JP7195568B2 - complex - Google Patents
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Description
本発明は、複合体に関する。 The present invention relates to composites.
慢性閉塞性肺疾患(Chronic Obstructive Pulmonary Disease:COPD)は、肺気腫と慢性気管支炎が合併した症状の総称であり、気流閉塞を特徴とする疾患である。この気流閉塞は末梢気道病変と気腫性病変(肺胞壁の破壊)が様々な割合で複合的に作用することで起こる。 Chronic Obstructive Pulmonary Disease (COPD) is a general term for symptoms in which pulmonary emphysema and chronic bronchitis are combined, and is a disease characterized by airflow obstruction. This airflow obstruction is caused by the complex action of small airway lesions and emphysematous lesions (destruction of alveolar walls) in varying proportions.
COPD患者の痰・血液・尿を加水分解処理し、高速液体クロマトグラフ-タンデム質量分析計(Liquid Chromatography-Tandem Mass Spectrometry:LC-MS/MS)で分析すると、肺胞の伸縮を司る弾性線維エラスチンの架橋アミノ酸であり下記式に示されるデスモシンおよびその異性体であり下記式に示されるイソデスモシンが観測される。健常者と比べて、COPD患者におけるそれらの存在量が異なることから、デスモシン類はCOPDのバイオマーカーとして有望視されている。 Sputum, blood, and urine of COPD patients were hydrolyzed and analyzed by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Desmosine represented by the following formula, which is a bridging amino acid, and isodesmosine represented by the following formula, which is an isomer thereof, are observed. Due to their different abundance in COPD patients compared to healthy subjects, desmosines are promising biomarkers for COPD.
一方、デスモシン類の定量法として、HPLCを用いた定量法の他に、酵素免疫測定法等の免疫化学的な手法が考えられるが、デスモシン類は、ハプテンであり、それ自体は免疫原性を有さない。そこで、非特許文献1において、デスモシン類とキャリアタンパク質との結合が検討されている。 On the other hand, as a quantification method for desmosines, in addition to the quantification method using HPLC, an immunochemical method such as an enzyme immunoassay method can be considered. don't have Therefore, in Non-Patent Document 1, the binding of desmosines and carrier proteins is studied.
ところが、非特許文献1においては、デスモシンとタンパク質を結合しようとした際に、デスモシンが4個のアミノ基とカルボキシ基を有しているため、デスモシンがタンパク質に多点で結合した種々の結合体が生じてしまい、抗体を安定的に作製できなかった。 However, in Non-Patent Document 1, when trying to bind desmosine to a protein, since desmosine has four amino groups and a carboxyl group, various conjugates in which desmosine is bound to the protein at multiple points was generated, and the antibody could not be produced stably.
そこで、本発明は、デスモシン類の簡便な定量法に用いることができる物質を提供する。 Accordingly, the present invention provides a substance that can be used for a simple quantitative method for desmosines.
本発明によれば、
デスモシンが、前記デスモシンのピリジン環の4位の側鎖末端において、直接または連結基を介してタンパク質に結合している、複合体が提供される。
According to the invention,
Conjugates are provided wherein desmosine is attached to a protein at the 4-side chain end of the pyridine ring of said desmosine, either directly or via a linking group.
本発明によれば、デスモシン類の簡便な定量法に用いることができる物質を提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, the substance which can be used for the simple quantification method of desmosines can be provided.
以下、本発明の実施形態を具体例に基づいて説明する。複数の実施形態に記載の態様を組み合わせて用いることもできる。 Hereinafter, embodiments of the present invention will be described based on specific examples. Aspects described in multiple embodiments can also be used in combination.
本実施形態において、複合体は、デスモシンが直接または連結基を介してタンパク質に結合した構造を有する。具体的には、本実施形態における複合体は、デスモシンが、デスモシンのピリジン環の4位の側鎖において、たとえばピリジン環の4位の側鎖末端において、直接または連結基を介してタンパク質に結合しているものである。 In this embodiment, the complex has a structure in which desmosine is bound directly or via a linking group to the protein. Specifically, in the conjugate of this embodiment, desmosine is bound to the protein at the 4-side chain of the pyridine ring of desmosine, for example, at the 4-side chain end of the pyridine ring, either directly or via a linking group. It is what we are doing.
