomethyl ester method, etc.), a method using Woodward reagent K, a carboimidazole method, a redox method, DCC-additive (HO
NB, HOBt, HOSu) method and the like, and can be applied to both solid phase synthesis and liquid phase synthesis.

【００２９】本発明のグルカゴンの製造において、アミ
ノ基を保護する場合に用いる保護基としては、通常用い
られているものを挙げることができ、例えばベンジルオ
キシカルボニル（Ｚ）、 t-ブトキシカルボニル（Ｂo
c）、t-アミルオキシカルボニル（Ａoc）、イソボニル
オキシカルボニル、p-メトキシベンジルオキシカルボニ
ル、2-クロル-ベンジルオキシカルボニル、アダマンチ
ルオキシカルボニル、トリフルオロアセチル、フタロイ
ル、ホルミル、o-ニトロフェニルスルフェニル、ジフェ
ニルホスフィノチオイルなどの基が挙げられる。アミノ
基への保護基の結合及び脱離は、上記のような従来公知
の方法で行うことができる。In the production of glucagon of the present invention, as the protecting group used for protecting the amino group, those commonly used can be mentioned, for example, benzyloxycarbonyl (Z), t-butoxycarbonyl (Bo
c), t-amyloxycarbonyl (Aoc), isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-chloro-benzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, o-nitrophenylsulfenyl And groups such as diphenylphosphinothioyl. The protecting group can be bound to and removed from the amino group by the conventionally known method as described above.

【００３０】また、本発明のグルカゴンの製造におい
て、カルボキシル基を保護する場合に用いる保護基とし
ては、通常用いられているものを挙げることができ、例
えば、メチルエステル、エチルエステル、プロピルエス
テル、ブチルエステル、tert−ブチルエステル等のアル
キルエステル、ベンジルエステル、p-ニトロベンジルエ
ステル、メチルベンジルエステル、p-クロロベンジルエ
ステル、ベンズヒドリルエステル、ベンジルオキシカル
ボニルヒドラジド、tert-ブチルオキシカルボニルヒド
ラジド、トリチルヒドラジド等が挙げられる。これらの
中で好ましいものは、である。カルボキシル基への保護
基の結合及び脱離は、上記のような従来公知の方法で行
うことができる。In the production of the glucagon of the present invention, the protecting group used for protecting the carboxyl group may be one commonly used, for example, methyl ester, ethyl ester, propyl ester and butyl. Ester, alkyl ester such as tert-butyl ester, benzyl ester, p-nitrobenzyl ester, methylbenzyl ester, p-chlorobenzyl ester, benzhydryl ester, benzyloxycarbonyl hydrazide, tert-butyloxycarbonyl hydrazide, trityl hydrazide, etc. Can be mentioned. Among these, preferred is. The protecting group can be attached to and removed from the carboxyl group by the conventionally known method as described above.

【００３１】ペプチド結合に関与するカルボキシル基の
活性化も、上記のような従来公知の方法にて行うことが
でき、用いられる試薬等も公知のものから適宜選択し得
る。例えば、カルボキシル基を活性化するために、該カ
ルボキシル基と種々の試薬を反応させて、例えば、対応
する酸クロライド、酸無水物又は混合酸無水物、アジ
ド、活性エステル（例えば、ペンタクロロフェノ−ル、
p-ニトロフェノ−ル、Ｎ−ヒドロキシコハク酸イミド、
Ｎ−ヒドロキシベンズトリアゾ−ル、Ｎ−ヒドロキシ−
５−ノルボルネン−２，３−ジカルボキシイミド等との
エステル）等を形成させればよい。The activation of the carboxyl group involved in the peptide bond can also be carried out by the conventionally known method as described above, and the reagents and the like used can be appropriately selected from the known ones. For example, in order to activate the carboxyl group, various reagents are reacted with the carboxyl group to form, for example, the corresponding acid chloride, acid anhydride or mixed acid anhydride, azide, active ester (for example, pentachloropheno- Le,
p-nitrophenol, N-hydroxysuccinimide,
N-hydroxybenztriazole, N-hydroxy-
5-norbornene-2,3-dicarboximide, etc.) and the like may be formed.

【００３２】本発明においてグルカゴンの製造に用いる
アミノ酸の中で、側鎖に官能基を有するものについて
は、ペプチド結合形成反応中はその官能基は保護されて
いるのが好ましく、特に、Ｈis、Ｔyr、Ｔhr、Ｌys、Ａ
sp、Ａrg及びＳerについては、その側鎖の官能基を保護
しておくのが好ましい。官能基の保護は、通常用いられ
ている方法で保護基を結合させることにより行われる。
グルカゴンの合成終了後、それらの保護基は脱離され
る。尚、側鎖カルボキシル基が保護されていない場合は
反応に関与すべき官能基をあらかじめ対称酸無水物法、
活性エステル法等で活性化し、ペプチド結合形成反応を
行うのが望ましい。Among the amino acids used for the production of glucagon in the present invention, those having a functional group in the side chain are preferably protected during the peptide bond formation reaction, particularly His and Tyr. , Thr, Lys, A
Regarding sp, Arg and Ser, it is preferable to protect the functional group of the side chain thereof. The protection of the functional group is carried out by attaching a protecting group by a commonly used method.
After the synthesis of glucagon is complete, those protecting groups are removed. Incidentally, when the side chain carboxyl group is not protected, the functional group to be involved in the reaction is previously symmetric acid anhydride method,
It is desirable to activate by an active ester method or the like to carry out a peptide bond forming reaction.

【００３３】Ｈisのイミノ基の保護基としては、例え
ば、ベンジルオキシメチル（Ｂｏｍ）、トシル（Ｔo
s）、ベンジル（Ｂzl）、ベンジルオキシカルボニル
（Ｚ）、トリチル等の基が挙げられる。Ｓer及びＴhrの
水酸基は、例えば、エステル化又はエ−テル化によって
保護することができるが、必ずしも保護する必要はな
い。エステル化によって導入される保護基としては、例
えば、アセチル等の低級アルカノイル基、ベンゾイル等
のアロイル基、ベンゾイルオキシカルボニル、エチルオ
キシカルボニル等の炭酸から誘導される基等が好適に用
いられる。またエ−テル化によって導入される保護基と
しては、例えば、ベンジル（Ｂzl）、テトラヒドロピラ
ニル、tert−ブチル等の基が好適に用いられる。Examples of protecting groups for the imino group of His include benzyloxymethyl (Bom) and tosyl (To
s), benzyl (Bzl), benzyloxycarbonyl (Z), trityl and the like. The hydroxyl groups of Ser and Thr can be protected by, for example, esterification or etherification, but they are not necessarily protected. As the protective group introduced by esterification, for example, a lower alkanoyl group such as acetyl, an aroyl group such as benzoyl, a group derived from carbonic acid such as benzoyloxycarbonyl, ethyloxycarbonyl and the like are preferably used. As the protective group introduced by etherification, for example, groups such as benzyl (Bzl), tetrahydropyranyl, tert-butyl are preferably used.

【００３４】Ｔyrの水酸基の保護基としては、例えばベ
ンジル（Ｂzl）、ブロモベンジルオキシカルボニル（Ｂ
ｒＺ）、ジクロロベンジルオキシ（Ｃｌ2-Ｂzl）、ベン
ジルオキシカルボニル（Ｚ）、アセチル、トシル（Ｔo
s）等の基が挙げられる。Ｌysのアミノ基の保護基とし
ては、例えば、ベンジルオキシカルボニル（Ｚ）、クロ
ロベンジルオキシカルボニル（Ｃｌ−Ｚ）、ジクロロベ
ンジルオキシ（Ｃｌ2-Ｂzl）、t-ブトキシカルボニル
（Ｂoc）、トシル（Ｔos）等の基が挙げられる。Examples of the protective group for the hydroxyl group of Tyr include benzyl (Bzl) and bromobenzyloxycarbonyl (B
rZ), dichlorobenzyloxy (Cl 2 -Bzl), benzyloxycarbonyl (Z), acetyl, tosyl (To
and groups such as s). Examples of the protecting group for the amino group of Lys include benzyloxycarbonyl (Z), chlorobenzyloxycarbonyl (Cl-Z), dichlorobenzyloxy (Cl 2 -Bzl), t-butoxycarbonyl (Boc), tosyl (Tos ) Etc. are mentioned.

【００３５】Ａrgのグアニジノ基の保護基としては、例
えば、トシル（Ｔos）、ニトロ、ベンジルオキシカルボ
ニル（Ｚ）、t-アミルオキシカルボニル等の基が挙げら
れる。Ａspのカルボキシル基の保護は、例えば、ベンジ
ルアルコ−ル、メタノ−ル、エタノ−ル、tert−ブタノ
−ル、シクロヘキサノール等によるエステル化により行
われる。Examples of the protecting group for the guanidino group of Arg include groups such as tosyl (Tos), nitro, benzyloxycarbonyl (Z) and t-amyloxycarbonyl. The carboxyl group of Asp is protected by, for example, esterification with benzyl alcohol, methanol, ethanol, tert-butanol, cyclohexanol or the like.

【００３６】また、必要に応じてＴrpのインドリル基も
保護される。Ｔrpの保護基としては、例えば、ホルミ
ル、ベンジルオキシカルボニル（Ｚ）、4-メトキシ-2,
3,6-トリメチルベンゼンスルホニル、2,2,2-トリクロロ
エチルオキシカルボニル等の基が挙げられる。また、必
要に応じてＭetのチオメチル基も保護される。Ｍetを保
護する方法としては、予めメチルスルホキシドにしてお
き、後に還元する方法がある。The indolyl group of Trp is also protected if necessary. Examples of the protecting group for Trp include formyl, benzyloxycarbonyl (Z), 4-methoxy-2,
Examples thereof include groups such as 3,6-trimethylbenzenesulfonyl and 2,2,2-trichloroethyloxycarbonyl. Further, the thiomethyl group of Met is also protected if necessary. As a method of protecting Met, there is a method of previously making methyl sulfoxide and then reducing it.

【００３７】尚、ペプチド結合の形成反応は、例えば、
ジシクロヘキシルカルボジイミド（ＤＣＣ）、カルボジ
イミダゾール等のカルボジイミド試薬やピロリン酸テト
ラエチル、ベンゾトリアゾ−ル−Ｎ−ヒドロキシトリス
ジメチルアミノホスホニウムヘキサフルオロリン化物塩
（Ｂｏｐ試薬）等の縮合剤の存在下に実施し得る場合も
ある。The peptide bond-forming reaction is, for example,
When it can be carried out in the presence of a carbodiimide reagent such as dicyclohexylcarbodiimide (DCC) or carbodiimidazole or a condensing agent such as tetraethyl pyrophosphate, benzotriazol-N-hydroxytrisdimethylaminophosphonium hexafluorophosphide salt (Bop reagent), etc. There is also.

【００３８】合成されたグルカゴンは、通常の方法に従
い脱塩、精製することができる。例えば、ＤＥＡＥ−セ
ルロ−ス等のイオン交換クロマトグラフィ−、セファデ
ックスＬＨ−２０、セファデックスＧ−２５等の分配ク
ロマトグラフィ−、シリカゲル等の順相クロマトグラフ
ィ−、ＯＤＳ−シリカゲル等の逆相クロマトグラフィ
−、高速液体クロマトグラフィ−等が挙げられる。The synthesized glucagon can be desalted and purified according to a usual method. For example, ion exchange chromatography such as DEAE-cellulose, partition chromatography such as Sephadex LH-20 and Sephadex G-25, normal phase chromatography such as silica gel, reverse phase chromatography such as ODS-silica gel, high speed. Liquid chromatography and the like can be mentioned.

【００３９】このようにして得られたグルカゴンは必要
に応じて医薬品として許容され得る塩、例えば、酢酸
塩、塩酸塩、リン酸塩等にすることができる。The glucagon thus obtained can be converted into a pharmaceutically acceptable salt, if necessary, such as acetate, hydrochloride, phosphate and the like.

【００４０】[0040]

【実施例】以下、本発明を実施例を挙げてより具体的に
説明するが、本発明はこれらに限定されるものではな
い。以下の実施例において、得られた純粋ペプチドの同
定は、高速液体クロマトグラフィ−（ＨＰＬＣ）の保持
時間の測定、旋光度の測定及びアミノ酸分析により行っ
た。これらの測定は、特に示さない限り、下記の測定法
及び測定条件により行った。 ・高速液体クロマトグラフィ−（ＨＰＬＣ） 高速液体クロマトグラフィ−分析には、ＬＣ−Ｍｏｄｕ
ｌｅ−１（日本ウォ−タ−ズ・リミテッド社製）を用い
た。EXAMPLES The present invention will now be described more specifically with reference to examples, but the present invention is not limited thereto. In the following examples, the obtained pure peptides were identified by high performance liquid chromatography- (HPLC) retention time measurement, optical rotation measurement and amino acid analysis. Unless otherwise specified, these measurements were performed by the following measuring methods and measuring conditions. -High performance liquid chromatography- (HPLC) For high performance liquid chromatography-analysis, LC-Modu
le-1 (manufactured by Nippon Waters Limited) was used.

【００４１】（ＨＰＬＣ分析条件） カラム：TSK GEL ODS-120T（4.6×250mm） 溶 媒：0.1%ＴＦＡ−アセトニトリル（アセトニトリル
を20％から50％に毎分１％変化させる直線勾配グラジェ
ント） 流 速：1ml/min 検出波長：２２０ｎｍ ・旋光度 旋光度の測定には、ＤＩＣ−３７０（日本分光工業社
製）を用いた。(HPLC analysis conditions) Column: TSK GEL ODS-120T (4.6 × 250 mm) Solvent: 0.1% TFA-acetonitrile (linear gradient gradient in which acetonitrile is changed from 20% to 50% by 1% per minute) Flow rate : 1 ml / min Detection wavelength: 220 nm-Optical rotation DIC-370 (manufactured by JASCO Corporation) was used to measure the optical rotation.

【００４２】（旋光度測定条件） 光線： Ｎａランプ 589nm 温度： ２０℃ 層長： 100mm 濃度： １％（０．１Ｍ−酢酸中） ・アミノ酸分析 アミノ酸分析は、得られたペプチドを6N-HCl（０．１％
フェノ−ル含有）中で110℃、20時間加水分解した後
に、日立アミノ酸分析装置Ｌ−８５００型（日立製作所
製）を用いて行った。 〔実施例１−１〕方法１によるグルカゴンの固相合成 まず、Ｂｏｃ−Ｔhr（Ｂzl）−ＰＡＭ樹脂（ペプチド研
究所社製、アミノ基含量：0.36 mmol/g、4-オキシメチ
ルフェニルアセトアミドメチル樹脂）２０ｇを、ＤＣＭ
200ml（２回）、50％ＴＦＡ含有ＤＣＭ溶液 300ml（３
０分）、イソプロピルアルコ−ル 200ml（１回）、Ｍｅ
ＯＨ200ml（２回）、10％ＴＥＡ含有ＤＣＭ 200ml（２
回）、ＭｅＯＨ 200ml（２回）、ＤＣＭ 200ml（２回）
の順に攪拌下で処理し、また、各処理毎に濾過を行っ
た。このようにして、Ｂｏｃ−Ｔhr（Ｂzl）−ＰＡＭ樹
脂の末端アミノ基の保護基を除去した。(Conditions for measuring optical rotation) Light ray: Na lamp 589 nm Temperature: 20 ° C. Layer length: 100 mm Concentration: 1% (in 0.1 M acetic acid) Amino acid analysis For amino acid analysis, the obtained peptide was 6N-HCl ( 0.1%
After hydrolysis in a phenol-containing solution) at 110 ° C. for 20 hours, it was carried out using a Hitachi amino acid analyzer L-8500 (manufactured by Hitachi, Ltd.). [Example 1-1] Solid phase synthesis of glucagon by method 1 First, Boc-Thr (Bzl) -PAM resin (manufactured by Peptide Institute, amino group content: 0.36 mmol / g, 4-oxymethylphenylacetamide methyl resin) ) 20 g of DCM
200 ml (twice), 300 ml of DCM solution containing 50% TFA (3
0 minutes), 200 ml of isopropyl alcohol (once), Me
200 ml of OH (twice), 200 ml of DCM containing 10% TEA (2
), MeOH 200 ml (2 times), DCM 200 ml (2 times)
In this order, the treatment was carried out under stirring, and filtration was performed after each treatment. In this way, the protecting group of the terminal amino group of the Boc-Thr (Bzl) -PAM resin was removed.

【００４３】次いで、得られたＴhr（Ｂzl）−ＰＡＭ樹
脂に、Ｂoc−Ａsn−ＯＨ 15mmol 、ＨＯＢｔ 15mmol 、
ＤＣＭ 100ml及びＤＭＦ 20mlを加えて攪拌し、さらに
ＤＣＣ（１Ｍ−ＤＣＭ溶液）15mlを加えて２時間反応さ
せた。その後、反応混合液を濾過し、ＭｅＯＨ 200ml
（２回）、ＤＣＭ 200ml（２回）の順に攪拌下で処理
し、また、各処理毎に濾過を行った。処理後、これに、
さらに、Ｂoc−Ａsn−ＯＨ8mmol 、ＨＯＢｔ 8mmol 、
ＤＣＭ 100ml及びＤＭＦ 20mlを加えて攪拌し、ＤＣＣ
（１Ｍ−ＤＣＭ溶液）8mlを加えて１時間反応させた。
その後、反応混合液を濾過し、ＭｅＯＨ 200ml（２
回）、ＤＣＭ 200ml（２回）の順に攪拌下で処理し、ま
た、各処理毎に濾過を行った。このようにして、Ｂoc−
Ａsn−Ｔhr（Ｂzl）−ＰＡＭ樹脂を得た。Then, to the obtained Thr (Bzl) -PAM resin, 15 mmol of Boc-Asn-OH, 15 mmol of HOBt,
100 ml of DCM and 20 ml of DMF were added and stirred, and 15 ml of DCC (1M-DCM solution) was further added and reacted for 2 hours. After that, the reaction mixture is filtered and MeOH 200 ml
(Twice), DCM (200 ml, twice) were treated in this order under stirring, and filtration was performed after each treatment. After processing,
Furthermore, Boc-Asn-OH 8 mmol, HOBt 8 mmol,
100 ml of DCM and 20 ml of DMF were added and stirred, and DCC was added.
8 ml of (1M-DCM solution) was added and reacted for 1 hour.
After that, the reaction mixture was filtered and 200 ml of MeOH (2
Times) and DCM 200 ml (twice) in this order under stirring, and filtration was performed after each treatment. In this way, Boc-
An Asn-Thr (Bzl) -PAM resin was obtained.

【００４４】尚、未反応のＴhr（Ｂzl）−ＰＡＭ樹脂
は、20％無水酢酸含有ＤＣＭ溶液100mlを加えて５分間
攪拌した後濾過して、次いでＭｅＯＨ 200ml（２回）、
ＤＣＭ200ml（２回）の順に攪拌下で処理した。各処理
毎に濾過した。このようにして副反応が進まないように
した。次に、得られたＢoc−Ａsn−Ｔhr（Ｂzl）−ＰＡ
Ｍ樹脂を 50％ＴＦＡ含有ＤＣＭ溶液 300ml（３０分）、
イソプロピルアルコ−ル 200ml（１回）、ＭｅＯＨ200ml
（２回）、10％ＴＥＡ含有ＤＣＭ 200ml（２回）、Ｍｅ
ＯＨ 200ml（２回）ＤＣＭ 200ml（２回）の順に攪拌下
で処理し、また、各処理毎に濾過を行った。このように
して、末端アミノ基の保護基を除去してＡsn−Ｔhr（Ｂ
zl）−ＰＡＭ樹脂を得た。Unreacted Thr (Bzl) -PAM resin was added with 100 ml of a 20% acetic anhydride-containing DCM solution, stirred for 5 minutes, filtered, and then 200 ml of MeOH (twice),
It was treated with stirring in the order of 200 ml DCM (twice). It was filtered after each treatment. In this way, side reactions were prevented. Then, the obtained Boc-Asn-Thr (Bzl) -PA
300 ml of DCM solution containing 50% TFA in DCM (30 minutes),
Isopropyl alcohol 200ml (1 time), MeOH 200ml
(Twice), DCM containing 10% TEA 200 ml (twice), Me
200 ml of OH (twice), 200 ml of DCM (twice) were treated in this order under stirring, and filtration was performed after each treatment. In this manner, the Asn-Thr (B
zl) -PAM resin was obtained.

【００４５】上記のようにして得られたＡsn−Ｔhr（Ｂ
zl）−ＰＡＭ樹脂に27位のアミノ酸残基に相当するＢoc
−Ｍet−ＯＨ 15mmol 、ＨＯＢｔ 15mmol 、ＤＣＭ 100
ml及びＤＭＦ 20mlを加えて攪拌し、さらにＤＣＣ（１
Ｍ−ＤＣＭ溶液）15mlを加えて２時間反応させた。その
後、反応混合液を濾過し、ＭｅＯＨ 200ml（２回）、Ｄ
ＣＭ 200ml（２回）の順に攪拌下で処理し、また、各処
理毎に濾過を行った。これに、さらに、Ｂoc−Ｍet−Ｏ
Ｈ 8mmol 、ＨＯＢｔ 8mmol 、ＤＣＭ 100ml及びＤＭＦ
20mlを加えて攪拌し、ＤＣＣ（１Ｍ−ＤＣＭ溶液）8ml
を加えて１時間反応させた。その後、反応混合液を濾過
し、ＭｅＯＨ 200ml（２回）、ＤＣＭ 200ml（２回）の
順に攪拌下で処理し、また、各処理毎に濾過を行った。
このようにして、Ｂoc−Ｍet−Ａsn−Ｔhr（Ｂzl）−Ｐ
ＡＭ樹脂を得た。Asn-Thr (B
zl) -Boc corresponding to the amino acid residue at position 27 in PAM resin
-Met-OH 15 mmol, HOBt 15 mmol, DCM 100
ml and DMF 20 ml were added and stirred, and further DCC (1
15 ml of M-DCM solution) was added and reacted for 2 hours. Then, the reaction mixture was filtered, 200 ml of MeOH (twice), D
CM 200 ml (twice) were treated in this order under stirring, and filtration was performed after each treatment. In addition to this, Boc-Met-O
H 8 mmol, HOBt 8 mmol, DCM 100 ml and DMF
20 ml was added and stirred, and 8 ml of DCC (1M-DCM solution)
Was added and reacted for 1 hour. Then, the reaction mixture was filtered, treated with 200 ml of MeOH (twice) and 200 ml of DCM (twice) in this order under stirring, and each treatment was filtered.
Thus, Boc-Met-Asn-Thr (Bzl) -P
An AM resin was obtained.

【００４６】尚、未反応のＡsn−Ｔhr（Ｂzl）−ＰＡＭ
樹脂は、20％無水酢酸含有ＤＣＭ溶液 100mlを加えて５
分間攪拌し、濾過した後ＭｅＯＨ 200ml（２回）、ＤＣ
Ｍ200ml（２回）の順に攪拌下で処理した。各処理毎に
濾過を行った。このようにして、副反応が進まないよう
にした。以下、下記に示すアミノ基保護アミノ酸を用い
て上記と同様の方法で順次26位から１位までのアミノ酸
残基をカップリングした。 アミノ アミノ基保護アミノ酸 使用量 酸順序 (mmol) ２６ Ｂoc−Ｌeu−ＯＨ １５＋８ ２５ Ｂoc−Ｔrp−ＯＨ １５＋８ ２４ Ｂoc−Ｇln（Ｘan）−ＯＨ １５＋８×２ ２３ Ｂoc−Ｖal−ＯＨ １５＋８×３ ２２ Ｂoc−Ｐhe−ＯＨ １５＋８×６ ２１ Ｂoc−Ａsp（ＯcＨｅx）−ＯＨ １５＋８×３ ２０ Ｂoc−Ｇln（Ｘan）−ＯＨ １５＋８×３ １９ Ｂoc−Ａla−ＯＨ １５＋８×３ １８ Ｂoc−Ａrg（Ｔos）−ＯＨ １５＋８×４ １７ Ｂoc−Ａrg（Ｔos）−ＯＨ １５＋８×４ １６ Ｂoc−Ｓer（Ｂzl ）−ＯＨ １５＋８×２ １５ Ｂoc−Ａsp（ＯcＨｅｘ）−ＯＨ １５＋８×２ １４ Ｂoc−Ｌeu−ＯＨ １５＋８×２ １３ Ｂoc−Ｔyr（Ｂr-Ｚ）−ＯＨ １５＋８×３ １２ Ｂoc−Ｌys（Ｃl-Ｚ）−ＯＨ １５＋８ １１ Ｂoc−Ｓer（Ｂzl ）−ＯＨ １５＋８×２ １０ Ｂoc−Ｔyr（Ｂr-Ｚ）−ＯＨ １５＋８×２ ９ Ｂoc−Ａsp（ＯcＨx）−ＯＨ １５＋８ ８ Ｂoc−Ｓer（Ｂzl ）−ＯＨ １５＋８ ７ Ｂoc−Ｔhr（Ｂzl ）−ＯＨ １５＋８ ６ Ｂoc−Ｐhe−ＯＨ １５＋８ ５ Ｂoc−Ｔhr（Ｂzl ）−ＯＨ １５＋８ ４ Ｂoc−Ｇly−ＯＨ １５＋８ ３ Ｂoc−Ｇln（Ｘan）−ＯＨ １５＋８ ２ Ｂoc−Ｓer（Ｂzl ）−ＯＨ １５＋８ １ Ｂoc−Ｈis（Ｂom）−ＯＨ １５＋８ このようにして、Ｈis(Ｂom）−Ｓer(Ｂzl）−Ｇln−Ｇ
ly−Ｔhr（Ｂzl）−Ｐhe−Ｔhr（Ｂzl）−Ｓer（Ｂzl）
−Ａsp（ＯcＨｅx）−Ｔyr（Ｂr-Ｚ）−Ｓer(Ｂzl）−
Ｌys(Ｃl-Ｚ）−Ｔyr(Ｂr-Ｚ）−Ｌeu−Ａsp（ＯcＨｅ
x）−Ｓer（Ｂzl）−Ａrg（Ｔos）−Ａrg（Ｔos）−Ａl
a−Ｇln−Ａsp（ＯcＨｅx）−Ｐhe−Ｖal-Ｇln−Ｔrp−
Ｌeu−Ｍet−Ａsn−Ｔhr（Ｂzl）−ＰＡＭ樹脂 50.93g
を得た。Unreacted Asn-Thr (Bzl) -PAM
The resin was added by adding 100 ml of DCM solution containing 20% acetic anhydride to 5
After stirring for 1 minute and filtering, 200 ml of MeOH (twice), DC
M 200 ml (twice) were treated in this order under stirring. Filtration was performed after each treatment. In this way, side reactions were prevented from proceeding. Thereafter, the amino acid-protected amino acids shown below were used to sequentially couple amino acid residues from position 26 to position 1 in the same manner as above. Amino Amino group-protected amino acid amount used Acid order (mmol) 26 Boc-Leu-OH 15 + 8 25 Boc-Trp-OH 15 + 8 24 Boc-Gln (Xan) -OH 15 + 8 × 2 23 Boc-Val-OH 15 + 8 × 3 22 Boc- Phe-OH 15 + 8 × 6 21 Boc-Asp (OcHex) -OH 15 + 8 × 3 20 Boc-Gln (Xan) -OH 15 + 8 × 3 19 Boc-Ala-OH 15 + 8 × 3 18 Boc-Arg (Tos) -OH 15 + 8 × 4 17 Boc-Arg (Tos) -OH 15 + 8 × 4 16 Boc-Ser (Bzl) -OH 15 + 8 × 2 15 Boc-Asp (OcHex) -OH 15 + 8 × 2 14 Boc-Leu-OH 15 + 8 × 2 13 Boc-Tyr (Br-Z) -OH 15 + 8 × 3 12 Boc-Lys (Cl-Z) -OH 15 + 8 11 Boc-Ser (Bzl) -OH 15 + 8 × 2 10 Boc-Tyr (Br-Z) -OH 1 5 + 8 × 29 Boc-Asp (OcHx) -OH 15 + 8 8 Boc-Ser (Bzl) -OH 15 + 8 7 Boc-Thr (Bzl) -OH 15 + 8 6 Boc-Phe-OH 15 + 8 5 Boc-Thr (B15 +)-OH 4 Boc-Gly-OH 15 + 8 3 Boc-Gln (Xan) -OH 15 + 8 2 Boc-Ser (Bzl) -OH 15 + 8 1 Boc-His (Bom) -OH 15 + 8 Thus, His (Bom) -Ser (Bzl) ) -Gln-G
ly-Thr (Bzl) -Phe-Thr (Bzl) -Ser (Bzl)
-Asp (OcHex) -Tyr (Br-Z) -Ser (Bzl)-
Lys (Cl-Z) -Tyr (Br-Z) -Leu-Asp (OcHe
x) -Ser (Bzl) -Arg (Tos) -Arg (Tos) -Al
a-Gln-Asp (OcHex) -Phe-Val-Gln-Trp-
Leu-Met-Asn-Thr (Bzl) -PAM resin 50.93g
I got

【００４７】上記の保護ペプチド−ＰＡＭ樹脂 50ｇに
アニソ−ル 75ml及び1,2-エタンジチオール 75mlを加
え、さらに無水フッ化水素 600mlを加えて０℃で１時間
撹拌した。その後、無水フッ化水素を減圧下留去後、残
渣をエ−テルで洗浄し、これに10％酢酸 1250mlを加え
てペプチドを抽出した。得られた抽出液を以下に示す条
件で逆相カラムクロマトグラフィーにより精製後、さら
に分取用高速液体クロマトグラフィーを用いたイオン交
換クロマトグラフィー及び逆相クロマトグラフィーによ
り精製した。得られた溶出液を凍結乾燥し、精製グルカ
ゴン 1.00gを得た。 ・逆相カラムクロマトグラフィー カラム：ＯＤＳ−１０２０ＴＴ（30×420mm） 溶 媒：平衡化 15％アセトニトリル−0.1％ＴＦＡ 溶出 35％アセトニトリル−0.1％ＴＦＡ 流 速：５ml/min ・イオン交換クロマトグラフィー（分取用高速液体クロ
マトグラフィー） カラム：TSK GEL SP-5PW（21.5×150mm） 溶 媒：35％イソプロピルアルコール−50mM酢酸緩衝液
（pH 5.3） 流 速：５ml/min ・逆相カラムクロマトグラフィー（分取用高速液体クロ
マトグラフィー） カラム：TSK GEL ODS-120T（550×600mm） 溶 媒：29％イソプロピルアルコール−0.1％ＴＦＡ 流 速：30ml/min また、上記精製ペプチドについて、高速液体クロマトグ
ラフィ−（ＨＰＬＣ）の保持時間の測定、旋光度の測定
及びアミノ酸分析を行った。その結果を下記に示す。 ・ＨＰＬＣの保持時間：２７．２分 ・［α］D ：−３５．３° ・アミノ酸分析 Asp(4) 3.94, Thr(3) 2.65, Ser(4) 2.90, Glu(3) 2.9
8, Gly(1) 1.07, Ala(1) 1.00, Val(1) 1.01, Met(1)
0.97, Leu(2) 2.06, Tyr(2) 1.93, Phe(2) 1.92,His(1)
1.05, Arg(2) 1.95 〔実施例１−２〕樹脂として、Ｂｏｃ−Ｔhr（Ｂzl）−
ＰＡＭ樹脂（渡辺化学工業株式会社製、アミノ基含有
量：0.74 mmol/g ）２０ｇを用い、アミノ基保護アミノ
酸として下記のものを用いた以外は実施例１−１と同様
の方法で保護ペプチド−ＰＡＭ樹脂66.19g を得た。 アミノ アミノ基保護アミノ酸 使用量 酸順序 (mmol) ２８ Ｂoc−Ａsn（Ｘan）−ＯＨ ３０＋１５ ２７ Ｂoc−Ｍet−ＯＨ ３０＋１５ ２６ Ｂoc−Ｌeu−ＯＨ ３０＋１５ ２５ Ｂoc−Ｔrp−ＯＨ ３０＋１５×２ ２４ Ｂoc−Ｇln（Ｘan）−ＯＨ ３０＋１５×２ ２３ Ｂoc−Ｖal−ＯＨ ３０＋１５×３ ２２ Ｂoc−Ｐhe−ＯＨ ３０＋１５×７ ２１ Ｂoc−Ａsp（ＯcＨex）−ＯＨ ３０＋１５×１２ ２０ Ｂoc−Ｇln（Ｘan）−ＯＨ ３０＋１５×７ １９ Ｂoc−Ａla−ＯＨ ３０＋１５×４ １８ Ｂoc−Ａrg（Ｔos）−ＯＨ ３０＋１５×６ １７ Ｂoc−Ａrg（Ｔos）−ＯＨ ３０＋１５×８ １６ Ｂoc−Ｓer（Ｂzl ）−ＯＨ ３０＋１５×３ １５ Ｂoc−Ａsp（ＯcＨex）−ＯＨ ３０＋１５×４ １４ Ｂoc−Ｌeu−ＯＨ ３０＋１５×２ １３ Ｂoc−Ｔyr（Ｂr-Ｚ）−ＯＨ ３０＋１５×３ １２ Ｂoc−Ｌys（Ｃl-Ｚ）−ＯＨ ３０＋１５×２ １１ Ｂoc−Ｓer（Ｂzl ）−ＯＨ ３０＋１５×２ １０ Ｂoc−Ｔyr（Ｂr-Ｚ）−ＯＨ ３０＋１５×２ ９ Ｂoc−Ａsp（ＯcＨex）−ＯＨ ３０＋１５ ８ Ｂoc−Ｓer（Ｂzl ）−ＯＨ ３０＋１５ ７ Ｂoc−Ｔhr（Ｂzl ）−ＯＨ ３０＋１５ ６ Ｂoc−Ｐhe−ＯＨ ３０＋１５ ５ Ｂoc−Ｔhr（Ｂzl ）−ＯＨ ３０＋１５ ４ Ｂoc−Ｇly−ＯＨ ３０＋１５ ３ Ｂoc−Ｇln（Ｘan）−ＯＨ ３０＋１５ ２ Ｂoc−Ｓer（Ｂzl ）−ＯＨ ３０＋１５ １ Ｂoc−Ｈis（Ｂom）−ＯＨ ３０＋１５×３ 得られた保護ペプチド−ＰＡＭ樹脂50ｇにアニソ−ル 8
8ml及び1,2-エタンジチオール 88mlを加え、さらに無水
フッ化水素 800mlを加えて０℃で１時間撹拌した。反応
後、無水フッ化水素を減圧下留去後、残渣をエ−テルで
洗浄し、これに 10％酢酸 250mlを加えてペプチドを抽
出した。To 50 g of the above protected peptide-PAM resin, 75 ml of anisole and 75 ml of 1,2-ethanedithiol were added, 600 ml of anhydrous hydrogen fluoride was further added, and the mixture was stirred at 0 ° C. for 1 hour. Then, anhydrous hydrogen fluoride was distilled off under reduced pressure, the residue was washed with ether, and 1250 ml of 10% acetic acid was added thereto to extract the peptide. The obtained extract was purified by reverse phase column chromatography under the conditions shown below, and further purified by ion exchange chromatography using preparative high performance liquid chromatography and reverse phase chromatography. The obtained eluate was freeze-dried to obtain 1.00 g of purified glucagon.・ Reverse phase column chromatography Column: ODS-1020TT (30 × 420 mm) Solvent: Equilibration 15% Acetonitrile-0.1% TFA Elution 35% Acetonitrile-0.1% TFA Flow rate: 5 ml / min ・ Ion exchange chromatography (preparation) High Performance Liquid Chromatography) Column: TSK GEL SP-5PW (21.5 x 150 mm) Solvent: 35% Isopropyl alcohol-50 mM acetate buffer (pH 5.3) Flow rate: 5 ml / min-Reverse phase column chromatography (for preparative separation) High Performance Liquid Chromatography) Column: TSK GEL ODS-120T (550 x 600 mm) Solvent: 29% Isopropyl alcohol-0.1% TFA Flow rate: 30 ml / min In addition, retention of high performance liquid chromatography (HPLC) for the above purified peptide Time measurement, optical rotation measurement, and amino acid analysis were performed. The results are shown below.・ HPLC retention time: 27.2 minutes ・ [α] D : -35.3 ° ・ Amino acid analysis Asp (4) 3.94, Thr (3) 2.65, Ser (4) 2.90, Glu (3) 2.9
8, Gly (1) 1.07, Ala (1) 1.00, Val (1) 1.01, Met (1)
0.97, Leu (2) 2.06, Tyr (2) 1.93, Phe (2) 1.92, His (1)
1.05, Arg (2) 1.95 [Example 1-2] As a resin, Boc-Thr (Bzl)-
PAM resin (manufactured by Watanabe Chemical Industry Co., Ltd., amino group content: 0.74 mmol / g) was used, and a protected peptide was prepared in the same manner as in Example 1-1, except that the following amino acids were used as amino group-protected amino acids. 66.19 g of PAM resin was obtained. Amino Amino group-protected amino acid amount used Acid order (mmol) 28 Boc-Asn (Xan) -OH 30 + 15 27 Boc-Met-OH 30 + 15 26 Boc-Leu-OH 30 + 15 25 Boc-Trp-OH 30 + 15 × 2 24 Boc-Gln ( Xan) -OH 30 + 15 × 2 23 Boc-Val-OH 30 + 15 × 3 22 Boc-Phe-OH 30 + 15 × 7 21 Boc-Asp (OcHex) -OH 30 + 15 × 12 20 Boc-Gln (Xan) -OH 30 + 15 × 7 19 Boc-Ala-OH 30 + 15 × 4 18 Boc-Arg (Tos) -OH 30 + 15 × 6 17 Boc-Arg (Tos) -OH 30 + 15 × 8 16 Boc-Ser (Bzl) -OH 30 + 15 × 3 15 Boc-Asp (OcHex ) -OH 30 + 15 × 4 14 Boc-Leu-OH 30 + 15 × 2 13 Boc-Tyr (Br-Z) -OH 30 + 15 × 3 12 Boc- ys (Cl-Z) -OH 30 + 15 × 2 11 Boc-Ser (Bzl) -OH 30 + 15 × 2 10 Boc-Tyr (Br-Z) -OH 30 + 15 × 2 9 Boc-Asp (OcHex) -OH 30 + 158 Boc- Ser (Bzl) -OH 30 + 157 Boc-Thr (Bzl) -OH 30 + 156 Boc-Phe-OH 30 + 155 Boc-Thr (Bzl) -OH 30 + 15 4 Boc-Gly-OH 30 + 153 Boc-Gln (Xan) -OH. 30 + 15 2 Boc-Ser (Bzl) -OH 30 + 151 Boc-His (Bom) -OH 30 + 15 × 3 50 g of the obtained protected peptide-PAM resin was anisole 8
8 ml and 1,2-ethanedithiol 88 ml were added, anhydrous hydrogen fluoride 800 ml was further added, and the mixture was stirred at 0 ° C. for 1 hour. After the reaction, anhydrous hydrogen fluoride was distilled off under reduced pressure, the residue was washed with ether, and 250 ml of 10% acetic acid was added to this to extract the peptide.

