TW401421B

TW401421B - A method of asymmeric synthezing of optically active peptides

Info

Publication number: TW401421B
Application number: TW83101938A
Authority: TW
Inventors: Jing-Jung Huang; Huei-Yau Huang; Da-Fu Dai; Jin-Tsai Fan
Original assignee: Syn Tech Chem & Pharm Co Ltd
Priority date: 1994-03-07
Filing date: 1994-03-07
Publication date: 2000-08-11

Abstract

This invention provides a method concerning the synthesis of optically active peptides, especially referring to a novel method regarding the synthesis of the optically active alkylamino dipeptide 3-acetylsulfur group-2-methylpropanic acid-amino acid, which combines the <alpha>, the <beta> unsaturated acetyl amine and amino acid to be the <alpha> and <beta> unsaturated carbonyl amino acid compound. Next, proceed the nucleophilic addition reaction by the nucleophilic agent (said as acetic acid sulfide etc): under suitable temperature, solvents, and amounts of additives to produce the unequal amounts of the diasteromers before the separation and the purification of the fractional crystallization that resulted in the optically active alkylamino dipeptide. It can largely elevate the proportion of the R.S-form, for example, when producing the caprolio and diminish the production of the undesied R.S-form and thus lower the cost largely with a novelness and industrial value.