本実施形態の複合体は、デスモシンが、デスモシンのピリジン環の4位の側鎖末端においてタンパク質に結合した構造であるため、デスモシン類がタンパク質に安定的に固定化されており、デスモシン類の簡便な定量法に用いることができる。たとえば、本実施形態における複合体を用いることにより、酵素免疫測定法等の免疫化学的な方法によるデスモシン類の検出または定量が可能となる。また、本実施形態における複合体を用いることにより、たとえば、抗デスモシン抗体を得ることも可能となる。
複合体を用いた抗体の製造および抗体を用いるデスモシン類の分析を安定的におこなう観点から、デスモシン1分子中のタンパク質との結合部位の数は、好ましくは1つである。
The complex of the present embodiment has a structure in which desmosine is bound to a protein at the 4-position side chain end of the pyridine ring of desmosine. It can be used for various quantitative methods. For example, the use of the conjugate of the present embodiment enables detection or quantification of desmosines by an immunochemical method such as enzyme immunoassay. Furthermore, by using the conjugate of this embodiment, it is possible to obtain, for example, an anti-desmosine antibody.
From the viewpoint of stably performing antibody production using the conjugate and analysis of desmosines using the antibody, the number of protein-binding sites in one molecule of desmosine is preferably one.
複合体において、デスモシンのタンパク質との結合部位として、たとえばアミノ基およびカルボキシル基からなる群から選択される1種が挙げられる。
好ましくは、デスモシンは、デスモシンのピリジン環の4位の側鎖末端のアミノ基においてタンパク質に結合している。
In the conjugate, the desmosine-protein binding site includes, for example, one selected from the group consisting of an amino group and a carboxyl group.
Preferably, desmosine is attached to the protein at the side chain terminal amino group at position 4 of the pyridine ring of desmosine.
また、複合体中のタンパク質(以下、「キャリアタンパク質」ともよぶ。)の具体例として、牛血清アルブミン(Bovine Serum Albumin:BSA)、スカシガイヘモシアニン(Keyhole Limpet Hemocyanin:KLH)、オブアルブミン(Ovalbumin:OVA)、チログロブリン、フィブリノーゲンからなる群から選択される1種が挙げられる。複合体を安定的に形成する観点から、キャリアタンパク質は、好ましくはBSA、KLHおよびOVAからなる群から選択される1種であり、より好ましくはBSAおよびKLHからからなる群から選択される1種である。 Further, specific examples of proteins in the complex (hereinafter also referred to as "carrier proteins") include bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), ovalbumin: OVA), thyroglobulin, and one selected from the group consisting of fibrinogen. From the viewpoint of stably forming a complex, the carrier protein is preferably one selected from the group consisting of BSA, KLH and OVA, more preferably one selected from the group consisting of BSA and KLH. is.
本実施形態における複合体は、好ましくはデスモシンとキャリアタンパク質P1とが直接または連結基を介して結合してなる。このとき、複合体は、たとえば下記一般式(1)または(2)に示される。 The complex in this embodiment is preferably formed by binding desmosine and carrier protein P1 directly or via a linking group. At this time, the complex is represented, for example, by the following general formula (1) or (2).
上記一般式(1)および(2)中、P1-は、キャリアタンパク質であるP1由来の1価の基であり、nは平均値であり、0より大きく100以下の数である。
また、上記一般式(1)中、L1は単結合または連結基である。上記一般式(2)中、L2は単結合または連結基である。
In the above general formulas (1) and (2), P1− is a monovalent group derived from the carrier protein P1, n is the average value, and is a number greater than 0 and 100 or less.
Further, in the above general formula (1), L1 is a single bond or a linking group. In general formula (2) above, L2 is a single bond or a linking group.