【００４８】抽出液から、実施例１−１と同様の方法で
精製グルカゴン 520mgを得た。また、上記精製グルカゴ
ンについて、高速液体クロマトグラフィ−（ＨＰＬＣ）
の保持時間の測定、旋光度の測定及びアミノ酸分析を行
った。その結果を下記に示す。 ・ＨＰＬＣの保持時間：２９．５分 ・［α］D ：−３５．４° ・アミノ酸分析 Asp(4) 3.92, Thr(3) 2.64, Ser(4) 2.89, Glu(3) 2.9
7, Gly(1) 1.07, Ala(1) 1.00, Val(1) 1.01, Met(1)
0.95, Leu(2) 2.06, Tyr(2) 1.91, Phe(2) 1.92,His(1)
1.05, Lys(1) 0.98, Arg(2) 1.93 〔実施例１−３〕樹脂として、Ｂｏｃ−Ｔhr（Ｂzl）−
ＰＡＭ樹脂（渡辺化学工業株式会社製、アミノ基含有
量：0.56 mmol/g ）２０ｇを用い、Ｂｏｃ−Ｍet−ＯＨ
及びＢｏｃ−Ｔrp−ＯＨのかわりにそれぞれＢｏＣ−Ｍ
et（Ｏ）−ＯＨ及びＢｏｃ−Ｔrp（ＣＨＯ）−ＯＨ用い
た以外は実施例１−１と同様の方法で保護ペプチド−Ｐ
ＡＭ樹脂58.98gを得た。From the extract, 520 mg of purified glucagon was obtained in the same manner as in Example 1-1. In addition, high performance liquid chromatography (HPLC) of the purified glucagon.
Retention time, optical rotation and amino acid analysis. The results are shown below. -HPLC retention time: 29.5 minutes- [α] D : -35.4 ° -Amino acid analysis Asp (4) 3.92, Thr (3) 2.64, Ser (4) 2.89, Glu (3) 2.9
7, Gly (1) 1.07, Ala (1) 1.00, Val (1) 1.01, Met (1)
0.95, Leu (2) 2.06, Tyr (2) 1.91, Phe (2) 1.92, His (1)
1.05, Lys (1) 0.98, Arg (2) 1.93 [Example 1-3] As a resin, Boc-Thr (Bzl)-
Using 20 g of PAM resin (Watanabe Chemical Co., Ltd., amino group content: 0.56 mmol / g), Boc-Met-OH
And BoC-M instead of Boc-Trp-OH
Protected peptide-P was prepared in the same manner as in Example 1-1, except that et (O) -OH and Boc-Trp (CHO) -OH were used.
58.98 g of AM resin was obtained.

【００４９】得られた保護ペプチド−ＰＡＭ樹脂 50ｇ
にp-クレゾール 26ｇ、ジメチルスルファイド 220ml、
及びp-チオクレゾール 8.6ｇを加え、さらに無水フッ化
水素 85mlを加えて０℃で２時間撹拌した。無水フッ化
水素を減圧下留去後、再度無水フッ化水素 700mlを加え
て０℃で１時間撹拌した。反応後、無水フッ化水素を減
圧下留去、残渣をエ−テルで洗浄し、これに 10％酢酸
250mlを加えてペプチドを抽出した。50 g of the obtained protected peptide-PAM resin
26 g of p-cresol, 220 ml of dimethyl sulfide,
And 8.6 g of p-thiocresol, 85 ml of anhydrous hydrogen fluoride were further added, and the mixture was stirred at 0 ° C. for 2 hours. The anhydrous hydrogen fluoride was distilled off under reduced pressure, 700 ml of anhydrous hydrogen fluoride was added again, and the mixture was stirred at 0 ° C. for 1 hour. After the reaction, anhydrous hydrogen fluoride was distilled off under reduced pressure, the residue was washed with ether, and 10% acetic acid was added thereto.
The peptide was extracted by adding 250 ml.

【００５０】抽出液から、実施例１−１と同様の方法で
精製ペプチド 610mgを得た。また、上記精製ペプチドに
ついて、高速液体クロマトグラフィ−（ＨＰＬＣ）の保
持時間の測定、旋光度の測定及びアミノ酸分析を行っ
た。その結果を下記に示す。 ・ＨＰＬＣの保持時間：２９．２分 ・［α］D ：−３５．２° ・アミノ酸分析 Asp(4) 3.93, Thr(3) 2.64, Ser(4) 2.90, Glu(3) 3.0
2, Gly(1) 1.08, Ala(1) 1.00, Val(1) 1.01, Met(1)
0.97, Leu(2) 2.05, Tyr(2) 1.92, Phe(2) 1.93,His(1)
1.07, Lys(1) 0.99, Arg(2) 1.94 〔実施例２〕方法２によるグルカゴンの固相合成 まず、ＢＨＡ樹脂（ペプチド研究所社製、アミノ基含有
量0.36 mmol/g、ベンズヒドリルアミン樹脂）２０ｇ
を、ＤＣＭ 200ml（２回）、10％ＴＥＡ含有ＤＣＭ 200
ml（２回）、ＭｅＯＨ 200ml（２回）、ＤＣＭ 200ml
（２回）の順に攪拌下で処理し、また、各処理毎に濾過
を行った。From the extract, 610 mg of purified peptide was obtained in the same manner as in Example 1-1. The purified peptide was subjected to high performance liquid chromatography (HPLC) retention time measurement, optical rotation measurement and amino acid analysis. The results are shown below.・ HPLC retention time: 29.2 minutes ・ [α] D : -35.2 ° ・ Amino acid analysis Asp (4) 3.93, Thr (3) 2.64, Ser (4) 2.90, Glu (3) 3.0
2, Gly (1) 1.08, Ala (1) 1.00, Val (1) 1.01, Met (1)
0.97, Leu (2) 2.05, Tyr (2) 1.92, Phe (2) 1.93, His (1)
1.07, Lys (1) 0.99, Arg (2) 1.94 [Example 2] Solid phase synthesis of glucagon by method 2 First, BHA resin (manufactured by Peptide Institute, amino group content 0.36 mmol / g, benzhydrylamine resin) ) 20 g
200 ml of DCM (twice), DCM 200 containing 10% TEA
ml (twice), MeOH 200 ml (twice), DCM 200 ml
The treatment was performed under stirring in the order of (twice), and filtration was performed for each treatment.

【００５１】上記のような処理を行った樹脂に、アミノ
酸順序27〜29位のアミノ酸残基に相当する下記式： Ｂoc−Ｍet−Ａsp−Ｔhr（Ｂzl）−ＯＢzl で表されるトリペプチド 15mmol 、ＨＯＢｔ 15mmol 、
ＤＣＭ 100ml及びＤＭＦ20mlを加えて攪拌し、さらにＤ
ＣＣ（１Ｍ−ＤＣＭ溶液）15mlを加えて２時間反応させ
た。その後、反応混合液を濾過し、ＭｅＯＨ 200ml（２
回）、ＤＣＭ 200ml（２回）の順に攪拌下で処理し、ま
た、各処理毎に濾過を行った。このようにして、Ｂoc−
Ｍet−Ａsp−Ｔhr（Ｂzl）−ＯＢzl−ＢＨＡ樹脂を得
た。15 mmol of a tripeptide represented by the following formula: Boc-Met-Asp-Thr (Bzl) -OBzl, which corresponds to the amino acid residues at the 27th to 29th amino acid positions, was added to the resin treated as described above. HOBt 15mmol,
DCM 100 ml and DMF 20 ml are added and stirred, and further D
15 ml of CC (1M-DCM solution) was added and reacted for 2 hours. After that, the reaction mixture was filtered and 200 ml of MeOH (2
Times) and DCM 200 ml (twice) in this order under stirring, and filtration was performed after each treatment. In this way, Boc-
A Met-Asp-Thr (Bzl) -OBzl-BHA resin was obtained.

【００５２】26位のアミノ酸残基からは、実施例１−１
と同様の方法で１位のアミノ酸残基まで１個ずつアミノ
酸残基を延長し、Ｈ−Ｈis(Ｂom）−Ｓer(Ｂzl）−Ｇln
−Ｇly−Ｔhr（Ｂzl）−Ｐhe−Ｔhr（Ｂzl）−Ｓer（Ｂ
zl）−Ａsp（ＯcＨｅx）−Ｔyr（Ｂr-Ｚ）−Ｓer(Ｂz
l）−Ｌys(Ｃl-Ｚ）−Ｔyr(Ｂr-Ｚ）−Ｌeu−Ａsp（Ｏc
Ｈｅx）−Ｓer（Ｂzl）−Ａrg（Ｔos）−Ａrg（Ｔos）
−Ａla−Ｇln−Ａsp（ＯcＨｅx）−Ｐhe−Ｖal-Ｇln−
Ｔrp−Ｌeu−Ｍet−Ａsp−Ｔhr（Ｂzl）−ＯＢzl−ＢＨ
Ａ樹脂 50.93gを得た。From the amino acid residue at position 26, Example 1-1
In the same manner as described above, the amino acid residue is extended one by one up to the amino acid residue at the 1-position, and H-His (Bom) -Ser (Bzl) -Gln is added.
-Gly-Thr (Bzl) -Phe-Thr (Bzl) -Ser (B
zl) -Asp (OcHex) -Tyr (Br-Z) -Ser (Bz
l) -Lys (Cl-Z) -Tyr (Br-Z) -Leu-Asp (Oc
Hex) -Ser (Bzl) -Arg (Tos) -Arg (Tos)
-Ala-Gln-Asp (OcHex) -Phe-Val-Gln-
Trp-Leu-Met-Asp-Thr (Bzl) -OBzl-BH
50.93 g of A resin was obtained.

【００５３】得られた保護ペプチド−ＢＨＡ樹脂から、
実施例１−１と同様の方法でペプチドの抽出及び精製を
行って、精製グルカゴン0.89gを得た。尚、上記で用い
たBoc-Met-Asp-Thr(Bzl)-OBzl は下記(1) 及び(2) の操
作によって製造した。 (1) Boc-Asp(OBut)-Thr(Bzl)-OBzl の合成 H-Thr(Bzl)-OBzl・1/2(COOH)2 6.89g(0.02mol)をTHF 10
0ml-DMF 100mlに溶解し、0℃で、Ｎ−メチルモルフォリ
ンを加えて中和した。From the resulting protected peptide-BHA resin,
The peptide was extracted and purified in the same manner as in Example 1-1 to obtain 0.89 g of purified glucagon. The Boc-Met-Asp-Thr (Bzl) -OBzl used above was produced by the following operations (1) and (2). (1) Synthesis of Boc-Asp (OBut) -Thr (Bzl) -OBzl H-Thr (Bzl) -OBzl ・ 1/2 (COOH) 2 6.89 g (0.02 mol) in THF 10
It was dissolved in 100 ml of 0 ml-DMF and neutralized by adding N-methylmorpholine at 0 ° C.

【００５４】Boc-Asp(OBut)-OH 5.79g(0.02mol) をTHF
100mlに溶解し、ドライアイス−エタノ−ルで-20℃に冷
却し、Ｎ−メチルモルフォリン 2.2ml(0.02mol)を滴下
した後クロロギ酸イソブチル 2.64ml(0.02mol)を滴下
し、-20℃で1分間撹拌して、混合酸無水物の溶液を調製
した。この溶液を前記の溶液に加え、0℃で5分間、室温
で30分間撹拌した後、減圧濃縮した。残渣に酢酸エチル
を加え、有機層を1N-HClで2回、飽和食塩水で1回、飽和
炭酸水素ナトリウム水溶液で2回、飽和食塩水で2回の順
で洗浄し、硫酸ナトリウムで乾燥し、減圧濃縮した。そ
の後、シリカゲルクロマトグラフィー｛ワコーゲルC-10
0(3x24cm) 、展開溶媒：酢酸エチル／ヘキサン(1/4)｝
にて精製を行った。目的物の溶出画分を集め減圧濃縮
し、真空乾燥を行って、油状物 8.95g(収率78%)を得
た。この油状物のＴＬＣのRf (BuOH/AcOH/H2O=4/1/5)
は0.92であった。このようにしてBoc-Asp(OBut)-Thr(Bz
l)-OBzl を合成した。 (2) Boc-Met-Asp-Thr(Bzl)-OBzl の合成 上記(1) で得られたBoc-Asp(OBut)-Thr(Bzl)-OBzl(油状
物) 8.95g(15.68mmol)を、DCM 8mlに溶解した。次いで0
℃でTFA 10mlを添加し、室温で30分間撹拌した後、エー
テル-ヘキサン(1:1)を加えた。生じた沈殿物を濾取、真
空乾燥することにより、粉末状のH-Asp-Thr(Bzl)-OBzlH
・TFAを得た。得られた粉末をTHF 100mlに溶解し、0℃
でＮ−メチルモルフォリンを加えて中和した。Boc-Asp (OBut) -OH 5.79 g (0.02 mol) in THF
Dissolve in 100 ml, cool to -20 ° C with dry ice-ethanol, add 2.2 ml (0.02 mol) of N-methylmorpholine, and then add 2.64 ml (0.02 mol) of isobutyl chloroformate at -20 ° C. The mixture was stirred for 1 minute to prepare a mixed acid anhydride solution. This solution was added to the above solution, stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes, and then concentrated under reduced pressure. Ethyl acetate was added to the residue, and the organic layer was washed twice with 1N-HCl, once with saturated brine, twice with saturated aqueous sodium hydrogen carbonate solution, and twice with saturated brine, and dried over sodium sulfate. , Concentrated under reduced pressure. Then, silica gel chromatography {Wako gel C-10
0 (3x24cm), developing solvent: ethyl acetate / hexane (1/4)}
Was purified. Elution fractions of the target compound were collected, concentrated under reduced pressure, and vacuum dried to obtain 8.95 g (yield 78%) of an oil. Rf of this oil TLC (BuOH / AcOH / H 2 O = 4/1/5)
Was 0.92. Thus Boc-Asp (OBut) -Thr (Bz
l) -OBzl was synthesized. (2) Synthesis of Boc-Met-Asp-Thr (Bzl) -OBzl Boc-Asp (OBut) -Thr (Bzl) -OBzl (oil) 8.95 g (15.68 mmol) obtained in (1) above, It was dissolved in 8 ml of DCM. Then 0
After adding 10 ml of TFA at 0 ° C and stirring at room temperature for 30 minutes, ether-hexane (1: 1) was added. The resulting precipitate was collected by filtration and vacuum dried to obtain powdered H-Asp-Thr (Bzl) -OBzlH.
・ I got TFA. Dissolve the obtained powder in 100 ml of THF, 0 ℃
Then, N-methylmorpholine was added to neutralize.

【００５５】Boc-Met-OH 3.91g(15.68mmol) をTHF50ml
に溶解し、ドライアイス−エタノ−ルで-20℃に冷却
し、Ｎ−メチルモルフォリン 1.8ml(15.68mmol)を滴下
した後クロロギ酸イソブチル2.07ml(15.68mmol) を滴下
して、-20℃で1分間撹拌して混合酸無水物の溶液を調製
した。この溶液を前記の溶液に加え、0℃で5分間、室温
で30分間撹拌した後、減圧濃縮した。残渣に酢酸エチル
を加え、有機層を1N-HClで2回、飽和食塩水で2回の順で
洗浄し、減圧濃縮した。その後真空乾燥を行って、油状
物 9.69g(収率95%)を得た。この油状物のＴＬＣのRf値
(BuOH/AcOH/H2O=4/1/5) は 0.85 であった。このように
してBoc-Met-Asp-Thr(Bzl)-OBzl を合成した。 〔実施例３〕方法２によるグルカゴンの固相合成 実施例１−１において得られる中間体 Ｈ−Ｌeu−Ｍet
−Ａsn−Ｔhr（Ｂzl）−ＰＡＭ樹脂 1.48gに、グルカゴ
ンのアミノ酸配列の19〜25位のアミノ酸残基に相当する
下記式： Ｂoc−Ａla−Ｇln−Ａsp（ＯＢzl）−Ｐhe−Ｖal−Ｇln
−Ｔrp−ＯＨ で表されるヘプタペプチド 15mmol 、ＨＯＢｔ 15mmol
、ＤＣＭ 100ml及びＤＭＦ 20mlを加えて攪拌し、さら
にＤＣＣ（１Ｍ−ＤＣＭ溶液）15mlを加えて２時間反応
させた。反応後、混合液を濾過し、ＭｅＯＨ 200ml（２
回）、ＤＣＭ 200ml（２回）の順に攪拌下で処理し、ま
た、各処理毎に濾過を行った。このようにして、Ｂoc−
Ａla−Ｇln−Ａsp（ＯcＨｅx）−Ｐhe−Ｖal-Ｇln−Ｔr
p−Ｌeu−Ｍet−Ａsn−Ｔhr（Ｂzl ）−ＰＡＭ樹脂を得
た。Boc-Met-OH (3.91 g, 15.68 mmol) was added to THF (50 ml).
Dissolved in water, cooled to -20 ° C with dry ice-ethanol, 1.8 ml (15.68 mmol) of N-methylmorpholine was added dropwise, and then 2.07 ml (15.68 mmol) of isobutyl chloroformate was added dropwise and -20 ° C. And stirred for 1 minute to prepare a mixed acid anhydride solution. This solution was added to the above solution, stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes, and then concentrated under reduced pressure. Ethyl acetate was added to the residue, the organic layer was washed twice with 1N-HCl and twice with saturated brine, and concentrated under reduced pressure. After that, vacuum drying was performed to obtain 9.69 g (yield 95%) of an oily substance. Rf value of TLC of this oil
(BuOH / AcOH / H 2 O = 4/1/5) was 0.85. In this way, Boc-Met-Asp-Thr (Bzl) -OBzl was synthesized. [Example 3] Solid phase synthesis of glucagon by method 2 Intermediate H-Leu-Met obtained in Example 1-1
1.48 g of -Asn-Thr (Bzl) -PAM resin, the following formula corresponding to the amino acid residues at positions 19 to 25 of the glucagon amino acid sequence: Boc-Ala-Gln-Asp (OBzl) -Phe-Val-Gln
-Trp-OH represented by heptapeptide 15 mmol, HOBt 15 mmol
, DCM 100 ml and DMF 20 ml were added and stirred, and DCC (1M-DCM solution) 15 ml was further added and reacted for 2 hours. After the reaction, the mixture was filtered, and 200 ml of MeOH (2
Times) and DCM 200 ml (twice) in this order under stirring, and filtration was performed after each treatment. In this way, Boc-
Ala-Gln-Asp (OcHex) -Phe-Val-Gln-Tr
A p-Leu-Met-Asn-Thr (Bzl) -PAM resin was obtained.

【００５６】上記樹脂に、実施例１−１の方法と同様に
して18位から１位までアミノ酸残基を順次１個ずつ結合
して延長し、Ｈ−Ｈis(Ｂom）−Ｓer(Ｂzl ）−Ｇln−
Ｇly−Ｔhr（Ｂzl ）−Ｐhe−Ｔhr（Ｂzl ）−Ｓer（Ｂ
zl ）−Ａsp（ＯcＨｅx）−Ｔyr（Ｂr-Ｚ）−Ｓer(Ｂzl
）−Ｌys(Ｃl-Ｚ）−Ｔyr(Ｂr-Ｚ）−Ｌeu−Ａsp（Ｏc
Ｈｅx）−Ｓer（Ｂzl ）−Ａrg（Ｔos）−Ａrg（Ｔos）
−Ａla−Ｇln−Ａsp（ＯＢzl）−Ｐhe−Ｖal-Ｇln−Ｔr
p−Ｌeu−Ｍet−Ａsn−Ｔhr（Ｂzl ）−ＰＡＭ樹脂50g
を得た。In the same manner as in Example 1-1, one amino acid residue from position 18 to position 1 was sequentially bonded to the above resin to extend it, and H-His (Bom) -Ser (Bzl)- Gln-
Gly-Thr (Bzl) -Phe-Thr (Bzl) -Ser (B
zl) -Asp (OcHex) -Tyr (Br-Z) -Ser (Bzl
) -Lys (Cl-Z) -Tyr (Br-Z) -Leu-Asp (Oc
Hex) -Ser (Bzl) -Arg (Tos) -Arg (Tos)
-Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Tr
p-Leu-Met-Asn-Thr (Bzl) -PAM resin 50g
I got

【００５７】得られた保護ペプチド−ＰＡＭ樹脂から、
実施例１−１と同様の方法でペプチドの抽出及び精製を
行って、精製グルカゴン0.89gを得た。尚、上記で用い
たBoc-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-OH は下記
(1) 〜(6) の操作によって製造した。 (1) Boc-Gln-Trp-OH の合成 H-Trp-OH 24.50g(0.12mmol)を水 200ml-DMF 100mlに溶
解し、0℃でＮ−メチルモルフォリン 13.2ml(0.12mol)
を加えて中和した。この溶液を、Boc-Gln-ONp36.73g(0.
1mol) のDMF溶液 200mlと混合し、最終液量を水 200ml-
DMF 500mlに調製した。次いで0℃で1時間、室温で一晩
撹拌した後減圧濃縮した。残渣に酢酸エチル、及び1N-H
Clを加えて水層を分離した後、有機層の沈殿物を濾取
し、エーテルで洗浄した。得られた粉末を90％MeOH−水
に溶解し、この溶液を陽イオン交換樹脂(AG 50W-x8)を
充填したカラムに通し、目的物の溶出画分を集め、減圧
濃縮した。残渣にエーテルを加えた後、生じた沈殿物を
濾取して真空乾燥を行って、Boc-Gln-Trp-OH 31.05g(
収率 72%)を得た。From the resulting protected peptide-PAM resin,
The peptide was extracted and purified in the same manner as in Example 1-1 to obtain 0.89 g of purified glucagon. The Boc-Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-OH used above was
It was manufactured by the operations of (1) to (6). (1) Synthesis of Boc-Gln-Trp-OH H-Trp-OH 24.50 g (0.12 mmol) was dissolved in 200 ml of water-DMF 100 ml, and N-methylmorpholine 13.2 ml (0.12 mol) at 0 ° C.
Was added to neutralize. This solution was added to Boc-Gln-ONp 36.73g (0.
(1mol) DMF solution (200ml) and the final volume is 200ml-
Prepared to 500 ml DMF. Then, the mixture was stirred at 0 ° C. for 1 hour and at room temperature overnight, and concentrated under reduced pressure. Ethyl acetate and 1N-H in the residue
After Cl was added to separate the aqueous layer, the precipitate of the organic layer was collected by filtration and washed with ether. The obtained powder was dissolved in 90% MeOH-water, and this solution was passed through a column packed with a cation exchange resin (AG 50W-x8), and elution fractions of the target compound were collected and concentrated under reduced pressure. After adding ether to the residue, the resulting precipitate was collected by filtration and vacuum dried to give Boc-Gln-Trp-OH 31.05g (
Yield 72%) was obtained.

【００５８】得られたBoc-Gln-Trp-OH の融点、旋光
度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：170.3 〜173.3℃ [α] D ：＋1.4920°(c=1.0, メタノール) Rf ：0.81 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ21Ｈ28Ｎ4Ｏ6 理論値 Ｃ:58.32% Ｈ:6.53% Ｎ:12.95% 測定値 Ｃ:58.40% Ｈ:6.48% Ｎ:12.78% (2) Boc-Val-Gln-Trp-OH の合成 上記(1) で得られたBoc-Gln-Trp-OH 31.05g(71.8mmol)
とエタンジチオール 3mlを0℃でTFA50ml に溶解し、室
温で30分間撹拌後、エーテル−ヘキサン(1:1)を加え、
生じた沈殿物を濾取、真空乾燥して、粉末状のH-Gln-Tr
p-OH・TFAを得た。得られた粉末をDMF300ml に溶解し、
0℃でＮ−メチルモルフォリンを加えて中和した。The melting point, optical rotation, Rf of TLC and elemental analysis values of the obtained Boc-Gln-Trp-OH are shown below. mp: 170.3 to 173.3 ° C [α] D : + 1.4920 ° (c = 1.0, methanol) Rf: 0.81 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 21 H 28 N 4 O 6 Theoretical value C: 58.32% H: 6.53% N: 12.95% Measured value C: 58.40% H: 6.48% N: 12.78% (2) Synthesis of Boc-Val-Gln-Trp-OH Obtained by the above (1) Boc-Gln-Trp-OH 31.05g (71.8mmol)
And 3 ml of ethanedithiol were dissolved in 50 ml of TFA at 0 ° C, and after stirring at room temperature for 30 minutes, ether-hexane (1: 1) was added,
The resulting precipitate was collected by filtration and dried in vacuum to obtain powdered H-Gln-Tr.
p-OH.TFA was obtained. Dissolve the obtained powder in 300 ml of DMF,
It was neutralized by adding N-methylmorpholine at 0 ° C.

【００５９】Boc-Val-OH 15.6g(71.8mmol)をDMF 150ml
に溶解し、ドライアイス−エタノールで-20℃に冷却
し、Ｎ−メチルモルフォリン 7.9ml(71.8mmol)を滴下
し、次いでクロロギ酸イソブチル 9.5ml(71.8mmol)を滴
下して、-20℃で1分間撹拌して混合酸無水物の溶液を調
製した。この溶液を前記の溶液に加えて0℃で5分間、室
温で30分間撹拌した後、減圧濃縮した。残渣に1N-HClを
加えて、生じた沈殿物を濾取して、水で洗浄した。得ら
れた粉末をDMF-エーテル-ヘキサンで再沈殿後、真空乾
燥を行い、Boc-Val-Gln-Trp-OH 29.71g(収率 78%)を得
た。15.6 g (71.8 mmol) of Boc-Val-OH was added to 150 ml of DMF.
Dissolved in water, cooled to −20 ° C. with dry ice-ethanol, 7.9 ml (71.8 mmol) of N-methylmorpholine was added dropwise, and then 9.5 ml (71.8 mmol) of isobutyl chloroformate was added dropwise at −20 ° C. A solution of mixed acid anhydride was prepared by stirring for 1 minute. This solution was added to the above solution, stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes, and then concentrated under reduced pressure. 1N-HCl was added to the residue, and the resulting precipitate was collected by filtration and washed with water. The obtained powder was reprecipitated with DMF-ether-hexane and then vacuum dried to obtain 29.71 g (yield 78%) of Boc-Val-Gln-Trp-OH.

【００６０】得られたBoc-Val-Gln-Trp-OH の融点、旋
光度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：171.2〜175.9℃ [α] D ：＋0.6754°(c=0.5, DMSO) Rf ：0.72 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ26Ｈ37Ｎ5Ｏ7 理論値 Ｃ:58.74% Ｈ:7.02% Ｎ:13.17% 測定値 Ｃ:58.53% Ｈ:7.37% Ｎ:12.99% (3) Boc-Phe-Val-Gln-Trp-OH の合成 上記(2) で得られたBoc-Val-Gln-Trp-OH 29.71g(55.9mm
ol)とエタンジチオール 3mlを0℃でTFA50ml に溶解し、
室温で30分間撹拌した。次いでエーテルを加えて、生じ
た沈殿物を濾取、真空乾燥して、粉末状のH-Val-Gln-Tr
p-OH・TFAを得た。得られた粉末をDMF 200mlに溶解し、
0℃でＮ−メチルモルフォリンを加えて中和した。The melting point, optical rotation, Tf Rf and elemental analysis values of the obtained Boc-Val-Gln-Trp-OH are shown below. mp: 171.2 to 175.9 ° C [α] D : + 0.6754 ° (c = 0.5, DMSO) Rf: 0.72 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 26 H 37 N 5 O 7 Theoretical value C: 58.74% H: 7.02% N: 13.17% Measured value C: 58.53% H: 7.37% N: 12.99% (3) Synthesis of Boc-Phe-Val-Gln-Trp-OH In the above (2) Obtained Boc-Val-Gln-Trp-OH 29.71 g (55.9 mm
ol) and 3 ml of ethanedithiol are dissolved in 50 ml of TFA at 0 ° C,
Stir for 30 minutes at room temperature. Then ether was added, and the resulting precipitate was collected by filtration and dried in vacuum to obtain powdery H-Val-Gln-Tr.
p-OH.TFA was obtained. Dissolve the obtained powder in 200 ml of DMF,
It was neutralized by adding N-methylmorpholine at 0 ° C.

【００６１】Boc-Phe-OH 14.9g(55.9mmol)をDMF 150ml
に溶解し、ドライアイス−エタノールで-20℃に冷却
し、Ｎ−メチルモルフォリン 6.2ml(55.9mmol)を滴下
し、ついでクロロギ酸イソブチル 7.4ml(55.9mmol)を滴
下し、-20℃で1分間撹拌して混合酸無水物の溶液を調製
した。この溶液を前記の溶液に加えて、0℃で5分間、室
温で30分間撹拌した後、減圧濃縮した。残渣に1N-HClを
加えて、生じた沈殿物を濾取して、水で洗浄した。得ら
れた粉末をDMF-エーテルで再沈殿後、真空乾燥を行っ
て、Boc-Phe-Val-Gln-Trp-OH 30.04g(収率 79%)を得
た。14.9 g (55.9 mmol) of Boc-Phe-OH was added to 150 ml of DMF.
Dissolve in water, cool to -20 ° C with dry ice-ethanol, add 6.2 ml (55.9 mmol) of N-methylmorpholine dropwise, then add 7.4 ml (55.9 mmol) of isobutyl chloroformate dropwise, and add 1 ml at -20 ° C. A solution of mixed acid anhydride was prepared by stirring for a minute. This solution was added to the above solution, stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes, and then concentrated under reduced pressure. 1N-HCl was added to the residue, and the resulting precipitate was collected by filtration and washed with water. The obtained powder was reprecipitated with DMF-ether and vacuum dried to obtain 30.04 g (yield 79%) of Boc-Phe-Val-Gln-Trp-OH.

【００６２】得られたBoc-Phe-Val-Gln-Trp-OH の融
点、旋光度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：145.7〜148.4℃（分解） [α] D ：＋4.2208°(c=1.0, DMSO) Rf ：0.74 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ35Ｈ46Ｎ6Ｏ8 理論値 Ｃ:61.93% Ｈ:6.83% Ｎ:12.38% 測定値 Ｃ:61.52% Ｈ:7.30% Ｎ:12.45% (4) Boc-Asp(OBzl)-Phe-Val-Gln-Trp-OH の合成 上記(3) で得られたBoc-Phe-Val-Gln-Trp-OH 29.95g(4
4.1mmol)とエタンジチオール 3mlを0℃でTFA 50mlに溶
解し、室温で30分間撹拌後、エーテルを加えて生じた沈
殿物を濾取、真空乾燥し、粉末状のH-Phe-Val-Gln-Trp-
OH・TFAを得た。得られた粉末をDMF200ml に溶解し、0
℃でＮ−メチルモルフォリンを加えて中和した。The melting point, optical rotation, Rf of TLC and elemental analysis values of the obtained Boc-Phe-Val-Gln-Trp-OH are shown below. mp: 145.7-148.4 ° C (decomposition) [α] D : + 4.2208 ° (c = 1.0, DMSO) Rf: 0.74 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 35 H 46 N 6 O 8 Theoretical value C: 61.93% H: 6.83% N: 12.38% Measured value C: 61.52% H: 7.30% N: 12.45% (4) Boc-Asp (OBzl) -Phe-Val-Gln-Trp- Synthesis of OH Boc-Phe-Val-Gln-Trp-OH obtained in the above (3) 29.95 g (4
4.1 mmol) and ethanedithiol (3 ml) were dissolved in TFA (50 ml) at 0 ° C., and the mixture was stirred at room temperature for 30 minutes, ether was added, and the resulting precipitate was collected by filtration and dried under vacuum to obtain powdery H-Phe-Val-Gln. -Trp-
I got OH / TFA. Dissolve the obtained powder in 200 ml of DMF and
Neutralization was carried out by adding N-methylmorpholine at 0 ° C.