Description

A6 ______B6 _ 五、發明説明（& (請先聞讀背面之注意事項再填寫本頁) 本發明係提供一種合成具光學活性之胜肽的方法•特別是一種合成具有光學活性之烷化胺基變胜肽3—乙醯硫基一 2 —甲基丙酸一胺基酸的削新方法。按，當胺基與羧基脫水镅合後即形成了具-C0-冊構造的醣胺鐽，而將一個胺基酸上的胺基與另一悝胺基酸上的羧基脫水縮合而成，則構成胜肽鍵的形態*埴個過程稱為「胜肽合成_( peptide synthesis )」。連结多個胺基酸則形成多肽，多肽的合成提供已知序列之胜肽，因此有肋於研究结構及生物活性間的關係，它也製造了特定序列之胜肽，供 X-ray晶趙编射，及建立多肽最適構肜的資料。大多数胜肽合成工作之目的在於製備如上所述之天然存在物或類似天然肽類结《之非天然胜肽。這些化合物一般為由具光學活性之胺基酸連结而成已知長度之鐽及已知系列之各殘基。由消旋性胺基酸姐成的多肽化合物，由於副產物為其異構物降低其纯度及生化活性，而不具實用的價值，因此宜採用具光學活性之反應物為原料或待合成消旋通後將產物進行光學分劄，來製備高纯度及高生理活性之化合物》胜肽的合成，蚤常用的方法為使用偁合劑（coupling a-gent )，堪稱最優良的氨基酸偶合反應是碳化二醚亞胺法 (carbodiiiide aethod, RN=C=NR, ) C Sheehan, J.C., Hess, G.P. : J. Aaer. Chen. Soc. 77,1067(1955)〕該反應可以在許多不同的溶劑内進行，二_•己基碳化二睡亞胺（dicyclohexylcarbodiinide)'是目前胜肽合成法最常用的。至於將胺基酸轉化為胺基醢氛（&(^-lanino acid chlorides ) C Katsoyannis, P. G., du Vigieaud, V:J. Aeer. Ch-en. Soc. 78,3114(1954)〕使其更具活性，再與胺基反應·此法因胺基醚氛上的α —氫原子極具酸性因此易為驗移去，而產生消旋化作用，以及易進行分子內的成環反懕而變成醯化甲 4 (210X297 公沒) A6 B6 401421 五、發明說明（’g 内鹿化物（azlactone )，因而使得其在合成具生化活性胜肽上的用途降低。酯類之胺解為製取胜肽之另一重要的方法，為使胜肋生成的速率加至最大且其產率最高，含有良好雄棄基的酯類是必須的，一般的例子有對一硝基苯酯（p~ Nitrophenyl ester )，五氛苯酷（Pentachlorophenyl ester )等。另一常用的方 >去為使用酵素具有專一之特性進行催化以合成胜肽键。酵素之加速反應的原理，如同催化劑，是藉著降低活化之自由能，使活化狀態的分子數量增加而增進反腥。此技術已愿用於阿斯巴甜（Aspartame)雙胜肽之製造|並已進行工桊化生產。由於多肽化合，物具有生物活性，有一定之结構及光學活性，因此在合成胜肽化合物時，如上所述需以具光學活性之反懕物及經遒當保護之胺基酸，方能合成已知願序之各殘基之胜肽化合物，因此需投入数多的人力與物力於前製作業。 _ _ __________________ 因此本發明乃使用較不相同之合成方式，來製造非天然胜肽* Μ合成具光學活性之烷代胺基雯胜肽，首先將a 、/3 —不飽和醢氛和胺基酸相结合為α、/3 —不胞和羧基胺基酸化合物，再利用親核性試爾（如硫ft醋酸等）進行親核性加成反懕，及在適當的添加劑作用下，經由部份立通選擇性（partially stereoselection)，獲得不等量的烷雯胜肽非對映異構物再Μ再结晶的方式進一步行光學分抑，可得具光學活性之烷代5胜肽（如式一）。此種方法亦可推展到參胜肽或肆胜肽、伍胜肽等。甲 4 (210X297 公沒) <請先閑讀背面之注意事項再瑱寫本百) •装. •打· 線· A6 B6 五、發明説明（3) R1 八 S 产丫'R2 R3 本發明的重黠在於利用親核性試劑（硫代醋酸等）對 α、/3 — 不鉋和续基化合物（a ，/3 -Unsaturated Carbonyl Compounds) 進行親核性加成反懕；親核性試劑加於（如圖一）碳一碳3鍵產生成陰離子（hybrid anion) I ，此陰離子自溶劑接受（如圖二）Μ難子產生最後生成物。此氫雜子可加在ct —碳上或氣上，於是獲得生成物之嗣（keto )或烯薄 (enol)式體；任一情形最後均得同一平衡混合物，其中Μ 酮式體為主產物，但是當肜成C_ Η鍵時若所需之S自由趙所提供*則S的位置含影銎其生成物之立體结播，如1 三時1在後測時形成S form，如圖四時氫在前方時則形成R form。A6 ______B6 _ V. Description of the invention (& Please read the notes on the back before filling out this page) The present invention provides a method for synthesizing peptides with optical activity • Especially an alkylamine with optical activity A new method for cleavage of peptides 3-acetamidine-2-methylpropanoic acid-amino acid. Pressing, when the amine group and the carboxyl group are dehydrated to form a glycosamine with a -C0-chain structure The dehydration-condensation of an amino group on one amino acid with a carboxyl group on the other amino acid forms the form of the peptide bond. This process is called "peptide synthesis". Multiple amino acids are linked to form a peptide. The synthesis of the peptide provides a peptide with a known sequence. Therefore, it is necessary to study the relationship between structure and biological activity. It also produces a peptide with a specific sequence for X-ray crystals. Edited by Zhao, and established information on the most suitable peptides for peptides. The purpose of most peptide synthesis work is to prepare natural peptides as described above or non-natural peptides similar to natural peptides. These compounds are generally made by optical Reactive amino acids are known Degree of residues and residues of known series. Polypeptide compounds formed from racemic amino acids, because the by-products of their isomers reduce their purity and biochemical activity, and are not of practical value, so An optically active reactant is used as a raw material or the product is optically separated after the racemic synthesis is performed to prepare a compound of high purity and high physiological activity. For the synthesis of peptides, a common method for fleas is to use a coupling agent (coupling a-gent). ), Called the best amino acid coupling reaction is the carbodiide aethod (RN = C = NR,) C Sheehan, JC, Hess, GP: J. Aaer. Chen. Soc. 77, 1067 (1955 )] The reaction can be carried out in many different solvents. Dicyclohexylcarbodiinide 'is currently the most commonly used method for peptide synthesis. As for the conversion of amino acids to amino groups (& (^ -lanino acid chlorides) C Katsoyannis, PG, du Vigieaud, V: J. Aeer. Ch-en. Soc. 78, 3114 (1954)] make it more active, and then react with amine groups The α-hydrogen atom on the ether ether atmosphere is extremely acidic and therefore easily removed for inspection. Produces racemization, and easily undergoes intramolecular ring formation and turns into tritiated formazan 4 (210X297 publicly available) A6 B6 401421 5. Description of the invention ('g azlactone), which makes it useful The use of biochemically active peptides is reduced. Aminolysis of esters is another important method for preparing peptides. In order to maximize the rate of production of ribs and the highest yield, the esters containing good androsteryl are Necessary, general examples are p-Nitrophenyl ester (P ~ Nitrophenyl ester), pentachlorophenyl ester (Pentachlorophenyl ester), etc. Another commonly used method is to use enzymes with specific properties for catalysis to synthesize peptide bonds. The principle of the accelerated reaction of enzymes, like the catalyst, is to increase the number of molecules in the activated state and increase the odor by reducing the free energy of activation. This technology is already ready for the production of Aspartame double peptide | and has been industrialized. Due to the combination of peptides, the material has biological activity, and has a certain structure and optical activity. Therefore, when synthesizing peptide compounds, as described above, it is necessary to use optically active anti-substances and amino acids that are protected by the amino acid before synthesis. The peptide compounds of each residue in the sequence are known. Therefore, a large amount of human and material resources need to be invested in pre-production operations. _ _ __________________ Therefore, the present invention uses a different synthesis method to produce unnatural peptides. * To synthesize optically active alkylamino peptides, firstly, a, / 3-unsaturated atmosphere and amine group The acid is combined into α, / 3-cell and carboxyamino acid compounds, and then nucleophilic tests (such as sulfur ft acetic acid) are used for nucleophilic addition reaction, and under the action of appropriate additives, Partially stereoselection can be obtained by re-crystallizing the diastereoisomers of alkaloid peptides in varying amounts by recrystallization, and optically active alkyl-5 peptides can be obtained ( As in formula one). This method can also be extended to reference peptides, vasoactive peptides, and wood peptides. A4 (210X297 public) < Please read the precautions on the back before copying a hundred copies) • Equipment. • Hit · Line · A6 B6 V. Description of the invention (3) R1 Eight S produced by 'R2 R3 The emphasis is on the use of nucleophilic reagents (thioacetic acid, etc.) for α, / 3 — unsaturated carbon compounds (a, / 3-Unsaturated Carbonyl Compounds) with nucleophilic addition reaction; At (Figure 1), the carbon-carbon 3 bond is formed into an anion (hybrid anion) I. This anion is accepted from the solvent (see Figure 2). This hydrogen can be added to ct-carbon or gas, so the keto or enol form of the product is obtained; in any case, the same equilibrium mixture is obtained, in which the ketone form is the main Product, but when the C_ key is pressed, if the required S is provided by Zhao *, then the position of S contains a three-dimensional broadcast of the product, such as 1 3: 1 when the S form is formed in the back test, as shown in the figure At four o'clock hydrogen is in the R form.