一般式(1)において、L1が連結基であるとき、連結基L1は、具体的には2価の基である。また、連結基L1の具体例として、-CO(CH2)x-L2-、-CO(CH2)x-、-(-CH2-CH2-O-)z-、または、下記式に示される基が挙げられる。上記連結基の具体例において、xは1以上の整数である。デスモシンとタンパク質との間に適度な空間を得る観点から、xは好ましくは2以上、より好ましくは3以上であり、また、好ましくは10以下であり、より好ましくは8以下、さらに好ましくは6以下、さらにより好ましくは5以下である。また、上記連結基の具体例において、zは1以上30以下の数である。 In general formula (1), when L1 is a linking group, the linking group L1 is specifically a divalent group. Specific examples of the linking group L1 include -CO(CH 2 ) x -L2-, -CO(CH 2 ) x -, -(-CH 2 -CH 2 -O-) z -, or groups shown. In specific examples of the above linking groups, x is an integer of 1 or more. From the viewpoint of obtaining an appropriate space between desmosine and protein, x is preferably 2 or more, more preferably 3 or more, and preferably 10 or less, more preferably 8 or less, and still more preferably 6 or less. , and more preferably 5 or less. Moreover, in the specific examples of the above linking group, z is a number of 1 or more and 30 or less.
(上記式中、*は結合手を示す。) (In the above formula, * indicates a bond.)
連結基L1は、縮合剤由来の構造を含んでいてもよい。縮合剤の例として、下記式40および41に記載の化合物が挙げられ、これらのいずれかを用いることができる。 The linking group L1 may contain a structure derived from the condensing agent. Examples of condensing agents include compounds described in formulas 40 and 41 below, any of which can be used.
また、他の縮合剤の具体例として、4-(4,6-ジメトキシ-1,3,5-トリアジン-2-イル)-4-メチルモルフォリニウムクロリドn-ハイドレート(DMT-MM)等のトリアジン系の縮合剤;ジシクロヘキシルカルボジイミド(DCC)、1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩(EDC)等のカルボジイミド系の縮合剤が挙げられる。 Further, specific examples of other condensing agents include 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM), etc. triazine-based condensing agents; carbodiimide-based condensing agents such as dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).
また、連結基L1は、スペーサーとして機能する構造を含んでいてもよい。スペーサーの具体例として、アルキル鎖、エチレングリコール鎖が挙げられる。 In addition, the linking group L1 may contain a structure that functions as a spacer. Specific examples of spacers include alkyl chains and ethylene glycol chains.
また、一般式(2)において、L2が連結基であるとき、連結基L2は、具体的には2価の基である。また、連結基L2の具体例として、ペプチド鎖が挙げられる。 Further, in general formula (2), when L2 is a linking group, the linking group L2 is specifically a divalent group. A specific example of the linking group L2 is a peptide chain.
一般式(1)および(2)において、P1-は、キャリアタンパク質であるP1由来の1価の基である。P1-の具体例として、P1中のアミノ基由来の基、P1中のカルボキシル基由来の基が挙げられる。複合体の製造容易性を高める観点から、P1-は、好ましくはP1中のアミノ基由来の基、または、P1中のカルボキシル基由来の基であり、より好ましくはP1中のアミノ基由来の基である。 In general formulas (1) and (2), P1- is a monovalent group derived from carrier protein P1. Specific examples of P1- include a group derived from an amino group in P1 and a group derived from a carboxyl group in P1. From the viewpoint of increasing the ease of production of the conjugate, P1- is preferably a group derived from an amino group in P1, or a group derived from a carboxyl group in P1, more preferably a group derived from an amino group in P1. is.
一般式(1)および(2)において、nは、1つの複合体中に導入されているデスモシンの数を示し、平均値である。複合体中のデスモシンの固定化密度を高める観点から、nは0より大きい数であり、好ましくは1以上、より好ましくは2以上、さらに好ましくは3以上、さらにより好ましくは4以上である。
また、複合体の製造安定性を高める観点から、nは、たとえば100以下であり、好ましくは50以下、より好ましくは30以下、さらに好ましくは20以下、さらにより好ましくは10以下である。
In general formulas (1) and (2), n indicates the number of desmosines introduced into one complex and is the average value. From the viewpoint of increasing the immobilization density of desmosine in the complex, n is a number greater than 0, preferably 1 or more, more preferably 2 or more, still more preferably 3 or more, and even more preferably 4 or more.
Also, from the viewpoint of enhancing the production stability of the composite, n is, for example, 100 or less, preferably 50 or less, more preferably 30 or less, even more preferably 20 or less, and even more preferably 10 or less.