【００６３】Boc-Asp(OBzl)-OH 14.3g(44.1mmol)をDMF
150mlに溶解し、ドライアイス−エタノールで-20℃に冷
却し、Ｎ−メチルモルフォリン 4.9ml(44.1mmol)を滴下
した後クロロギ酸イソブチル 5.9ml(44.1mmol)を滴下
し、-20℃で1分間撹拌して混合酸無水物の溶液を調製し
た。この溶液を前記の溶液に加え0℃で5分間、室温で30
分間撹拌した後、減圧濃縮した。残渣に1N-HClを加え
て、生じた沈殿物を濾取して、水で洗浄した。得られた
粉末をDMF-エーテルで再沈殿後、真空乾燥を行って、Bo
c-Asp(OBzl)-Phe-Val-Gln-Trp-OH 32.55g(収率 79%)を
得た。14.3 g (44.1 mmol) of Boc-Asp (OBzl) -OH was added to DMF.
Dissolve in 150 ml, cool to -20 ° C with dry ice-ethanol, add 4.9 ml (44.1 mmol) of N-methylmorpholine dropwise, then add 5.9 ml (44.1 mmol) of isobutyl chloroformate and add 1 at -20 ° C. A solution of mixed acid anhydride was prepared by stirring for a minute. Add this solution to the above solution for 5 minutes at 0 ° C and 30 minutes at room temperature.
After stirring for 1 minute, the mixture was concentrated under reduced pressure. 1N-HCl was added to the residue, and the resulting precipitate was collected by filtration and washed with water. After reprecipitation of the obtained powder with DMF-ether, vacuum drying was performed to
32.55 g (yield 79%) of c-Asp (OBzl) -Phe-Val-Gln-Trp-OH was obtained.

【００６４】得られたBoc-Asp(OBzl)-Phe-Val-Gln-Trp-
OH の融点、旋光度、ＴＬＣのRf及び元素分析値を下記
に示す。 m.p. ：178.6〜182.0℃（分解） [α] D ：−9.5106°(c=0.5, DMSO) Rf ：0.86 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ46Ｈ57Ｎ7Ｏ11 理論値 Ｃ:62.50% Ｈ:6.50% Ｎ:11.09% 測定値 Ｃ:62.11% Ｈ:7.93% Ｎ:11.02% (5) Boc-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-OH の合成 上記(4) で得られたBoc-Asp(OBzl)-Phe-Val-Gln-Trp-OH
33.40g(37.8mmol)とエタンジチオール 3mlを0℃でTFA
50mlに溶解し、室温で30分間撹拌後、エーテルを加え
て、生じた沈殿物を濾取、真空乾燥し、粉末状のH-Asp
(OBzl)-Phe-Val-Gln-Trp-OH・TFAを得た。得られた粉末
をDMF 200mlに溶解し、0℃でＮ−メチルモルフォリンを
加えて中和した。The obtained Boc-Asp (OBzl) -Phe-Val-Gln-Trp-
The melting point of OH, optical rotation, Rf of TLC and elemental analysis values are shown below. mp: 178.6-182.0 ° C (decomposition) [α] D : -9.5106 ° (c = 0.5, DMSO) Rf: 0.86 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 46 H 57 N 7 O 11 Theoretical value C: 62.50% H: 6.50% N: 11.09% Measured value C: 62.11% H: 7.93% N: 11.02% (5) Boc-Gln-Asp (OBzl) -Phe-Val-Gln-Trp Synthesis of -OH Boc-Asp (OBzl) -Phe-Val-Gln-Trp-OH obtained in (4) above
33.40 g (37.8 mmol) and 3 ml of ethanedithiol were added to TFA at 0 ° C.
After dissolving in 50 ml and stirring at room temperature for 30 minutes, ether was added, and the resulting precipitate was collected by filtration and dried under vacuum to give powdery H-Asp.
(OBzl) -Phe-Val-Gln-Trp-OH.TFA was obtained. The obtained powder was dissolved in 200 ml of DMF, and N-methylmorpholine was added at 0 ° C. to neutralize.

【００６５】この溶液と、Boc-Gln-ONp 13.9g(37.8mmo
l)のDMF溶液 100mlを混合し、0℃で1時間、室温で一晩
撹拌した後、減圧濃縮した。残渣に1N-HClを加えて、生
じた沈殿物を濾取して、水で洗浄した。得られた粉末を
DMF-エーテルで再沈殿後、真空乾燥を行って、Boc-Gln-
Asp(OBzl)-Phe-Val-Gln-Trp-OH 31.70g(収率 83%)を得
た。This solution and Boc-Gln-ONp 13.9g (37.8mmo
l) DMF solution (100 ml) was mixed, stirred at 0 ° C. for 1 hour and at room temperature overnight, and then concentrated under reduced pressure. 1N-HCl was added to the residue, and the resulting precipitate was collected by filtration and washed with water. The obtained powder
After reprecipitation with DMF-ether, vacuum drying was performed and Boc-Gln-
31.70 g (yield 83%) of Asp (OBzl) -Phe-Val-Gln-Trp-OH was obtained.

【００６６】得られたBoc-Gln-Asp(OBzl)-Phe-Val-Gln-
Trp-OH の融点、旋光度、ＴＬＣのRf及び元素分析値を
下記に示す。 m.p. ：165.5〜168.6℃（分解） [α] D ：−10.967°(c=1.0, DMSO) Rf ：0.79 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ51Ｈ65Ｎ9Ｏ13 理論値 Ｃ:60.52% Ｈ:6.47% Ｎ:12.45% 測定値 Ｃ:60.20% Ｈ:6.80% Ｎ:11.90% (6) Boc-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-OH の合
成 上記(5) で得られたBoc-Gln-Asp(OBzl)-Phe-Val-Gln-Tr
p-OH 31.70g(31.3mmol)とエタンジチオール 3mlを0℃で
TFA 50mlに溶解し、室温で30分間撹拌後、エーテルを加
えて生じた沈殿物を濾取、真空乾燥し、粉末状のH-Gln-
Asp(OBzl)-Phe-Val-Gln-Trp-OH・TFAを得た。得られた
粉末をDMF 200mlに溶解し、0℃でＮ−メチルモルフォリ
ンを加えて中和した。The obtained Boc-Gln-Asp (OBzl) -Phe-Val-Gln-
The melting point, optical rotation, Tf Rf, and elemental analysis values of Trp-OH are shown below. mp: 165.5 to 168.6 ° C (decomposition) [α] D : -10.967 ° (c = 1.0, DMSO) Rf: 0.79 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 51 H 65 N 9 O 13 Theoretical value C: 60.52% H: 6.47% N: 12.45% Measured value C: 60.20% H: 6.80% N: 11.90% (6) Boc-Ala-Gln-Asp (OBzl) -Phe-Val-Gln Synthesis of -Trp-OH Boc-Gln-Asp (OBzl) -Phe-Val-Gln-Tr obtained in (5) above
31.70 g (31.3 mmol) of p-OH and 3 ml of ethanedithiol at 0 ° C
After dissolving in 50 ml of TFA and stirring for 30 minutes at room temperature, ether was added to form a precipitate, which was collected by filtration and dried under vacuum to obtain powdery H-Gln-
Asp (OBzl) -Phe-Val-Gln-Trp-OH.TFA was obtained. The obtained powder was dissolved in 200 ml of DMF, and N-methylmorpholine was added at 0 ° C. to neutralize.

【００６７】Boc-Ala-OH 5.92g(31.3mmol)をDMF 150ml
に溶解し、ドライアイス−エタノールで-20℃に冷却
し、Ｎ−メチルモルフォリン 3.4ml(31.3mmol)を滴下し
た後クロロギ酸イソブチル 4.1ml(31.3mmol)を滴下し
て、-20℃で1分間撹拌して混合酸無水物の溶液を調製し
た。この溶液を前記の溶液に加え0℃で5分間、室温で30
分間撹拌した後、減圧濃縮した。残渣に1N-HClを加え
て、生じた沈殿物を濾取して、水で洗浄した。得られた
粉末をDMF-酢酸エチルで再沈殿、エーテルで洗浄後、真
空乾燥を行って、Boc-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-
Trp-OH 24.59g(収率73%)を得た。Boc-Ala-OH 5.92 g (31.3 mmol) was added to DMF 150 ml.
Dissolve in water, cool to -20 ° C with dry ice-ethanol, add 3.4 ml (31.3 mmol) of N-methylmorpholine dropwise, and add 4.1 ml (31.3 mmol) of isobutyl chloroformate dropwise, and add 1 ml at -20 ° C. A solution of mixed acid anhydride was prepared by stirring for a minute. Add this solution to the above solution for 5 minutes at 0 ° C and 30 minutes at room temperature.
After stirring for 1 minute, the mixture was concentrated under reduced pressure. 1N-HCl was added to the residue, and the resulting precipitate was collected by filtration and washed with water. The obtained powder was reprecipitated with DMF-ethyl acetate, washed with ether, and then vacuum dried to give Boc-Ala-Gln-Asp (OBzl) -Phe-Val-Gln-.
24.59 g (yield 73%) of Trp-OH was obtained.

【００６８】得られたBoc-Gln-Asp(OBzl)-Phe-Val-Gln-
Trp-OH の融点、旋光度、ＴＬＣのRf及び元素分析値を
下記に示す。 m.p. ：190.33〜194.37℃（分解） [α] D ：−15.652°(c=1.0, DMSO) Rf ：0.82 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ54Ｈ70Ｎ10Ｏ14 理論値 Ｃ:59.88% Ｈ:6.51% Ｎ:12.93% 測定値 Ｃ:59.40% Ｈ:6.71% Ｎ:12.19% 〔実施例４〕方法２によるグルカゴンの固相合成 実施例１−１において得られた中間体 Ｈ−Ｌeu−Ｍet
−Ａsn−Ｔhr（Ｂzl）−ＰＡＭ樹脂 1.48gに、アミノ酸
配列の17〜25位のアミノ酸残基に相当する下記式： Ｂoc−Ａrg（Ｔｏｓ）−Ａrg（Ｔｏｓ）−Ａla−Ｇln−
Ａsp（ＯＢzl）−Ｐhe−Ｖal−Ｇln−Ｔrp−ＯＨ で表されるノナペプチド 15mmol 、ＨＯＢｔ 15mmol 、
ＤＣＭ 100ml及びＤＭＦ20mlを加えて攪拌し、さらにＤ
ＣＣ（１Ｍ−ＤＣＭ溶液）15mlを加えて２時間反応させ
た。反応後、反応混合液を濾過し、ＭｅＯＨ 200ml（２
回）、ＤＣＭ 200ml（２回）の順に攪拌下で処理した。
尚、各処理毎に濾過を行った。このようにして、Ｂoc−
Ａrg（Ｔｏｓ）−Ａrg（Ｔｏｓ）−Ａla−Ｇln−Ａsp
（ＯcＨｅx）−Ｐhe−Ｖal-Ｇln−Ｔrp−Ｌeu−Ｍet−
Ａsn−Ｔhr（Ｂzl）−ＰＡＭ樹脂を得た。The obtained Boc-Gln-Asp (OBzl) -Phe-Val-Gln-
The melting point, optical rotation, Tf Rf, and elemental analysis values of Trp-OH are shown below. mp: 190.33 to 194.37 ° C (decomposition) [α] D : −15.652 ° (c = 1.0, DMSO) Rf: 0.82 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 54 H 70 N 10 O 14 Theoretical value C: 59.88% H: 6.51% N: 12.93% Measured value C: 59.40% H: 6.71% N: 12.19% [Example 4] Solid-phase synthesis of glucagon by method 2 In Example 1-1 Obtained intermediate H-Leu-Met
-Asn-Thr (Bzl) -PAM resin 1.48 g has the following formula corresponding to the amino acid residues at positions 17 to 25 of the amino acid sequence: Boc-Arg (Tos) -Arg (Tos) -Ala-Gln-
15 mmol of nonapeptide represented by Asp (OBzl) -Phe-Val-Gln-Trp-OH, HOBt 15 mmol,
DCM 100 ml and DMF 20 ml are added and stirred, and further D
15 ml of CC (1M-DCM solution) was added and reacted for 2 hours. After the reaction, the reaction mixture was filtered, and 200 ml of MeOH (2
Times) and DCM 200 ml (twice) in that order under stirring.
In addition, filtration was performed for each treatment. In this way, Boc-
Arg (Tos) -Arg (Tos) -Ala-Gln-Asp
(OcHex) -Phe-Val-Gln-Trp-Leu-Met-
An Asn-Thr (Bzl) -PAM resin was obtained.

【００６９】上記樹脂に、実施例１−１の方法と同様に
して16位から１位までアミノ酸残基を順次１個ずつ結合
して延長し、Ｈ−Ｈis(Ｂom）−Ｓer(Ｂzl）−Ｇln−Ｇ
ly−Ｔhr（Ｂzl）−Ｐhe−Ｔhr（Ｂzl）−Ｓer（Ｂzl）
−Ａsp（ＯcＨｅx）−Ｔyr（Ｂr-Ｚ）−Ｓer(Ｂzl）−
Ｌys(Ｃl-Ｚ）−Ｔyr(Ｂr-Ｚ）−Ｌeu−Ａsp（ＯcＨｅ
x）−Ｓer（Ｂzl）−Ａrg（Ｔos）−Ａrg（Ｔos）−Ａl
a−Ｇln−Ａsp（ＯＢzl）−Ｐhe−Ｖal-Ｇln−Ｔrp−Ｌ
eu−Ｍet−Ａsn−Ｔhr（Ｂzl）−ＰＡＭ樹脂50gを得
た。To the above resin, amino acid residues from position 16 to position 1 were sequentially linked and extended one by one in the same manner as in Example 1-1, and H-His (Bom) -Ser (Bzl)- Gln-G
ly-Thr (Bzl) -Phe-Thr (Bzl) -Ser (Bzl)
-Asp (OcHex) -Tyr (Br-Z) -Ser (Bzl)-
Lys (Cl-Z) -Tyr (Br-Z) -Leu-Asp (OcHe
x) -Ser (Bzl) -Arg (Tos) -Arg (Tos) -Al
a-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-L
50 g of eu-Met-Asn-Thr (Bzl) -PAM resin was obtained.

【００７０】得られた保護ペプチド−ＰＡＭ樹脂から、
実施例１−１と同様の方法でペプチドの抽出及び精製を
行って、精製グルカゴン0.89gを得た。尚、上記で用い
たBoc-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val-
Gln-Trp-OH は下記(1) 及び(2) の操作によって製造し
た。 (1) Boc-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp
-OH の合成 上記実施例３の(6) で得られたBoc-Ala-Gln-Asp(OBzl)-
Phe-Val-Gln-Trp-OH 24.59g(22.7mmol)とエタンジチオ
ール 3mlを0℃でTFA 50mlに溶解し、室温で30分間撹拌
後、エーテルを加えて、生じた沈殿物を濾取、真空乾燥
し、粉末状のH-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-OH
・TFAを得た。得られた粉末をDMF 200mlに溶解し、0℃
でＮ−メチルモルフォリンを加えて中和した。From the resulting protected peptide-PAM resin,
The peptide was extracted and purified in the same manner as in Example 1-1 to obtain 0.89 g of purified glucagon. Incidentally, Boc-Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-Val-used above
Gln-Trp-OH was produced by the following operations (1) and (2). (1) Boc-Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Trp
Synthesis of -OH Boc-Ala-Gln-Asp (OBzl)-obtained in (6) of Example 3 above.
24.59 g (22.7 mmol) of Phe-Val-Gln-Trp-OH and 3 ml of ethanedithiol were dissolved in 50 ml of TFA at 0 ° C., and after stirring at room temperature for 30 minutes, ether was added, and the resulting precipitate was collected by filtration and vacuumed. Dry, powdered H-Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-OH
・ I got TFA. Dissolve the obtained powder in 200 ml of DMF and
Then, N-methylmorpholine was added to neutralize.

【００７１】Boc-Arg(Tos)-OH 9.80g(22.7mmol)をDMF 1
50mlに溶解し、ドライアイス−エタノールで-20℃に冷
却し、Ｎ−メチルモルフォリン 2.5ml(22.7mmol)を滴下
した後クロロギ酸イソブチル 3.0ml(22.7mmol)を滴下し
て、-20℃で1分間撹拌し、混合酸無水物の溶液を調製し
た。この溶液を前記の溶液に加えて、0℃で5分間、室温
で30分間撹拌した後、減圧濃縮した。残渣に1N-HClを加
えて、生じた沈殿物を濾取して、水で洗浄した。得られ
た粉末をDMF-酢酸エチルで再沈殿させ、沈殿物を濾取し
てエーテルで洗浄後、真空乾燥を行って、Boc-Arg(Tos)
-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-OH 24.11g(収率
76%)を得た。9.80 g (22.7 mmol) of Boc-Arg (Tos) -OH was added to DMF 1
Dissolve in 50 ml, cool to -20 ° C with dry ice-ethanol, add 2.5 ml (22.7 mmol) of N-methylmorpholine dropwise, and then add 3.0 ml (22.7 mmol) of isobutyl chloroformate at -20 ° C. After stirring for 1 minute, a mixed acid anhydride solution was prepared. This solution was added to the above solution, stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes, and then concentrated under reduced pressure. 1N-HCl was added to the residue, and the resulting precipitate was collected by filtration and washed with water. The obtained powder was reprecipitated with DMF-ethyl acetate, the precipitate was collected by filtration, washed with ether, and then vacuum dried to give Boc-Arg (Tos).
-Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-OH 24.11 g (yield
76%).

【００７２】得られたBoc-Arg(Tos)-Ala-Gln-Asp(OBzl)
-Phe-Val-Gln-Trp-OH の融点、旋光度、ＴＬＣのRf及び
元素分析値を下記に示す。 m.p. ：194.2 〜197.6 ℃（分解） [α] D ：−7.8009°(c=1.0, DMSO) Rf ：0.77 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ67Ｈ88Ｎ14Ｏ17S 理論値 Ｃ:57.75% Ｈ:6.36% Ｎ:14.07% 測定値 Ｃ:57.92% Ｈ:6.66% Ｎ:13.11% (2) Boc-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Va
l-Gln-Trp-OH の合成 上記(1) で得られたBoc-Arg(Tos)-Ala-Gln-Asp(OBzl)-P
he-Val-Gln-Trp-OH 24.11g(17.3mmol)とエタンジチオー
ル 3mlを0℃でTFA 50mlに溶解し、室温で30分間撹拌
後、エーテルを加えて、生じた沈殿物を濾取、真空乾燥
し、粉末状のH-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val-G
ln-Trp-OH・TFAを得た。得られた粉末をDMF 200mlに溶
解し、0℃でＮ−メチルモルフォリンを加えて中和し
た。Boc-Arg (Tos) -Ala-Gln-Asp (OBzl) obtained
The melting point, optical rotation, Rf of TLC, and elemental analysis values of -Phe-Val-Gln-Trp-OH are shown below. mp: 194.2 to 197.6 ° C (decomposition) [α] D : −7.8009 ° (c = 1.0, DMSO) Rf: 0.77 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 67 H 88 N 14 O 17 S Theoretical value C: 57.75% H: 6.36% N: 14.07% Measured value C: 57.92% H: 6.66% N: 13.11% (2) Boc-Arg (Tos) -Arg (Tos) -Ala-Gln -Asp (OBzl) -Phe-Va
Synthesis of l-Gln-Trp-OH Boc-Arg (Tos) -Ala-Gln-Asp (OBzl) -P obtained in (1) above
He-Val-Gln-Trp-OH 24.11 g (17.3 mmol) and ethanedithiol 3 ml were dissolved in TFA 50 ml at 0 ° C., stirred at room temperature for 30 minutes, ether was added, and the resulting precipitate was collected by filtration and vacuumed. Dried, powdered H-Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-Val-G
ln-Trp-OH.TFA was obtained. The obtained powder was dissolved in 200 ml of DMF, and N-methylmorpholine was added at 0 ° C. to neutralize.

【００７３】Boc-Arg(Tos)-OH 7.41g(17.3mmol)をDMF 1
50mlに溶解し、ドライアイス−エタノールで-20℃に冷
却し、Ｎ−メチルモルフォリン 1.9ml(17.3mmol)を滴下
した後クロロギ酸イソブチル 2.3ml(17.3mmol)を滴下し
て、-20℃で1分間撹拌し、混合酸無水物の溶液を調製し
た。この溶液を前記の溶液に加えて、0℃で5分間、室温
で30分間撹拌した後、減圧濃縮した。残渣に1N-HClを加
えて、生じた沈殿物を濾取して、水で洗浄した。得られ
た粉末をDMF-酢酸エチルで再沈殿させ、沈殿物をエーテ
ルで洗浄後、真空乾燥を行って、Boc-Arg(Tos)-Arg(To
s)-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-OH 24.70g( 収
率 84%)を得た。7.41 g (17.3 mmol) of Boc-Arg (Tos) -OH was added to DMF 1
Dissolve in 50 ml, cool to -20 ° C with dry ice-ethanol, add 1.9 ml (17.3 mmol) of N-methylmorpholine dropwise, and then add 2.3 ml (17.3 mmol) of isobutyl chloroformate at -20 ° C. After stirring for 1 minute, a mixed acid anhydride solution was prepared. This solution was added to the above solution, stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes, and then concentrated under reduced pressure. 1N-HCl was added to the residue, and the resulting precipitate was collected by filtration and washed with water. The obtained powder was reprecipitated with DMF-ethyl acetate, the precipitate was washed with ether, and then vacuum dried to obtain Boc-Arg (Tos) -Arg (To
s) -Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-OH (24.70 g, yield 84%) was obtained.

【００７４】得られたBoc-Arg(Tos)-Ala-Gln-Asp(OBzl)
-Phe-Val-Gln-Trp-OH の融点、旋光度、ＴＬＣのRf及び
元素分析値を下記に示す。 m.p. ：192.2 〜194.4 ℃（分解） [α] D ：−8.5057°(c=1.0, DMSO) Rf ：0.81 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ80Ｈ106Ｎ18Ｏ20S2 理論値 Ｃ:56.39% Ｈ:6.36% Ｎ:14.80% 測定値 Ｃ:56.48% Ｈ:6.71% Ｎ:14.15% 〔実施例５〕方法２によるグルカゴンの固相合成 実施例１−１において得られた中間体 Ｈ−Ｔrp−Ｌeu
−Ｍet−Ａsn−Ｔhr（Ｂzl）−ＰＡＭ樹脂 1.48gに、グ
ルカゴンのアミノ酸配列の19〜24位のアミノ酸残基に相
当する下記式： Ｂoc−Ａla−Ｇln−Ａsp（ＯＢzl）−Ｐhe−Ｖal−Ｇln
−ＯＨ で表されるヘキサペプチド 15mmol 、ＨＯＢｔ 15mmol
、ＤＣＭ 100ml及びＤＭＦ 20mlを加えて攪拌し、さら
にＤＣＣ（１Ｍ−ＤＣＭ溶液）15mlを加えて２時間反応
させた。反応後、反応混合液を濾過して、ＭｅＯＨ 200
ml（２回）、ＤＣＭ 200ml（２回）の順に攪拌下で処理
した。尚、各処理毎に濾過を行った。このようにして、
Ｂoc−Ａla−Ｇln−Ａsp（ＯcＨｅx）−Ｐhe−Ｖal-Ｇl
n−Ｔrp−Ｌeu−Ｍet−Ａsn−Ｔhr（Ｂzl ）−ＰＡＭ樹
脂を得た。Boc-Arg (Tos) -Ala-Gln-Asp (OBzl) obtained
The melting point, optical rotation, Rf of TLC, and elemental analysis values of -Phe-Val-Gln-Trp-OH are shown below. mp: 192.2 to 194.4 ° C (decomposition) [α] D : −8.5057 ° (c = 1.0, DMSO) Rf: 0.81 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 80 H 106 N 18 O 20 S 2 Theoretical value C: 56.39% H: 6.36% N: 14.80% Measured value C: 56.48% H: 6.71% N: 14.15% [Example 5] Solid phase synthesis of glucagon by Method 2 Example 1- 1 obtained in 1 H-Trp-Leu
-Met-Asn-Thr (Bzl) -PAM resin 1.48 g, the following formula corresponding to amino acid residues 19-24 of the amino acid sequence of glucagon: Boc-Ala-Gln-Asp (OBzl) -Phe-Val- Gln
Hexapeptide represented by -OH 15 mmol, HOBt 15 mmol
, DCM 100 ml and DMF 20 ml were added and stirred, and DCC (1M-DCM solution) 15 ml was further added and reacted for 2 hours. After the reaction, the reaction mixture is filtered to remove MeOH 200
ml (twice), DCM 200 ml (twice) in that order under stirring. In addition, filtration was performed for each treatment. In this way,
Boc-Ala-Gln-Asp (OcHex) -Phe-Val-Gl
An n-Trp-Leu-Met-Asn-Thr (Bzl) -PAM resin was obtained.

【００７５】上記樹脂に、実施例１−１の方法と同様に
して18位から１位までアミノ酸残基を順次１個ずつ結合
して延長し、Ｈ−Ｈis(Ｂom）−Ｓer(Ｂzl）−Ｇln−Ｇ
ly−Ｔhr（Ｂzl）−Ｐhe−Ｔhr（Ｂzl）−Ｓer（Ｂzl）
−Ａsp（ＯcＨｅx）−Ｔyr（Ｂr-Ｚ）−Ｓer(Ｂzl）−
Ｌys(Ｃl-Ｚ）−Ｔyr(Ｂr-Ｚ）−Ｌeu−Ａsp（ＯcＨｅ
x）−Ｓer（Ｂzl）−Ａrg（Ｔos）−Ａrg（Ｔos）−Ａl
a−Ｇln−Ａsp（ＯＢzl）−Ｐhe−Ｖal-Ｇln−Ｔrp−Ｌ
eu−Ｍet−Ａsn−Ｔhr（Ｂzl）−ＰＡＭ樹脂50gを得
た。In the same manner as in Example 1-1, the above resin was sequentially extended with one amino acid residue from position 18 to position 1 by coupling with H-His (Bom) -Ser (Bzl)-. Gln-G
ly-Thr (Bzl) -Phe-Thr (Bzl) -Ser (Bzl)
-Asp (OcHex) -Tyr (Br-Z) -Ser (Bzl)-
Lys (Cl-Z) -Tyr (Br-Z) -Leu-Asp (OcHe
x) -Ser (Bzl) -Arg (Tos) -Arg (Tos) -Al
a-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-L
50 g of eu-Met-Asn-Thr (Bzl) -PAM resin was obtained.

【００７６】得られた保護ペプチド−ＰＡＭ樹脂から、
実施例１−１と同様の方法でペプチドの抽出及び精製を
行って、精製グルカゴン0.89gを得た。尚、上記で用い
たBoc-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-OH は下記(1)
〜(5)の操作によって製造した。 (1) Z-Val-Gln-OH の合成 Z-Val-OH 25.13g(0.1mol) をTHF 200mlに溶解し、ドラ
イアイス−エタノールで-20℃に冷却し、Ｎ−メチルモ
ルフォリン 11ml(0.1mol)を滴下した後、クロロギ酸イ
ソブチル13.2ml(0.1mol) を滴下して、-20℃で1分間攪
拌して混合酸無水物の溶液を調製した。さらに、NHS 1
2.08g(0.105mol)のTHF溶液 150mlと混合し、室温で30分
間攪拌した。得られた反応混合液を、Ｎ−メチルモルフ
ォリン 33ml(0.3mol)で中和したH-Gln-OH 21.92g(0.15m
ol)の水溶液 500mlと混合し、室温で一晩撹拌して、THF
を留去した。エーテル、酢酸エチルの順で洗浄後、1N-H
ClでpH3に調製し、残渣に酢酸エチルを加え、1N-HClで1
回、水で2回の順で洗浄した。有機層を減圧濃縮し、残
渣にエーテルを加えて、生じた沈殿物を濾取し、真空乾
燥を行って、Z-Val-Gln-OH 21.63g(収率 57％)を得
た。From the resulting protected peptide-PAM resin,
The peptide was extracted and purified in the same manner as in Example 1-1 to obtain 0.89 g of purified glucagon. In addition, Boc-Ala-Gln-Asp (OBzl) -Phe-Val-Gln-OH used above is the following (1)
It was produced by the operation of (5). (1) Synthesis of Z-Val-Gln-OH 25.13 g (0.1 mol) of Z-Val-OH was dissolved in 200 ml of THF, cooled to -20 ° C with dry ice-ethanol, and 11 ml (0.1 ml of N-methylmorpholine). mol), followed by dropwise addition of isobutyl chloroformate 13.2 ml (0.1 mol) and stirring at -20 ° C for 1 minute to prepare a mixed acid anhydride solution. In addition, NHS 1
2.08 g (0.105 mol) of a THF solution (150 ml) was mixed and stirred at room temperature for 30 minutes. The obtained reaction mixture was neutralized with 33 ml (0.3 mol) of N-methylmorpholine, 21.92 g (0.15 m) of H-Gln-OH.
ol) in 500 ml of water and stirred at room temperature overnight to remove THF.
Was distilled off. After washing with ether and ethyl acetate in that order, 1N-H
Adjust to pH 3 with Cl, add ethyl acetate to the residue, and add 1N-HCl to 1
And washed twice with water. The organic layer was concentrated under reduced pressure, ether was added to the residue, the resulting precipitate was collected by filtration, and vacuum dried to obtain 21.63 g (yield 57%) of Z-Val-Gln-OH.

【００７７】得られたZ-Val-Gln-OH の融点、旋光度、
ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：187.9 〜190.03℃ [α] D ：+0.9341°(c=1.0, DMF) Rf ：0.68 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ18Ｈ25Ｎ3Ｏ6 理論値 Ｃ:56.98% Ｈ:6.64% Ｎ:11.08% 測定値 Ｃ:56.80% Ｈ:7.04% Ｎ:10.92% (2) Z-Phe-Val-Gln-OH の合成 上記(1) で得られたZ-Val-Gln-OH 8.47g(22.3mmol) を
メタノール100ml-50%酢酸300mlの混合溶液に溶解し、Pd
-C 1.2gを加え、窒素ガスを通した後、空気を置換して
水素ガスを導入し、接触還元を行った。これを室温で5.
5時間撹拌し、濾過、減圧濃縮後、残渣にエーテルを加
えて、生じた沈殿物を濾取し、真空乾燥を行って、粉末
状のH-Val-Gln-OH・AcOHを得た。得られた粉末を水 200
mlに溶解し、0℃でＮ−メチルモルフォリンを加えて中
和した。The melting point and optical rotation of the obtained Z-Val-Gln-OH
The Rf and elemental analysis values of TLC are shown below. mp: 187.9 to 190.03 ° C [α] D : + 0.9341 ° (c = 1.0, DMF) Rf: 0.68 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 18 H 25 N 3 O 6 Theoretical value C: 56.98% H: 6.64% N: 11.08% Measured value C: 56.80% H: 7.04% N: 10.92% (2) Synthesis of Z-Phe-Val-Gln-OH Obtained by the above (1) 8.47 g (22.3 mmol) of Z-Val-Gln-OH was dissolved in a mixed solution of 100 ml of methanol and 300 ml of 50% acetic acid to obtain Pd.
After adding 1.2 g of -C and passing nitrogen gas, the air was replaced and hydrogen gas was introduced to carry out catalytic reduction. This at room temperature 5.
After stirring for 5 hours, filtration and concentration under reduced pressure, ether was added to the residue, the resulting precipitate was collected by filtration, and vacuum dried to obtain powdery H-Val-Gln-OH.AcOH. The obtained powder is water 200
It was dissolved in ml and neutralized by adding N-methylmorpholine at 0 ° C.

【００７８】Z-Phe-OH 10.01g(33.45mmol)をTHF 100ml
に溶解し、ドライアイス−エタノールで-20℃に冷却
し、Ｎ−メチルモルフォリン 3.7ml(33.45mmol)を滴下
した後クロロギ酸イソブチル 4.4ml(33.45mmol)を滴下
して、-20℃で1分間撹拌して混合酸無水物の溶液を調製
した。これを、NHS 4.23g(36.80mmol)のTHF溶液 50mlと
混合し、室温で30分間攪拌した。この溶液を、前記の水
溶液と混合し、室温で一晩撹拌し、THFを留去した。残
渣をエーテルで2回洗浄した後、1N-HClでpH3に調製し
た。生じた沈殿物を濾取し、水で洗浄した。濾取物をDM
F−エーテルで再沈殿させ、真空乾燥を行って、Z-Phe-V
al-Gln-OH 8.22g(収率 70%)を得た。Z-Phe-OH 10.01 g (33.45 mmol) was added to THF 100 ml.
Dissolved in water, cooled to −20 ° C. with dry ice-ethanol, 3.7 ml (33.45 mmol) of N-methylmorpholine was added dropwise, and then 4.4 ml (33.45 mmol) of isobutyl chloroformate was added dropwise, and 1 at -20 ° C. A solution of mixed acid anhydride was prepared by stirring for a minute. This was mixed with 50 ml of a THF solution containing 4.23 g (36.80 mmol) of NHS and stirred at room temperature for 30 minutes. This solution was mixed with the above aqueous solution, stirred at room temperature overnight, and THF was distilled off. The residue was washed twice with ether and then adjusted to pH 3 with 1N-HCl. The resulting precipitate was collected by filtration and washed with water. DM the filtered material
Re-precipitate with F-ether and vacuum dry to remove Z-Phe-V
8.22 g (yield 70%) of al-Gln-OH was obtained.

【００７９】得られたZ-Val-Gln-OH の融点、旋光度、
ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：231.7 〜233.5 ℃ [α] D ：-24.222°(c=1.0, メタノール) Rf ：0.73 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ27Ｈ34Ｎ4Ｏ7 理論値 Ｃ:61.58% Ｈ:6.51% Ｎ:10.64% 測定値 Ｃ:61.45% Ｈ:6.78% Ｎ: 9.96% (3) Boc-Asp(OBzl)-Phe-Val-Gln-OH の合成 上記(2) で得られたZ-Phe-Val-Gln-OH 5.39g(10.2mmol)
をメタノール100ml-50%酢酸150mlの混合溶液に溶解
し、Pd-C 0.15gを加え、窒素ガスを通して空気を置換し
た後、水素ガスを導入し接触還元を行った。これを室温
で一晩撹拌し、濾過、減圧濃縮後、残渣にエーテルを加
えて。生じた沈殿物を濾取し、真空乾燥を行って、粉末
状のH-Phe-Val-Gln-OH・AcOHを得た。得られた粉末をDM
F100ml- 水300mlの混合溶媒に溶解し、0℃でＮ−メチル
モルフォリンを加えて中和した。The melting point and optical rotation of the obtained Z-Val-Gln-OH
The Rf and elemental analysis values of TLC are shown below. mp: 231.7-233.5 ° C [α] D : -24.222 ° (c = 1.0, methanol) Rf: 0.73 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis C 27 H 34 N 4 O 7 Theoretical value C: 61.58% H: 6.51% N: 10.64% Measured value C: 61.45% H: 6.78% N: 9.96% (3) Synthesis of Boc-Asp (OBzl) -Phe-Val-Gln-OH Above (2 ) Obtained Z-Phe-Val-Gln-OH 5.39 g (10.2 mmol)
Was dissolved in a mixed solution of 100 ml of methanol and 150 ml of 50% acetic acid, 0.15 g of Pd-C was added, nitrogen gas was replaced with air, and then hydrogen gas was introduced to carry out catalytic reduction. This was stirred at room temperature overnight, filtered, concentrated under reduced pressure, and ether was added to the residue. The resulting precipitate was collected by filtration and vacuum dried to obtain powdery H-Phe-Val-Gln-OH.AcOH. DM the obtained powder
F100 ml-dissolved in a mixed solvent of 300 ml of water, and neutralized by adding N-methylmorpholine at 0 ° C.