III I '1 I —C=C—c=o + :Z -^ ~^—C~C~〇III I '1 I —C = C—c = o +: Z-^ ~ ^ —C ~ C ~ 〇

圖- ZFigure-Z

I I I 一C一C=C 一0一H / Enol (請先聞讀背面之注意事項再填寫本頁) •打. .線. 二圖 ¾ c li c、 I -C-IzI I I One C One C = C One 0 One H / Enol (please read the notes on the back before filling out this page) • Hit .. Line. Two Figure ¾ c li c, I -C-Iz

HH

-5 —0—H I 丨〇丨z-5 —0—H I 丨〇丨 z

K 甲 4 (210X297 公簷) 40142.1 A6 B6 五、發明説明（4)K A 4 (210X297 public eaves) 40142.1 A6 B6 V. Description of the invention (4)

AcAc

ΟΟ

Η 二 S-Form ο Ac*Η Two S-Form ο Ac *

R οR ο

R (請先W讀背面之注意事項再填寫本頁) 圖四在進行親核性加成 L一胺基酸或其衍生因有立體阻此法稱為立的高低端視間的距雜而而溶劑及添選擇性。因此本成反應時，及RS前驅物件，將所得 ization )可光學活性之礙的差異性體選擇法（原先不對稱定，兩者愈加劑的加入發明乃.利用不同的溶劑比值的影響的前锞物，將SS前羅物烷化胺基胜 R-Form 反應，當第一酒不對稱中心的存在物），而形成第二個不對稱中心時，將獲得不等量的非對映異構物， stereoselective synthesis)。立通選擇性中心與新形成第二個不對稱中心之近立通選擇性愈高，反之則愈低* ，可加大此差異性，使其更具立體上述釗作新構想，再探討親核性加，添加劑及反應條件，對產物中SS ，以尋找最通合SS前驅物生成的條利用分段结晶法（fractional crystal 1-结晶分離（ee=91.2)，如此可提高具肽的產率。 •線· 甲 4(210X297 公沒) 401421 A6 __B6__ .五、發明説明（5) (請先閲讀背面之注意事項再淇寫本百) 其中一種重要的烷代胺基雯胜肽，為卡普利歐（〇3?1；-opril, Physicians Desk Reference, 1985, Page 1768)全名為 1-〔（2S) -3-necapto-2-Biethylpropionyl〕-L-proline.式(二〉〇R (please read the precautions on the reverse side before filling out this page) Figure 4: The distance between the upper and lower end-views is referred to as the nucleophilic addition of L-amino acid or its derivative due to steric hindrance. And the solvent and Tim selectivity. Therefore, during the reaction, and the RS precursor object, the obtained) can be optically affected by the differential body selection method (originally asymmetric, the addition of the two more additives was invented to make use of the effects of different solvent ratios. For the reaction of the SS precursor, alkylating the amino group with R-Form, when the asymmetric center of the first wine exists, and the second asymmetric center is formed, an unequal amount of diastereomers will be obtained. Structure, stereoselective synthesis). The higher the Litong selectivity center and the newly formed second asymmetric center, the higher the Litong selectivity, and vice versa *, which can increase this difference and make it more three-dimensional. Addition of nucleus, additives and reaction conditions to SS in the product to find the most compatible SS precursors. The use of fractional crystallization (fractional crystal 1-crystallization separation (ee = 91.2)) can improve the production of peptides. Rate. • Line A 4 (210X297 publicly available) 401421 A6 __B6__. V. Description of the invention (5) (Please read the notes on the back before writing a hundred) One of the important alkylamino peptides is a card Puliou (〇3? 1; -opril, Physicians Desk Reference, 1985, Page 1768) The full name is 1-[(2S) -3-necapto-2-Biethylpropionyl] -L-proline. Formula (2> 〇)

為心臟血管用藥〔M.A. Ondetti et al.，U.S.P. 4,046,889(1977)] ，其作用為血管緊缩素轉化酵素（angiotensin converting enzyne) 的抑制劑，阻止血管緊缩素I轉化為血管緊縮素H，由於具高度安全性及無不良副作用，因此受到廣泛使用。卡普利歐為一具光學活性之雙胜肽，因此如何藺易獲得其具光學活性的前驅物，乃整傾合成之重心所在。有關卡普利歐的合成可分為化學法〔J. Iwao, et al.，Jpn. Kokai Tokkyo Koho 79:151912(1979)； H. -J. -P. Marie. Jpn. Kokai Tokkyo Koho 80-,38386(1980); N. Ohashi, et al., Jpn. Kokai. Tokkyo Roho 81:7756(1981); J. Fishet et al·, U. S. P. 4,332,725(1982)〕及酵素法〔A. Safcimae et al., U. S. P. No. 4,629,701(1986); C. J. Sih. PCT iio. Wo 87/05328 ]。不論化學法或酵素法合成卡普利歐，均先合成消旋性3-acylt-hio-2-niet;hylpropionic acid或其酸氛衍生物，再與Μ胺酸進行偁合，但這些方法或多或少均有其困難點，例如：旋光度维持不易或分雜純化之問題多少限制其工業化之可行性，或大大的影坌生產成本。甲 4(210X297 公沒) 經濟部中央標準局*:工消贲合作杜印« 401421 A6 B6 五、發明説明（6 ) Η将本發明之合成步驟，簡列反懕式如下：Cardiovascular medication [MA Ondetti et al., USP 4,046,889 (1977)], which acts as an inhibitor of angiotensin converting enzyne, preventing the conversion of angiotensin I to angiotensin H, because With high safety and no adverse side effects, it is widely used. Capriol is an optically active double peptide. Therefore, how to easily obtain its optically active precursor is the focus of the synthesis. The synthesis of Capriol can be divided into chemical methods [J. Iwao, et al., Jpn. Kokai Tokkyo Koho 79: 151912 (1979); H. -J. -P. Marie. Jpn. Kokai Tokkyo Koho 80- , 38386 (1980); N. Ohashi, et al., Jpn. Kokai. Tokkyo Roho 81: 7756 (1981); J. Fishet et al., USP 4,332,725 (1982)] and the enzyme method [A. Safcimae et al. , USP No. 4,629,701 (1986); CJ Sih. PCT iio. Wo 87/05328]. Regardless of the chemical or enzymatic synthesis of Capriol, racemic 3-acylt-hio-2-niet; hylpropionic acid or its acid derivative is first synthesized, and then combined with M amino acid, but these methods or There are more or less difficulties, such as the difficulty of maintaining optical rotation or the problem of purification of impurities, which limits the feasibility of industrialization, or greatly affects the production cost. A 4 (210X297 public) Central Standards Bureau of the Ministry of Economic Affairs *: Industrial and consumer cooperation cooperation Du Yin «401421 A6 B6 V. Description of the invention (6) Η The synthesis steps of the present invention are summarized as follows:

R,為C, 一 G之烷基。Ra為C，一 C?之烷基（或含苯基、雜環）或含有下列官能基，胺基、羧基、硫基、酚羥基。 b為羥基、苄氧基或C*_ Ci之烷氧基及L或D form之天然胺基酸或其多肽及其酯類化合物^蚪為苯基、苄基、甲基或乙基。買際懕用於各種有光學活太烷代雙胜肽之製造。實施例如下：實施例一R is C, an alkyl group of G. Ra is C, alkyl group (or phenyl group, heterocyclic ring) containing C? Or contains the following functional groups, amine group, carboxyl group, thio group, and phenolic hydroxyl group. b is a hydroxy group, a benzyloxy group or an alkoxy group of C * _Ci and a natural amino acid of L or D form or a polypeptide thereof and an ester compound thereof; 蚪 is a phenyl group, a benzyl group, a methyl group or an ethyl group. Maijiye is used in the production of various optically active thioalkylated bipeptides. The implementation example is as follows:

Methacryloyl-L-proline benzyl ester (甲基丙烯基一 L —脯胺酸一苯甲基酯）之合成 98 咖ole L-proline benzyl ester 溶於 450ml DCM 中，加入 200咖ole ------------------------装------tr-------.¾ (請先閲为背面之洼悉寧項再堝罵本頁) 本纸任尺度通用t國國家標準（CNS)甲4规格（210 X 297公；f ) 沒濟.部中央櫺準局8工消賁合作.吐印焚 A6 __B6__ 五、發明説明（7 ) 三乙基胺，室溫搜伴1小時，於冰浴下，加人12〇mmole meth-acryloylchloride ，攪伴30分鐘，於室溫下缰反應24小-，經 5 % Hcl流液，5 % NaHC03溶液及5 % NaCl溶液各洗三次，經硫酸鎂乾煉，濃揃得63.4_le產物。其’HNMR如附件一中之附圖(一）(5 ppm:2.0 (3H,s), 2.0(3H，n), 2.25(lH，q), 3.65(2H,t), 4.45(q，d,d)， 5.1(lH，d,d)，5.2 (2H，q), 5.3(lH，cl，d), 7.35(5H.s)，產率為 64.7 涔。萁陁洌二Synthesis of Methacryloyl-L-proline benzyl ester (methacryl-L-proline monobenzyl ester) 98 kale L-proline benzyl ester is dissolved in 450ml DCM and 200 kale is added ------ ------------------ 装 ------ tr -------. ¾ (please read this article for the back side of the Sining item and then scold it) Page) This paper is a standard of the National Standard (CNS) A4 specification (210 X 297 male; f). It is useless. The Ministry of Central Standards and Quarantine Bureau cooperates with the 8 workers. Print A6 __B6__ 5. Description of the invention (7 ) Triethylamine, search for 1 hour at room temperature, add 120 mmole meth-acryloylchloride in an ice bath, stir for 30 minutes, and react for 24 hours at room temperature. After passing 5% Hcl, 5 % NaHC03 solution and 5% NaCl solution were washed three times each, and dried with magnesium sulfate, and concentrated to obtain 63.4_le product. The 'HNMR is shown in the attached figure (I) (5 ppm: 2.0 (3H, s), 2.0 (3H, n), 2.25 (lH, q), 3.65 (2H, t), 4.45 (q, d , d), 5.1 (lH, d, d), 5.2 (2H, q), 5.3 (lH, cl, d), 7.35 (5H.s), yield 64.7 4.7. 萁陁洌二

Methacryioyl-L-phenylalanine benzyl ester (甲基丙嫌基 tL-苯丙胺酸一苯甲基酯）之合成將 L-proline benzyl ester 改為 L-phenylalanine benzyl ester，其餘方法同實施洌一，產率為S8_2 %。萁施例三Synthesis of Methacryioyl-L-phenylalanine benzyl ester (L-proline benzyl ester) was changed to L-phenylalanine benzyl ester. %. Example 3

Methacryloyl-L-tyrosine benzyl ester (甲基丙稀基-L-色胺酸一苯甲基酯）之合成將 L-proline benzyl ester 改為 L-tyrosine benzyl ester，其餘方法同S施例一，產率為62.5 %。實施例四 l-(3-acetylthio-2-aethylpropanoyl)-L-proline benzyl ester 〔1- ( 3-乙醱磺基-2-甲基丙_) -L-腕胺酸-苯甲基酯〕之合成取 36.6nmole netliacryloyl-L-proline benzyl ester，加入 IOObI Di-oxane、l83miole 硫代釋酸及 500mg eserine (毒扁豆驗），於 5〇 t：下反應48小時，缉HPLC測定，產率為47.0¾，其SS/RS+SS比值為 0.42 。黃施例五· C S,S ] 1-(3-acetylthio-2-methylpropanoly)-L-phenylalanine benzyl ester〔1-(3-乙醢硫基-2-甲基丙酸）-L-***酸-苯甲基酯]之合成Synthesis of Methacryloyl-L-tyrosine benzyl ester (L-proline benzyl ester) was changed to L-tyrosine benzyl ester. The rate is 62.5%. Example 4 l- (3-acetylthio-2-aethylpropanoyl) -L-proline benzyl ester [1- (3-ethylsulfonyl-2-methylpropanyl) -L-carnitine-benzyl ester] The synthesis takes 36.6nmole netliacryloyl-L-proline benzyl ester, adds 100bI Di-oxane, l83miole thio acid and 500mg eserine (Poison Lentil Test), and reacts at 50t for 48 hours. The yield is determined by HPLC. 47.0¾, with an SS / RS + SS ratio of 0.42. Example 5 of Huang · CS, S] 1- (3-acetylthio-2-methylpropanoly) -L-phenylalanine benzyl ester [1- (3-ethylsulfanyl-2-methylpropanoic acid) -L-phenylalanine- Synthesis of benzyl ester]