また、本実施形態における複合体は、デスモシン類の側鎖末端のアミノ基と、キャリアタンパク質中のアミノ基とが結合する構造であってもよい。このとき、複合体は、たとえば下記一般式(3)に示される。 Moreover, the complex in this embodiment may have a structure in which the amino group at the side chain end of the desmosine and the amino group in the carrier protein are bound. At this time, the complex is represented, for example, by the following general formula (3).
(上記一般式(3)中、P2-NH-は、キャリアタンパク質であるP2-NH2由来の1価の基であり、mは平均値であり、0より大きく100以下の数であり、yは2以上15以下の整数である。) (In the above general formula (3), P2-NH- is a monovalent group derived from the carrier protein P2-NH 2 , m is an average value, a number greater than 0 and 100 or less, and y is an integer of 2 or more and 15 or less.)
一般式(3)において、キャリアタンパク質P2-NH2は、リシン残基またはN末端に存在するアミノ基において、デスモシンと結合している。 In general formula (3), carrier protein P2-NH 2 is bound to desmosine at a lysine residue or an amino group present at the N-terminus.
一般式(3)において、mは、1つの複合体中に導入されているデスモシンの数を示し、平均値である。複合体中のデスモシンの固定化密度を高める観点から、mは0より大きい数であり、好ましくは1以上、より好ましくは2以上、さらに好ましくは3以上、さらにより好ましくは4以上である。
また、複合体の製造安定性を高める観点から、mは、たとえば100以下であり、好ましくは50以下、より好ましくは30以下、さらに好ましくは20以下、さらにより好ましくは10以下である。
In general formula (3), m represents the number of desmosines introduced into one complex, and is the average value. From the viewpoint of increasing the immobilization density of desmosine in the complex, m is a number greater than 0, preferably 1 or more, more preferably 2 or more, still more preferably 3 or more, and even more preferably 4 or more.
Also, from the viewpoint of enhancing the production stability of the composite, m is, for example, 100 or less, preferably 50 or less, more preferably 30 or less, even more preferably 20 or less, and even more preferably 10 or less.
また、一般式(3)において、yは、キャリアタンパク質とデスモシンとの間に適度な空間を得る観点から、たとえば2以上であり、好ましくは3以上、より好ましくは4以上であり、また、たとえば15以下であり、好ましくは12以下、より好ましくは10以下、さらに好ましくは8以下、さらにより好ましくは6以下である。 In general formula (3), y is, for example, 2 or more, preferably 3 or more, more preferably 4 or more, from the viewpoint of obtaining an appropriate space between the carrier protein and desmosine. It is 15 or less, preferably 12 or less, more preferably 10 or less, even more preferably 8 or less, and even more preferably 6 or less.
次に、本実施形態における複合体の製造方法を説明する。
本実施形態において、複合体の製造方法は、たとえば、デスモシン類の側鎖末端のアミノ基とタンパク質とを直接または連結基を介して結合させて、複合体を得る工程を含む。
さらに具体的には、複合体の製造方法は、以下の工程を含む。
(工程1)デスモシンの4位の側鎖に、反応性基が選択的に導入されたデスモシン類誘導体を準備する工程
(工程2)デスモシン類誘導体とタンパク質またはその誘導体とを接触させて、複合体を形成する工程
Next, a method for manufacturing a composite according to this embodiment will be described.
In the present embodiment, the method for producing a conjugate includes, for example, a step of binding an amino group at the side chain end of a desmosine to a protein directly or via a linking group to obtain a conjugate.
More specifically, the method for manufacturing the composite includes the following steps.
(Step 1) Step of preparing a desmosine derivative in which a reactive group is selectively introduced to the 4-position side chain of desmosine (Step 2) Contacting the desmosine derivative with a protein or a derivative thereof to form a complex the process of forming
工程1においては、デスモシン類誘導体を準備する。デスモシン類誘導体を合成する場合、たとえば、特開2015-178957号公報に記載の方法を用いて得ることができる。
ここで、デスモシンの4位の側鎖においてタンパク質と選択的に結合させる観点から、デスモシン類誘導体は、好ましくは、デスモシンの4位の側鎖に、反応性基が選択的に導入された化合物である。
反応性基の具体例として、マレイミド基が挙げられる。複合体を安定的に得る観点から、反応性基は、好ましくはマレイミド基である。
たとえば、デスモシンの4位側鎖のアミノ基に反応性基が選択的に導入されたデスモシン誘導体の例として、下記一般式(4)に示される化合物が挙げられる。
In step 1, a desmosine derivative is prepared. When synthesizing a desmosine derivative, it can be obtained, for example, using the method described in JP-A-2015-178957.