【００８０】Boc-Asp(OBzl)-OH 4.0g(12.24mmol)をDMF
50mlに溶解し、ドライアイス−エタノ−ルで-20℃に冷
却し、Ｎ−メチルモルフォリン 1.35ml(12.24mmol)を滴
下した後クロロギ酸イソブチル 1.6ml(12.24mmol)を滴
下して、-20℃で1分間撹拌して混合酸無水物の溶液を調
製した。この溶液を、NHS 2.1g(18.36mmol)のDMF溶液50
mlと混合し、室温で30分間攪拌した。得られた混合溶液
を、前記のH-Phe-Val-Gln-OH・AcOHの溶液と混合し、室
温で一晩撹拌した後減圧濃縮した。残渣に1N-HClを加え
て、生じた沈殿物を濾取して水で洗浄し、濾取物をDMF
−エーテルで再沈殿させ、真空乾燥を行った。さらに、
1N-HClで3回、水で1回の順で洗浄し、濾取物をメタノー
ル−エーテルで再沈殿させ、真空乾燥を行って、Boc-As
p(OBzl)-Phe-Val-Gln-OH 5.55g(収率 78%)を得た。4.0 g (12.24 mmol) of Boc-Asp (OBzl) -OH was added to DMF.
It was dissolved in 50 ml, cooled to -20 ° C with dry ice-ethanol, 1.35 ml (12.24 mmol) of N-methylmorpholine was added dropwise, and then 1.6 ml (12.24 mmol) of isobutyl chloroformate was added dropwise, and -20 A solution of mixed acid anhydride was prepared by stirring at 0 ° C for 1 minute. This solution was added with NHS 2.1 g (18.36 mmol) in DMF 50
It was mixed with ml and stirred at room temperature for 30 minutes. The obtained mixed solution was mixed with the above H-Phe-Val-Gln-OH.AcOH solution, stirred overnight at room temperature, and then concentrated under reduced pressure. 1N-HCl was added to the residue, and the resulting precipitate was collected by filtration and washed with water.
-Reprecipitated with ether and vacuum dried. further,
It was washed 3 times with 1N-HCl and once with water in this order, and the filtered material was reprecipitated with methanol-ether and vacuum dried to obtain Boc-As.
5.55 g (yield 78%) of p (OBzl) -Phe-Val-Gln-OH was obtained.

【００８１】得られたBoc-Asp(OBzl)-Phe-Val-Gln-OH
の融点、旋光度、ＴＬＣのRf及び元素分析値を下記に示
す。 m.p. ：189.37 〜190.67℃ [α] D ：-17.422°(c=1.0, DMF ) Rf ：0.79 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ35Ｈ47Ｎ5Ｏ10 理論値 Ｃ:60.25% Ｈ:6.79% Ｎ:10.04% 測定値 Ｃ:60.10% Ｈ:6.90% Ｎ:10.23% (4) Boc-Ala-Gln-OH の合成 Boc-Ala-OH 18.92g(0.1mol)をTHF 200mlに溶解し、ドラ
イアイス−エタノールで-20℃に冷却し、Ｎ−メチルモ
ルフォリン 11ml(0.1mol)を滴下した後クロロギ酸イソ
ブチル 13.2ml(0.1mol)を滴下して、-20℃で１分間攪拌
して混合酸無水物の溶液を調製した。この溶液をNHS 1
2.08g(0.105mol)のTHF溶液 100mlと混合し、室温で30分
間攪拌した。得られた混合液を、Ｎ−メチルモルフォリ
ン 33ml(0.3mol)で中和したH-Gln-OH 21.92g(0.15mol)
の水溶液500mlと混合し、室温で一晩撹拌した後1N-HCl
でpH3に調製した。水層を酢酸エチルで2回洗浄し、ブタ
ノール抽出後、減圧濃縮して真空乾燥した後、シリカゲ
ルクロマトグラフィー｛ワコーゲルC-100(8×13cm),展
開溶媒:5%メタノール-酢酸エチル｝にて精製を行った。
目的物の溶出画分を集め、減圧濃縮し、残渣にエーテル
を加えて、生じた沈殿物を濾取し、真空乾燥を行って、
Boc-Ala-Gln-OH 22.63g(収率 71%)を得た。Obtained Boc-Asp (OBzl) -Phe-Val-Gln-OH
The melting point, optical rotation, Tf Rf, and elemental analysis values of are shown below. mp: 189.37 to 190.67 ° C [α] D : -17.422 ° (c = 1.0, DMF) Rf: 0.79 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 35 H 47 N 5 O 10 Theoretical value C: 60.25% H: 6.79% N: 10.04% Measured value C: 60.10% H: 6.90% N: 10.23% (4) Synthesis of Boc-Ala-Gln-OH Boc-Ala-OH 18.92 g (0.1 mol) ) Was dissolved in 200 ml of THF, cooled to -20 ° C with dry ice-ethanol, 11 ml (0.1 mol) of N-methylmorpholine was added dropwise, and 13.2 ml (0.1 mol) of isobutyl chloroformate was added dropwise, -20 The solution of the mixed acid anhydride was prepared by stirring at 0 ° C. for 1 minute. This solution is NHS 1
2.08 g (0.105 mol) of THF solution (100 ml) was mixed and stirred at room temperature for 30 minutes. The obtained mixture was neutralized with 33 ml (0.3 mol) of N-methylmorpholine, 21.92 g (0.15 mol) of H-Gln-OH.
Was mixed with 500 ml of an aqueous solution of, and stirred overnight at room temperature, then 1N-HCl
The pH was adjusted to 3 with. The aqueous layer was washed twice with ethyl acetate, extracted with butanol, concentrated under reduced pressure, and vacuum dried, followed by silica gel chromatography {Wakogel C-100 (8 × 13 cm), developing solvent: 5% methanol-ethyl acetate}. Purified.
The elution fractions of the target substance were collected, concentrated under reduced pressure, ether was added to the residue, the resulting precipitate was collected by filtration, and vacuum dried,
22.63 g (yield 71%) of Boc-Ala-Gln-OH was obtained.

【００８２】得られたBoc-Ala-Gln-OHの融点、旋光度、
ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：65.53〜68.77℃ [α] D ：-21.272°(c=1.0, メタノール) Rf ：0.47 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ13Ｈ23Ｎ3Ｏ6 理論値 Ｃ:49.20% Ｈ:7.31% Ｎ:13.24% 測定値 Ｃ:49.09% Ｈ:7.34% Ｎ:12.73% (5) Boc-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-OH の合成 上記(3) で得られたBoc-Asp(OBzl)-Phe-Val-Gln-OH 4.1
1g(5.89mmol) を、0℃でTFA 10mlに溶解し、室温で30分
間撹拌後、エーテルを加えて、生じた沈殿物を濾取、真
空乾燥して粉末状のH-Asp(OBzl)-Phe-Val-Gln-OH・TFA
を得た。得られた粉末をDMF40ml に溶解し、0℃で、Ｎ
−メチルモルフォリンを加えて中和した。The melting point and optical rotation of the obtained Boc-Ala-Gln-OH,
The Rf and elemental analysis values of TLC are shown below. mp: 65.53 to 68.77 ° C [α] D : -21.272 ° (c = 1.0, methanol) Rf: 0.47 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 13 H 23 N 3 O 6 Theoretical value C: 49.20% H: 7.31% N: 13.24% Measured value C: 49.09% H: 7.34% N: 12.73% (5) Boc-Ala-Gln-Asp (OBzl) -Phe-Val-Gln-OH Synthesis Boc-Asp (OBzl) -Phe-Val-Gln-OH 4.1 obtained in (3) above
1 g (5.89 mmol) was dissolved in 10 ml of TFA at 0 ° C., stirred at room temperature for 30 minutes, ether was added, and the resulting precipitate was collected by filtration and vacuum dried to obtain powdery H-Asp (OBzl)-. Phe-Val-Gln-OH ・ TFA
I got The obtained powder was dissolved in 40 ml of DMF, and N was added at 0 ° C.
-Methylmorpholine was added to neutralize.

【００８３】上記(4) で得られたBoc-Ala-Gln-OH 2.8g
(8.84mmol)をDMF 40mlに溶解し、ドライアイス−エタノ
−ルで-20℃に冷却し、Ｎ−メチルモルフォリン 0.98ml
(8.84mmol)を滴下した後クロロギ酸イソブチル 1.17ml
(8.84mmol)を滴下して、-20℃で1分間撹拌して、混合酸
無水物の溶液を調製した。この溶液を前記の溶液に加え
て、0℃で5分間、室温で30分間撹拌した後、減圧濃縮し
た。残渣に1N-HClを加えて、生じた沈殿物を濾取して、
水で洗浄した。得られた粉末をDMF−酢酸エチルで再沈
殿させ、エーテルで洗浄後、真空乾燥を行って、Boc-Al
a-Gln-Asp(OBzl)-Phe-Val-Gln-OH 5.13g( 収率 97%)を
得た。2.8 g of Boc-Ala-Gln-OH obtained in the above (4)
(8.84 mmol) was dissolved in 40 ml of DMF, cooled to -20 ° C with dry ice-ethanol, and 0.98 ml of N-methylmorpholine.
(8.84 mmol) was added dropwise and isobutyl chloroformate 1.17 ml
(8.84 mmol) was added dropwise, and the mixture was stirred at -20 ° C for 1 minute to prepare a mixed acid anhydride solution. This solution was added to the above solution, stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes, and then concentrated under reduced pressure. 1N-HCl was added to the residue, the resulting precipitate was collected by filtration,
Washed with water. The obtained powder was reprecipitated with DMF-ethyl acetate, washed with ether, and then vacuum dried to obtain Boc-Al.
5.13 g (yield 97%) of a-Gln-Asp (OBzl) -Phe-Val-Gln-OH was obtained.

【００８４】得られたBoc-Ala-Gln-Asp(OBzl)-Phe-Val-
Gln-OHの融点、旋光度、ＴＬＣのRf及び元素分析値を下
記に示す。 m.p. ：230.0 〜235.0℃(分解) [α] D ：-10.799°(c=0.5, DMF） Rf ：0.63 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ43Ｈ60Ｎ8Ｏ13 理論値 Ｃ:57.58% Ｈ:6.74% Ｎ:12.49% 測定値 Ｃ:57.34% Ｈ:6.92% Ｎ:12.70% また、上記(3) で合成したBoc-Asp(OBzl)-Phe-Val-Gln-
OH は、下記の(6) 〜(8) によっても合成することがで
きる。 (6) Boc-Val-Gln-OH の合成 H-Gln-OH 14.60ｇ(0.1mol)を水 500mlに溶解し、０℃で
Ｎ−メチルモルフォリン22ml(0.2mol)を添加して中和し
た。得られた水溶液をBoc-Val-OSu 15.72ｇ(50mmol)の
THF 溶液 300mlと混合し、最終液量を1500ml(THF：水＝
１：１) に調整した後、室温で一晩攪拌した。THF を留
去後、水層をエーテルで洗浄し、１Ｎ塩酸でpH３に調節
して、酢酸エチル−ブタノール(2:1) で抽出した。有機
層を水で２回洗浄した後、減圧濃縮した。次いで残渣に
エーテルを加えて生じた沈殿物を濾取し、真空乾燥し
た。このようにして、Boc-Val-Gln-OH 11.74ｇ（収率66
％）を得た。The obtained Boc-Ala-Gln-Asp (OBzl) -Phe-Val-
The melting point of Gln-OH, optical rotation, Rf of TLC and elemental analysis values are shown below. mp: 230.0 to 235.0 ° C (decomposition) [α] D : -10.799 ° (c = 0.5, DMF) Rf: 0.63 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 43 H 60 N 8 O 13 Theoretical value C: 57.58% H: 6.74% N: 12.49% Measured value C: 57.34% H: 6.92% N: 12.70% Also, Boc-Asp (OBzl) -Phe-Val synthesized in (3) above. -Gln-
OH can also be synthesized by the following (6) to (8). (6) Synthesis of Boc-Val-Gln-OH 14.60 g (0.1 mol) of H-Gln-OH was dissolved in 500 ml of water, and 22 ml (0.2 mol) of N-methylmorpholine was added at 0 ° C. for neutralization. . The obtained aqueous solution was added with 15.72 g (50 mmol) of Boc-Val-OSu.
Mix with 300 ml of THF solution to make a final volume of 1500 ml (THF: water =
After adjusting to 1: 1), the mixture was stirred overnight at room temperature. After removing THF, the aqueous layer was washed with ether, adjusted to pH 3 with 1N hydrochloric acid, and extracted with ethyl acetate-butanol (2: 1). The organic layer was washed twice with water and then concentrated under reduced pressure. Next, ether was added to the residue, and the resulting precipitate was collected by filtration and dried under vacuum. Thus, Boc-Val-Gln-OH 11.74 g (yield 66
%) Was obtained.

【００８５】得られたBoc-Val-Gln-OH の融点、旋光
度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：88.7〜92.13 ℃ [α] D ：−19.848°(c=1.0, メタノール) Rf ：0.77 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ15Ｈ27Ｎ3Ｏ6 理論値 Ｃ:52.16% Ｈ:7.88% Ｎ:12.17% 測定値 Ｃ:52.09% Ｈ:7.63% Ｎ:11.91% (7) Boc-Phe-Val-Gln-OH の合成 上記(6) で得られたBoc-Val-Gln-OH 10.44g(28.6mmol)
を０℃でTFA 15mlに溶解し、室温で30分間攪拌した。次
いでエーテルを添加し、生じた沈殿物を濾取して、真空
乾燥した。このようにして粉末状のH-Val-Gln-OH・TFA
を得た。得られた粉末を、水 500mlに溶解し、０℃でＮ
−メチルモルフォリンを加えて中和した。The melting point, optical rotation, Rf of TLC and elemental analysis values of the obtained Boc-Val-Gln-OH are shown below. mp: 88.7 to 92.13 ° C [α] D : -19.848 ° (c = 1.0, methanol) Rf: 0.77 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 15 H 27 N 3 O 6 Theoretical value C: 52.16% H: 7.88% N: 12.17% Measured value C: 52.09% H: 7.63% N: 11.91% (7) Synthesis of Boc-Phe-Val-Gln-OH Obtained by the above (6) Boc-Val-Gln-OH 10.44g (28.6mmol)
Was dissolved in 15 ml of TFA at 0 ° C. and stirred at room temperature for 30 minutes. Then ether was added and the resulting precipitate was filtered off and dried under vacuum. In this way, powdered H-Val-Gln-OH ・ TFA
I got The obtained powder is dissolved in 500 ml of water, and N is added at 0 ° C.
-Methylmorpholine was added to neutralize.

【００８６】上記で得られた水溶液を、Boc-Phe-OSu 2
0.70ｇ（57.2mmol) のTHF 溶液 500mlと混合し、室温で
一晩攪拌した。THF を留去後、水層をエーテルで洗浄
し、１Ｎ塩酸でpH３に調整し、酢酸エチル−ブタノール
(2:1) で抽出した。有機層を水で２回洗浄した後、減圧
濃縮した。残渣にエーテルを加えて生じた沈殿物を濾取
し、真空乾燥を行った。このようにして、Boc-Phe-Val-
Gln-OH 9.93ｇ（収率70％）を得た。The aqueous solution obtained above was treated with Boc-Phe-OSu 2
It was mixed with 0.70 g (57.2 mmol) of THF solution (500 ml) and stirred at room temperature overnight. After the THF was distilled off, the aqueous layer was washed with ether, adjusted to pH 3 with 1N hydrochloric acid, and ethyl acetate-butanol was added.
Extracted with (2: 1). The organic layer was washed twice with water and then concentrated under reduced pressure. Ether was added to the residue and the resulting precipitate was collected by filtration and vacuum dried. In this way, Boc-Phe-Val-
9.93 g (70% yield) of Gln-OH was obtained.

【００８７】得られたBoc-Phe-Val-Gln-OHの融点、旋光
度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：163.0 〜165.5 ℃ [α] D ：−22.210°(c=1.0, メタノール) Rf ：0.75 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ24Ｈ36Ｎ4Ｏ7 理論値 Ｃ:58.52% Ｈ:7.37% Ｎ:11.37% 測定値 Ｃ:58.42% Ｈ:7.29% Ｎ:11.31% (8) Boc-Asp(OBzl)-Phe-Val-Gln-OH の合成 上記(7) で得られたBoc-Phe-Val-Gln-OH 8.93ｇ(18.1m
mol)を０℃でTFA 15mlに溶解し、室温で30分間攪拌し
た。次いでエーテルを添加して生じた沈殿物を濾取し、
真空乾燥した。このようにして、粉末状のH-Phe-Val-Gl
n-OH・TFA を得た。得られた粉末をDMF 100 ml−水 300
mlの混合溶媒に溶解し、０℃でＮ−メチルモルフォリン
を加えて中和した。The melting point, optical rotation, Tf Rf and elemental analysis values of the obtained Boc-Phe-Val-Gln-OH are shown below. mp: 163.0 to 165.5 ° C [α] D : -22.210 ° (c = 1.0, methanol) Rf: 0.75 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 24 H 36 N 4 O 7 Theoretical value C: 58.52% H: 7.37% N: 11.37% Measured value C: 58.42% H: 7.29% N: 11.31% (8) Synthesis of Boc-Asp (OBzl) -Phe-Val-Gln-OH Above (7 ) Boc-Phe-Val-Gln-OH 8.93 g (18.1 m)
mol) was dissolved in 15 ml of TFA at 0 ° C. and stirred at room temperature for 30 minutes. Then ether was added and the resulting precipitate was filtered off,
Vacuum dried. In this way, powdered H-Phe-Val-Gl
n-OHTFA was obtained. The obtained powder is DMF 100 ml-water 300
It was dissolved in a mixed solvent of ml and neutralized by adding N-methylmorpholine at 0 ° C.

【００８８】上記で得られた溶液を、Boc-Asp(OBzl)-OS
u 11.4 ｇ（27.2mmol) のDMF 溶液500mlと混合し、室
温で一晩攪拌した。得られた反応混合液を減圧濃縮し
た。残渣に１Ｎ塩酸を添加して、生じた沈殿物を濾取
し、水洗した。更にDMF −エーテルで再沈殿させ、沈殿
物を濾取し、真空乾燥を行った。このようにして、Boc-
Asp(OBzl)-Phe-Val-Gln-OH 9.80 ｇ（収率78％）を得
た。The solution obtained above was treated with Boc-Asp (OBzl) -OS.
The mixture was mixed with 500 ml of a DMF solution containing 11.4 g (27.2 mmol) of u and stirred overnight at room temperature. The resulting reaction mixture was concentrated under reduced pressure. 1N Hydrochloric acid was added to the residue, and the resulting precipitate was collected by filtration and washed with water. Further, the precipitate was reprecipitated with DMF-ether, the precipitate was collected by filtration, and vacuum dried. In this way, Boc-
9.80 g (yield 78%) of Asp (OBzl) -Phe-Val-Gln-OH was obtained.

【００８９】得られたBoc-Asp(OBzl)-Phe-Val-Gln-OHの
融点、旋光度、ＴＬＣのRf及び元素分析値を下記に示
す。 m.p. ：181.53 〜184.03℃ [α] D ：−29.919°(c=1.0, メタノール) Rf ：0.79 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ35Ｈ47Ｎ5Ｏ10 理論値 Ｃ:60.25% Ｈ:6.79% Ｎ:10.04% 測定値 Ｃ:59.94% Ｈ:6.85% Ｎ:10.37% 〔実施例６〕方法２によるグルカゴンの固相合成 実施例１−１において得られた中間体 Ｈ−Ｔrp−Ｌeu
−Ｍet−Ａsn−Ｔhr（Ｂzl）−ＰＡＭ樹脂 1.48gに、グ
ルカゴンのアミノ酸配列の17〜24位のアミノ酸残基に相
当する下記式： Ｂoc−Ａrg（Ｔｏｓ）−Ａrg（Ｔｏｓ）−Ａla−Ｇln−
Ａsp（ＯＢzl）−Ｐhe−Ｖal−Ｇln−ＯＨ で表されるオクタペプチド 15mmol 、ＨＯＢｔ 15mmol
、ＤＣＭ 100ml及びＤＭＦ 20mlを加えて攪拌し、さら
にＤＣＣ（１Ｍ−ＤＣＭ溶液）15mlを加えて２時間反応
させた。反応後、反応混合液を濾過し、ＭｅＯＨ 200ml
（２回）、ＤＣＭ200ml（２回）の順に攪拌下で処理し
た。尚各処理毎に濾過を行った。このようにして、Ｂoc
−Ａrg（Ｔｏｓ）−Ａrg（Ｔｏｓ）−Ａla−Ｇln−Ａsp
（ＯcＨｅx）−Ｐhe−Ｖal-Ｇln−Ｔrp−Ｌeu−Ｍet−
Ａsn−Ｔhr（Ｂzl）−ＰＡＭ樹脂を得た。The melting point, optical rotation, Tf Rf and elemental analysis values of the obtained Boc-Asp (OBzl) -Phe-Val-Gln-OH are shown below. mp: 181.53 to 184.03 ° C [α] D : −29.919 ° (c = 1.0, methanol) Rf: 0.79 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 35 H 47 N 5 O 10 Theoretical value C: 60.25% H: 6.79% N: 10.04% Measured value C: 59.94% H: 6.85% N: 10.37% [Example 6] Solid phase synthesis of glucagon by method 2 Obtained in Example 1-1. Intermediate H-Trp-Leu
-Met-Asn-Thr (Bzl) -PAM resin 1.48 g, the following formula corresponding to the amino acid residues at positions 17 to 24 in the amino acid sequence of glucagon: Boc-Arg (Tos) -Arg (Tos) -Ala-Gln −
15 mmol octapeptide represented by Asp (OBzl) -Phe-Val-Gln-OH, 15 mmol HOBt
, DCM 100 ml and DMF 20 ml were added and stirred, and DCC (1M-DCM solution) 15 ml was further added and reacted for 2 hours. After the reaction, the reaction mixture was filtered and MeOH 200ml
(Twice), then 200 ml of DCM (twice) in this order. Note that filtration was performed after each treatment. In this way, Boc
-Arg (Tos) -Arg (Tos) -Ala-Gln-Asp
(OcHex) -Phe-Val-Gln-Trp-Leu-Met-
An Asn-Thr (Bzl) -PAM resin was obtained.

【００９０】上記樹脂に、実施例１−１の方法と同様に
して16位から１位までアミノ酸残基を順次１個ずつ結合
して延長し、Ｈ−Ｈis(Ｂom）−Ｓer(Ｂzl）−Ｇln−Ｇ
ly−Ｔhr（Ｂzl）−Ｐhe−Ｔhr（Ｂzl）−Ｓer（Ｂzl）
−Ａsp（ＯcＨｅx）−Ｔyr（Ｂr-Ｚ）−Ｓer(Ｂzl）−
Ｌys(Ｃl-Ｚ）−Ｔyr(Ｂr-Ｚ）−Ｌeu−Ａsp（ＯcＨｅ
x）−Ｓer（Ｂzl）−Ａrg（Ｔos）−Ａrg（Ｔos）−Ａl
a−Ｇln−Ａsp（ＯＢzl）−Ｐhe−Ｖal-Ｇln−Ｔrp−Ｌ
eu−Ｍet−Ａsn−Ｔhr（Ｂzl）−ＰＡＭ樹脂50gを得
た。H-His (Bom) -Ser (Bzl)- Gln-G
ly-Thr (Bzl) -Phe-Thr (Bzl) -Ser (Bzl)
-Asp (OcHex) -Tyr (Br-Z) -Ser (Bzl)-
Lys (Cl-Z) -Tyr (Br-Z) -Leu-Asp (OcHe
x) -Ser (Bzl) -Arg (Tos) -Arg (Tos) -Al
a-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-L
50 g of eu-Met-Asn-Thr (Bzl) -PAM resin was obtained.

【００９１】得られた保護ペプチド−ＰＡＭ樹脂から、
実施例１−１と同様の方法でペプチドの抽出及び精製を
行って、精製グルカゴン0.89gを得た。尚、上記で用い
たBoc-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val-
Gln-OHは下記(1) 及び(2) の操作によって製造した。 (1) Boc-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-OH
の合成 実施例５の(5) で得られたBoc-Ala-Gln-Asp(OBzl)-Phe-
Val-Gln-OH 5.13g(5.72mmol) を0℃でTFA 10mlに溶解
し、室温で30分間撹拌した後、エーテルを加えて、生じ
た沈殿物を濾取、真空乾燥して粉末状のH-Ala-Gln-Asp
(OBzl)-Phe-Val-Gln-OH・TFAを得た。得られた粉末をDM
F 100mlに溶解し、0℃でＮ−メチルモルフォリンを加え
て中和した。From the resulting protected peptide-PAM resin,
The peptide was extracted and purified in the same manner as in Example 1-1 to obtain 0.89 g of purified glucagon. Incidentally, Boc-Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-Val-used above
Gln-OH was produced by the following operations (1) and (2). (1) Boc-Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-Val-Gln-OH
Synthesis of Boc-Ala-Gln-Asp (OBzl) -Phe- obtained in (5) of Example 5
Val-Gln-OH (5.13 g, 5.72 mmol) was dissolved in TFA (10 ml) at 0 ° C., and the mixture was stirred at room temperature for 30 min, ether was added, and the resulting precipitate was collected by filtration and dried in vacuo to give powdery H -Ala-Gln-Asp
(OBzl) -Phe-Val-Gln-OH.TFA was obtained. DM the obtained powder
It was dissolved in 100 ml of F and neutralized by adding N-methylmorpholine at 0 ° C.

【００９２】Boc-Arg(Tos)-OH 3.75g(8.84mmol) をDMF4
0mlに溶解し、ドライアイス−エタノ−ルで-20℃に冷却
し、Ｎ−メチルモルフォリン 0.98ml(8.84mmol)を滴下
した後クロロギ酸イソブチル 1.17ml(8.84mmol)を滴下
して、-20℃で1分間撹拌して混合酸無水物の溶液を調製
した。この溶液を前記の溶液に加えて、0℃で5分間、室
温で30分間撹拌した後、減圧濃縮した。残渣に1N-HClを
加えて、生じた沈殿物を濾取して、水で洗浄した。得ら
れた粉末を、DMF−酢酸エチル、さらにDMF−エーテルで
再沈殿させた後、真空乾燥を行って、Boc-Arg(Tos)-Ala
-Gln-Asp(OBzl)-Phe-Val-Gln-OH 4.89g(収率 69%)を得
た。3.75 g (8.84 mmol) of Boc-Arg (Tos) -OH was added to DMF4.
It was dissolved in 0 ml, cooled to -20 ° C with dry ice-ethanol, 0.98 ml (8.84 mmol) of N-methylmorpholine was added dropwise, and then 1.17 ml (8.84 mmol) of isobutyl chloroformate was added dropwise, and -20 A solution of mixed acid anhydride was prepared by stirring at 0 ° C for 1 minute. This solution was added to the above solution, stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes, and then concentrated under reduced pressure. 1N-HCl was added to the residue, and the resulting precipitate was collected by filtration and washed with water. The obtained powder was reprecipitated with DMF-ethyl acetate and further with DMF-ether, and then vacuum dried to obtain Boc-Arg (Tos) -Ala.
4.89 g (69% yield) of -Gln-Asp (OBzl) -Phe-Val-Gln-OH was obtained.

【００９３】得られたBoc-Arg(Tos)-Ala-Gln-Asp(OBzl)
-Phe-Val-Gln-OH の融点、旋光度、ＴＬＣのRf及び元素
分析値を下記に示す。 m.p. ：229.9 〜233.2 ℃(分解) [α] D ：+6.3232°(c=1.0, DMF) Rf ：0.70 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ56Ｈ78Ｎ12Ｏ16Ｓ 理論値 Ｃ:55.71% Ｈ:6.51% Ｎ:13.92% 測定値 Ｃ:56.01% Ｈ:6.56% Ｎ:13.22% (2) Boc-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Va
l-Gln-OH の合成 上記(1) で得られたBoc-Arg(Tos)-Ala-Gln-Asp(OBzl)-P
he-Val-Gln-OH 4.89g(4.05mmol)を0℃でTFA 10mlに溶解
し、室温で30分間撹拌した後エーテルを加えて、生じた
沈殿物を濾取、真空乾燥して粉末状のH-Arg(Tos)-Ala-G
ln-Asp(OBzl)-Phe-Val-Gln-OH・TFAを得た。得られた粉
末をDMF 200mlに溶解し、0℃で、Ｎ−メチルモルフォリ
ンを加えて中和した。Boc-Arg (Tos) -Ala-Gln-Asp (OBzl) obtained
The melting point, optical rotation, Rf of TLC and elemental analysis values of -Phe-Val-Gln-OH are shown below. mp: 229.9 to 233.2 ℃ (decomposition) [α] D : + 6.3232 ° (c = 1.0, DMF) Rf: 0.70 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 56 H 78 N 12 O 16 S Theoretical value C: 55.71% H: 6.51% N: 13.92% Measured value C: 56.01% H: 6.56% N: 13.22% (2) Boc-Arg (Tos) -Arg (Tos) -Ala-Gln -Asp (OBzl) -Phe-Va
Synthesis of l-Gln-OH Boc-Arg (Tos) -Ala-Gln-Asp (OBzl) -P obtained in (1) above
4.89 g (4.05 mmol) of he-Val-Gln-OH was dissolved in 10 ml of TFA at 0 ° C., stirred at room temperature for 30 minutes, ether was added, and the resulting precipitate was collected by filtration and dried in vacuum to give a powder. H-Arg (Tos) -Ala-G
ln-Asp (OBzl) -Phe-Val-Gln-OH.TFA was obtained. The obtained powder was dissolved in 200 ml of DMF, and N-methylmorpholine was added thereto for neutralization at 0 ° C.

【００９４】Boc-Arg(Tos)-OH 2.61g(6.08mmol) をDMF4
0mlに溶解し、ドライアイス−エタノ−ルで-20℃に冷却
し、Ｎ−メチルモルフォリン 0.67ml(6.08mmol)を滴下
した後クロロギ酸イソブチル 0.802ml(6.08mmol)を滴下
し、-20℃で1分間撹拌して混合酸無水物の溶液を調製し
た。この溶液を前記の溶液に加え、0℃で5分間、室温で
30分間撹拌した後、減圧濃縮した。残渣に1N-HClを加え
て、生じた沈殿物を濾取して、水で洗浄した。得られた
粉末をDMF−エーテルで再沈殿させ沈殿物を濾取して水
で洗浄する操作を3回繰り返した。その後、真空乾燥を
行って、Boc-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Ph
e-Val-Gln-OH 3.60g(収率 59%)を得た。2.61 g (6.08 mmol) of Boc-Arg (Tos) -OH was added to DMF4.
It was dissolved in 0 ml, cooled to -20 ° C with dry ice-ethanol, 0.67 ml (6.08 mmol) of N-methylmorpholine was added dropwise, and then 0.802 ml (6.08 mmol) of isobutyl chloroformate was added dropwise, and -20 ° C. And stirred for 1 minute to prepare a mixed acid anhydride solution. Add this solution to the above solution and at 0 ° C for 5 minutes at room temperature.
After stirring for 30 minutes, the mixture was concentrated under reduced pressure. 1N-HCl was added to the residue, and the resulting precipitate was collected by filtration and washed with water. The operation of reprecipitating the obtained powder with DMF-ether and collecting the precipitate by filtration and washing with water was repeated 3 times. After that, vacuum drying is performed, and Boc-Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBzl) -Ph.
3.60 g (yield 59%) of e-Val-Gln-OH was obtained.

【００９５】得られたBoc-Arg(Tos)-Arg(Tos)-Ala-Gln-
Asp(OBzl)-Phe-Val-Gln-OH の融点、旋光度、ＴＬＣのR
f及び元素分析値を下記に示す。 m.p. ：242.0 〜245.0 ℃(分解) [α] D ：-14.551°(c=1.0, DMF) Rf ：0.68 (BuOH/AcOH/H2O=4/1/5) 元素分析値 Ｃ69Ｈ96Ｎ16Ｏ19Ｓ2 理論値 Ｃ:54.60% Ｈ:6.38% Ｎ:14.77% 測定値 Ｃ:54.72% Ｈ:6.48% Ｎ:14.61% 〔実施例７〕方法３によるグルカゴンの合成 (1) グルカゴンのアミノ酸配列の１位から12位のアミノ
酸残基に相当する下記式：Ｆｍｏｃ−Ｈis−Ｓer−Ｇln
−Ｇly−Ｔhr−Ｐhe−Ｔhr−Ｓer−Ａsp−Ｔyr−Ｓer−
Ｌys−ＯＨで表されるペプチドの製造 Ｂｏｃ−Ｌys（Ｃｌ−Ｚ）−ＰＡＭ樹脂（渡辺化学社
製、アミノ基含有量： 0.5mmol/g）1.2ｇに、実施例１
−１の方法と同様にして上記ペプチドのアミノ酸配列に
したがって順次アミノ酸残基をカップリングし、Ｆｍｏ
ｃ−Ｈis（Ｔｏｓ）−Ｓer（Ｂzl）−Ｇln−Ｇly−Ｔhr
（Ｂzl）−Ｐhe−Ｔhr（Ｂzl）−Ｓer−Ａsp（ＯｃＨｅ
ｘ）−Ｔyr（Ｂｒ-Ｚ）−Ｓer（Ｂzl）−Ｌys（Ｃｌ-
Ｚ）−ＰＡＭ樹脂 2.4gを得た。Obtained Boc-Arg (Tos) -Arg (Tos) -Ala-Gln-
Asp (OBzl) -Phe-Val-Gln-OH melting point, optical rotation, TLC R
The f and elemental analysis values are shown below. mp: 242.0 to 245.0 ℃ (decomposition) [α] D : -14.551 ° (c = 1.0, DMF) Rf: 0.68 (BuOH / AcOH / H 2 O = 4/1/5) Elemental analysis value C 69 H 96 N 16 O 19 S 2 Theoretical value C: 54.60% H: 6.38% N: 14.77% Measured value C: 54.72% H: 6.48% N: 14.61% [Example 7] Synthesis of glucagon by method 3 (1) Amino acid of glucagon The following formula corresponding to amino acid residues 1 to 12 of the sequence: Fmoc-His-Ser-Gln
-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-
Production of Peptide Represented by Lys-OH Boc-Lys (Cl-Z) -PAM resin (Watanabe Chemical Co., Ltd., amino group content: 0.5 mmol / g)
In the same manner as in -1, the amino acid residues were sequentially coupled according to the amino acid sequence of the peptide, and Fmo
c-His (Tos) -Ser (Bzl) -Gln-Gly-Thr
(Bzl) -Phe-Thr (Bzl) -Ser-Asp (OcHe
x) -Tyr (Br-Z) -Ser (Bzl) -Lys (Cl-
2.4 g of Z) -PAM resin was obtained.