V 本紙張又度通用中3國家漂準（CNS)甲4規^备（210 X 297公;Ϊ ) ------------------------^------，玎------線 (请先兄才背面之^念^項再埤耳夂开) 401421 A6 B6 沒濟部中央樣弟局费工消費合作社印- 五、發明説明（8 將 Methacryloyi-L-proline benzyl ester 改為 Methacryloyl-IrPhenyla-lanine benzyl ester ·:其合成方法同3施例四，產率為丨50.8% · SS/RS+SS=0.52，將所得產物經矽膠管柱纯化，再烴分段结晶 •可得[S.S.] WS-acetylthio-Z-neUiylpropanoyD-L-phenylalanirte benzyl * ·. ester 其 HMMR 如附件:一中之附圖(二），δ PPn:l.l5(3H，d，CHd，2·30 (3H,s,C0CH?) > 2.37(lH,i,CH in the side chain) - 2.99(2H,ih,SCHJ · 3.12 (2H,m,in the phenylalanine) » 4.90(lH,q,in the phenylalanine) > 5.11(2H,q, in the benzyl ester) * 5.98(lH,d,NH) * 7.l5(5H,m, in the phenylalanine) · 7.30(5H,a,in the benzyl ester) ° 實施洌六 l-(3-acetylthio-2-iethylpropanoyl)-L-tyrosine benzyl ester C 1- (3-乙豳碲基-2-甲基丙酸）胺酸一苯甲基酯]之合成將 Methacryloyl-L-proline benzyl ester 改為 Methacryloyl-L-tyrosine benzyl ester，其合成方法同實施例四。產率為43.3%，SS/RS+ SS=0.48，將所得產物經矽膠管柱純化，再经分段结晶，可得[S，S]l-(3-acetylthio-2-nethyipr*opanoyl)-L-tjrosine benzyl ester 其’HNMR 如附件一中之附 _(三），3 ppn:l.13(311，(1.(；恥），2.30(3H，s，C0CHy， 2.38(lH,ni,CH in the side chain) * 2.92(2H,m,SCHJ » 3.10(2Η,ιπ,ίίΐ the t-yrosine) » 4.90(lH,q,in the tyrosine) » 5.15(2H,q, in the benzyl ester) * 5.47(lH,s,0H) » 6.04(lH,d,NH) » 6.8Q(4H,q,in the tyrosine) · 7.30(5H,jn, in the benzyl ester) ° r' ' 買施例七〜十三改愛三级胺渾種類，其餘均和霣例四相同，结果列於附件二中之表一〇 SIS例十四〜十t 改變反愿溶劑種類，其餘均和實例十二相同，结果列於附件二中之表二。一一 i纸張Λ及通用中3困家標準（CNS)甲4说格（210 x 297公坌） ------------------------#------‘玎------'^ (请先閱4背面之;15?項再埤3夂頁) 401421 A6 __B6_ 五、發明説明（9 ) (請先閲讀背面之注意事項再填寫衣頁) 實施例十八V This paper is also universally used in China 3 National Standards (CNS) A4 regulations ^ (210 X 297 male; Ϊ) ----------------------- -^ ------, 玎 ------ line (please read the ^ items ^ on the back of your brother, and then open your ears) 401421 A6 B6 Printed by the Consumer Procurement Cooperative of the Central Sample Bureau of the Ministry of Health- V. Description of the invention (8 Change Methacryloyi-L-proline benzyl ester to Methacryloyl-IrPhenyla-lanine benzyl ester ·: The synthesis method is the same as that in Example 4 with a yield of 50.8% · SS / RS + SS = 0.52. The obtained product was purified by a silica gel column, and then the hydrocarbon was partially crystallized. [SS] WS-acetylthio-Z-neUiylpropanoyD-L-phenylalanirte benzyl * ·. Ester The HMMR is shown in the appendix: Figure (2) in the first, δ PPn: l.l5 (3H, d, CHd, 2 · 30 (3H, s, C0CH?) &Gt; 2.37 (lH, i, CH in the side chain)-2.99 (2H, ih, SCHJ · 3.12 (2H, m, in the phenylalanine) »4.90 (lH, q, in the phenylalanine) > 5.11 (2H, q, in the benzyl ester) * 5.98 (lH, d, NH) * 7.l5 (5H, m, in the phenylalanine) 7.30 (5H, a, in the benzyl ester) ° Implementing l- (3-acetylthio-2-iethylpropanoyl) -L-tyrosine benzyl ester C 1- (3-ethylfluorenyl telluryl-2-methyl Synthesis of propyl propionate) amino acid monobenzyl ester] Methacryloyl-L-proline benzyl ester was changed to Methacryloyl-L-tyrosine benzyl ester, and the synthesis method was the same as in Example 4. The yield was 43.3%, SS / RS + SS = 0.48, the obtained product was purified through a silica gel column, and then fractionally crystallized to obtain [S, S] l- (3-acetylthio-2-nethyipr * opanoyl) -L-tjrosine benzyl ester.中之附 _ (三), 3 ppn: l.13 (311, (1. (; shame), 2.30 (3H, s, COCHy, 2.38 (lH, ni, CH in the side chain) * 2.92 (2H, m, SCHJ »3.10 (2Η, ιπ, ίί the t-yrosine)» 4.90 (lH, q, in the tyrosine) »5.15 (2H, q, in the benzyl ester) * 5.47 (lH, s, 0H)» 6.04 (lH, d, NH) »6.8Q (4H, q, in the tyrosine) · 7.30 (5H, jn, in the benzyl ester) ° r '' Buy Example 7 ~ 13 change the tertiary ammonium species, The rest are the same as those in Example 4. The results are listed in Table 1 in Appendix II. SIS Examples 14 to 10 t The type of anti-wish solvent is changed. The rest are the same as in Example 12. The results are listed in Table 2 in Annex II. One-by-one paper Λ and GM 3 Standards (CNS) A 4 grid (210 x 297 cm) ----------------------- -# ------ '玎 ------' ^ (please read 4 on the back of the page first; 15? On the 3rd page) 401421 A6 __B6_ 5. Description of the invention (9) (Please read the back first (Notes should be filled in the clothing page) Example 18