Here, from the viewpoint of selectively binding to proteins at the 4-position side chain of desmosine, the desmosine derivative is preferably a compound in which a reactive group is selectively introduced to the 4-position side chain of desmosine. be.
A specific example of a reactive group is a maleimide group. From the viewpoint of stably obtaining the complex, the reactive group is preferably a maleimide group.
Examples of desmosine derivatives in which a reactive group is selectively introduced to the amino group at the 4-position side chain of desmosine include compounds represented by the following general formula (4).
(上記一般式(4)中、yは、前述した一般式(3)におけるyと同じである。)
一般式(4)において、yの好ましい範囲は、一般式(3)において前述した範囲と同じである。
(In general formula (4) above, y is the same as y in general formula (3) described above.)
In general formula (4), the preferred range of y is the same as the range described above in general formula (3).
また、デスモシンの4位の側鎖に反応性基が選択的に導入されたデスモシン類誘導体は、たとえば、デスモシンの4位の側鎖の所定の基を前述した縮合剤と反応させることにより得ることもできる。また、デスモシンの4位の側鎖に、前述したスペーサーを介して反応性基が結合していてもよい。 A desmosine derivative in which a reactive group is selectively introduced into the side chain at the 4-position of desmosine can be obtained, for example, by reacting a predetermined group of the side chain at the 4-position of desmosine with the aforementioned condensing agent. can also Moreover, a reactive group may be bound to the 4-position side chain of desmosine through the aforementioned spacer.
工程2においては、デスモシン類とタンパク質またはその誘導体とを接触させて、タンパク質にデスモシン類を導入することにより、複合体を得る。
タンパク質誘導体の例として、たとえば、タンパク質に、工程1で得られるデスモシン類誘導体における反応性基と反応する基が導入されたものが挙げられる。
たとえば、デスモシン類誘導体における反応性基がマレイミド基である場合、タンパク質中の1級アミノ基をチオール化し、このチオール基とマレイミド基とを反応させて複合体を形成してもよい。1級アミノ基のチオール化の例として、タンパク質と、2-イミノチオラン塩酸塩(Traut's試薬)との反応が挙げられる。
In step 2, a complex is obtained by contacting desmosines with a protein or a derivative thereof to introduce desmosines into the protein.
Examples of protein derivatives include proteins into which groups reactive with reactive groups in the desmosine derivative obtained in step 1 have been introduced.
For example, when the reactive group in the desmosine derivative is a maleimide group, the primary amino group in the protein may be thiolated, and the thiol group and the maleimide group may be reacted to form a complex. An example of thiolation of primary amino groups is the reaction of proteins with 2-iminothiolane hydrochloride (Traut's reagent).
なお、固定化の際には、必要に応じて、N-ヒドロキシスクシンイミド(NHS)、p-ニトロフェノール、ペンタフルオロフェノール等の活性エステル類を併用してもよい。 For immobilization, active esters such as N-hydroxysuccinimide (NHS), p-nitrophenol, and pentafluorophenol may be used in combination, if necessary.
以上の手順により、複合体が得られる。得られた複合体においては、デスモシン類がタンパク質に、デスモシンのピリジン環の4位の側鎖末端において選択的に固定化されているため、デスモシン類の検出や定量に好ましく用いることができる。 A complex is obtained by the above procedure. In the resulting complex, the desmosines are selectively immobilized on the protein at the 4-position side chain terminal of the pyridine ring of desmosines, and therefore can be preferably used for detection and quantification of desmosines.