【００９６】得られた保護ペプチド−ＰＡＭ樹脂 2.4g
にアニソ−ル 3mlを加え、さらに無水フッ化水素25mlを
加え、０℃で１時間撹拌した。反応後、無水フッ化水素
を減圧下留去後、残渣をエ−テルで洗浄し、これに 10%
酢酸 200mlを加えてペプチドを抽出した。抽出液はＯＤ
Ｓ−１０２０ＴＴ（富士デビソン社製）を用いた逆相カ
ラムクロマトグラフィーにより精製し、目的とするペプ
チド 89.7mgを得た。このペプチドについて高速液体ク
ロマトグラフィー（ＨＰＬＣ）の保持時間の測定、旋光
度の測定及びアミノ酸分析を行った。その結果を下記に
示す。 ・ＨＰＬＣ 分析条件 カラム：TSK GEL ODS-120T（4.6×250mm） 溶 媒：30%アセトニトリル−0.1%ＴＦＡ 流 速：1ml/min 検出波長：２２０ｎｍ ＨＰＬＣの保持時間：９．６分 ・ [α] D ：−７．５° ・アミノ酸分析 Asp(1) 1.10, Thr(2) 2.04, Ser(3) 2.29, Glu(1) 1.1
3, Gly(1) 1.20,Tyr(1) 0.94, Phe(1) 1.04, Lys(1) 1.
12, His(1) 1.14 (2) グルカゴンのアミノ酸配列の13位から29位のアミノ
酸残基に相当する下記式：Ｈ−Ｔyr−Ｌeu−Ａsp−Ｓer
−Ａrg−Ａrg−Ａla−Ｇln−Ａsp−Ｐhe−Ｖal−Ｇln−
Ｔrp−Ｌeu−Ｍet−Ａsn−Ｔhr−ＯＨで表されるペプチ
ドの製造 実施例１−１において得られる中間体 Ｈ−Ｔyr（Ｂｒ
Ｚ）−Ｌeu−Ａsp(ＯｃＨｅｘ）−Ｓer（Ｂzl）−Ａrg
（Ｔｏｓ）−Ａrg（Ｔｏｓ）−Ａla−Ｇln−Ａsp（Ｏｃ
Ｈｅｘ）−Ｐhe−Ｖal−Ｇln−Ｔrp−Ｌeu−Ｍet−Ａsn
−Ｔhr（Ｂzl）−ＰＡＭ樹脂 1.2gに、アニソ−ル 1ml
及びエタンジチオール 1mlを加え、さらに無水フッ化水
素 10mlを加え、０℃で１時間撹拌した。反応後、無水
フッ化水素を減圧下留去後、残渣をエ−テルで洗浄し、
これに10%酢酸 70mlを加えてペプチドを抽出した。抽出
液を凍結乾燥して 396mgの白色粉末を得た。次に得られ
た粉末を40%酢酸に溶解した後、ＯＤＳ−１０２０ＴＴ
（富士デビソン社製）を用いた逆相カラムクロマトグラ
フィーにより精製し、目的とするペプチド 149mgを得
た。このペプチドについて高速液体クロマトグラフィー
（ＨＰＬＣ）の保持時間の測定、旋光度の測定及びアミ
ノ酸分析を行った。その結果を下記に示す。 ・ＨＰＬＣ 分析条件 カラム：TSK GEL ODS-120T（4.6×250mm） 溶 媒：36%アセトニトリル−0.1%ＴＦＡ 流 速：1ml/min 検出波長：２２０ｎｍ ＨＰＬＣの保持時間：９．７分 ・ [α] D ：−２１．２° ・アミノ酸分析 Asp(3) 3.23, Thr(1) 0.85, Ser(1) 0.48, Glu(2) 2.2
2, Ala(1) 1.07,Val(1) 1.07, Met(1) 0.69, Leu(2) 2.
32, Tyr(1) 0.92, Phe(1) 1.11,Arg(2) 2.03 (3) 下記式：Ｆｍｏｃ−Ｈis−Ｓer−Ｇln−Ｇly−Ｔhr
−Ｐhe−Ｔhr−Ｓer−Ａsp−Ｔyr−Ｓer−Ｌys−Ｔyr−
Ｌeu−Ａsp−Ｓer−Ａrg−Ａrg−Ａla−Ｇln−Ａsp−Ｐ
he−Ｖal−Ｇln−Ｔrp−Ｌeu−Ｍet−Ａsn−Ｔhr−ＯＨ
で表される末端アミノ基保護ペプチドの製造 上記(1) で得られたペプチド 89.7mg(56.8μmol)、上記
(2) で得られたペプチド 149mg(69.5μmol)及び 50%Ｄ
ＭＳＯを含む0.2Mトリス緩衝液（ｐＨ 6.5）50mlに、ト
リプシン 50mgを加えて、37℃で 24時間振盪した。氷酢
酸 2mlを加えた後、生成物を濾取して水で洗浄した。次
いで五酸化リン存在下、デシケーター中で減圧乾燥し
た。得られた粉末に 10%酢酸 200mlを加えてペプチドを
抽出した。2.4 g of the obtained protected peptide-PAM resin
3 ml of anisole was added to the mixture, 25 ml of anhydrous hydrogen fluoride was further added, and the mixture was stirred at 0 ° C. for 1 hour. After the reaction, the anhydrous hydrogen fluoride was distilled off under reduced pressure, the residue was washed with ether, and 10% of this was washed.
The peptide was extracted by adding 200 ml of acetic acid. Extract is OD
Purification by reverse phase column chromatography using S-1020TT (manufactured by Fuji Devison Co., Ltd.) gave 89.7 mg of the desired peptide. This peptide was subjected to high performance liquid chromatography (HPLC) retention time measurement, optical rotation measurement and amino acid analysis. The results are shown below.・ HPLC analysis conditions Column: TSK GEL ODS-120T (4.6 × 250 mm) Solvent: 30% acetonitrile-0.1% TFA Flow rate: 1 ml / min Detection wavelength: 220 nm HPLC retention time: 9.6 minutes ・ [α] D : -7.5 ° ・ Amino acid analysis Asp (1) 1.10, Thr (2) 2.04, Ser (3) 2.29, Glu (1) 1.1
3, Gly (1) 1.20, Tyr (1) 0.94, Phe (1) 1.04, Lys (1) 1.
12, His (1) 1.14 (2) The following formula corresponding to amino acid residues 13 to 29 of the amino acid sequence of glucagon: H-Tyr-Leu-Asp-Ser
-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-
Preparation of peptide represented by Trp-Leu-Met-Asn-Thr-OH Intermediate H-Tyr (Br
Z) -Leu-Asp (OcHex) -Ser (Bzl) -Arg
(Tos) -Arg (Tos) -Ala-Gln-Asp (Oc
Hex) -Phe-Val-Gln-Trp-Leu-Met-Asn
-Thr (Bzl) -PAM resin 1.2g, Anisole 1ml
And 1 ml of ethanedithiol were added, 10 ml of anhydrous hydrogen fluoride was further added, and the mixture was stirred at 0 ° C. for 1 hour. After the reaction, anhydrous hydrogen fluoride was distilled off under reduced pressure, and the residue was washed with ether,
To this, 70 ml of 10% acetic acid was added to extract the peptide. The extract was freeze-dried to obtain 396 mg of white powder. Next, the obtained powder was dissolved in 40% acetic acid and then ODS-1020TT
The product was purified by reverse phase column chromatography using (Fuji Devison) to obtain 149 mg of the desired peptide. This peptide was subjected to high performance liquid chromatography (HPLC) retention time measurement, optical rotation measurement and amino acid analysis. The results are shown below.・ HPLC analysis conditions Column: TSK GEL ODS-120T (4.6 × 250 mm) Solvent: 36% acetonitrile-0.1% TFA Flow rate: 1 ml / min Detection wavelength: 220 nm HPLC retention time: 9.7 minutes ・ [α] D : -21.2 ° ・ Amino acid analysis Asp (3) 3.23, Thr (1) 0.85, Ser (1) 0.48, Glu (2) 2.2
2, Ala (1) 1.07, Val (1) 1.07, Met (1) 0.69, Leu (2) 2.
32, Tyr (1) 0.92, Phe (1) 1.11, Arg (2) 2.03 (3) The following formula: Fmoc-His-Ser-Gln-Gly-Thr
-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-
Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-P
he-Val-Gln-Trp-Leu-Met-Asn-Thr-OH
The production of a peptide protected with a terminal amino group represented by
149mg (69.5μmol) of the peptide obtained in (2) and 50% D
50 mg of trypsin was added to 50 ml of 0.2 M Tris buffer (pH 6.5) containing MSO, and the mixture was shaken at 37 ° C. for 24 hours. After adding 2 ml of glacial acetic acid, the product was collected by filtration and washed with water. Then, it was dried under reduced pressure in a desiccator in the presence of phosphorus pentoxide. The peptide was extracted by adding 200 ml of 10% acetic acid to the obtained powder.

【００９７】抽出液をＯＤＳ−１０２０ＴＴ（30×420m
m）カラムを用いた逆相カラムクロマトグラフィーによ
り精製し、目的とするペプチド 12mg（収率 4%）を得
た。このペプチドについて高速液体クロマトグラフィー
（ＨＰＬＣ）の保持時間の測定、旋光度の測定及びアミ
ノ酸分析を行った。その結果を下記に示す。 ・ＨＰＬＣ 分析条件 カラム：TSK GEL ODS-120T（4.6×250mm） 溶 媒：40%アセトニトリル−0.1%ＴＦＡ 流 速：1ml/min 検出波長：２２０ｎｍ ＨＰＬＣの保持時間：１０．２分 ・ [α] D ：−３４．２° ・アミノ酸分析 Asp(4) 3.93, Thr(3) 2.63, Ser(4) 2.89, Glu(3) 3.0
0, Gly(1) 1.07,Ala(1) 1.01, Val(1) 1.00, Met(1) 0.
95, Leu(2) 2.06, Tyr(2) 1.92,Phe(2) 1.92, His(1)
1.05, Lys(1) 0.88, Arg(2) 1.94 (4) グルカゴンの製造 上記(3) で得られた末端アミノ基保護ペプチド 12mg
に、氷冷下 10%ジエチルアミン／ＤＭＦ 50mlを加えて
溶解させ、室温で 1.5時間攪拌した。反応液を減圧濃縮
した後、エーテルを加えて固化し、実施例１−１と同様
の操作により精製し、精製グルカゴン 5.2mg（収率 2
%）を得た。この精製グルカゴンについて高速液体クロ
マトグラフィー（ＨＰＬＣ）の保持時間の測定、旋光度
の測定及びアミノ酸分析を行った。その結果を下記に示
す。 ・ＨＰＬＣ 分析条件 カラム：TSK GEL ODS-120T（4.6×250mm） 溶 媒：35%アセトニトリル−0.1%ＴＦＡ 流 速：1ml/min 検出波長：２２０ｎｍ ＨＰＬＣの保持時間：１０．４分 ・ [α] D ：−３４．４° ・アミノ酸分析 Asp(4) 3.91, Thr(3) 2.63, Ser(4) 2.89, Glu(3) 2.9
8, Gly(1) 1.07,Ala(1) 1.00, Val(1) 1.00, Met(1) 0.
97, Leu(2) 2.06, Tyr(2) 1.92,Phe(2) 1.92, His(1)
1.05, Lys(1) 0.99, Arg(2) 1.94 〔実施例８〕方法４によるグルカゴンの製造 (1) グルカゴンのアミノ酸配列の１位から18位のアミノ
酸残基に相当する下記式：Ｆｍｏｃ−Ｈis−Ｓer−Ｇln
−Ｇly−Ｔhr−Ｐhe−Ｔhr−Ｓer−Ａsp−Ｔyr−Ｓer−
Ｌys−Ｔyr−Ｌeu−Ａsp−Ｓer−Ａrg−Ａrg−ＯＨで表
されるペプチドの製造 Ｂｏｃ−Ａrg（Ｔｏｓ）−ＰＡＭ樹脂（渡辺化学社製、
アミノ基 0.44mmol/g）5.43ｇに、実施例１−１の方法
と同様にして上記ペプチドのアミノ酸配列にしたがって
順次アミノ酸残基をカップリングし、Ｆｍｏｃ−Ｈis
（Ｔｏｓ）−Ｓer（Ｂzl）−Ｇln−Ｇly−Ｔhr（Ｂzl）
−Ｐhe−Ｔhr（Ｂzl）−Ｓer−Ａsp(ＯｃＨｅｘ）−Ｔy
r（Ｂｒ-Ｚ）−Ｓer（Ｂzl）−Ｌys（Ｃｌ-Ｚ）−Ｔyr
（ＢｒＺ）−Ｌeu−Ａsp（ＯｃＨｅｘ）−Ｓer（Ｂzl）
−Ａrg（Ｔｏｓ）−Ａrg（Ｔｏｓ）−ＰＡＭ樹脂 12.6g
を得た。The extract was added to ODS-1020TT (30 × 420 m
m) Purification was carried out by reverse phase column chromatography using a column to obtain 12 mg of the desired peptide (yield 4%). This peptide was subjected to high performance liquid chromatography (HPLC) retention time measurement, optical rotation measurement and amino acid analysis. The results are shown below.・ HPLC analysis conditions Column: TSK GEL ODS-120T (4.6 × 250 mm) Solvent: 40% acetonitrile-0.1% TFA Flow rate: 1 ml / min Detection wavelength: 220 nm HPLC retention time: 10.2 minutes ・ [α] D : −34.2 ° ・ Amino acid analysis Asp (4) 3.93, Thr (3) 2.63, Ser (4) 2.89, Glu (3) 3.0
0, Gly (1) 1.07, Ala (1) 1.01, Val (1) 1.00, Met (1) 0.
95, Leu (2) 2.06, Tyr (2) 1.92, Phe (2) 1.92, His (1)
1.05, Lys (1) 0.88, Arg (2) 1.94 (4) Production of glucagon 12 mg of terminal amino group-protected peptide obtained in (3) above
Under ice-cooling, 10% of diethylamine / DMF (50 ml) was added to and dissolved, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, solidified by adding ether, and purified by the same operation as in Example 1-1. Purified glucagon 5.2 mg (yield 2
%). The purified glucagon was subjected to high performance liquid chromatography (HPLC) retention time measurement, optical rotation measurement and amino acid analysis. The results are shown below.・ HPLC analysis conditions Column: TSK GEL ODS-120T (4.6 × 250 mm) Solvent: 35% acetonitrile-0.1% TFA Flow rate: 1 ml / min Detection wavelength: 220 nm HPLC retention time: 10.4 minutes ・ [α] D : −34.4 ° ・ Amino acid analysis Asp (4) 3.91, Thr (3) 2.63, Ser (4) 2.89, Glu (3) 2.9
8, Gly (1) 1.07, Ala (1) 1.00, Val (1) 1.00, Met (1) 0.
97, Leu (2) 2.06, Tyr (2) 1.92, Phe (2) 1.92, His (1)
1.05, Lys (1) 0.99, Arg (2) 1.94 [Example 8] Production of glucagon by method 4 (1) The following formula corresponding to amino acid residues 1 to 18 in the amino acid sequence of glucagon: Fmoc-His -Ser-Gln
-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-
Production of peptide represented by Lys-Tyr-Leu-Asp-Ser-Arg-Arg-OH Boc-Arg (Tos) -PAM resin (Watanabe Chemical Co., Ltd.,
Amino groups (0.44 mmol / g) 5.43 g were sequentially coupled with amino acid residues according to the amino acid sequence of the above peptide in the same manner as in Example 1-1, and Fmoc-His
(Tos) -Ser (Bzl) -Gln-Gly-Thr (Bzl)
-Phe-Thr (Bzl) -Ser-Asp (OcHex) -Ty
r (Br-Z) -Ser (Bzl) -Lys (Cl-Z) -Tyr
(BrZ) -Leu-Asp (OcHex) -Ser (Bzl)
-Arg (Tos) -Arg (Tos) -PAM resin 12.6g
I got

【００９８】得られた保護ペプチド−ＰＡＭ樹脂 5.0g
にアニソ−ル 8mlを加え、さらに無水フッ化水素25mlを
加えて、０℃で１時間撹拌した。反応後、無水フッ化水
素を減圧下留去後、残渣をエ−テルで洗浄し、これに 1
0%酢酸 200mlを加えてペプチドを抽出した。抽出液はＯ
ＤＳ−１０２０ＴＴ（富士デビソン社製）を用いた逆相
カラムクロマトグラフィーにより精製し、目的とするペ
プチド 456mgを得た。このペプチドについて高速液体ク
ロマトグラフィー（ＨＰＬＣ）の保持時間の測定、旋光
度の測定及びアミノ酸分析を行った。その結果を下記に
示す。 ・ＨＰＬＣ 分析条件 カラム：TSK GEL ODS-120T（4.6×250mm） 溶 媒：32%アセトニトリル−0.1%ＴＦＡ 流 速：1ml/min 検出波長：２２０ｎｍ ＨＰＬＣの保持時間：８．８分 ・ [α] D ：−５．２° ・アミノ酸分析 Asp(2) 2.08, Thr(2) 2.02, Ser(4) 3.27, Glu(1) 1.1
3, Gly(1) 1.22,Leu(1) 1.08, Tyr(2) 1.87, Phe(1) 1.
00, Lys(1) 1.10, His(1) 1.18,Arg(2) 2.05 (2) グルカゴンのアミノ酸配列の19〜29位のアミノ酸残
基に相当する下記式：Ｈ−Ａla−Ｇln−Ａsp−Ｐhe−Ｖ
al−Ｇln−Ｔrp−Ｌeu−Ｍet−Ａsn−Ｔhr−ＯＨで表さ
れるペプチドの製造 実施例１−１において得られた中間体 Ｈ−Ａla−Ｇln
−Ａsp（ＯｃＨｅｘ）−Ｐhe−Ｖal−Ｇln−Ｔrp−Ｌeu
− Ｍet−Ａsn−Ｔhr（Ｂzl）−ＰＡＭ樹脂 1.48gに、
アニソ−ル 1ml及びエタンジチオール 1mlを加え、さら
に無水フッ化水素 7mlを加えて、０℃で１時間撹拌し
た。反応後、無水フッ化水素を減圧下留去後、残渣をエ
−テルで洗浄し、これに 50%酢酸 70mlを加えてペプチ
ドを抽出した。抽出液を凍結乾燥して 430mgの白色粉末
を得た。次に得られた粉末を0.1%酢酸に溶解した後、Ｏ
ＤＳ−１０２０ＴＴ（富士デビソン社製）を用いた逆相
カラムクロマトグラフィーにより精製し、目的とするペ
プチド 101mgを得た。このペプチドについて高速液体ク
ロマトグラフィー（ＨＰＬＣ）の保持時間の測定、旋光
度の測定及びアミノ酸分析を行った。その結果を下記に
示す。 ・ＨＰＬＣ 分析条件 カラム：TSK GEL ODS-120T（4.6×250mm） 溶 媒：36%アセトニトリル−0.1%ＴＦＡ 流 速：1ml/min 検出波長：２２０ｎｍ ＨＰＬＣの保持時間：１０．４分 ・ [α] D ：−２３．３° ・アミノ酸分析 Asp(2) 2.10, Thr(1) 0.80, Glu(2) 2.05, Ala(1) 1.0
2, Val(1) 1.00,Met(1) 0.77, Leu(1) 1.23, Phe(1) 1.
04 (3) 下記式：Ｆｍｏｃ−Ｈis−Ｓer−Ｇln−Ｇly−Ｔhr
−Ｐhe−Ｔhr−Ｓer−Ａsp−Ｔyr−Ｓer−Ｌys−Ｔyr−
Ｌeu−Ａsp−Ｓer−Ａrg−Ａrg−Ａla−Ｇln−Ａsp−Ｐ
he−Ｖal−Ｇln−Ｔrp−Ｌeu− Ｍet−Ａsn−Ｔhr−Ｏ
Ｈで表される末端アミノ基保護ペプチドの製造 上記(1) で製造したペプチド 200mg(80μmol)、上記(2)
で製造したペプチド50mg(40μmol)及び 50%ＤＭＳＯを
含む0.2Mトリス緩衝液（ｐＨ 6.5）50mlに、トリプシン
30mgを加え、37℃で 24時間振盪した。次いで氷酢酸 2
mlを加えた後、生成物を濾取し、水で洗浄した後、五酸
化リン存在下、デシケーター中で減圧乾燥した。得られ
た粉末に10%酢酸 200mlを加えてペプチドを抽出した。5.0 g of the obtained protected peptide-PAM resin
8 ml of anisole was added to the mixture, 25 ml of anhydrous hydrogen fluoride was further added, and the mixture was stirred at 0 ° C for 1 hour. After the reaction, the anhydrous hydrogen fluoride was distilled off under reduced pressure, and the residue was washed with ether.
The peptide was extracted by adding 200 ml of 0% acetic acid. The extract is O
Purification by reverse-phase column chromatography using DS-1020TT (manufactured by Fuji Devison) gave 456 mg of the desired peptide. This peptide was subjected to high performance liquid chromatography (HPLC) retention time measurement, optical rotation measurement and amino acid analysis. The results are shown below.・ HPLC analysis conditions Column: TSK GEL ODS-120T (4.6 × 250 mm) Solvent: 32% acetonitrile-0.1% TFA Flow rate: 1 ml / min Detection wavelength: 220 nm HPLC retention time: 8.8 minutes ・ [α] D : -5.2 ° -Amino acid analysis Asp (2) 2.08, Thr (2) 2.02, Ser (4) 3.27, Glu (1) 1.1
3, Gly (1) 1.22, Leu (1) 1.08, Tyr (2) 1.87, Phe (1) 1.
00, Lys (1) 1.10, His (1) 1.18, Arg (2) 2.05 (2) The following formula corresponding to the amino acid residues at positions 19 to 29 of the amino acid sequence of glucagon: H-Ala-Gln-Asp-Phe -V
Preparation of peptide represented by al-Gln-Trp-Leu-Met-Asn-Thr-OH The intermediate H-Ala-Gln obtained in Example 1-1.
-Asp (OcHex) -Phe-Val-Gln-Trp-Leu
-To 1.48 g of Met-Asn-Thr (Bzl) -PAM resin,
1 ml of anisole and 1 ml of ethanedithiol were added, 7 ml of anhydrous hydrogen fluoride was further added, and the mixture was stirred at 0 ° C. for 1 hour. After the reaction, anhydrous hydrogen fluoride was distilled off under reduced pressure, the residue was washed with ether, and 70 ml of 50% acetic acid was added to this to extract the peptide. The extract was freeze-dried to obtain 430 mg of white powder. Next, the obtained powder was dissolved in 0.1% acetic acid, and then O
Purification was performed by reverse phase column chromatography using DS-1020TT (manufactured by Fuji Devison Co., Ltd.) to obtain 101 mg of the desired peptide. This peptide was subjected to high performance liquid chromatography (HPLC) retention time measurement, optical rotation measurement and amino acid analysis. The results are shown below.・ HPLC analysis conditions Column: TSK GEL ODS-120T (4.6 × 250 mm) Solvent: 36% acetonitrile-0.1% TFA Flow rate: 1 ml / min Detection wavelength: 220 nm HPLC retention time: 10.4 minutes ・ [α] D : −23.3 ° ・ Amino acid analysis Asp (2) 2.10, Thr (1) 0.80, Glu (2) 2.05, Ala (1) 1.0
2, Val (1) 1.00, Met (1) 0.77, Leu (1) 1.23, Phe (1) 1.
04 (3) The following formula: Fmoc-His-Ser-Gln-Gly-Thr
-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-
Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-P
he-Val-Gln-Trp-Leu-Met-Asn-Thr-O
Production of peptide protected with terminal amino group represented by H 200 mg (80 μmol) of peptide produced in (1) above, (2) above
Trypsin was added to 50 ml of 0.2M Tris buffer (pH 6.5) containing 50 mg (40 μmol) of the peptide prepared in 1. and 50% DMSO.
30 mg was added and shaken at 37 ° C. for 24 hours. Then glacial acetic acid 2
After adding ml, the product was collected by filtration, washed with water, and dried under reduced pressure in a desiccator in the presence of phosphorus pentoxide. 200 ml of 10% acetic acid was added to the obtained powder to extract the peptide.

【００９９】抽出液をＯＤＳ−１０２０ＴＴ（30×420m
m）カラムを用いた逆相カラムクロマトグラフィーによ
り精製し、目的とするペプチド 8mg（収率 5%）を得
た。このペプチドについて高速液体クロマトグラフィー
（ＨＰＬＣ）の保持時間の測定、旋光度の測定及びアミ
ノ酸分析を行った。その結果を下記に示す。 ・ＨＰＬＣ 分析条件 カラム：TSK GEL ODS-120T（4.6×250mm） 溶 媒：40%アセトニトリル−0.1%ＴＦＡ 流 速：1ml/min 検出波長：２２０ｎｍ ＨＰＬＣの保持時間：１０．２分 ・ [α] D ：−３４．１° ・アミノ酸分析 Asp(4) 3.88, Thr(3) 2.75, Ser(4) 2.89, Glu(3) 2.8
2, Gly(1) 1.15,Ala(1) 1.01, Val(1) 1.06, Met(1) 0.
89, Leu(2) 2.14, Tyr(2) 2.13,Phe(2) 2.07, His(1)
1.11, Lys(1) 1.01, Arg(2) 2.09 (4) グルカゴンの製造 上記(3) で得られた末端アミノ基保護ペプチド 8mgに、
氷冷下 10%ジエチルアミン／ＤＭＦ 50mlを加えて溶解
し、室温で 1.5時間攪拌した。反応液を減圧濃縮した
後、エーテルを加えて固化し、実施例１−１と同様の操
作により精製し、精製グルカゴン 5mg（収率 3%）を得
た。この精製グルカゴンについて高速液体クロマトグラ
フィー（ＨＰＬＣ）の保持時間の測定、旋光度の測定及
びアミノ酸分析を行った。その結果を下記に示す。 ・ＨＰＬＣ 分析条件 カラム：TSK GEL ODS-120T（4.6×250mm） 溶 媒：35%アセトニトリル−0.1%ＴＦＡ 流 速：1ml/min 検出波長：２２０ｎｍ ＨＰＬＣの保持時間：１０．４分 ・ [α] D ：−３５．４° ・アミノ酸分析 Asp(4) 3.84, Thr(3) 2.73, Ser(4) 2.86, Glu(3) 2.7
9, Gly(1) 1.13,Ala(1) 1.00, Val(1) 1.05, Met(1) 0.
88, Leu(2) 2.11, Tyr(2) 2.11,Phe(2) 2.05, His(1)
1.10, Lys(1) 1.00, Arg(2) 2.07 〔実施例９〕方法５によるグルカゴンの合成 (1) グルカゴンのアミノ酸配列の１位から18位のアミノ
酸残基に相当する下記式：Ｆｍｏｃ−Ｈis−Ｓer−Ｇln
−Ｇly−Ｔhr−Ｐhe−Ｔhr−Ｓer−Ａsp−Ｔyr−Ｓer−
Ｌys（Ｆｍｏｃ）−Ｔyr−Ｌeu−Ａsp−Ｓer−Ａrg−Ａ
rg−ＯＨで表されるペプチドの製造 Ｂｏｃ−Ａrg（Ｔｏｓ）−ＰＡＭ樹脂（渡辺化学社製、
アミノ基 0.44mmol/g）1.0ｇに、実施例１−１の方法と
同様にして上記ペプチドのアミノ酸配列にしたがって順
次アミノ酸残基をカップリングし、Ｆｍｏｃ−Ｈis（Ｔ
ｏｓ）−Ｓer（Ｂzl）−Ｇln−Ｇly−Ｔhr（Ｂzl）−Ｐ
he−Ｔhr（Ｂzl）−Ｓer−Ａsp(ＯｃＨｅｘ）−Ｔyr
（Ｂｒ-Ｚ）−Ｓer（Ｂzl）−Ｌys（Ｆｍｏｃ）−Ｔyr
（ＢｒＺ）−Ｌeu−Ａsp（ＯｃＨｅｘ）−Ｓer（Ｂzl）
−Ａrg（Ｔｏｓ）−Ａrg（Ｔｏｓ）−ＰＡＭ樹脂 2.4g
を得た。The extract was added to ODS-1020TT (30 × 420 m
m) Purification was carried out by reverse phase column chromatography using a column to obtain 8 mg of the desired peptide (yield 5%). This peptide was subjected to high performance liquid chromatography (HPLC) retention time measurement, optical rotation measurement and amino acid analysis. The results are shown below.・ HPLC analysis conditions Column: TSK GEL ODS-120T (4.6 × 250 mm) Solvent: 40% acetonitrile-0.1% TFA Flow rate: 1 ml / min Detection wavelength: 220 nm HPLC retention time: 10.2 minutes ・ [α] D : −34.1 ° ・ Amino acid analysis Asp (4) 3.88, Thr (3) 2.75, Ser (4) 2.89, Glu (3) 2.8
2, Gly (1) 1.15, Ala (1) 1.01, Val (1) 1.06, Met (1) 0.
89, Leu (2) 2.14, Tyr (2) 2.13, Phe (2) 2.07, His (1)
1.11, Lys (1) 1.01, Arg (2) 2.09 (4) Production of glucagon To the terminal amino group-protected peptide 8 mg obtained in (3) above,
Under ice cooling, 50 ml of 10% diethylamine / DMF was added and dissolved, and the mixture was stirred at room temperature for 1.5 hours. After the reaction solution was concentrated under reduced pressure, ether was added to solidify it, and the mixture was purified by the same procedure as in Example 1-1 to obtain 5 mg of purified glucagon (yield 3%). The purified glucagon was subjected to high performance liquid chromatography (HPLC) retention time measurement, optical rotation measurement and amino acid analysis. The results are shown below.・ HPLC analysis conditions Column: TSK GEL ODS-120T (4.6 × 250 mm) Solvent: 35% acetonitrile-0.1% TFA Flow rate: 1 ml / min Detection wavelength: 220 nm HPLC retention time: 10.4 minutes ・ [α] D : -35.4 ° ・ Amino acid analysis Asp (4) 3.84, Thr (3) 2.73, Ser (4) 2.86, Glu (3) 2.7
9, Gly (1) 1.13, Ala (1) 1.00, Val (1) 1.05, Met (1) 0.
88, Leu (2) 2.11, Tyr (2) 2.11, Phe (2) 2.05, His (1)
1.10, Lys (1) 1.00, Arg (2) 2.07 [Example 9] Synthesis of glucagon by method 5 (1) The following formula corresponding to amino acid residues 1 to 18 in the amino acid sequence of glucagon: Fmoc-His -Ser-Gln
-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-
Lys (Fmoc) -Tyr-Leu-Asp-Ser-Arg-A
Production of peptide represented by rg-OH Boc-Arg (Tos) -PAM resin (Watanabe Chemical Co., Ltd.,
Amino groups 0.44 mmol / g) 1.0 g were sequentially coupled with amino acid residues according to the amino acid sequence of the above peptide in the same manner as in Example 1-1, and Fmoc-His (T
os) -Ser (Bzl) -Gln-Gly-Thr (Bzl) -P
he-Thr (Bzl) -Ser-Asp (OcHex) -Tyr
(Br-Z) -Ser (Bzl) -Lys (Fmoc) -Tyr
(BrZ) -Leu-Asp (OcHex) -Ser (Bzl)
-Arg (Tos) -Arg (Tos) -PAM resin 2.4g
I got

【０１００】得られた保護ペプチド−ＰＡＭ樹脂 2.4g
に、アニソ−ル 1mlを加え、さらに無水フッ化水素25ml
を加え、０℃で１時間撹拌した。反応後、無水フッ化水
素を減圧下留去後、残査をエ−テルで洗浄し、これに 3
0%酢酸 200mlを加えてペプチドを抽出した。抽出液はＯ
ＤＳ−１０２０ＴＴ（富士デビソン社製）を用いた逆相
カラムクロマトグラフィーにより精製し、目的とするペ
プチド 216mgを得た。このペプチドについて高速液体ク
ロマトグラフィー（ＨＰＬＣ）の保持時間の測定、旋光
度の測定及びアミノ酸分析を行った。その結果を下記に
示す。 ・ＨＰＬＣ 分析条件 カラム：TSK GEL ODS-120T（4.6×250mm） 溶 媒：40%アセトニトリル−0.1%ＴＦＡ 流 速：1ml/min 検出波長：２２０ｎｍ ＨＰＬＣの保持時間：１０．２分 ・ [α] D ：−５．１° ・アミノ酸分析 Asp(2) 2.16, Thr(2) 1.87, Ser(4) 3.20, Glu(1) 1.2
5, Gly(1) 1.20,Leu(1) 1.33, Tyr(2) 1.84, Phe(1) 1.
25, Lys(1) 0.97, His(1) 1.01,Arg(2) 1.82 (2) 下記式：Ｆｍｏｃ−Ｈis−Ｓer−Ｇln−Ｇly−Ｔhr
−Ｐhe−Ｔhr−Ｓer−Ａsp−Ｔyr−Ｓer−Ｌys（Ｆｍｏ
ｃ）−Ｔyr−Ｌeu−Ａsp−Ｓer−Ａrg−Ａrg−Ａla−Ｇ
ln−Ａsp−Ｐhe−Ｖal−Ｇln−Ｔrp−Ｌeu−Ｍet−Ａsn
−Ｔhr−ＯＨで表される末端アミノ基保護ペプチドの製
造 上記(1) で得られたペプチド 216mg(80μmol)、上記実
施例８の(2) で得られたペプチド 50mg(40μmol)を用い
て、実施例８の(3) の方法と同様にして目的とするペプ
チド 5mg（収率 3%）を得た。このペプチドについて高
速液体クロマトグラフィー（ＨＰＬＣ）の保持時間の測
定、旋光度の測定及びアミノ酸分析を行った。その結果
を下記に示す。 ・ＨＰＬＣ 分析条件 カラム：TSK GEL ODS-120T（4.6×250mm） 溶 媒：40%アセトニトリル−0.1%ＴＦＡ 流 速：1ml/min 検出波長：２２０ｎｍ ＨＰＬＣの保持時間：１０．５分 ・ [α] D ：−３４．１° ・アミノ酸分析 Asp(4) 3.92, Thr(3) 2.63, Ser(4) 2.89, Glu(3) 3.0
0, Gly(1) 1.07,Ala(1) 1.01, Val(1) 1.01, Met(1) 0.
95, Leu(2) 2.05, Tyr(2) 1.91,Phe(2) 1.91, His(1)
0.99, Lys(1) 1.04, Arg(2) 1.92 (3) グルカゴンの製造 上記(2) で得られた末端アミノ基保護ペプチド 5mgに、
氷冷下 10%ジエチルアミン／ＤＭＦ 50mlを加えて溶解
し、室温で 1.5時間攪拌した。反応液を減圧濃縮した
後、エーテルを加えて固化し、実施例１−１と同様の操
作により精製し、精製グルカゴン 3mg（収率 1%）を得
た。このペプチドについて高速液体クロマトグラフィー
（ＨＰＬＣ）の保持時間の測定、旋光度の測定及びアミ
ノ酸分析を行った。その結果を下記に示す。 ・ＨＰＬＣ 分析条件 カラム：TSK GEL ODS-120T（4.6×250mm） 溶 媒：35%アセトニトリル−0.1%ＴＦＡ 流 速：1ml/min 検出波長：２２０ｎｍ ＨＰＬＣの保持時間：１０．４分 ・ [α] D ：−３５．４° ・アミノ酸分析Ａｓｐ（４） ３．９２， Ｔｈｒ
（３） ３．１３， Ｓｅｒ（４） ３．００， Ｇｌ
ｕ（３） ２．９２， Ｇｌｙ（１） １．１０，Ａｌ
ａ（１） １．０２， Ｖａｌ（１） ０．９８， Ｍ
ｅｔ（１） ０．９０， Ｌｅｕ（２） ２．０５，
Ｔｙｒ（２） １．９８，Ｐｈｅ（２） １．９２，
Ｈｉｓ（１） １．０５， Ｌｙｓ（１） ０．９６，
Ａｒｇ（２） ２．０３ 〔実施例10〕方法６によるグルカゴンの液相合成 (1) Boc-Asn-Thr(Bzl)-OBzl の合成 Boc-Asn-OH 34.8ｇ、H-Thr(Bzl)-OBzl 41.65ｇ及びHO
BT 19.25 ｇを、DMF200mlとTHF 450mlとの混合溶媒に
溶解した。得られた溶液に、０℃に冷却しながらWSC 2
3.25 mlを添加して２時間攪拌した後、室温で一夜攪拌
した。次いで、０℃に冷却しながらN-メチルモルフォリ
ンを用いてpH７に調節した後、さらにWSC 23.25mlを添
加して攪拌した。2.4 g of the obtained protected peptide-PAM resin
1 ml of anisole was added to 25 ml of anhydrous hydrogen fluoride.
Was added and the mixture was stirred at 0 ° C. for 1 hour. After the reaction, the anhydrous hydrogen fluoride was distilled off under reduced pressure, and the residue was washed with ether.
The peptide was extracted by adding 200 ml of 0% acetic acid. The extract is O
Purification by reverse phase column chromatography using DS-1020TT (manufactured by Fuji Davisson) to obtain 216 mg of the desired peptide. This peptide was subjected to high performance liquid chromatography (HPLC) retention time measurement, optical rotation measurement and amino acid analysis. The results are shown below.・ HPLC analysis conditions Column: TSK GEL ODS-120T (4.6 × 250 mm) Solvent: 40% acetonitrile-0.1% TFA Flow rate: 1 ml / min Detection wavelength: 220 nm HPLC retention time: 10.2 minutes ・ [α] D : -5.1 ° ・ Amino acid analysis Asp (2) 2.16, Thr (2) 1.87, Ser (4) 3.20, Glu (1) 1.2
5, Gly (1) 1.20, Leu (1) 1.33, Tyr (2) 1.84, Phe (1) 1.
25, Lys (1) 0.97, His (1) 1.01, Arg (2) 1.82 (2) The following formula: Fmoc-His-Ser-Gln-Gly-Thr
-Phe-Thr-Ser-Asp-Tyr-Ser-Lys (Fmo
c) -Tyr-Leu-Asp-Ser-Arg-Arg-Ala-G
ln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn
-Preparation of a peptide protected with a terminal amino group represented by -Thr-OH Using 216 mg (80 µmol) of the peptide obtained in (1) above and 50 mg (40 µmol) of the peptide obtained in (2) of Example 8 above, 5 mg of the desired peptide (yield 3%) was obtained in the same manner as in the method (3) of Example 8. This peptide was subjected to high performance liquid chromatography (HPLC) retention time measurement, optical rotation measurement and amino acid analysis. The results are shown below.・ HPLC analysis conditions Column: TSK GEL ODS-120T (4.6 × 250 mm) Solvent: 40% acetonitrile-0.1% TFA Flow rate: 1 ml / min Detection wavelength: 220 nm HPLC retention time: 10.5 minutes ・ [α] D : -34.1 ° ・ Amino acid analysis Asp (4) 3.92, Thr (3) 2.63, Ser (4) 2.89, Glu (3) 3.0
0, Gly (1) 1.07, Ala (1) 1.01, Val (1) 1.01, Met (1) 0.
95, Leu (2) 2.05, Tyr (2) 1.91, Phe (2) 1.91, His (1)
0.99, Lys (1) 1.04, Arg (2) 1.92 (3) Production of glucagon To the terminal amino group-protected peptide 5 mg obtained in (2) above,
Under ice cooling, 50 ml of 10% diethylamine / DMF was added and dissolved, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, solidified by adding ether, and purified by the same operation as in Example 1-1 to obtain 3 mg of purified glucagon (yield 1%). This peptide was subjected to high performance liquid chromatography (HPLC) retention time measurement, optical rotation measurement and amino acid analysis. The results are shown below.・ HPLC analysis conditions Column: TSK GEL ODS-120T (4.6 × 250 mm) Solvent: 35% acetonitrile-0.1% TFA Flow rate: 1 ml / min Detection wavelength: 220 nm HPLC retention time: 10.4 minutes ・ [α] D : -35.4 ° -Amino acid analysis Asp (4) 3.92, Thr
(3) 3.13, Ser (4) 3.00, Gl
u (3) 2.92, Gly (1) 1.10, Al
a (1) 1.02, Val (1) 0.98, M
et (1) 0.90, Leu (2) 2.05
Tyr (2) 1.98, Phe (2) 1.92,
His (1) 1.05, Lys (1) 0.96
Arg (2) 2.03 [Example 10] Liquid phase synthesis of glucagon by method 6 (1) Synthesis of Boc-Asn-Thr (Bzl) -OBzl Boc-Asn-OH 34.8 g, H-Thr (Bzl)- OBzl 41.65g and HO
19.25 g of BT was dissolved in a mixed solvent of 200 ml of DMF and 450 ml of THF. The resulting solution was cooled to 0 ° C with WSC 2
After adding 3.25 ml and stirring for 2 hours, the mixture was stirred overnight at room temperature. Next, the pH was adjusted to 7 with N-methylmorpholine while cooling to 0 ° C., and then 23.25 ml of WSC was added and stirred.