Methacryloyl-L-proline 之合成取 lOnmole Hethacrylojd-proline benzyl ester 溶於甲酵，K NaOH行水解反應，得 9.2nunole Methacryloyl-L-proline，其 ’HNMR 如附件 ~ 中之附圖(四），3 PPm:2.0(3H,s)，2.0(3H,ai)，2.25(lH,q)，3_65(2fl，t) ，4.55(lH，t) ， 5.25(lH,s) ， 5.35(lH，s)，產率 98 % 。貢施例十九The synthesis of Methacryloyl-L-proline takes lOnmole Hethacrylojd-proline benzyl ester, which is dissolved in formazan, and K NaOH is hydrolyzed to obtain 9.2nunole Methacryloyl-L-proline. Its' HNMR is shown in the appendix ~ (4), 3 PPm : 2.0 (3H, s), 2.0 (3H, ai), 2.25 (lH, q), 3_65 (2fl, t), 4.55 (lH, t), 5.25 (lH, s), 5.35 (lH, s), Yield: 98%. Gong Shi Example 19

Hethacryioyi-L-phenyialanine 之合成 M Methacryloyi-L-phenyalanine benzyl ester 取代 Methacryloyl-L-pr-oline benzyl ester，其餘方法同實施例十八，產率為94%。篱施例廿Synthesis of Hethacryioyi-L-phenyialanine M Methacryloyi-L-phenyalanine benzyl ester was used in place of Methacryloyl-L-pr-oline benzyl ester. The rest of the method was the same as in Example 18 with a yield of 94%. Example

Hethacryioyl-L-tyr〇sine 之合成 M Methacryloyl-L-tyrosine benzyl ester 取代 Methacryloyl-L-proline benzyl ester，其餘方法同實施洌十八，產率為92%。萁施洌廿一 l-(3-acetylthio-2-aiethacryloyl)-L-proline 之合成取 0.2mmole metha-cryloyl-L-proline溶於Ιπιΐ二氛甲烧中，於冰浴下授伴，將0.3ia-nole硫代醋酸加入，在室溫下反應24小時，取出經HPLC测定，產率為78.0 %，其SS/RS+SS比值為0.45。實施例廿二〜廿六 ’ L ，Synthesis of Hethacryioyl-L-tyrosoline M Methacryloyl-L-tyrosine benzyl ester was used in place of Methacryloyl-L-proline benzyl ester.萁施洌廿一-(3-acetylthio-2-aiethacryloyl) -L-proline Synthesis 0.2 mmole metha-cryloyl-L-proline was dissolved in Ιπιΐ diazepam, and accompany in an ice bath. 0.3 ia-nole thioacetic acid was added, and the reaction was performed at room temperature for 24 hours. The yield was determined by HPLC, and the yield was 78.0%. The SS / RS + SS ratio was 0.45. Embodiments Twenty Two to Twenty-six 'L,

經濟部中央櫺準局员工消費合作社印S 加入路易斯酸為添加劑，其餘均和實例廿一相同，其结果列於附件二中之表三。 S施例廿七〜四十七改變反懕溶劑種類，及添加BFy其餘方法均和實施洌廿一相同，其结果列於附件二中之表四。實施例四十七〜七十二改雯BF#添加量，其餘方法均和實施例廿一相同，其〜 10〜太纸張尺度迺用中國國家標準（CNS)甲4規格（210 X 297公;Ϊ ).Addition of Lewis acid as an additive by the Consumer Cooperatives Cooperative of the Central Bureau of Standards and Quarantine of the Ministry of Economic Affairs, and the rest are the same as in Example 1. The results are shown in Table 3 in Annex II. Example Example 7-7: Forty-seven to change the type of anti-solvent, and the other methods of adding BFy are the same as those in the first implementation. The results are listed in Table 4 in Annex 2. Forty-seven to seventy-two modified Wen BF # addition amount, the rest of the methods are the same as the first example, which is ~ 10 ~ too paper size, using China National Standard (CNS) A 4 specifications (210 X 297) ; Ϊ).