以上、本発明の実施形態について述べたが、これらは本発明の例示であり、上記以外の様々な構成を採用することもできる。
以下、参考形態の例を付記する。
1. デスモシンが、前記デスモシンのピリジン環の4位の側鎖末端において、直接または連結基を介してタンパク質に結合している、複合体。
2. 前記デスモシンが、前記4位の側鎖末端のアミノ基において前記タンパク質に結合している、1.に記載の複合体。
3. 前記タンパク質が、牛血清アルブミン、スカシガイヘモシアニン、オブアルブミン、チログロブリン、フィブリノーゲンからなる群から選択される1種である、1.または2.に記載の複合体。
4. 当該複合体が下記一般式(1)に示される、1.乃至3.いずれか1つに記載の複合体。
(上記一般式(1)中、L1は単結合または連結基であり、P1-は、キャリアタンパク質であるP1由来の1価の基であり、nは平均値であり、0より大きく100以下の数である。)
5. 当該複合体が下記一般式(2)に示される、4.に記載の複合体。
(上記一般式(2)中、P1-およびnは、ぞれぞれ、前記一般式(1)におけるP1-およびnと同じであり、L2は単結合または連結基である。)
6. 当該複合体が下記一般式(3)に示される、1.乃至5.いずれか1つに記載の複合体。
(上記一般式(3)中、P2-NH-は、キャリアタンパク質であるP2-NH2由来の1価の基であり、mは平均値であり、0より大きく100以下の数であり、yは2以上15以下の整数である。)
7. 前記キャリアタンパク質が、牛血清アルブミン、スカシガイヘモシアニンおよびオブアルブミンからなる群から選択される1種である、4.乃至6.いずれか1つに記載の複合体。
Although the embodiments of the present invention have been described above, these are examples of the present invention, and various configurations other than those described above can also be adopted.
Examples of reference forms are added below.
1. A conjugate wherein desmosine is attached to a protein at the side chain end of position 4 of the pyridine ring of said desmosine, either directly or via a linking group.
2. 1. The desmosine is bound to the protein at the amino group of the side chain terminal of the 4-position; A complex as described in .
3. 1. The protein is one selected from the group consisting of bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, thyroglobulin, and fibrinogen. or 2. A complex as described in .
4. 1. The complex is represented by the following general formula (1); to 3. A conjugate according to any one of the preceding claims.
(In the above general formula (1), L1 is a single bond or a linking group, P1- is a monovalent group derived from P1, which is a carrier protein, n is an average value, greater than 0 and 100 or less number.)
5. 4. The complex is represented by the following general formula (2); A complex as described in .
(In general formula (2) above, P1- and n are the same as P1- and n in general formula (1) above, respectively, and L2 is a single bond or a linking group.)
6. 1. The complex is represented by the following general formula (3); to 5. A conjugate according to any one of the preceding claims.
(In the above general formula (3), P2-NH- is a monovalent group derived from the carrier protein P2-NH 2 , m is an average value, a number greater than 0 and 100 or less, and y is an integer of 2 or more and 15 or less.)
7. 3. the carrier protein is one selected from the group consisting of bovine serum albumin, keyhole limpet hemocyanin and ovalbumin; to 6. A conjugate according to any one of the preceding claims.