【０１０１】得られた反応混合液を減圧濃縮した後、残
渣に酢酸エチル 500mlを添加して、１Ｎ塩酸 200mlで２
回、飽和重曹水 200mlで２回、飽和食塩水で２回の順で
洗浄した。有機層を無水硫酸ナトリウムで乾燥した後、
減圧濃縮した。残渣を酢酸エチル−ヘキサンに溶解して
再結晶化させた。この再結晶化の操作は３回繰り返し
た。得られた結晶を、できるだけ少量のクロロホルムに
溶解し、クロロホルム−メタノール−酢酸（５：１：
５）を用いたシリカゲルクロマトグラフィーにより精製
した。このようにして、Boc-Asn-Thr(Bzl)-OBzl 60.1ｇ
（収率78.6％）を得た。The obtained reaction mixture was concentrated under reduced pressure, 500 ml of ethyl acetate was added to the residue, and 2 ml of 200 ml of 1N hydrochloric acid was added.
The extract was washed twice with 200 ml of saturated aqueous sodium hydrogen carbonate and twice with saturated saline. After drying the organic layer with anhydrous sodium sulfate,
It was concentrated under reduced pressure. The residue was dissolved in ethyl acetate-hexane and recrystallized. This recrystallization operation was repeated 3 times. The obtained crystals are dissolved in as little chloroform as possible, and then chloroform-methanol-acetic acid (5: 1:
Purified by silica gel chromatography using 5). In this way, 60.1 g of Boc-Asn-Thr (Bzl) -OBzl
(Yield 78.6%) was obtained.

【０１０２】得られたBoc-Asn-Thr(Bzl)-OBzl の融点、
旋光度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：54〜56℃ [α] D ：−17.5°(c=1, DMF) Rf ：0.51(CHCl3/EtOH/酢酸エチル＝5/2/5) 元素分析値 Ｃ27Ｈ35Ｎ3Ｏ7 理論値 Ｃ:63.14% Ｈ:6.87% Ｎ:8.18% 測定値 Ｃ:63.05% Ｈ:6.90% Ｎ:7.98% (2) Boc-Met-Asn-Thr(Bzl)-OBzl の合成 上記(1) で得られたBoc-Asn-Thr(Bzl)-OBzl 56.1ｇに、
氷冷しながらTFA 100ml を添加して室温で30分間攪拌し
た後、減圧濃縮してTFA を留去した。残渣にエーテルを
添加して、生じた沈殿物を濾取した後、乾燥してH-Asn-
Thr(Bzl)-OBzl・TFA を得た。Melting point of the obtained Boc-Asn-Thr (Bzl) -OBzl,
The optical rotation, Rf of TLC and elemental analysis values are shown below. mp: 54-56 ° C [α] D : -17.5 ° (c = 1, DMF) Rf: 0.51 (CHCl 3 / EtOH / ethyl acetate = 5/2/5) Elemental analysis value C 27 H 35 N 3 O 7 Theoretical value C: 63.14% H: 6.87% N: 8.18% Measured value C: 63.05% H: 6.90% N: 7.98% (2) Synthesis of Boc-Met-Asn-Thr (Bzl) -OBzl In the above (1) 56.1 g of the obtained Boc-Asn-Thr (Bzl) -OBzl,
100 ml of TFA was added while cooling with ice, and the mixture was stirred at room temperature for 30 minutes, and then concentrated under reduced pressure to evaporate TFA. Ether was added to the residue, and the resulting precipitate was collected by filtration, dried, and dried with H-Asn-
We got Thr (Bzl) -OBzl.TFA.

【０１０３】一方、Boc-Met-OH（ペプチド研究所製）2
7.4ｇをTHF 50mlに溶解し、ドライアイス−エタノール
で-20℃に冷却し、Ｎ−メチルモルフォリン 12.1ml を
滴下した後、クロロギ酸イソブチル14.52ml(0.1mol)を
滴下して、-20℃で1分間攪拌して混合酸無水物の溶液を
調製した。得られた溶液を、Ｎ−メチルモルフォリンで
中和した上記H-Asn-Thr(Bzl)-OBzl ・TFA のTHF 溶液 2
00mlと混合し、０℃で５分間、室温で30分間攪拌した
後、減圧濃縮した。残渣に酢酸エチル 500mlを加え、1N
塩酸 200mlで２回、飽和重曹水 200mlで２回、飽和食塩
水で２回の順で洗浄した。有機層を無水硫酸ナトリウム
で乾燥した後、減圧濃縮した。残渣を酢酸エチル−ヘキ
サンに溶解して、再結晶化させた。この再結晶化の操作
は４回繰り返した。このようにして、Boc-Met-Asn-Thr
(Bzl)-OBzl 56.18 ｇ（収率79.8％）を得た。On the other hand, Boc-Met-OH (manufactured by Peptide Institute) 2
Dissolve 7.4 g in 50 ml THF, cool to -20 ° C with dry ice-ethanol, add 12.1 ml N-methylmorpholine, and then add 14.52 ml (0.1 mol) isobutyl chloroformate and -20 ° C. A mixed acid anhydride solution was prepared by stirring for 1 minute. The obtained solution was neutralized with N-methylmorpholine to obtain a solution of H-Asn-Thr (Bzl) -OBzl.TFA in THF 2
The mixture was mixed with 00 ml, stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes, and concentrated under reduced pressure. Add 500 ml of ethyl acetate to the residue and add 1N.
The extract was washed twice with 200 ml of hydrochloric acid, twice with 200 ml of saturated aqueous sodium hydrogen carbonate and twice with saturated saline. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The residue was dissolved in ethyl acetate-hexane and recrystallized. This recrystallization operation was repeated 4 times. In this way, Boc-Met-Asn-Thr
56.18 g (yield 79.8%) of (Bzl) -OBzl was obtained.

【０１０４】得られたBoc-Met-Asn-Thr(Bzl)-OBzl の融
点、旋光度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：89〜91℃ [α] D ：−15.0°(c=1, DMF) Rf ：0.61(CHCl3/EtOH/酢酸エチル＝5/2/5) 元素分析値 Ｃ32Ｈ44Ｎ4Ｏ8Ｓ 理論値 Ｃ:59.61% Ｈ:6.88% Ｎ:8.69% 測定値 Ｃ:59.48% Ｈ:6.35% Ｎ:8.60% (3) Boc-Leu-Met-Asn-Thr(Bzl)-OBzl の合成 上記(2) で得られたBoc-Met-Asn-Thr(Bzl)-OBzl 54.4ｇ
に、氷冷しながらTFA60ml及びエタンジチオール２mlを
添加して室温で30分間攪拌した後、減圧濃縮してTFA を
留去した。残渣にエーテルを添加して、生じた沈殿物を
濾取した後、乾燥してH-Met-Asn-Thr(Bzl)-OBzl ・TFA
を得た。The melting point, optical rotation, Rf of TLC and elemental analysis values of the obtained Boc-Met-Asn-Thr (Bzl) -OBzl are shown below. mp: 89 to 91 ° C [α] D : -15.0 ° (c = 1, DMF) Rf: 0.61 (CHCl 3 / EtOH / ethyl acetate = 5/2/5) Elemental analysis value C 32 H 44 N 4 O 8 S Theoretical value C: 59.61% H: 6.88% N: 8.69% Measured value C: 59.48% H: 6.35% N: 8.60% (3) Synthesis of Boc-Leu-Met-Asn-Thr (Bzl) -OBzl Above ( 54.4 g of Boc-Met-Asn-Thr (Bzl) -OBzl obtained in 2)
While cooling with ice, 60 ml of TFA and 2 ml of ethanedithiol were added, and the mixture was stirred at room temperature for 30 minutes, and then concentrated under reduced pressure to distill off TFA. Ether was added to the residue, and the resulting precipitate was collected by filtration and dried to give H-Met-Asn-Thr (Bzl) -OBzl.TFA.
I got

【０１０５】上記で得られたH-Met-Asn-Thr(Bzl)-OBzl
・TFA に、HOBT 13.77ｇ及びBoc-Leu-OH・H2O 25.5 ｇ
を添加した。次いで、０℃に冷却しながらWSC 18.5mlを
添加して室温で一夜攪拌した。得られた反応混合液を減
圧濃縮した後残渣に氷水を添加した。生じた沈殿物を濾
取し、酢酸エチルに加熱溶解した。これにベンゼンを添
加して共沸により水を除去した。この操作を３回繰り返
した後、エタノール−−エーテルを用いて再結晶化させ
た。このようにして、Boc-Leu-Met-Asn-Thr(Bzl)-OBzl
52.46 ｇ（収率82.3％）を得た。H-Met-Asn-Thr (Bzl) -OBzl obtained above
・ For TFA, 13.77g of HOBT and 25.5g of Boc-Leu-OH ・ H 2 O
Was added. Then, 18.5 ml of WSC was added while cooling to 0 ° C., and the mixture was stirred at room temperature overnight. The obtained reaction mixture was concentrated under reduced pressure, and ice water was added to the residue. The generated precipitate was collected by filtration and dissolved in ethyl acetate with heating. Benzene was added to this and water was removed azeotropically. After repeating this operation 3 times, it was recrystallized using ethanol-ether. Thus, Boc-Leu-Met-Asn-Thr (Bzl) -OBzl
52.46 g (yield 82.3%) was obtained.

【０１０６】得られたBoc-Leu-Met-Asn-Thr(Bzl)-OBzl
の融点、旋光度、ＴＬＣのRf及び元素分析値を下記に示
す。 m.p. ：172 〜173 ℃ [α] D ：−20.0°(c=1, DMF) Rf ：0.72(CHCl3/EtOH/酢酸エチル＝5/2/5) 元素分析値 Ｃ38Ｈ55Ｎ5Ｏ9Ｓ 理論値 Ｃ:60.22% Ｈ:7.31% Ｎ:9.24% 測定値 Ｃ:60.10% Ｈ:7.35% Ｎ:9.16% (4) Boc-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Va
l-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-OBzl の合成 上記(3) で得られたBoc-Leu-Met-Asn-Thr(Bzl)-OBzl 4
8.75 ｇに、氷冷しながらTFA 50ml及びエタンジチオー
ル 1.2mlを添加して室温で30分間攪拌した後、減圧濃縮
してTFA を留去した。残渣にエーテルを添加して、生じ
た沈殿物を濾取した後、乾燥してH-Leu-Met-Asn-Thr(Bz
l)-OBzl ・TFA を得た。The Boc-Leu-Met-Asn-Thr (Bzl) -OBzl obtained
The melting point, optical rotation, Tf Rf, and elemental analysis values of are shown below. mp: 172-173 ° C [α] D : -20.0 ° (c = 1, DMF) Rf: 0.72 (CHCl 3 / EtOH / ethyl acetate = 5/2/5) Elemental analysis value C 38 H 55 N 5 O 9 S theoretical value C: 60.22% H: 7.31% N: 9.24% measured value C: 60.10% H: 7.35% N: 9.16% (4) Boc-Arg (Tos) -Arg (Tos) -Ala-Gln-Asp ( OBzl) -Phe-Va
Synthesis of l-Gln-Trp-Leu-Met-Asn-Thr (Bzl) -OBzl Boc-Leu-Met-Asn-Thr (Bzl) -OBzl 4 obtained in (3) above
TFA (50 ml) and ethanedithiol (1.2 ml) were added to 8.75 g with ice cooling, the mixture was stirred at room temperature for 30 minutes, and concentrated under reduced pressure to remove TFA. Ether was added to the residue, and the resulting precipitate was collected by filtration, dried, and dried with H-Leu-Met-Asn-Thr (Bz
l) -OBzl ・ TFA was obtained.

【０１０７】上記のH-Leu-Met-Asn-Thr(Bzl)-OBzl ・TF
A をDMF に溶解して、得られた溶液に、HOBT 10.53ｇ、
実施例４の(2) で得られたBoc-Arg(Tos)-Arg(Tos)-Ala-
Gln-Asp(OBzl)-Phe-Val-Gln-Trp-OH 151.58g 、及びWS
C 4.2ml を添加して、N-メチルモルフォリンを用いてpH
７に調節し、室温で一夜攪拌した。得られた反応混合液
を減圧濃縮し、残渣に氷水を添加した。生じた沈殿物を
濾取し、水洗し、乾燥した後、エタノール−ジエチルエ
ーテルを用いて再結晶化させた。再結晶化の操作は２回
繰り返した。このようにして、Boc-Arg(Tos)-Arg(Tos)-
Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr
(Bzl)-OBzl 192.6 ｇ（収率95.6％）を得た。The above H-Leu-Met-Asn-Thr (Bzl) -OBzl.TF
A was dissolved in DMF, and the obtained solution was added with HOBT 10.53 g,
Boc-Arg (Tos) -Arg (Tos) -Ala- obtained in (2) of Example 4
Gln-Asp (OBzl) -Phe-Val-Gln-Trp-OH 151.58g, and WS
Add 4.2 ml of C and pH with N-methylmorpholine
Adjusted to 7 and stirred at room temperature overnight. The obtained reaction mixture was concentrated under reduced pressure, and ice water was added to the residue. The generated precipitate was collected by filtration, washed with water, dried, and recrystallized with ethanol-diethyl ether. The recrystallization operation was repeated twice. In this way, Boc-Arg (Tos) -Arg (Tos)-
Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-Leu-Met-Asn-Thr
192.6 g (yield 95.6%) of (Bzl) -OBzl was obtained.

【０１０８】得られたペプチドのアミノ酸分析値を下記
に示す。 Asp(2) 2.10, Thr(1) 0.96, Ala(1) 1.06, Glu(2) 2.0
3, Val(1) 0.98,Met(1) 0.96, Leu(1) 1.09, Phe(1) 1.
01, Arg(2) 2.11 (5) Boc-Ser(Bzl)-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBz
l)-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-OBzl の合
成 上記(4) で得られたBoc-Arg(Tos)-Arg(Tos)-Ala-Gln-As
p(OBzl)-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-OBzl
186.3 ｇに、氷冷しながらTFA 200ml 及びエタンジチオ
ール10mlを添加して室温で30分間攪拌した後、減圧濃縮
してTFA を留去した。残渣にエーテルを添加して、生じ
た沈殿物を濾取した後、乾燥してH-Arg(Tos)-Arg(Tos)-
Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr
(Bzl)-OBzl ・TFA を得た。The amino acid analysis values of the obtained peptides are shown below. Asp (2) 2.10, Thr (1) 0.96, Ala (1) 1.06, Glu (2) 2.0
3, Val (1) 0.98, Met (1) 0.96, Leu (1) 1.09, Phe (1) 1.
01, Arg (2) 2.11 (5) Boc-Ser (Bzl) -Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBz
l) -Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (Bzl) -OBzl synthesis Boc-Arg (Tos) -Arg (Tos) -Ala-Gln-As obtained in (4) above.
p (OBzl) -Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (Bzl) -OBzl
To 186.3 g, 200 ml of TFA and 10 ml of ethanedithiol were added while cooling with ice, and the mixture was stirred at room temperature for 30 minutes and then concentrated under reduced pressure to remove TFA. Ether was added to the residue, the resulting precipitate was collected by filtration, dried and then H-Arg (Tos) -Arg (Tos)-.
Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-Leu-Met-Asn-Thr
(Bzl) -OBzl-TFA was obtained.

【０１０９】一方、Boc-Ser(Bzl)-OH （ペプチド研究所
製）10.04 ｇをTHF 100ml に溶解し、ドライアイス−エ
タノールで-20℃に冷却し、Ｎ−メチルモルフォリン 6.
6mlを滴下した後、クロロギ酸イソブチル7.92mlを滴下
して、-20℃で1分間攪拌して混合酸無水物の溶液を調製
した。得られた溶液を、Ｎ−メチルモルフォリンで中和
した上記H-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-
Val-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-OBzl ・TFA のTHF
溶液 200mlと混合し、０℃で５分間、室温で30分間攪拌
した後、減圧濃縮した。残渣に氷水を添加した。生じた
沈殿物を濾取し、水洗し、メタノール−エーテルを用い
て再結晶化させた。再結晶化の操作は２回繰り返した。
このようにして、Boc-Ser(Bzl)-Arg(Tos)-Arg(Tos)-Ala
-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(Bz
l)-OBzl 173.83ｇ（収率88.6％）を得た。On the other hand, 10.04 g of Boc-Ser (Bzl) -OH (manufactured by Peptide Laboratories) was dissolved in 100 ml of THF, cooled to -20 ° C with dry ice-ethanol, and N-methylmorpholine 6.
After dropwise adding 6 ml, 7.92 ml of isobutyl chloroformate was added dropwise and stirred at -20 ° C for 1 minute to prepare a mixed acid anhydride solution. The obtained solution was neutralized with N-methylmorpholine to the above H-Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-.
Val-Gln-Trp-Leu-Met-Asn-Thr (Bzl) -OBzl ・ TFA THF
The mixture was mixed with 200 ml of the solution, stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes, and concentrated under reduced pressure. Ice water was added to the residue. The resulting precipitate was collected by filtration, washed with water, and recrystallized with methanol-ether. The recrystallization operation was repeated twice.
Thus, Boc-Ser (Bzl) -Arg (Tos) -Arg (Tos) -Ala
-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (Bz
l) -OBzl (173.83 g, yield 88.6%) was obtained.

【０１１０】得られたペプチドのアミノ酸分析値を下記
に示す。 Asp(2) 2.10, Thr(1) 0.96, Ser(1) 0.92, Ala(1) 1.0
6, Glu(2) 2.04,Val(1) 0.98, Met(1) 0.96, Leu(1) 1.
08, Phe(1) 1.00, Arg(2) 2.10 (6) Boc-Asp(OBzl)-Ser(Bzl)-Arg(Tos)-Arg(Tos)-Ala-G
ln-Asp(OBzl)-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-
OBzl の合成 上記(5) で得られたBoc-Ser(Bzl)-Arg(Tos)-Arg(Tos)-A
la-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(B
zl)-OBzl 163.5ｇに、氷冷しながらTFA 200ml及びエタ
ンジチオール４mlを添加して室温で30分間攪拌した後、
減圧濃縮してTFA を留去した。残渣にエーテルを添加し
て、生じた沈殿物を濾取した後、乾燥してH-Ser(Bzl)-A
rg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp
-Leu-Met-Asn-Thr(Bzl)-OBzl・TFA を得た。The amino acid analysis values of the obtained peptides are shown below. Asp (2) 2.10, Thr (1) 0.96, Ser (1) 0.92, Ala (1) 1.0
6, Glu (2) 2.04, Val (1) 0.98, Met (1) 0.96, Leu (1) 1.
08, Phe (1) 1.00, Arg (2) 2.10 (6) Boc-Asp (OBzl) -Ser (Bzl) -Arg (Tos) -Arg (Tos) -Ala-G
ln-Asp (OBzl) -Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (Bzl)-
Synthesis of OBzl Boc-Ser (Bzl) -Arg (Tos) -Arg (Tos) -A obtained in (5) above
la-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (B
zl) -OBzl To 163.5 g, 200 ml of TFA and 4 ml of ethanedithiol were added while cooling with ice and stirred at room temperature for 30 minutes,
After concentration under reduced pressure, TFA was distilled off. Ether was added to the residue, the resulting precipitate was collected by filtration, dried and then H-Ser (Bzl) -A.
rg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Trp
-Leu-Met-Asn-Thr (Bzl) -OBzl.TFA was obtained.

【０１１１】一方、Boc-Asp(OBzl)-OH（ペプチド研究所
製）16.9ｇをTHF 100ml に溶解し、ドライアイス−エタ
ノールで-20℃に冷却し、Ｎ−メチルモルフォリン 5.5m
lを滴下した後、クロロギ酸イソブチル6.6ml を滴下し
て、-20℃で1分間攪拌して混合酸無水物の溶液を調製し
た。得られた溶液を、Ｎ−メチルモルフォリンで中和し
た上記H-Ser(Bzl)-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBz
l)-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-OBzl・TFA
のTHF 溶液 200mlと混合し、０℃で５分間、室温で30分
間攪拌した後、減圧濃縮した。残渣に氷水を添加した。
生じた沈殿物を濾取し、水洗し、メタノール−エーテル
を用いて再結晶化させた。再結晶化の操作は２回繰り返
した。このようにして、Boc-Asp(OBzl)-Ser(Bzl)-Arg(T
os)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp- Le
u-Met-Asn-Thr(Bzl)-OBzl 160.7ｇ（収率92.1％）を得
た。On the other hand, 16.9 g of Boc-Asp (OBzl) -OH (manufactured by Peptide Laboratories) was dissolved in 100 ml of THF and cooled to -20 ° C with dry ice-ethanol to obtain 5.5 m of N-methylmorpholine.
After dropwise adding l, 6.6 ml of isobutyl chloroformate was added dropwise and stirred at -20 ° C for 1 minute to prepare a mixed acid anhydride solution. The resulting solution was neutralized with N-methylmorpholine to the above H-Ser (Bzl) -Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBz
l) -Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (Bzl) -OBzl ・ TFA
Was mixed with 200 ml of a THF solution of 1: 1, stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes, and then concentrated under reduced pressure. Ice water was added to the residue.
The resulting precipitate was collected by filtration, washed with water, and recrystallized with methanol-ether. The recrystallization operation was repeated twice. Thus, Boc-Asp (OBzl) -Ser (Bzl) -Arg (T
os) -Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Trp- Le
160.7 g (yield 92.1%) of u-Met-Asn-Thr (Bzl) -OBzl was obtained.

【０１１２】得られたペプチドのアミノ酸分析値を下記
に示す。 Asp(3) 3.12, Thr(1) 0.95, Ser(1) 0.91, Ala(1) 1.0
5, Glu(2) 2.04,Val(1) 0.98, Met(1) 0.95, Leu(1) 1.
08, Phe(1) 1.01, Arg(2) 2.05 (7) Boc-Tyr-Leu-OMe の合成 Boc-Tyr-OH 42.2ｇ、H-Leu-OMe 21.6ｇ及びHOBT 19.25
ｇを乾燥DMF 200 mlと乾燥THF 450 mlの混合溶媒に溶解
した。得られた溶液に、０℃に冷却しながらWSC23.25ml
を添加して２時間攪拌した後室温で一夜攪拌した。得ら
れた反応混合液を減圧濃縮した後残渣を酢酸エチル 600
mlに溶解した。次いで、１Ｎ塩酸 200mlで２回、飽和重
曹水 200mlで２回、飽和食塩水 200mlで２回の順で洗浄
した。有機層を無水硫酸ナトリウムで乾燥した後、減圧
濃縮した。残渣を酢酸エチル−エーテルを用いて再結晶
化させた。この再結晶の操作は２回繰り返した。このよ
うにして、Boc-Tyr-Leu-OMe 52.0ｇ（収率85.3％）を得
た。The amino acid analysis values of the obtained peptides are shown below. Asp (3) 3.12, Thr (1) 0.95, Ser (1) 0.91, Ala (1) 1.0
5, Glu (2) 2.04, Val (1) 0.98, Met (1) 0.95, Leu (1) 1.
08, Phe (1) 1.01, Arg (2) 2.05 (7) Synthesis of Boc-Tyr-Leu-OMe Boc-Tyr-OH 42.2 g, H-Leu-OMe 21.6 g and HOBT 19.25
g was dissolved in a mixed solvent of 200 ml of dry DMF and 450 ml of dry THF. 23.25 ml of WSC was added to the resulting solution while cooling to 0 ° C.
Was added and the mixture was stirred for 2 hours and then stirred at room temperature overnight. The obtained reaction mixture was concentrated under reduced pressure and the residue was treated with ethyl acetate 600
dissolved in ml. Then, the mixture was washed twice with 200 ml of 1N hydrochloric acid, twice with 200 ml of saturated aqueous sodium hydrogen carbonate and twice with 200 ml of saturated saline. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The residue was recrystallized with ethyl acetate-ether. This recrystallization operation was repeated twice. In this way, 52.0 g (yield 85.3%) of Boc-Tyr-Leu-OMe was obtained.

【０１１３】得られたBoc-Tyr-Leu-OMe の融点、旋光
度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：106 〜108 ℃ [α] D ：−14.7°(c=1, DMF) Rf ：0.73(CHCl3/MeOH/AcOH＝9/1/0.5) 元素分析値 Ｃ21Ｈ32Ｎ2Ｏ6 理論値 Ｃ:61.74% Ｈ:7.90% Ｎ:6.86% 測定値 Ｃ:61.86% Ｈ:7.73% Ｎ:6.84% (8) Boc-Lys(Z)-Tyr-Leu-OMeの合成 上記(7) で得られたBoc-Tyr-Leu-OMe 50.0ｇに、TFA 50
mlを添加して30分間攪拌した後、減圧濃縮してTFA を留
去した。残渣にエーテルを添加して、生じた沈殿物を濾
取した後、乾燥THF 250ml に溶解した。The melting point, optical rotation, Tf Rf, and elemental analysis values of the obtained Boc-Tyr-Leu-OMe are shown below. mp: 106-108 ° C [α] D : -14.7 ° (c = 1, DMF) Rf: 0.73 (CHCl 3 /MeOH/AcOH=9/1/0.5) Elemental analysis value C 21 H 32 N 2 O 6 theory Value C: 61.74% H: 7.90% N: 6.86% Measured value C: 61.86% H: 7.73% N: 6.84% (8) Synthesis of Boc-Lys (Z) -Tyr-Leu-OMe Obtained in (7) above. Boc-Tyr-Leu-OMe (50.0 g), TFA 50
After adding ml and stirring for 30 minutes, TFA was distilled off by concentration under reduced pressure. Ether was added to the residue, the resulting precipitate was collected by filtration, and then dissolved in 250 ml of dry THF.

【０１１４】得られた溶液に、HOBT 19.44ｇ及びBoc-Ly
s(Z)-OH 45.6 ｇを添加した。次いで、０℃に冷却しな
がらWSC 26.2mlを添加して室温で一夜攪拌した。得られ
た反応混合液を減圧濃縮した後残渣を酢酸エチル 800ml
に溶解した。次いで、１Ｎ塩酸 200mlで２回、飽和重曹
水 200mlで２回、飽和食塩水 200mlで２回の順で洗浄し
た。有機層を無水硫酸ナトリウムで乾燥した後、減圧乾
固した。残渣を酢酸エチル−エーテルを用いて再結晶化
させた。この再結晶の操作は２回繰り返した。このよう
にして、Boc-Lys(Z)-Tyr-Leu-OMe 72.36ｇ（収率90.0
％）を得た。To the resulting solution, 19.44 g of HOBT and Boc-Ly
45.6 g of s (Z) -OH was added. Then, 26.2 ml of WSC was added while cooling to 0 ° C., and the mixture was stirred at room temperature overnight. The obtained reaction mixture was concentrated under reduced pressure and the residue was diluted with 800 ml of ethyl acetate.
Dissolved in. Then, the mixture was washed twice with 200 ml of 1N hydrochloric acid, twice with 200 ml of saturated aqueous sodium hydrogen carbonate and twice with 200 ml of saturated saline. The organic layer was dried over anhydrous sodium sulfate and then dried under reduced pressure. The residue was recrystallized with ethyl acetate-ether. This recrystallization operation was repeated twice. In this way, 72.36 g of Boc-Lys (Z) -Tyr-Leu-OMe (yield 90.0
%) Was obtained.

【０１１５】得られたBoc-Lys(Z)-Tyr-Leu-OMe の融
点、旋光度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：110 〜111 ℃ [α] D ：−23.1°(c=1, DMF) Rf ：0.53(CHCl3/MeOH/AcOH＝9/1/0.5) 元素分析値 Ｃ35Ｈ50Ｎ4Ｏ9 理論値 Ｃ:62.67% Ｈ:7.51% Ｎ:8.35% 測定値 Ｃ:62.39% Ｈ:7.77% Ｎ:8.09% (9) Boc-Ser-Lys(Z)-Tyr-Leu-OMeの合成 上記(8) で得られたBoc-Lys(Z)-Tyr-Leu-OMe 70.3 ｇ
に、TFA 100ml を添加して30分間攪拌した後、減圧濃縮
してTFA を留去した。残渣にエーテルを添加して、生じ
た沈殿物を濾取した後、乾燥し、THF 250ml に溶解し
た。The melting point, optical rotation, Tf Rf and elemental analysis values of the obtained Boc-Lys (Z) -Tyr-Leu-OMe are shown below. mp: 110-111 ° C [α] D : -23.1 ° (c = 1, DMF) Rf: 0.53 (CHCl 3 /MeOH/AcOH=9/1/0.5) Elemental analysis value C 35 H 50 N 4 O 9 theory Value C: 62.67% H: 7.51% N: 8.35% Measured value C: 62.39% H: 7.77% N: 8.09% (9) Synthesis of Boc-Ser-Lys (Z) -Tyr-Leu-OMe Above (8) 70.3 g of Boc-Lys (Z) -Tyr-Leu-OMe obtained in
To the mixture, 100 ml of TFA was added, and the mixture was stirred for 30 minutes, concentrated under reduced pressure, and TFA was distilled off. Ether was added to the residue and the resulting precipitate was collected by filtration, dried and dissolved in 250 ml of THF.

【０１１６】得られた溶液に、HOBT 17.01ｇ及びBoc-Se
r-OH 21.5ｇを添加した。次いで、０℃に冷却しながら
WSC 22.93ml を添加して室温で一夜攪拌した。得られた
反応混合液を減圧濃縮した後残渣に氷水を添加した。生
じた沈殿物を濾取し、水洗し、メタノール−エーテルを
用いて再結晶化させた。この再結晶の操作は２回繰り返
した。このようにして、Boc-Ser-Lys(Z)-Tyr-Leu-OMe 6
9.53ｇ（収率87.6％）を得た。To the obtained solution, 17.01 g of HOBT and Boc-Se were added.
21.5 g of r-OH was added. Then, while cooling to 0 ° C
WSC (22.93 ml) was added and the mixture was stirred at room temperature overnight. The obtained reaction mixture was concentrated under reduced pressure, and ice water was added to the residue. The resulting precipitate was collected by filtration, washed with water, and recrystallized with methanol-ether. This recrystallization operation was repeated twice. Thus, Boc-Ser-Lys (Z) -Tyr-Leu-OMe 6
9.53 g (yield 87.6%) was obtained.

【０１１７】得られたBoc-Ser-Lys(Z)-Tyr-Leu-OMeの融
点、旋光度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：109 〜111 ℃ [α] D ：−24.2°(c=1, DMF) Rf ：0.59(CHCl3/MeOH/AcOH＝9/1/0.5) 元素分析値 Ｃ38Ｈ55Ｎ5Ｏ11 理論値 Ｃ:58.82% Ｈ:7.41% Ｎ:9.03% 測定値 Ｃ:59.02% Ｈ:7.08% Ｎ:9.15% (10)Boc-Ser-Lys(Z)-Tyr-Leu-NHNH2の合成 上記(9) で得られたBoc-Ser-Lys(Z)-Tyr-Leu-OMe 50.0
ｇをメタノール 250mlに溶解し、抱水ヒドラジン20.8ml
を添加して、室温で20時間攪拌した。得られた反応混合
液に水を添加し、沈殿物を濾取し、乾燥してメタノール
で洗浄した。このようにして、Boc-Ser-Lys(Z)-Tyr-Leu
-NHNH2 41.72ｇ（収率92.1％）を得た。The melting point, optical rotation, Tf Rf and elemental analysis values of the obtained Boc-Ser-Lys (Z) -Tyr-Leu-OMe are shown below. mp: 109-111 ° C [α] D : −24.2 ° (c = 1, DMF) Rf: 0.59 (CHCl 3 /MeOH/AcOH=9/1/0.5) Elemental analysis value C 38 H 55 N 5 O 11 theory Value C: 58.82% H: 7.41% N: 9.03% Measured value C: 59.02% H: 7.08% N: 9.15% (10) Synthesis of Boc-Ser-Lys (Z) -Tyr-Leu-NHNH 2 Above (9 ) Obtained Boc-Ser-Lys (Z) -Tyr-Leu-OMe 50.0
g dissolved in 250 ml of methanol, 20.8 ml hydrazine hydrate
Was added and stirred at room temperature for 20 hours. Water was added to the obtained reaction mixture, and the precipitate was collected by filtration, dried and washed with methanol. Thus, Boc-Ser-Lys (Z) -Tyr-Leu
41.72 g (yield 92.1%) of NHNH 2 was obtained.