Cl D7 五、創作説明（/〇 ) 结果列於附件二中之表五。實施例七十三〜七十七其他添加劑對產率及比值之影響，添加量為5 % ,其反應濃度為〇.123mniole methacryloy卜L-proline 和 0,615mmole 硫代醋酸於lml二氮甲烷中反應，其结果列於附件二中之表六。實施例七+八〜八+三 l-(3-acetylthi〇-2-met.hylpropanoyl)-L-phenylalanine 之合成。 K Methacryloy卜L-phenylalanine 取代 Methacryloy卜L-proline，其餘方法同1-(3-3061?11：11丨〇-2-脈1^^-13「(^311071)-1^1'〇1〗脱之合成，其结果列於附件二中之表七。實施例八十四 1-(3^)6脱0711；11丨0-2-11161；}^1?「0?3110丫丨）-11-?「0丨丨脱之合成〇取 0.2mmole methacryloy卜L-proline 溶於 lml 二氛甲院，於室溫下，加入0.6mmole硫代苯甲酸，及5mg eserine，繼續攪拌反應 24小時，經HPLC測定產率為88.4%，其SS/RS+SS=0.62。實施例八十五卡普利歐之合成取 120g(0.656 mole) Methacryloy卜L-proline，加入二氛甲院 1311ml 及0.13 mole三氟化硼，攪拌30分鐘，將75g (0.984 mole)硫代醋酸媛媛加入，室溫下反應48小時後，加入冰水中，取下層經濟部中央標準局員工消費合作社印裝 (請先閱讀背面之注意事項再填寫本頁) 線液，再Μ二氮甲烷萃取兩次，有機層濃縮得162g AMP-L-proli- ne ’將162g AHP-L-proline溶於.200ml水中，加熱至溶解後，再靜置，可得 48.6g S-AHP-L-p「oline晶體（SS/RS+SS=0.96, [a 1^=-162 (c=2 in ethanol}，其’冊MR如附件一中之附圖(五）。加入132ml HJ) ’ 29ml濃复水，反應一小時，完全後經乙酸乙酯萃取，濃縮得34.4gCl D7 V. Creative Instructions (/ 〇) The results are listed in Table 5 in Annex II. Example seventy-three to seventy-seven. The effect of other additives on the yield and ratio. The added amount is 5%, and the reaction concentration is 0.123 mniole methacryloy. L-proline and 0,615 mmole thioacetic acid are reacted in 1 ml of diazomethane. The results are listed in Table 6 in Annex II. Example 7 Synthesis of 8 + 8 ~ 8 + 3 l- (3-acetylthi〇-2-met.hylpropanoyl) -L-phenylalanine. K Methacryloy and L-phenylalanine replace Methacryloy and L-proline. The rest of the method is the same as 1- (3-3061? 11: 11 丨〇-2-pulse 1 ^^-13 "(^ 311071) -1 ^ 1'〇1〗 The results of synthesis are listed in Table 7 in Annex 2. Example 84 1- (3 ^) 6 Extract 0711; 11 丨 0-2-11161;} ^ 1? "0? 3110ā 丨)- 11-? "Synthesis of 0. Take 0.2mmole of methacryloy and L-proline and dissolve it in 1ml of Erfangjiayuan. At room temperature, add 0.6mmole of thiobenzoic acid and 5mg of eserine, and continue to stir for 24 hours. The yield determined by HPLC was 88.4%, and its SS / RS + SS = 0.62. Example 85 Synthesis of Capriol 120 g (0.656 mole) Methacryloy Bu L-proline was added to 1311ml and 0.13 mole of Erfangjiayuan Boron trifluoride, stir for 30 minutes, add 75g (0.984 mole) of thioacetic acid, and after 48 hours of reaction at room temperature, add ice water, remove the lower layer printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read first Note on the back, please fill in this page again.) Thread solution, extract twice with M diazomethane, and concentrate the organic layer to 162g AMP-L-proli-ne 'Dissolve 162g AHP-L-proline in .200ml water After being heated to dissolve, and then left to stand, 48.6g of S-AHP-Lp "oline crystal (SS / RS + SS = 0.96, [a 1 ^ =-162 (c = 2 in ethanol}) is obtained. Attachment (five) in Annex I. Add 132ml HJ) '29ml concentrated water and react for one hour. After completion, extract with ethyl acetate and concentrate to obtain 34.4g

，經乙酸乙酷/正己烷再結晶，得25.5g卡普利歐，ni.P.=104°C ，〔a〕tr=-128.0(C=l in ethanol)，其 ’HNMR〔如附件一中之附圖(六）δ ppm:1.18(3H，d, CH>，1.57 (lH,t,SH)，2.20(4H, m，3,4-0^0^After recrystallization from ethyl acetate / n-hexane, 25.5 g of Capriol were obtained, ni.P. = 104 ° C, [a] tr = -128.0 (C = 1 in ethanol), and its' HNMR [as shown in Annex I Figure (6) δ ppm: 1.18 (3H, d, CH >, 1.57 (lH, t, SH), 2.20 (4H, m, 3,4-0 ^ 0 ^

, 1 I 本紙張尺度適用中國國家樣準（CNS ) A4規格（210X297公釐） C7 D7 401421 五、發明説明(1r ) in proline) > 2.48(lH,ra,CH in the side chain) > 2.86(211,10,113^^) > 3.65 (211,1,2-011^ ΐη the proline) > 4.62(lH,ni,4-CH in the proline) > 7.75(1H, s.COOH)] 〇實施例八十六 l-(3-thio-2-methylpropanoyl)-L-pheny〗.alanine之合成 K Met.hacryloyl -1-phenylalanine取代 Met.hacry卜L-proline_，其餘方法同實施例八十五，產率 38 %，m.p.=122Tj，[α t =+46(c=l in CHC1>) ，HNMR5ppra:l_16(3H，d，CI^),i.57(lH，t，S-H),2.4〜3.0(ffl,5H)，48(g，lH), 7.0〜7.4(m,6H) 實施例八十七 l-(3-thii)-2-methylpropanoyl)-L-tyrosine之合成 Μ Methacyloyl-1-tyrosiiie 取代 Methacyl-L-proline其餘方法同實施例八十五，產率 41 %, m.p_=155°C，[a =+32(c=l in Et.oh) Ή NMR δ PPm:l.m3iI，d，CH今），1.57(lH，t，S-H)，2.4〜3.0(m，5H)， 6.6(d,2H), 6.9(d,2H), 7.2(S,1H) (請先閲讀背面之注意事項再填寫本頁) 經濟部中央標準局負工消費合作社印策〜 12〜本紙張尺度逋用中國困家標準（CNS ) A4規格（210X2S»7公釐〉, 1 I This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) C7 D7 401421 V. Description of the invention (1r) in proline) > 2.48 (lH, ra, CH in the side chain) > 2.86 (211,10,113 ^^) > 3.65 (211,1,2-011 ^ ΐη the proline) > 4.62 (lH, ni, 4-CH in the proline) > 7.75 (1H, s.COOH)] 〇 Example eighty-six l- (3-thio-2-methylpropanoyl) -L-pheny〗. Synthesis of alanine K Met. Hacryloyl -1-phenylalanine instead of Met. Hacry Bu L-proline_, the rest of the method is the same as in Example eighty V. Yield 38%, mp = 122Tj, [α t = + 46 (c = l in CHC1 >), HNMR5ppra: l_16 (3H, d, CI ^), i.57 (lH, t, SH), 2.4 ~ 3.0 (ffl, 5H), 48 (g, lH), 7.0 ~ 7.4 (m, 6H) Example 87 Synthesis of l- (3-thii) -2-methylpropanoyl) -L-tyrosine M Methacyloyl-1 -tyrosiiie replaces Methacyl-L-proline and the other methods are the same as those in Example 85. Yield 41%, m.p_ = 155 ° C, [a = + 32 (c = l in Et.oh) Ή NMR δ PPm: l .m3iI, d, CH now), 1.57 (lH, t, SH), 2.4 to 3.0 (m, 5H), 6.6 (d, 2H), 6.9 (d, 2H), 7.2 (S, 1H) (please first (Read the notes on the back and fill out this page) Printed by the Central Standards Bureau of the Ministry of Economic Affairs, Consumer Cooperatives ～ 12 ～ This paper size uses the Chinese Standard for Household Standards (CNS) A4 (210X2S »7mm>