以下において、非水系反応は、とくに記載のない場合、いずれも、窒素雰囲気下、マグネチックスターラーで撹拌しながらおこなった。t-ブタノール(tBuOH)は、CaH2を用いた蒸留により乾燥し、活性モレキュラーシーブ上に保存した。DMFはMgSO4を用いた蒸留により乾燥し、活性モレキュラーシーブ上に保存した。トリエチルアミン(Et3N)、ジイソプロピルエチルアミン(iPr2NEt)およびBF3・OEt2は、CaH2上で乾燥した。ジクロロメタン(CH2Cl2)、メタノール(MeOH)、アセトニトリル(MeCN)、ジエチルエーテル(Et2O)、テトラヒドロフラン(THF)、エタノール(EtOH)等の他の溶媒については、市販品を用い、活性モレキュラーシーブ上に保存した。PBS(pH7.4)は、PBSタブレットを200mLの2回蒸留水に溶解して調製した。PBS(pH8.4)は、46mgのNaH2PO4 2H2Oおよび674mgのNa2HPO4を100mLの2回蒸留水に溶解して調製した。ホウ酸緩衝液(pH8.0)は、87mgのB(OH)3および57mgのNa2B4O710H2Oを10mLの2回蒸留水に溶解して調製した。すべての試薬は、とくに記載のない場合、市販品をさらに精製することなく用いた。N,N-ジメチル-4-アミノピリジン(DMAP)およびトリフェニルホスフィン(PPh3)はトルエンを用いて再結晶化した。高光学純度(>99.5%)のアミノ酸保護体、出発物質であるヨードアミノ酸(化合物9および化合物32)ならびにアルキン(化合物8)は渡辺化学工業社より購入した。薄層クロマトグラフィー(TLC)分析は、Merck社製、シリカゲル60 F254プレートを用いておこなった。カラムクロマトグラフィーは、関東化学製、酸性のシリカゲル60(球状、40~50μm)または中性のシリカゲル60N(球状、40~50μm)を用いておこなった。脱塩には、Zebaスピン脱塩カラム(Thermo Fisher Scientific社製、7K MWCO、10mL)またはNAP 5 columns(GEヘルスケア社製)を用いた。インキュベーションおよび濃縮は、バイオクロマト社製のSmart Evaporatorを用いておこなった。 In the following, all non-aqueous reactions were carried out under a nitrogen atmosphere while stirring with a magnetic stirrer, unless otherwise specified. t-Butanol (tBuOH) was dried by distillation with CaH 2 and stored over activated molecular sieves. DMF was dried by distillation with MgSO4 and stored over activated molecular sieves. Triethylamine (Et 3 N), diisopropylethylamine (iPr 2 NEt) and BF 3 .OEt 2 were dried over CaH 2 . Other solvents, such as dichloromethane (CH 2 Cl 2 ), methanol (MeOH), acetonitrile (MeCN), diethyl ether (Et 2 O), tetrahydrofuran (THF), ethanol (EtOH), etc., are commercially available, and active molecular Stored on sieves. PBS (pH 7.4) was prepared by dissolving a PBS tablet in 200 mL of double distilled water. PBS (pH 8.4) was prepared by dissolving 46 mg NaH 2 PO 4 2H 2 O and 674 mg Na 2 HPO 4 in 100 mL double distilled water. Borate buffer (pH 8.0) was prepared by dissolving 87 mg of B (OH) 3 and 57 mg of Na2B4O710H2O in 10 mL of double distilled water. All reagents were obtained from commercial sources without further purification unless otherwise stated. N,N-dimethyl-4-aminopyridine (DMAP) and triphenylphosphine (PPh 3 ) were recrystallized using toluene. Protected amino acids with high optical purity (>99.5%), starting iodoamino acids (compounds 9 and 32) and alkynes (compound 8) were purchased from Watanabe Kagaku Kogyo. Thin layer chromatography (TLC) analysis was performed using Merck silica gel 60 F254 plates. Column chromatography was performed using Kanto Kagaku's acidic silica gel 60 (spherical, 40-50 μm) or neutral silica gel 60N (spherical, 40-50 μm). For desalting, Zeba spin desalting columns (manufactured by Thermo Fisher Scientific, 7K MWCO, 10 mL) or NAP 5 columns (manufactured by GE Healthcare) were used. Incubation and concentration were performed using a Biochromato Smart Evaporator.
UVスペクトルは、JASCO V-730BIO紫外/可視分光光度計(日本分光社製)により測定した。旋光度は、JASCO P-2200デジタル旋光計(日本分光社製)を用い、ナトリウムランプ(λ=589nm)D線にて測定し、本明細書中以下のように表記する:[α]D T(c g/100mL、溶媒)。スピン脱塩カラムは、Sorvall Legend XTR 遠心分離機(Thermo Fisher Scientific社製)にて使用した。1Hおよび13C核磁気共鳴(NMR)スペクトルは日本電子社製JNM-EXC 300分光計(300MHz)または日本電子社製JNM-ECA 500分光計(500MHz)により測定した。1H NMRのデータは、本明細書中以下のように表記する:化学シフト(δ、ppm)、積分値、***(s:一重線、d:二重線、t:三重線、q:四重線、m:多重線)、カップリング定数J(Hz)、帰属。13C NMRのデータは、本明細書中化学シフト(δ、ppm)にて表記する。マトリックス支援レーザー脱離イオン化-飛行時間質量分析(MALDI-TOFMS)スペクトルは、島津製作所社製AXIMA performance装置により測定し、本明細書中、質量電荷比(m/z)で示す。エレクトロスプレーイオン化質量分析(ESI-MS)スペクトルは、日本分光社製JMS-T100LC装置により測定し、本明細書中、質量電荷比(m/z)で示す。 