【０１１８】得られたBoc-Ser-Lys(Z)-Tyr-Leu-NHNH2の
融点、旋光度、ＴＬＣのRf及び元素分析値を下記に示
す。 m.p. ：130 〜135 ℃ [α] D ：−26.2°(c=1, DMF) Rf ：0.60(CHCl3/MeOH/AcOH＝9/1/0.5) 元素分析値 Ｃ37Ｈ55Ｎ7Ｏ10 理論値 Ｃ:58.64% Ｈ:7.31% Ｎ:12.94% 測定値 Ｃ:58.70% Ｈ:7.25% Ｎ:12.94% (11) Boc-Ser-Lys(Z)-Tyr-Leu-Asp(OBzl)-Ser-Arg(Tos)
-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-Leu-Me
t-Asn-Thr(Bzl)-0Bzlの合成 上記(6) で得られたBoc-Asp(OBzl)-Ser(Bzl)-Arg(Tos)-
Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-Leu-Met
-Asn-Thr(Bzl)-0Bzl 104.5g に、氷冷しながらTFA 150m
l 及びエタンジチオール４mlを添加して、室温で30分間
攪拌した後、減圧濃縮下してTFAを留去した。残渣にエ
ーテルを添加して、生じた沈殿物を濾取した後、乾燥し
てH-Asp(OBzl)-Ser(Bzl)-Arg(Tos)-Arg(Tos)-Ala-Gln-A
sp(0Bzl)-Phe-Val-Gln-Leu-Met-Asn-Thr(Bzl)-OBzl・TFA
を得た。The melting point, optical rotation, Tf Rf, and elemental analysis values of the obtained Boc-Ser-Lys (Z) -Tyr-Leu-NHNH 2 are shown below. mp: 130-135 ° C [α] D : -26.2 ° (c = 1, DMF) Rf: 0.60 (CHCl 3 /MeOH/AcOH=9/1/0.5) Elemental analysis value C 37 H 55 N 7 O 10 theory Value C: 58.64% H: 7.31% N: 12.94% Measured value C: 58.70% H: 7.25% N: 12.94% (11) Boc-Ser-Lys (Z) -Tyr-Leu-Asp (OBzl) -Ser- Arg (Tos)
-Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-Leu-Me
Synthesis of t-Asn-Thr (Bzl) -0Bzl Boc-Asp (OBzl) -Ser (Bzl) -Arg (Tos) -obtained in (6) above
Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-Leu-Met
-Asn-Thr (Bzl) -0Bzl 104.5g, TFA 150m while cooling with ice
1 and 4 ml of ethanedithiol were added, the mixture was stirred at room temperature for 30 minutes, and then concentrated under reduced pressure to remove TFA by distillation. Ether was added to the residue, the resulting precipitate was collected by filtration, dried and then H-Asp (OBzl) -Ser (Bzl) -Arg (Tos) -Arg (Tos) -Ala-Gln-A.
sp (0Bzl) -Phe-Val-Gln-Leu-Met-Asn-Thr (Bzl) -OBzl ・ TFA
I got

【０１１９】一方、上記(10)で得られたBoc-Ser-Lys(Z)
-Tyr-Leu-NHNH2 22.68ｇをDMFに溶解し、ドライアイス
−エタノールで−20℃に冷却し、６N HCl-ジオキサン1
8.07ml 及び亜硝酸イソアミル3.９mlを添加してアジド
化合物とした。これに、更にトリエチルアミン15.15ml
を添加した後中和した混液を、上記H-Asp(OBzl)-Ser(Bz
l)-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(0Bzl)-Phe-Val-Gln
-Leu-Met-Asn-Thr(Bzl)-OBzl・TFAのDMF溶液に移し、−2
0℃で２時間、４℃で20時間放置した。得られた反応混
合液を減圧濃縮し、残渣に氷水を加えた。生じた沈殿物
を濾取し、水洗した後、メタノール−エーテルを用いて
再結晶化させた。再結晶化の操作は２回繰り返した。こ
のようにして、Boc-Ser-Lys(Z)-Tyr-Leu-Asp(OBzl)-Ser
-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-T
rp-Leu-Met-Asn-Thr(Bzl)-0Bzl 101.9g （収率82.5％)
を得た。On the other hand, Boc-Ser-Lys (Z) obtained in (10) above
22.68 g of -Tyr-Leu-NHNH 2 was dissolved in DMF, cooled to -20 ° C with dry ice-ethanol, and 6N HCl-dioxane 1 was added.
8.07 ml and 3.9 ml of isoamyl nitrite were added to give an azide compound. In addition to this, 15.15 ml of triethylamine
Was added and then the mixture was neutralized to obtain the above H-Asp (OBzl) -Ser (Bz
l) -Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (0Bzl) -Phe-Val-Gln
-Leu-Met-Asn-Thr (Bzl) -OBzl-transferred to DFA solution of TFA, -2
It was left at 0 ° C. for 2 hours and at 4 ° C. for 20 hours. The obtained reaction mixture was concentrated under reduced pressure, and ice water was added to the residue. The generated precipitate was collected by filtration, washed with water, and recrystallized using methanol-ether. The recrystallization operation was repeated twice. Thus, Boc-Ser-Lys (Z) -Tyr-Leu-Asp (OBzl) -Ser
-Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-Val-Gln-T
rp-Leu-Met-Asn-Thr (Bzl) -0Bzl 101.9g (82.5% yield)
I got

【０１２０】得られたペプチドのアミノ酸分析値を下記
に示す。 Asp(3) 3.15, Thr(1) 0.94, Ser(2) 1.80, Ala(1) 1.0
5, Glu(2) 2.03,Val(1) 0.98, Met(1) 0.95, Leu(2) 1.
08, Tyr(1) 0.96, Phe(1) 1.01,Lys(1) 1.01, Arg(2)
2.13 (12) Boc-Asp(OBzl)-Tyr-OH の合成 Boc-Asp(OBzl)-OH（ペプチド研究所製）9.69g をTHF 10
0ml に溶解し、ドライアイス−エタノールで−20℃に冷
却し、Ｎ−メチルモルフォリン3.３mlを滴下した後、ク
ロロギ酸イソブチル3.96mlを滴下して、−20℃で１分間
攪拌して混合酸無水物を調整した。得られた溶液を、NH
SのTHF 200ml 溶液と混合し、０℃で５分間、室温で30
分間攪拌した。これを、H-Tyr-OH 5.43gを２N NaOHに溶
解した溶液に加えて室温で一夜攪拌した。得られた反応
混合液を減圧濃縮し、残渣を酢酸エチル800ml に溶解し
た。１Ｎ塩酸200ml で２回、飽和食塩水200ml で２回の
順で洗浄し、有機層を無水硫酸ナトリウムで乾燥した
後、減圧乾固した。残渣を酢酸エチル−ヘキサンに溶解
して再結晶化させた。この再結晶化の操作は３回繰り返
した。得られた結晶をできるだけ少量のクロロホルムに
溶かし、クロロホルム−メタノール−酢酸（20：１：0.
5)を用いたシリカゲルクロマトグラフィーにより精製し
た。このようにしてBoc-Asp(OBzl)-Tyr-OH 13.41g (収
率 92.0％) を得た。The amino acid analysis values of the obtained peptides are shown below. Asp (3) 3.15, Thr (1) 0.94, Ser (2) 1.80, Ala (1) 1.0
5, Glu (2) 2.03, Val (1) 0.98, Met (1) 0.95, Leu (2) 1.
08, Tyr (1) 0.96, Phe (1) 1.01, Lys (1) 1.01, Arg (2)
2.13 (12) Synthesis of Boc-Asp (OBzl) -Tyr-OH Boc-Asp (OBzl) -OH (manufactured by Peptide Laboratories) 9.69 g in THF 10
Dissolve in 0 ml, cool to -20 ° C with dry ice-ethanol, add 3.3 ml of N-methylmorpholine, then add 3.96 ml of isobutyl chloroformate and stir at -20 ° C for 1 minute to mix. The acid anhydride was prepared. The resulting solution is NH
Mix with 200 ml of THF in S, mix at 0 ° C for 5 minutes and room temperature for 30 minutes.
Stir for minutes. This was added to a solution of 5.43 g of H-Tyr-OH dissolved in 2N NaOH, and the mixture was stirred overnight at room temperature. The resulting reaction mixture was concentrated under reduced pressure and the residue was dissolved in 800 ml of ethyl acetate. The organic layer was washed twice with 200 ml of 1N hydrochloric acid and twice with 200 ml of saturated saline, dried over anhydrous sodium sulfate, and then dried under reduced pressure. The residue was dissolved in ethyl acetate-hexane and recrystallized. This recrystallization operation was repeated 3 times. The crystals obtained were dissolved in as little chloroform as possible, and chloroform-methanol-acetic acid (20: 1: 0.
Purified by silica gel chromatography using 5). Thus, 13.41 g (yield 92.0%) of Boc-Asp (OBzl) -Tyr-OH was obtained.

【０１２１】得られたBoc-Asp(OBzl)-Tyr-OHの融点、旋
光度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：75〜77℃ [α] D ：−3.4°(c=1, DMF) Rf ： 0.50 (CHCl3/MeOH/AcOH=9/1/0.5) 元素分析値 Ｃ25Ｈ30Ｎ2Ｏ8 理論値 Ｃ：59.51％ Ｈ：6.39％ Ｎ：5.55％ 測定値 Ｃ：59.66％ Ｈ：6.13％ Ｎ：5.47％ (13) Boc-Asp(OBzl)-Tyr-Set-Lys(Z)-Tyr-Leu-Asp(OBz
l)-Ser-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val
-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-OBzlの合成 上記(11)で得られたBoc-Ser-Lys(Z)-Tyr-Leu-Asp(OBzl)
-Ser-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val-G
ln-Trp-Leu-Met-Asn-Thr(Bzl)-0Bzl 100.0g に、氷冷し
ながら TFA 100ml、アニソール10ml及びエタンジチオー
ル５mlを添加して、室温で30分間攪拌した後、減圧濃縮
してTFAを留去した。残渣にエーテルを添加して、生じ
た沈殿物を濾取した後、乾燥してH-Ser-Lys(Z)-Tyr-Leu
-Asp(OBzl)-Ser-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)
-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-0Bzl・TFA を
得た。The melting point, optical rotation, Rf of TLC and elemental analysis values of the obtained Boc-Asp (OBzl) -Tyr-OH are shown below. mp: 75-77 ° C [α] D : -3.4 ° (c = 1, DMF) Rf: 0.50 (CHCl 3 /MeOH/AcOH=9/1/0.5) Elemental analysis value C 25 H 30 N 2 O 8 theory Value C: 59.51% H: 6.39% N: 5.55% Measured value C: 59.66% H: 6.13% N: 5.47% (13) Boc-Asp (OBzl) -Tyr-Set-Lys (Z) -Tyr-Leu- Asp (OBz
l) -Ser-Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-Val
-Gln-Trp-Leu-Met-Asn-Thr (Bzl) -OBzl Synthesis Boc-Ser-Lys (Z) -Tyr-Leu-Asp (OBzl) obtained in (11) above.
-Ser-Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-Val-G
To 100.0 g of ln-Trp-Leu-Met-Asn-Thr (Bzl) -0Bzl, 100 ml of TFA, 10 ml of anisole and 5 ml of ethanedithiol were added while cooling with ice, and the mixture was stirred at room temperature for 30 minutes and concentrated under reduced pressure. The TFA was distilled off. Ether was added to the residue, and the resulting precipitate was collected by filtration and dried to give H-Ser-Lys (Z) -Tyr-Leu.
-Asp (OBzl) -Ser-Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBzl)
-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (Bzl) -0Bzl.TFA was obtained.

【０１２２】この生成物をDMFに溶解し、HOBT 3.24g、B
oc-Asp(OBzl)-Tyr-OH 9.42g及びWSC4.36mlを添加して、
Ｎ−メチルモルフォリンを用いてpH７に調節した後、室
温で一夜攪拌した。得られた反応混合液を減圧濃縮し、
残渣に氷水を加えた。生じた沈殿物を濾取し、水洗し、
乾燥した後、エタノール−エーテルに溶解して再結晶化
させた。この再結晶化の操作は２回繰り返した。このよ
うにして、Boc-Asp(OBzl)-Tyr-Set-Lys(Z)-Tyr-Leu-Asp
(OBzl)-Ser-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe
-Val-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-OBzl 81.18g (収
率90.5％) を得た。This product was dissolved in DMF and HOBT 3.24 g, B
Add oc-Asp (OBzl) -Tyr-OH 9.42g and WSC 4.36ml,
After adjusting to pH 7 with N-methylmorpholine, the mixture was stirred overnight at room temperature. The reaction mixture obtained was concentrated under reduced pressure,
Ice water was added to the residue. The resulting precipitate is collected by filtration, washed with water,
After drying, it was dissolved in ethanol-ether and recrystallized. This recrystallization operation was repeated twice. Thus, Boc-Asp (OBzl) -Tyr-Set-Lys (Z) -Tyr-Leu-Asp
(OBzl) -Ser-Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe
81.18 g (yield 90.5%) of -Val-Gln-Trp-Leu-Met-Asn-Thr (Bzl) -OBzl was obtained.

【０１２３】得られたペプチドのアミノ酸分析値を下記
に示す。 Asp(4) 4.20, Thr(1) 0.93, Ser(2) 1.79, Ala(1) 1.0
3, Glu(2) 2.03,Val(1) 0.99, Met(1) 0.95, Leu(2) 1.
07, Tyr(2) 1.95, Phe(1) 1.00,Lys(1) 1.01, Arg(2)
2.11 (14) Z-Thr-Ser-OMeの合成 Z-Thr-OH 42.2g、H-Ser-OMe 21.6g及びHOBT 19.25gをDM
F 200mlとTHF 450 mlの混合溶媒に溶解した。得られた
溶液に、０℃に冷却しながらWSC 23.25mlを添加して２
時間攪拌した後、室温で一夜攪拌した。得られた反応混
合液を減圧濃縮した後、残渣を酢酸エチル800ml に溶解
した。次いで１Ｎ塩酸200ml で２回、飽和重曹水200ml
で２回、飽和食塩水200ml で２回の順で洗浄した。有機
層を無水硫酸ナトリウムで乾燥した後、減圧乾固した。
残渣を酢酸エチル−ヘキサンに溶解して再結晶化させ
た。この再結晶化の操作は２回繰り返した。このように
して、Z-Thr-Ser-OMe 47.21g (収率 88.9％) を得た。The amino acid analysis values of the obtained peptides are shown below. Asp (4) 4.20, Thr (1) 0.93, Ser (2) 1.79, Ala (1) 1.0
3, Glu (2) 2.03, Val (1) 0.99, Met (1) 0.95, Leu (2) 1.
07, Tyr (2) 1.95, Phe (1) 1.00, Lys (1) 1.01, Arg (2)
2.11 (14) Synthesis of Z-Thr-Ser-OMe DM Z-Thr-OH 42.2g, H-Ser-OMe 21.6g and HOBT 19.25g
It was dissolved in a mixed solvent of F 200 ml and THF 450 ml. 23.25 ml of WSC was added to the resulting solution while cooling to 0 ° C, and 2
After stirring for an hour, the mixture was stirred at room temperature overnight. The obtained reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 800 ml of ethyl acetate. Next, 200 ml of saturated sodium bicarbonate water twice with 200 ml of 1N hydrochloric acid.
It was washed twice with a saturated saline solution (200 ml) in this order. The organic layer was dried over anhydrous sodium sulfate and then dried under reduced pressure.
The residue was dissolved in ethyl acetate-hexane and recrystallized. This recrystallization operation was repeated twice. Thus, 47.21 g (yield 88.9%) of Z-Thr-Ser-OMe was obtained.

【０１２４】得られたZ-Thr-Ser-OMe の融点、旋光度、
ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：134〜137℃ [α] D ：−6.9°(c=1, メタノール) Rf ：0.60 (CHCl3/MeOH/H2O=8/3/1) 元素分析値 Ｃ16Ｈ22Ｎ7Ｏ2 理論値 Ｃ：54.23％ Ｈ：6.26％ Ｎ：7.91％ 測定値 Ｃ：54.26％ Ｈ：6.25％ Ｎ：7.89％ (15) Boc-Thr-Phe-NHNH2 の合成 Boc-Thr-OH 42.2g(0.15mol)、H-Phe-OMe 21.6g(0.15mo
l)及びHOBT 19.25g(0.15mol)を、DMF 200ml とTHF 450m
l の混合溶媒に溶解した。得られた溶液に、０℃に冷却
しながらWSC 23.25ml を添加して２時間攪拌した後、室
温で一夜攪拌した。得られた反応混合液を減圧濃縮し、
残渣を酢酸エチル800ml に溶解した。次いで、１Ｎ塩酸
200ml で２回、飽和重曹水200ml で２回、飽和食塩水20
0ml で２回の順で洗浄した。有機層を無水硫酸ナトリウ
ムで乾燥した後、減圧乾固した。残渣を酢酸エチル−ヘ
キサンに溶解し、再結晶化させた。この再結晶化の操作
は２回繰り返した。得られた結晶をメタノール250ml に
溶解し、抱水ヒドラジン48mlを添加して室温で20時間攪
拌した。得られた反応液に水を添加した後、濾取して、
乾燥し、メタノール−エーテルに溶解して再結晶化させ
た。このようにして、Boc-Thr-Phe-NHNH2 52.49g (収率
92.1％) を得た。The melting point and optical rotation of the obtained Z-Thr-Ser-OMe,
The Rf and elemental analysis values of TLC are shown below. mp: 134-137 ° C. [α] D : −6.9 ° (c = 1, methanol) Rf: 0.60 (CHCl 3 / MeOH / H 2 O = 8/3/1) Elemental analysis value C 16 H 22 N 7 O 2 Theoretical value C: 54.23% H: 6.26% N: 7.91% Measured value C: 54.26% H: 6.25% N: 7.89% (15) Synthesis of Boc-Thr-Phe-NHNH 2 Boc-Thr-OH 42.2g ( 0.15mol), H-Phe-OMe 21.6g (0.15mo
l) and HOBT 19.25g (0.15mol), DMF 200ml and THF 450m
It was dissolved in a mixed solvent of l. WSC (23.25 ml) was added to the resulting solution while cooling to 0 ° C., and the mixture was stirred for 2 hours and then at room temperature overnight. The reaction mixture obtained was concentrated under reduced pressure,
The residue was dissolved in 800 ml of ethyl acetate. Then 1N hydrochloric acid
200 ml twice, saturated sodium bicarbonate water 200 ml twice, saturated saline solution 20
It was washed with 0 ml twice in this order. The organic layer was dried over anhydrous sodium sulfate and then dried under reduced pressure. The residue was dissolved in ethyl acetate-hexane and recrystallized. This recrystallization operation was repeated twice. The obtained crystals were dissolved in 250 ml of methanol, 48 ml of hydrazine hydrate was added, and the mixture was stirred at room temperature for 20 hours. After adding water to the obtained reaction solution, it was collected by filtration,
It was dried, dissolved in methanol-ether and recrystallized. Thus, 52.49 g of Boc-Thr-Phe-NHNH 2 (yield
92.1%).

【０１２５】得られたBoc-Thr-Phe-NHNH2の融点、旋光
度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：191〜195℃ [α] D ：−12.3°(c=1, DMF) Rf ：0.64 (CHCl3/MeOH/H2O=8/3/1) 元素分析値 Ｃ18Ｈ28Ｎ4Ｏ5 理論値 Ｃ：56.16％ Ｈ：7.46％ Ｎ：14.17％ 測定値 Ｃ：56.13％ Ｈ：7.57％ Ｎ：14.38％ (16) Boc-Thr-Phe-Thr-Ser-OMeの合成 上記(14)で得られたZ-Thr-Ser-OMe 46.2g をメタノール
に溶解し、１Ｎ塩酸4.76ml及びPd-C 6.5g を添加して、
窒素ガスを通して空気を置換した後、水素ガスを導入し
接触還元を行った。これを室温で６時間攪拌し、濾過
し、減圧濃縮した。残渣にエーテルを加えて、生じた沈
殿物を濾取し、真空乾燥してH-Thr-Ser-OMe・HClを得
た。The melting point, optical rotation, Tf Rf and elemental analysis values of the obtained Boc-Thr-Phe-NHNH 2 are shown below. mp: 191-195 ° C [α] D : -12.3 ° (c = 1, DMF) Rf: 0.64 (CHCl 3 / MeOH / H 2 O = 8/3/1) Elemental analysis value C 18 H 28 N 4 O 5 Theoretical value C: 56.16% H: 7.46% N: 14.17% Measured value C: 56.13% H: 7.57% N: 14.38% (16) Synthesis of Boc-Thr-Phe-Thr-Ser-OMe In (14) above. 46.2 g of the obtained Z-Thr-Ser-OMe was dissolved in methanol, 4.76 ml of 1N hydrochloric acid and 6.5 g of Pd-C were added,
After replacing the air with nitrogen gas, hydrogen gas was introduced to carry out catalytic reduction. This was stirred at room temperature for 6 hours, filtered and concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected by filtration and vacuum dried to obtain H-Thr-Ser-OMe.HCl.

【０１２６】一方、上記(15)で得られたZ-Thr-Phe-NHNH
2 49.4gをDMFに溶解し、ドライアイス−エタノールで
−20℃に冷却し、６N HCl-ジオキサン78.3ml及び亜硝酸
イソアミル16.9mlを添加してアジド化合物とした。これ
に、更にトリエチルアミン65.6mlを添加して中和した混
合液を、上記H-Thr-Ser-OMe・HClのDMF溶液に移し、−2
0℃で２時間、４℃で20時間放置した。得られた反応混
合液を減圧濃縮し、残渣に氷水を加えた。生じた沈殿物
を濾取し、水洗し、メタノール−エーテルを用いて再結
晶化させた。この再結晶化の操作は２回繰り返した。こ
のようにして、Boc-Thr-Phe-Thr-Ser-OMe 68.3g (収率
92.5％) を得た。On the other hand, Z-Thr-Phe-NHNH obtained in (15) above
2 49.4 g was dissolved in DMF, cooled to −20 ° C. with dry ice-ethanol, and 68.3 HCl-dioxane 78.3 ml and isoamyl nitrite 16.9 ml were added to give an azide compound. To this, a mixture of 65.6 ml of triethylamine and neutralization was transferred to the DMF solution of H-Thr-Ser-OMe.HCl,
It was left at 0 ° C. for 2 hours and at 4 ° C. for 20 hours. The obtained reaction mixture was concentrated under reduced pressure, and ice water was added to the residue. The resulting precipitate was collected by filtration, washed with water, and recrystallized with methanol-ether. This recrystallization operation was repeated twice. In this way, 68.3 g of Boc-Thr-Phe-Thr-Ser-OMe (yield
92.5%).

【０１２７】得られたBoc-Thr-Phe-Thr-Ser-OMe の融
点、旋光度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：169〜172℃ [α] D ：−3.7°(c=1, DMF) Rf ：Rf 0.64 (CHCl3/MeOH/H2O=8/3/1) 元素分析値 Ｃ26Ｈ40Ｎ4Ｏ10 理論値 Ｃ：54.92％ Ｈ：7.09％ Ｎ：9.85％ 測定値 Ｃ：54.56％ Ｈ：7.57％ Ｎ：9.41％ (17) Boc-Thr-Phe-Thr-Ser-NHNH2 上記(16)で得られたBoc-Thr-Phe-Thr-Ser-OMe 62.5g を
メタノール250ml に溶解し、抱水ヒドラジン35.2mlを添
加して室温で20時間攪拌した。得られた反応混合液に水
を添加して、沈殿物を濾取し、乾燥して、メタノール−
エーテルに溶解して再結晶化させた。このようにして、
Boc-Thr-Phe-Thr-Ser-NHNH2 57.54g (収率 92.1％) を
得た。The melting point, optical rotation, TLC Rf and elemental analysis values of the obtained Boc-Thr-Phe-Thr-Ser-OMe are shown below. mp: 169 to 172 ° C. [α] D : −3.7 ° (c = 1, DMF) Rf: Rf 0.64 (CHCl 3 / MeOH / H 2 O = 8/3/1) Elemental analysis value C 26 H 40 N 4 O 10 theoretical value C: 54.92% H: 7.09% N: 9.85% Measured value C: 54.56% H: 7.57% N: 9.41% (17) Boc-Thr-Phe-Thr-Ser-NHNH 2 In the above (16) 62.5 g of the obtained Boc-Thr-Phe-Thr-Ser-OMe was dissolved in 250 ml of methanol, 35.2 ml of hydrazine hydrate was added, and the mixture was stirred at room temperature for 20 hours. Water was added to the obtained reaction mixture, the precipitate was collected by filtration, dried and methanol-
It was dissolved in ether and recrystallized. In this way,
57.54 g (yield 92.1%) of Boc-Thr-Phe-Thr-Ser-NHNH 2 was obtained.

【０１２８】得られたBoc-Thr-Phe-Thr-Ser-NHNH2の融
点、旋光度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：179〜184℃ [α] D ：−4.4°(c=1, DMF) Rf ：0.64 (CHCl3/MeOH/H2O=8/3/1) 元素分析値 Ｃ25Ｈ40Ｎ6Ｏ9 理論値 Ｃ：52.81％ Ｈ：7.09％ Ｎ：14.78％ 測定値 Ｃ：52.33％ Ｈ：7.57％ Ｎ：14.88％ (18) Boc-Thr-Phe-Thr-Ser-Asp(OBzl)-Tyr-Ser-Lys(Z)-
Tyr-Leu-Asp(OBzl)-Ser-Arg(Tos)-Arg(Tos)-Ala-Gln-As
p(OBzl)-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-OBzl
の合成 上記(13)で得られたBoc-Asp(OBzl)-Tyr-Ser-Lys(Z)-Tyr
-Leu-Asp(OBzl)-Ser(Bzl)-Arg(Tos)-Arg(Tos)-Ala-Gln-
Asp(OBzl)-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-OBz
80gに、氷冷しながらTFA 160ml 、アニソール８ml及び
エタンジチオール４mlを添加して溶解し、室温で30分間
攪拌した後、減圧濃縮してTFAを留去した。残渣にエー
テルを添加して、生じた沈殿物を濾取した後、乾燥して
H-Asp(OBzl)-Tyr-Ser-Lys(Z)-Tyr-Leu-Asp(OBzl)-Ser(B
zl)-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val-Gl
n-Leu-Met-Asn-Thr(Bzl)-OBzl ・TFAを得た。The melting point, optical rotation, Rf of TLC and elemental analysis values of the obtained Boc-Thr-Phe-Thr-Ser-NHNH 2 are shown below. mp: 179 to 184 ° C. [α] D : −4.4 ° (c = 1, DMF) Rf: 0.64 (CHCl 3 / MeOH / H 2 O = 8/3/1) Elemental analysis value C 25 H 40 N 6 O 9 Theoretical value C: 52.81% H: 7.09% N: 14.78% Measured value C: 52.33% H: 7.57% N: 14.88% (18) Boc-Thr-Phe-Thr-Ser-Asp (OBzl) -Tyr-Ser -Lys (Z)-
Tyr-Leu-Asp (OBzl) -Ser-Arg (Tos) -Arg (Tos) -Ala-Gln-As
p (OBzl) -Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (Bzl) -OBzl
Synthesis of Boc-Asp (OBzl) -Tyr-Ser-Lys (Z) -Tyr obtained in (13) above
-Leu-Asp (OBzl) -Ser (Bzl) -Arg (Tos) -Arg (Tos) -Ala-Gln-
Asp (OBzl) -Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (Bzl) -OBz
While cooling with ice, 160 ml of TFA, 8 ml of anisole and 4 ml of ethanedithiol were added to and dissolved in 80 g, and the mixture was stirred at room temperature for 30 minutes and then concentrated under reduced pressure to remove TFA by distillation. Ether was added to the residue and the resulting precipitate was collected by filtration and dried.
H-Asp (OBzl) -Tyr-Ser-Lys (Z) -Tyr-Leu-Asp (OBzl) -Ser (B
zl) -Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-Val-Gl
n-Leu-Met-Asn-Thr (Bzl) -OBzl.TFA was obtained.

【０１２９】上記(17)で得られたBoc-Thr-Phe-Thr-Ser-
NHNH2 11.36g をDMF 50mlに溶解し、ドライアイス−エ
タノールで−20℃に冷却して、６N HCl-ジオキサン10.2
ml、及び亜硝酸イソアミル2.６mlを添加してアジド化合
物とした。これに、更にトリエチルアミン8.54mlを添加
して中和した混合液を、上記H-Asp(OBzl)-Tyr-Ser-Lys
(Z)-Tyr-Leu-Asp(OBzl)-Ser(Bzl)-Arg(Tos)-Arg(Tos)-A
la-Gln-Asp(OBzl)-Phe-Val-Gln-Leu-Met-Asn-Thr(Bzl)-
OBzl ・TFAのDMF溶液に移し、−20℃で２時間、４℃で2
0時間放置した。得られた反応混合液を減圧濃縮し、残
渣に氷水を加えた。生じた沈殿物を濾取し、水洗して、
乾燥した後、エタノール−エーテルに溶解して再結晶化
させた。この再結晶化の操作は２回繰り返した。このよ
うにして、Boc-Thr-Phe-Thr-Ser-Asp(OBzl)-Tyr-Ser-Ly
s(Z)-Tyr-Leu-Asp(OBzl)-Ser-Arg(Tos)-Arg(Tos)-Ala-G
ln-Asp(OBzl)-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-
OBzl89.74g (収率 96.5％) を得た。Boc-Thr-Phe-Thr-Ser-obtained in (17) above
Dissolve 11.36 g of NHNH 2 in 50 ml of DMF, cool to −20 ° C. with dry ice-ethanol, and add 6N HCl-dioxane 10.2
ml and 2.6 ml of isoamyl nitrite were added to obtain an azide compound. To this, a mixed solution obtained by further adding 8.54 ml of triethylamine and neutralizing was added to the above H-Asp (OBzl) -Tyr-Ser-Lys.
(Z) -Tyr-Leu-Asp (OBzl) -Ser (Bzl) -Arg (Tos) -Arg (Tos) -A
la-Gln-Asp (OBzl) -Phe-Val-Gln-Leu-Met-Asn-Thr (Bzl)-
OBzl-Transfer to TFA in DMF solution, 2 hours at -20 ℃, 2 at 4 ℃
Left for 0 hours. The obtained reaction mixture was concentrated under reduced pressure, and ice water was added to the residue. The resulting precipitate is collected by filtration, washed with water,
After drying, it was dissolved in ethanol-ether and recrystallized. This recrystallization operation was repeated twice. Thus, Boc-Thr-Phe-Thr-Ser-Asp (OBzl) -Tyr-Ser-Ly
s (Z) -Tyr-Leu-Asp (OBzl) -Ser-Arg (Tos) -Arg (Tos) -Ala-G
ln-Asp (OBzl) -Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (Bzl)-
89.74 g (yield 96.5%) of OBzl was obtained.

【０１３０】得られたペプチドのアミノ酸分析値を下記
に示す。 Asp(4) 4.20, Thr(3) 2.81, Ser(3) 2.69, Ala(1) 1.0
3, Glu(2) 2.04,Val(1) 0.99, Met(1) 0.95, Leu(2) 1.
07, Tyr(2) 1.95, Phe(1) 1.00,Lys(1) 1.01, Arg(2)
2.11 (19) Z-Gln-Gly-OButの合成 H-Gly-OBut・HCl 16.7gをTHF 100ml に溶解して０℃に
冷却し、Ｎ−メチルモルフォリンを用いて中和した。得
られた溶液にZ-Gln-ONp 40.14gのTHF溶液 200mlを添加
して、０℃で１時間、室温で一晩攪拌した後、減圧濃縮
した。残渣に酢酸エチル−ブタノール（１：１) を加
え、水で２回洗浄した。有機層を減圧濃縮し、残渣にエ
ーテルを加えて、生じた沈殿物を濾取した。濾取物をメ
タノール−エーテルで再沈殿させ、真空乾燥を行って、
Z-Gln-Gly-OBut 28.37g (収率 72.1％）を得た。The amino acid analysis values of the obtained peptides are shown below. Asp (4) 4.20, Thr (3) 2.81, Ser (3) 2.69, Ala (1) 1.0
3, Glu (2) 2.04, Val (1) 0.99, Met (1) 0.95, Leu (2) 1.
07, Tyr (2) 1.95, Phe (1) 1.00, Lys (1) 1.01, Arg (2)
2.11 (19) Synthesis of Z-Gln-Gly-OBut 16.7 g of H-Gly-OBut.HCl was dissolved in 100 ml of THF, cooled to 0 ° C., and neutralized with N-methylmorpholine. 200 ml of a THF solution containing 40.14 g of Z-Gln-ONp was added to the obtained solution, and the mixture was stirred at 0 ° C. for 1 hour and at room temperature overnight, and concentrated under reduced pressure. Ethyl acetate-butanol (1: 1) was added to the residue, and the mixture was washed twice with water. The organic layer was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was collected by filtration. The filtered material was reprecipitated with methanol-ether, vacuum dried,
28.37 g (yield 72.1%) of Z-Gln-Gly-OBut was obtained.

【０１３１】得られたZ-Gln-Gly-OButの融点、旋光度、
ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：168.5〜170.1℃ [α] D ：−6.9°(c=0.9, DMF) Rf ：0.63 (CHCl3/MeOH/AcOH=9/1/0.5) 元素分析値 Ｃ19Ｈ27Ｎ3Ｏ6 理論値 Ｃ：58.00％ Ｈ：6.92％ Ｎ：10.63％ 測定値 Ｃ：57.71％ Ｈ：6.99％ Ｎ：10.60％ (20) Z-Ser-Gln-Gly-OButの合成 上記(19)で得られたZ-Gln-Gly-OBut 27.20g(69.1mmol)
をメタノールに溶解し、Pd-C 3.5g を加え、窒素ガスを
通して空気を置換した後、水素ガスを導入して接触還元
を行った。これを室温で６時間攪拌した後、濾過し、減
圧濃縮して、残渣にエーテルを加えた。生じた沈殿物を
濾取し、真空乾燥を行ってH-Gln-Gly-OButを得た。Melting point, optical rotation of the obtained Z-Gln-Gly-OBut,
The Rf and elemental analysis values of TLC are shown below. mp: 168.5 to 170.1 ° C [α] D : −6.9 ° (c = 0.9, DMF) Rf: 0.63 (CHCl 3 /MeOH/AcOH=9/1/0.5) Elemental analysis value C 19 H 27 N 3 O 6 theory Value C: 58.00% H: 6.92% N: 10.63% Measured value C: 57.71% H: 6.99% N: 10.60% (20) Synthesis of Z-Ser-Gln-Gly-OBut Z obtained in (19) above -Gln-Gly-OBut 27.20g (69.1mmol)
Was dissolved in methanol, 3.5 g of Pd-C was added, and after replacing the air by passing nitrogen gas, hydrogen gas was introduced to carry out catalytic reduction. This was stirred at room temperature for 6 hours, then filtered, concentrated under reduced pressure, and ether was added to the residue. The resulting precipitate was collected by filtration and vacuum dried to obtain H-Gln-Gly-OBut.