Claims

公告本 ABCD 六、申請專利範圍 )1421 , 4 一播合成具光學活性之胜肽的方法，该以親核性試劑（例如硫代醋酸）和a ，y3 —不飽和锻基胺基酸化合物在 0〜50Γ之反應溫度，含氛鼠酮、if或酯官能基之有婊溶 _及添加大於0且小於20 %之路易斯酸或三级胺的情狀下，行親核性加成反應* K合成不等虽非對掌異構物的新方法，用來製造具光學活性烷代胺基之雙胜肽；其中，非對掌異構物〔卡#利歐（Captopril)之前猥物〕之结構如式(一），其中Ri為苯基、甲基、乙基，R*為L或D form 之夭然胺基酸或其多肽及其酯類化合物，h為Ο〜（^之烷基。 (請先間讀背面之注意事項再填寫本頁) 0 RVAnnouncement ABCD VI. Application patent scope) 1421, 4 A method for synthesizing optically active peptides, which uses nucleophilic reagents (such as thioacetic acid) and a, y3-unsaturated fordylamino acid compounds in Reaction temperature of 0 ~ 50Γ, nucleophilic addition reaction under the condition of dissolving humorone, if or ester functional group and adding Lewis acid or tertiary amine greater than 0 and less than 20% * K A new method for synthesizing non-para-isomers, used to make bis-peptides with optically active alkylamino groups; among them, non-para-isomers [pre-Captopril] The structure is as shown in formula (I), where Ri is phenyl, methyl, ethyl, R * is L or D form of the natural amino acid or its polypeptide and its ester compound, and h is an alkyl group of 0 ~ (^ (Please read the notes on the back before filling out this page) 0 RV

R2 :經濟部中捸準 :Ά .工消 t 合 η 社印裝 R3R2: Ministry of Economic Affairs, China Standard: Ά.Industrial Consumers' Printing Co., Ltd. R3

R2 :經濟部中捸準 :Ά .工消 t 合 η 社印裝 R3 A8 B8 C8 D8 六、申請專利範圍 ⑵ 方法，其中，反應溶劑為二氮甲烷、三氛甲烷、四氮甲烷、丙酮、甲乙嗣、氰甲烷、四氫呋哺、乙酸乙酯等沸點低於150f之一般有機溶劑。 5. 如申請專利範圍第1項所述之合成具光學活性之胜&的方法，其中，添加劑為氛化鋅、四氛化錫、三氛化鐵、三氛化鋁、三氟化硼、四氛化钛等之路易斯酸（Lewis add) 酒石酸、醋酸、撺樣酸等有機酸或三乙基胺、百路新 (brucine)、組織胺酸（histidine)、奎寧（quinine)三级胺胺或生物驗*添加劑與反應物之莫耳比為> 0:1〜1:1。 6. —種製造卡普利歐的方法，係將nethacryloyl-proline依1-5項所逑方法Μ親核劑（硫代醋酸或硫代苯甲酸）進行親核性加成反懕，而獲得非等量之非對掌異構物，將此異構物以水為溶劑，經數次分段结晶進行光學分割，可得SS forn之卡普利歐前驅物，最後再將硫上之醢基除去，而得具光學活性之卡普利歐之方法。 7. 如申請專利範圍第6項所述之製造卡普利歐的方法，其中Μ水為溶劑而结晶之方法，係將162g AMP-L-p「o丨ine溶於 200nl水中，加熱至溶解後再靜置，gp可得48.6g之晶體。 (請先閲讀背面之注意If項再填寫本頁) 訂 ...¾R2: Standards of the Ministry of Economic Affairs: Ά. Industrial consumer printing and printing R3 A8 B8 C8 D8 VI. Patent application scope ⑵ Method, in which the reaction solvent is diazomethane, trifluoromethane, tetrazomethane, acetone, A common organic solvent with a boiling point of less than 150f, such as methyl ethyl cyanide, methyl cyanide, tetrahydrofuran, and ethyl acetate. 5. The method for synthesizing optically active wins & as described in item 1 of the scope of patent application, wherein the additives are aluminized zinc, tetraammonium tin, triammonium iron, triammonium aluminum, boron trifluoride Lewis acid such as Teflon, Titanium, etc. Organic acids such as tartaric acid, acetic acid, osmotic acid or triethylamine, brucine, histidine, quinine, etc. Mole ratio of amine or bioassay * additive to reactant is > 0: 1 ~ 1: 1. 6. —A method for manufacturing Capriol, which is obtained by adding nethacryloyl-proline nucleophilic agent (thioacetic acid or thiobenzoic acid) according to the method 1-5 described in item 1-5. Non-equivalent non-palladium isomers. This isomer is water-solvent, and is optically divided by several fractional crystallizations to obtain the Capriol precursor of SS forn. Removal of the substrate to obtain an optically active Capriol method. 7. The method of manufacturing Capriol as described in item 6 of the scope of the patent application, wherein the method of crystallizing M water as a solvent is to dissolve 162 g of AMP-Lp o oine in 200 nl of water and heat until dissolved. Let it stand, gp can get 48.6g crystals. (Please read the Note If on the back before filling this page) Order ... ¾

娌濟部中央揉率扃另工消費合作社印袈本紙伕尺度適用中國國家樣準（CNS ) Λ4規格（210X297公釐）Central Ministry of Economic Affairs's rubbing rate (printed by another consumer and consumer cooperative) The standard of this paper is applicable to China National Standard (CNS) Λ4 specification (210X297 mm)