【０１３２】Z-Ser-OH 16.53g とHOBt 11.11gをDMFに溶
解し、上記H-Gln-Gly-OButのDMF溶液と混合し、０℃でW
SC 12.89ml を加えた後、０℃で１時間、室温で一晩攪
拌した。次いで減圧濃縮し、残渣に酢酸エチル−ブタノ
ール (１：１) を加え、水で２回洗浄した。有機層を減
圧濃縮し、残渣にエーテルを加えて生じた沈殿物を濾取
した。濾取物をメタノール−エーテルで再沈殿させ、真
空乾燥を行って、Z-Ser-Gln-Gly-OBut 24.24g(収率70.
3％）を得た。16.53 g of Z-Ser-OH and 11.11 g of HOBt were dissolved in DMF, mixed with the above DMF solution of H-Gln-Gly-OBut, and mixed with W at 0 ° C.
After adding 12.89 ml of SC, the mixture was stirred at 0 ° C. for 1 hour and at room temperature overnight. Then, the mixture was concentrated under reduced pressure, ethyl acetate-butanol (1: 1) was added to the residue, and the mixture was washed twice with water. The organic layer was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was collected by filtration. The filtered material was reprecipitated with methanol-ether and vacuum dried to obtain 24.24 g of Z-Ser-Gln-Gly-OBut (yield 70.
3%) was obtained.

【０１３３】得られたZ-Ser-Gln-Gly-OButの融点、旋光
度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：158.0〜161.1℃ [α] D ：−0.3°(c=1.0, DMF) Rf ：0.30 (CHCl3/MeOH/AcOH=9/1/0.5) 元素分析値 Ｃ22Ｈ32Ｎ4Ｏ8 理論値 Ｃ：54.99％ Ｈ：6.71％ Ｎ：11.66％ 測定値 Ｃ：54.79％ Ｈ：6.92％ Ｎ：11.31％ (21) Z-His-Ser-Gln-Gly-OButの合成 上記(20)で得られたZ-Ser-Gln-Gly-OBut 22.10g(46.0mm
ol) をメタノールに溶解し、Pd-C 2.3g を加え、窒素ガ
スを通して空気を置換した後、水素ガスを導入して接触
還元を行った。これを室温で８時間攪拌した後、濾過し
て、減圧濃縮した。残渣にエーテルを加えて、生じた沈
殿物を濾取し、真空乾燥を行って、H-Ser-Gln-Gly-OBut
を得た。The melting point, optical rotation, Rf of TLC and elemental analysis values of the obtained Z-Ser-Gln-Gly-OBut are shown below. mp: 158.0 to 161.1 ° C [α] D : -0.3 ° (c = 1.0, DMF) Rf: 0.30 (CHCl 3 /MeOH/AcOH=9/1/0.5) Elemental analysis value C 22 H 32 N 4 O 8 theory Value C: 54.99% H: 6.71% N: 11.66% Measured value C: 54.79% H: 6.92% N: 11.31% (21) Synthesis of Z-His-Ser-Gln-Gly-OBut Obtained by the above (20). Z-Ser-Gln-Gly-OBut 22.10g (46.0mm
ol) was dissolved in methanol, 2.3 g of Pd-C was added, the atmosphere was replaced with nitrogen gas, and then hydrogen gas was introduced to carry out catalytic reduction. This was stirred at room temperature for 8 hours, filtered, and concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected by filtration and vacuum dried to give H-Ser-Gln-Gly-OBut.
I got

【０１３４】Z-His-OH 13.31gとHOBt 7.40gをDMFに溶解
し、上記H-Ser-Gln-Gly-OButのDMF溶液と混合し、０℃
でWSC 8.79mlを加えて０℃で１時間、室温で一晩攪拌し
た。次いで減圧濃縮し、残渣に酢酸エチル−ブタノール
（１：１）を加えて、水で２回洗浄した。有機層を減圧
濃縮し、残渣にエーテルを加えて、生じた沈殿物を濾取
した。濾取物をメタノール−エーテルで再沈殿させ、真
空乾燥を行って、Z-His-Ser-Gln-Gly-OBut 26.20g(収
率 92.2％）を得た。13.31 g of Z-His-OH and 7.40 g of HOBt were dissolved in DMF, mixed with the above DMF solution of H-Ser-Gln-Gly-OBut, and the mixture was mixed at 0 ° C.
Then, 8.79 ml of WSC was added, and the mixture was stirred at 0 ° C. for 1 hour and at room temperature overnight. Then, the mixture was concentrated under reduced pressure, ethyl acetate-butanol (1: 1) was added to the residue, and the mixture was washed twice with water. The organic layer was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was collected by filtration. The filtered material was reprecipitated with methanol-ether and vacuum dried to obtain 26.20 g (yield 92.2%) of Z-His-Ser-Gln-Gly-OBut.

【０１３５】得られたZ-His-Ser-Gln-Gly-OButの融点、
旋光度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：118.1〜120.7℃ [α] D ：−5.0°(c=1.0, DMF) Rf ：0.60 (CHCl3/MeOH/AcOH=9/1/0.5) 元素分析値 Ｃ28Ｈ39Ｎ7Ｏ9 理論値 Ｃ：54.45％ Ｈ：6.36％ Ｎ：15.87％ 測定値 Ｃ：54.44％ Ｈ：6.54％ Ｎ：16.01％ (22) Z-His-Ser-Gln-Gly-OHの合成 上記(21)で得られたZ-His-Ser-Gln-Gly-OBut 24.50g を
０℃でTFA 50mlに溶解し、室温で30分間攪拌した。次い
でエーテルを加え、生じた沈殿物を濾取し、真空乾燥し
てZ-His-Ser-Gln-Gly-OH 20.84g(収率 93.5％) を得
た。Melting point of the obtained Z-His-Ser-Gln-Gly-OBut,
The optical rotation, Rf of TLC and elemental analysis values are shown below. mp: 118.1-120.7 ° C [α] D : -5.0 ° (c = 1.0, DMF) Rf: 0.60 (CHCl 3 /MeOH/AcOH=9/1/0.5) Elemental analysis value C 28 H 39 N 7 O 9 theory Value C: 54.45% H: 6.36% N: 15.87% Measured value C: 54.44% H: 6.54% N: 16.01% (22) Synthesis of Z-His-Ser-Gln-Gly-OH Obtained in (21) above. 24.50 g of Z-His-Ser-Gln-Gly-OBut was dissolved in 50 ml of TFA at 0 ° C. and stirred at room temperature for 30 minutes. Then, ether was added, and the resulting precipitate was collected by filtration and vacuum dried to obtain 20.84 g (yield 93.5%) of Z-His-Ser-Gln-Gly-OH.

【０１３６】得られたZ-His-Ser-Gln-Gly-OHの融点、旋
光度、ＴＬＣのRf及び元素分析値を下記に示す。 m.p. ：165.5〜169.1℃ [α] D ：−34.8°(c=1.0, H2O) Rf ：0.65 (CHCl3/MeOH/AcOH=9/1/0.5) 元素分析値 Ｃ24Ｈ31Ｎ7Ｏ9 理論値 Ｃ：51.33％ Ｈ：5.56％ Ｎ：17.46％ 測定値 Ｃ：51.32％ Ｈ：5.78％ Ｎ：17.11％ (23) Z-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp(OBzl)-T
yr-Ser-Lys(Z)-Tyr-Leu-Asp(OBzl)-Ser-Arg(Tos)-Arg(T
os)-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-Leu-Met-Asn-
Thr(Bzl)-OBzlの合成 上記(18)で得られたBoc-Thr-Phe-Thr-Ser-Asp(OBzl)-Ty
r-Ser-Lys(Z)-Tyr-Leu-Asp(OBzl)-Ser-Arg(Tos)-Arg(To
s)-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-Leu-Met-Asn-T
hr(Bzl)-OBzl 46.2gに、氷冷しながら、TFA 50ml、アニ
ソール、及びエタンジチオール２mlを加えて溶解し、室
温で30分間攪拌した後、減圧濃縮してTFAを留去した。
残渣にエーテルを加え、生じた沈殿物を濾取した後、乾
燥して H-Thr-Phe-Thr-Ser-Asp(OBzl)-Tyr-Ser-Lys(Z)-
Tyr-Leu-Asp(OBzl)-Ser-Arg(Tos)-Arg(Tos)-Ala-Gln-As
p(OBzl)-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-OBzl
・TFAを得た。The melting point, optical rotation, Tf Rf and elemental analysis values of the obtained Z-His-Ser-Gln-Gly-OH are shown below. mp: 165.5 to 169.1 ° C. [α] D : −34.8 ° (c = 1.0, H 2 O) Rf: 0.65 (CHCl 3 /MeOH/AcOH=9/1/0.5) Elemental analysis value C 24 H 31 N 7 O 9 Theoretical value C: 51.33% H: 5.56% N: 17.46% Measured value C: 51.32% H: 5.78% N: 17.11% (23) Z-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser- Asp (OBzl) -T
yr-Ser-Lys (Z) -Tyr-Leu-Asp (OBzl) -Ser-Arg (Tos) -Arg (T
os) -Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-Leu-Met-Asn-
Synthesis of Thr (Bzl) -OBzl Boc-Thr-Phe-Thr-Ser-Asp (OBzl) -Ty obtained in (18) above
r-Ser-Lys (Z) -Tyr-Leu-Asp (OBzl) -Ser-Arg (Tos) -Arg (To
s) -Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Trp-Leu-Met-Asn-T
TFA (50 ml), anisole and ethanedithiol (2 ml) were added to hr (Bzl) -OBzl (46.2 g) with ice cooling to dissolve the mixture. The mixture was stirred at room temperature for 30 minutes and concentrated under reduced pressure to remove TFA.
Ether was added to the residue, the resulting precipitate was collected by filtration, dried and then H-Thr-Phe-Thr-Ser-Asp (OBzl) -Tyr-Ser-Lys (Z)-
Tyr-Leu-Asp (OBzl) -Ser-Arg (Tos) -Arg (Tos) -Ala-Gln-As
p (OBzl) -Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (Bzl) -OBzl
・ I got TFA.

【０１３７】上記生成物をDMFに溶解し、これにHOBT 1.
8g 、上記(22)で得られたZ-His-Ser-Gln-Gly-OH6.35g
及びWSC 2.46mlを加え、Ｎ−メチルモルフォリンを用い
てpH７に調節した後、室温で一夜攪拌した。得られた反
応混合液を減圧濃縮し、残渣に氷水を加えた。生じた沈
殿物を濾取し、水洗し、乾燥した後、エタノール−エー
テルに溶解して再結晶化させた。再結晶化の操作は２回
繰り返した。このようにして、Z-His-Ser-Gln-Gly-Thr-
Phe-Thr-Ser-Asp(OBzl)-Tyr-Ser-Lys(Z)-Tyr-Leu-Asp(O
Bzl)-Ser-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-V
al-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-OBzl 44.9g (収率 8
2.35％) を得た。The above product was dissolved in DMF and HOBT 1.
8g, Z-His-Ser-Gln-Gly-OH 6.35g obtained in (22) above
And WSC (2.46 ml) were added, and the pH was adjusted to 7 with N-methylmorpholine, and the mixture was stirred at room temperature overnight. The obtained reaction mixture was concentrated under reduced pressure, and ice water was added to the residue. The resulting precipitate was collected by filtration, washed with water, dried, and then dissolved in ethanol-ether for recrystallization. The recrystallization operation was repeated twice. Thus, Z-His-Ser-Gln-Gly-Thr-
Phe-Thr-Ser-Asp (OBzl) -Tyr-Ser-Lys (Z) -Tyr-Leu-Asp (O
Bzl) -Ser-Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-V
al-Gln-Trp-Leu-Met-Asn-Thr (Bzl) -OBzl 44.9 g (yield 8
2.35%).

【０１３８】得られたペプチドのアミノ酸分析値を下記
に示す。 Asp(4) 4.23, Thr(3) 0.85, Ser(4) 3.60, Glu(3) 3.0
7, Gly(1) 1.01,Ala(1) 1.07, Val(1) 1.07, Met(1) 0.
69, Leu(2) 2.32, Tyr(2) 1.92,Phe(2) 2.11, Arg(2)
2.03, His(1) 0.99, Lys(1) 1.01 (24) H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser
-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-G
ln-Trp-Leu-Met-Asn-Thr-OH の合成 上記(23)で得られたZ-His-Ser-Gln-Gly-Thr-Phe-Thr-Se
r-Asp(OBzl)-Tyr-Ser-Lys(Z)-Tyr- Leu-Asp(OBzl)-Ser-
Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Tr
p-Leu-Met-Asn-Thr(Bzl)-OBzl 10g にアニソール30ml及
びエタンジオール５mlを加え、さらに無水弗化水素 250
mlを加え、０℃で１時間攪拌した。反応後、無水弗化水
素を減圧下留去し、残渣をエーテルで洗浄し、次いでこ
れに10％酢酸 350mlを加えてペプチドを抽出した。得ら
れた抽出液は実施例１−１と同様の操作で精製して精製
グルカゴン1.5gを得た。The amino acid analysis values of the obtained peptides are shown below. Asp (4) 4.23, Thr (3) 0.85, Ser (4) 3.60, Glu (3) 3.0
7, Gly (1) 1.01, Ala (1) 1.07, Val (1) 1.07, Met (1) 0.
69, Leu (2) 2.32, Tyr (2) 1.92, Phe (2) 2.11, Arg (2)
2.03, His (1) 0.99, Lys (1) 1.01 (24) H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser
-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-G
Synthesis of ln-Trp-Leu-Met-Asn-Thr-OH Z-His-Ser-Gln-Gly-Thr-Phe-Thr-Se obtained in (23) above
r-Asp (OBzl) -Tyr-Ser-Lys (Z) -Tyr- Leu-Asp (OBzl) -Ser-
Arg (Tos) -Arg (Tos) -Ala-Gln-Asp (OBzl) -Phe-Val-Gln-Tr
To p-Leu-Met-Asn-Thr (Bzl) -OBzl 10g, add 30 ml of anisole and 5 ml of ethanediol, and add 250 ml of anhydrous hydrogen fluoride.
ml was added, and the mixture was stirred at 0 ° C for 1 hr. After the reaction, anhydrous hydrogen fluoride was distilled off under reduced pressure, the residue was washed with ether, and then 350 ml of 10% acetic acid was added thereto to extract the peptide. The obtained extract was purified in the same manner as in Example 1-1 to obtain 1.5 g of purified glucagon.

【０１３９】得られた精製グルカゴンのアミノ酸分析値
を下記に示す。 Asp(4) 4.23, Thr(3) 0.85, Ser(4) 3.60, Glu(3) 3.0
7, Gly(1) 1.01,Ala(1) 1.07, Val(1) 1.07, Met(1) 0.
69, Leu(2) 2.32, Tyr(2) 1.92,Phe(2) 2.11, Arg(2)
2.03, His(1) 0.99, Lys(1) 1.01 〔実施例11〕グルカゴンの活性の測定 上記実施例１〜10で合成したグルカゴンの活性を下記の
方法にて測定し、天然グルカゴンの活性と比較した。The amino acid analysis values of the obtained purified glucagon are shown below. Asp (4) 4.23, Thr (3) 0.85, Ser (4) 3.60, Glu (3) 3.0
7, Gly (1) 1.01, Ala (1) 1.07, Val (1) 1.07, Met (1) 0.
69, Leu (2) 2.32, Tyr (2) 1.92, Phe (2) 2.11, Arg (2)
2.03, His (1) 0.99, Lys (1) 1.01 [Example 11] Measurement of activity of glucagon The activity of glucagon synthesized in Examples 1 to 10 was measured by the following method, and compared with the activity of natural glucagon. did.

【０１４０】ウサギを用いたグルカゴンの血糖値上昇作
用測定法 体重 2.0〜3.0kgの健康なウサギを用い、その血糖値を
測定することにより、グルカゴンの活性値を求めた。ま
ず、測定試料溶液を 2用量（高用量試料溶液Ｔ H 及び低
用量試料溶液ＴL ，公比 2）、活性値既知のグルカゴン
標準溶液 2用量（高用量標準溶液ＳH 及び低用量標準溶
液ＳL ，公比 2）を作製した（生理食塩水に 0.1N HCl
を適量加え、pHを 3とし、これを溶媒に用いた。）。動
物は 1群当たり 6羽以上、計 4群に群分けした。グルカ
ゴン投与48時間前に 0.75%コルチゾン／0.5%カルボキシ
メチルセルロースを投与し、16時間以上絶食の前処置を
行った。投与は 2日行った。下記の表１（一例）のよう
に 1日目と 2日目の試料溶液及び標準溶液、高用量及び
低用量を違えて群毎に投与した。[0140]Glucagon elevated blood glucose level using rabbits
Measuring method Using a healthy rabbit weighing 2.0 to 3.0 kg,
By measuring, the activity value of glucagon was obtained. Well
The measurement sample solution at 2 doses (high dose sample solution T HAnd low
Dose sample solution TL, Common ratio 2), Glucagon with known activity value
2 doses of standard solution (high dose standard solution SHAnd low dose standard dissolution
Liquid SL, Common ratio 2) was prepared (0.1N HCl in physiological saline)
Was added to adjust the pH to 3, and this was used as a solvent. ). Movement
Items were grouped into 4 groups, 6 or more per group. Gurka
0.75% cortisone / 0.5% carboxy 48 hours before gon administration
Administer methylcellulose and prepare for fasting for 16 hours or more.
went. The administration was performed for 2 days. As shown in Table 1 (example) below
On day 1 and day 2, sample and standard solutions, high doses and
Different low doses were given per group.

【０１４１】[0141]

【表１】 [Table 1]

【０１４２】グルカゴン投与後 20分及び 60 分に動物
の耳介周辺の静脈から採血し、比色法により血糖値を定
量した。2日間の各群の血糖値の合計値を用いて試料グ
ルカゴンの活性値を求めた。その結果を表２に示す。ま
た、天然グルカゴンの活性値は1.03u/mgであった。Blood was collected from the vein around the ear of the animal 20 minutes and 60 minutes after the administration of glucagon, and the blood glucose level was quantified by a colorimetric method. The activity value of the sample glucagon was obtained using the total blood glucose value of each group for 2 days. The results are shown in Table 2. The activity value of natural glucagon was 1.03u / mg.

【０１４３】[0143]

【表２】 [Table 2]

【０１４４】以上の結果から、本発明の方法で合成した
グルカゴンは、いずれも天然のグルカゴンと同等の活性
を示すことが確認された。From the above results, it was confirmed that the glucagons synthesized by the method of the present invention all showed activity equivalent to that of natural glucagons.

【０１４５】[0145]

【発明の効果】本発明によれば、化学合成手法により、
グルカゴンを効率よく大量に、しかも安価に製造するこ
とができる。したがって、グルカゴンの工業的生産性が
向上する。According to the present invention, by the chemical synthesis method,
Glucagon can be efficiently manufactured in large quantities and at low cost. Therefore, the industrial productivity of glucagon is improved.

─────────────────────────────────────────────────────
─────────────────────────────────────────────────── ───

【手続補正書】[Procedure amendment]

【提出日】平成７年６月１６日[Submission date] June 16, 1995

【手続補正１】[Procedure Amendment 1]

【補正対象書類名】明細書[Document name to be amended] Statement

【補正対象項目名】００１２[Correction target item name] 0012

【補正方法】変更[Correction method] Change

【補正内容】[Correction content]

【００１２】更に、本発明は、上記式（１）で表される
グルカゴンの製造方法であって、前記グルカゴンを少な
くとも２個のフラグメントに分けて各フラグメントを液
相中で合成し、次いで液相中で各フラグメントを反応さ
せてカップリングすることを特徴とする製造方法（方法
６）を提供する。本明細書において、アミノ酸、ペプチ
ド、保護基、溶媒その他に関して略号で表示する場合、
国際純正及び応用化学連合（ＩＵＰＡＣ）、国際生化学
連合（ＩＵＢ）の規定或いは該当分野における慣用記号
に従うものとする。ただしアミノ酸等に関し光学異性体
がありうる場合は、特に明示しなければＬ体を示すもの
とする。以下、その例を示す。Further, the present invention is a method for producing glucagon represented by the above formula (1), wherein the glucagon is divided into at least two fragments and each fragment is synthesized in a liquid phase, and then the liquid phase is synthesized. There is provided a production method (method 6), which comprises reacting and coupling each fragment therein. In the present specification, when an abbreviation is used for amino acids, peptides, protecting groups, solvents, etc.,
The rules of the International Union of Pure and Applied Chemistry (IUPAC), the International Union of Biochemistry ( IUB ) or the conventional symbols in the relevant fields shall be followed. However, when an amino acid and the like may have optical isomers, the L form is shown unless otherwise specified. The example is shown below.

───────────────────────────────────────────────────── フロントページの続き (72)発明者 高畠 ひかり 茨城県北相馬郡守谷町久保ヶ丘１丁目２番 伊藤ハム株式会社中央研究所内 (72)発明者 富崎 欣也 茨城県北相馬郡守谷町久保ヶ丘１丁目２番 伊藤ハム株式会社中央研究所内 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Hikari Takahata, Hikari Takahata 1-2 Kubogaoka, Moriya-cho, Kitasoma-gun, Ibaraki Prefecture, Central Research Laboratory, Itoham Co., Ltd. (72) Kinya Tomisaki Kubo, Moriya-cho, Kitasoma-gun, Ibaraki Prefecture 1-2, Kugaoka Central Research Laboratory, Itoham Co., Ltd.

Claims (11)

【特許請求の範囲】[Claims] 【請求項１】 下記式(1) ： H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- (1) Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH で表されるグルカゴンの製造方法であって、カルボキシ
ル基と結合可能な官能基を有する不溶性樹脂に、アミノ
基保護スレオニンのカルボキシル基を結合させて前記樹
脂に29位のスレオニン残基を導入する工程、該スレオニ
ン残基の保護基を除去して得られた遊離のアミノ基に、
上記式(1) のアミノ酸配列にしたがってアミノ基保護ア
ミノ酸を順次ペプチド結合させて28位のアスパラギン残
基から１位のヒスチジン残基まで一つずつ延長する工
程、及び得られたペプチドを前記樹脂から脱離させる工
程を含む製造方法。1. The following formula (1): H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala. -Gln- (1) A method for producing glucagon represented by Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH, in which an insoluble resin having a functional group capable of binding to a carboxyl group is used. , A step of introducing a threonine residue at position 29 into the resin by binding a carboxyl group of an amino group-protected threonine, to a free amino group obtained by removing the protecting group of the threonine residue,
According to the amino acid sequence of the above formula (1), amino-protected amino acids are sequentially peptide-bonded to extend one by one from the asparagine residue at the 28th position to the histidine residue at the 1st position, A manufacturing method including a step of desorbing. 【請求項２】 下記式(1) ： H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- (1) Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH で表されるグルカゴンの製造方法であって、前記グルカ
ゴンを構成する１又は複数のフラグメント（ここで、フ
ラグメントを構成するアミノ酸残基の総数は２〜28個で
ある。）であって末端アミノ基を保護したものを合成す
る工程、カルボキシル基と結合可能な官能基を有する不
溶性樹脂に、アミノ基保護スレオニン又は29位のスレオ
ニン残基を含む末端アミノ基保護フラグメントのカルボ
キシル基を結合させる工程、前記樹脂に結合したスレオ
ニン残基又はフラグメントの末端アミノ基の保護基を除
去して得られた遊離のアミノ基に、上記式(1) のアミノ
酸配列にしたがって末端アミノ基保護フラグメント及び
／又は末端アミノ基保護アミノ酸を順次ペプチド結合さ
せて１位のヒスチジン残基まで延長する工程、及び得ら
れたペプチドを前記樹脂から脱離させる工程を含む製造
方法。2. The following formula (1): H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala. -Gln- (1) A method for producing glucagon represented by Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH, which comprises one or more fragments (wherein , The total number of amino acid residues constituting the fragment is 2 to 28), and a step of synthesizing a product in which the terminal amino group is protected, the amino group is added to the insoluble resin having a functional group capable of binding to a carboxyl group. A step of attaching a carboxyl group of a terminal amino group-protected fragment containing a protected threonine or threonine residue at position 29, a free group obtained by removing the protecting group of the terminal amino group of the threonine residue or fragment bound to the resin. The amino group has a terminal amino group-protected fragment and / or a terminal according to the amino acid sequence of the above formula (1). Manufacturing method comprising the amino group steps protected amino acids were sequentially peptide bonds extended to position 1 histidine residue, and the resulting peptides steps desorbed from the resin. 【請求項３】 末端アミノ基保護フラグメントが、29位
のアミノ酸残基を含む下記一般式(2) ： Ｒ1-Ｘ-Asp-Thr( Ｒ3)-Ｒ2 (2) （式中、Ｒ1は末端アミノ基の保護基であり、Ｒ2は末端
カルボキシル基の保護基であり、Ｒ3はスレオニン残基
の側鎖の水酸基の保護基であり、Ｘは化学結合、Met、
又はLeu-Met である。）で表されるペプチドを含み、該
ペプチドのアスパラギン酸残基の側鎖のカルボキシル基
を不溶性樹脂に結合させることを特徴とする、請求項２
に記載のグルカゴンの製造方法。3. The terminal amino group-protected fragment contains an amino acid residue at position 29, represented by the following general formula (2): R 1 -X-Asp-Thr (R 3 ) -R 2 (2) (wherein R is 1 is a terminal amino group protecting group, R 2 is a terminal carboxyl group protecting group, R 3 is a side chain hydroxyl group protecting group of a threonine residue, X is a chemical bond, Met,
Or Leu-Met. 3. A peptide represented by the formula (1), wherein the carboxyl group of the side chain of the aspartic acid residue of the peptide is bound to an insoluble resin.
The method for producing glucagon according to 1. 【請求項４】 末端アミノ基保護フラグメントが、19位
から25位のアミノ酸残基に相当する下記一般式(3) ： Ｒ1-Ala-Gln-Asp(Ｒ4)-Phe-Val-Gln-Trp-OH (3) （式中、Ｒ1は末端アミノ基の保護基であり、Ｒ4はアス
パラギン酸残基の側鎖のカルボキシル基の保護基であ
る。）で表されるヘプタペプチドを含む、請求項２又は
３に記載のグルカゴンの製造方法。4. The terminal amino group-protected fragment corresponds to the amino acid residues at positions 19 to 25, represented by the following general formula (3): R 1 -Ala-Gln-Asp (R 4 ) -Phe-Val-Gln- Trp-OH (3), wherein R 1 is a terminal amino group protecting group and R 4 is a side chain carboxyl group protecting group of an aspartic acid residue. The method for producing glucagon according to claim 2 or 3. 【請求項５】 末端アミノ基保護フラグメントが、17位
から25位のアミノ酸残基に相当する下記一般式(4) ： Ｒ1-Arg(Ｒ5)-Arg(Ｒ5)-Ala-Gln-Asp(Ｒ4)-Phe-Val-Gln-Trp-OH (4) （式中、Ｒ1は末端アミノ基の保護基であり、Ｒ4はアス
パラギン酸残基の側鎖のカルボキシル基の保護基であ
り、Ｒ5はアルギニン残基の側鎖のグアニジノ基の保護
基である。）で表されるノナペプチドを含む、請求項２
又は３に記載のグルカゴンの製造方法。5. The terminal amino group-protected fragment corresponds to the amino acid residues at positions 17 to 25, represented by the following general formula (4): R 1 -Arg (R 5 ) -Arg (R 5 ) -Ala-Gln- Asp (R 4 ) -Phe-Val-Gln-Trp-OH (4) (In the formula, R 1 is a protecting group for the terminal amino group, and R 4 is a protecting group for the carboxyl group on the side chain of the aspartic acid residue. And R 5 is a protecting group for the guanidino group of the side chain of the arginine residue.).
Or the method for producing glucagon according to item 3. 【請求項６】 末端アミノ基保護フラグメントが、19位
から24位のアミノ酸残基に相当する下記一般式(5) ： Ｒ1-Ala-Gln-Asp(Ｒ4)-Phe-Val-Gln-OH (5) （式中、Ｒ1は末端アミノ基の保護基であり、Ｒ4はアス
パラギン酸残基の側鎖のカルボキシル基の保護基であ
る。）で表されるヘキサペプチドを含む、請求項２又は
３に記載のグルカゴンの製造方法。6. The terminal amino group-protected fragment corresponds to the amino acid residues at positions 19 to 24, represented by the following general formula (5): R 1 -Ala-Gln-Asp (R 4 ) -Phe-Val-Gln- A compound containing a hexapeptide represented by OH (5) (wherein R 1 is a protecting group for a terminal amino group and R 4 is a protecting group for a carboxyl group of a side chain of an aspartic acid residue). Item 4. A method for producing glucagon according to Item 2 or 3. 【請求項７】 末端アミノ基保護フラグメントが、17位
から24位のアミノ酸残基に相当する下記一般式(6) ： Ｒ1-Arg(Ｒ5)-Arg(Ｒ5)-Ala-Gln-Asp( Ｒ4)-Phe-Val-Gln-OH (6) （式中、Ｒ1は末端アミノ基の保護基であり、Ｒ4はアス
パラギン酸残基の側鎖のカルボキシル基の保護基であ
り、Ｒ5はアルギニン残基の側鎖のグアニジノ基の保護
基である。）で表されるオクタペプチドを含む、請求項
２又は３に記載のグルカゴンの製造方法。7. The terminal amino group-protected fragment has the following general formula (6) corresponding to amino acid residues at positions 17 to 24: R 1 -Arg (R 5 ) -Arg (R 5 ) -Ala-Gln- Asp (R 4 ) -Phe-Val-Gln-OH (6) (wherein R 1 is a protecting group for the terminal amino group, R 4 is a protecting group for the carboxyl group of the side chain of the aspartic acid residue) , R 5 is a protecting group for the guanidino group of the side chain of the arginine residue.), The method for producing glucagon according to claim 2 or 3. 【請求項８】 下記式(1) ： H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- (1) Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH で表されるグルカゴンの製造方法であって、１位から12
位のアミノ酸残基に相当する下記一般式(7) ： Ｒ1-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-OH (7) （式中、Ｒ1は末端アミノ基の保護基である。）で表さ
れるペプチドと、13位から29位のアミノ酸残基に相当す
る下記式(8) ： Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr- OH (8) で表されるペプチドとを、トリプシン又はトリプシン様
酵素の存在下で反応させることを特徴とする製造方法。8. The following formula (1): H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala. -Gln- (1) A method for producing glucagon represented by Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH, wherein 1 to 12
Corresponding to the amino acid residue at position (7): R 1 -His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-OH (7) (wherein R 1 is a terminal amino group-protecting group) and a compound represented by the following formula (8) corresponding to the amino acid residues at positions 13 to 29: Tyr-Leu-Asp-Ser-Arg-Arg- Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH (8) is characterized by reacting with a peptide represented by trypsin or trypsin-like enzyme. Production method. 【請求項９】 下記式(1) ： H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- (1) Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH で表されるグルカゴンの製造方法であって、１位から18
位のアミノ酸残基に相当する下記一般式(9) ： Ｒ1-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-OH (9) （式中、Ｒ1は末端アミノ基の保護基である。）で表さ
れるペプチドと、19位から29位のアミノ酸残基に相当す
る下記式(10)： H-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH (10) で表されるペプチドとを、トリプシン又はトリプシン様
酵素の存在下で反応させることを特徴とする製造方法。9. The following formula (1): H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala. -Gln- (1) A method for producing glucagon represented by Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH.
The following general formula (9) corresponding to the amino acid residue at position: R 1 -His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser- A peptide represented by Arg-Arg-OH (9) (wherein R 1 is a terminal amino group-protecting group) and the following formula (10) corresponding to the amino acid residues at positions 19 to 29: H-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH (10) is characterized by reacting with a peptide represented by trypsin or trypsin-like enzyme And manufacturing method. 【請求項１０】 下記式(1) ： H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- (1) Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH で表されるグルカゴンの製造方法であって、１位から18
位のアミノ酸残基に相当する下記一般式(11)： Ｒ1-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys(Ｒ6)-Tyr-Leu-Asp-Ser-Arg-Arg-OH (11) （式中、Ｒ1は末端アミノ基の保護基であり、Ｒ6はリジ
ンの側鎖のアミノ基の保護基である。）で表されるペプ
チドと、19位から29位のアミノ酸残基に相当する下記式
(10)： H-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH (10) で表されるペプチドとを、トリプシン又はトリプシン様
酵素の存在下で反応させることを特徴とする製造方法。10. The following formula (1): H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala. -Gln- (1) A method for producing glucagon represented by Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH.
The following general formula (11) corresponding to the amino acid residue at position: R 1 -His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys (R 6 ) -Tyr-Leu- A peptide represented by Asp-Ser-Arg-Arg-OH (11) (in the formula, R 1 is a terminal amino group protecting group, and R 6 is a side chain amino group protecting group of lysine). And the following formula corresponding to the amino acid residues at positions 19 to 29
(10): H-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH (10) reacted with the peptide represented by trypsin or trypsin-like enzyme A manufacturing method characterized by: 【請求項１１】 下記式(1) ： H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- (1) Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH で表されるグルカゴンの製造方法であって、前記グルカ
ゴンを少なくとも２個のフラグメントに分けて各フラグ
メントを液相中で合成し、次いで液相中で各フラグメン
トを反応させてカップリングすることを特徴とする製造
方法。11. The following formula (1): H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala. -Gln- (1) A method for producing glucagon represented by Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH, wherein the glucagon is divided into at least two fragments, and each fragment is divided into fragments. Is synthesized in a liquid phase, and then each fragment is reacted and coupled in the liquid phase.
JP7146255A 1995-06-13 1995-06-13 Production of glucagon Pending JPH08337600A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP7146255A JPH08337600A (en) 1995-06-13 1995-06-13 Production of glucagon

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP7146255A JPH08337600A (en) 1995-06-13 1995-06-13 Production of glucagon

Publications (1)

Publication Number Publication Date
JPH08337600A true JPH08337600A (en) 1996-12-24

Family

ID=15403609

Family Applications (1)

Application Number Title Priority Date Filing Date
JP7146255A Pending JPH08337600A (en) 1995-06-13 1995-06-13 Production of glucagon

Country Status (1)

Country Link
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001206897A (en) * 2000-01-26 2001-07-31 Nippon Seibutsu Seizai:Kk Hydroxyproline derivative
JP2002539219A (en) * 1999-03-15 2002-11-19 ノボ ノルディスク アクティーゼルスカブ Ion exchange chromatography separation of GLP-1 and related peptides
US7749955B2 (en) 2003-08-21 2010-07-06 Novo Nordisk A/S Separation of polypeptides comprising a racemized amino acid
US10323708B2 (en) 2015-04-27 2019-06-18 Akebono Brake Industry Co., Ltd. Friction material composition, friction material and production method thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002539219A (en) * 1999-03-15 2002-11-19 ノボ ノルディスク アクティーゼルスカブ Ion exchange chromatography separation of GLP-1 and related peptides
JP2001206897A (en) * 2000-01-26 2001-07-31 Nippon Seibutsu Seizai:Kk Hydroxyproline derivative
US7749955B2 (en) 2003-08-21 2010-07-06 Novo Nordisk A/S Separation of polypeptides comprising a racemized amino acid
US10323708B2 (en) 2015-04-27 2019-06-18 Akebono Brake Industry Co., Ltd. Friction material composition, friction material and production method thereof

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