The UV spectrum was measured with a JASCO V-730BIO ultraviolet/visible spectrophotometer (manufactured by JASCO Corporation). The optical rotation is measured using a JASCO P-2200 digital polarimeter (manufactured by JASCO Corporation) under the D line of a sodium lamp (λ = 589 nm), and is represented herein as follows: [α] D T (c g/100 mL, solvent). Spin desalting columns were used in a Sorvall Legend XTR centrifuge (Thermo Fisher Scientific). 1 H and 13 C nuclear magnetic resonance (NMR) spectra were measured with a JEOL JNM-EXC 300 spectrometer (300 MHz) or a JEOL JNM-ECA 500 spectrometer (500 MHz). 1 H NMR data are represented herein as follows: chemical shift (δ, ppm), integral, splitting (s: singlet, d: doublet, t: triplet, q: quadruple doublet, m: multiplet), coupling constant J (Hz), assignment. 13 C NMR data are expressed herein in terms of chemical shifts (δ, ppm). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOFMS) spectra were measured with an AXIMA performance instrument manufactured by Shimadzu Corporation and are presented herein in terms of mass-to-charge ratio (m/z). Electrospray ionization mass spectrometry (ESI-MS) spectra were measured with a JMS-T100LC instrument manufactured by JASCO Corporation, and are shown in the specification as mass-to-charge ratios (m/z).
ピリジニウム化合物のNMRにおける炭素の番号付けは、以下のマレイミド活性化デスモシン(化合物2)に対応する。 The carbon numbering in the NMR of the pyridinium compounds corresponds to the maleimide-activated desmosine (compound 2) below.
(合成例1)
(2S)-2-[(Benzyloxycarbonyl)amino]-3-hydroxypropanoic acid(化合物12)の合成
(Synthesis example 1)
Synthesis of (2S)-2-[(Benzyloxycarbonyl)amino]-3-hydroxypropanoic acid (compound 12)
クロロギ酸ベンジル(50wt%溶液, トルエン中, 0.42 mL, 2.82 mmol, 1.48 eq)を、飽和NaHCO3 (7.6 mg, 0.25M)水溶液中のL-セリン(化合物11)溶液(200 mg, 1.96 mmol, 1.0 eq)に加えた。混合物を室温にて4時間激しく撹拌し、水層をエーテルで抽出した。水溶液を1M HClにて酸性化して酢酸エチルにて抽出し、有機層をNa2SO4上で乾燥した。シリカゲルカラムクロマトグラフィー(CH2Cl2/MeOH = 20:1)にて精製することにより、生成物である化合物12を無色固体(249.6 mg, 1.03 mmol, <69%)として得た; Rf 0.1 (hexane/EtOAc = 2:1); 1H NMR (300 MHz, CDCl3) δ 7.36 (5H, m, Bn), 5.11 (2H, s, CH2Ph), 4.28 (1H, t, J = 4.2 Hz, CH), 3.88 (2H, m, CH2)。 Benzyl chloroformate (50 wt% solution, in toluene, 0.42 mL, 2.82 mmol, 1.48 eq) was added to a solution of L - serine (compound 11) (200 mg, 1.96 mmol, 1.0 eq). The mixture was vigorously stirred at room temperature for 4 hours and the aqueous layer was extracted with ether. The aqueous solution was acidified with 1M HCl, extracted with ethyl acetate and the organic layer was dried over Na 2 SO 4 . Purification by silica gel column chromatography (CH 2 Cl 2 /MeOH = 20:1) gave the product compound 12 as a colorless solid (249.6 mg, 1.03 mmol, <69%); R f 0.1. (hexane/EtOAc = 2:1); 1 H NMR (300 MHz, CDCl 3 ) δ 7.36 (5H, m, Bn), 5.11 (2H, s, CH 2 Ph), 4.28 (1H, t, J = 4.2 Hz, CH), 3.88 (2H, m, CH2 ).
(合成例2)
(S)-tert-Butyl 2-(((benzyloxy)carbonyl)amino)-3-hydroxypropanoate(化合物13)の合成
(Synthesis example 2)
Synthesis of (S)-tert-Butyl 2-(((benzyloxy)carbonyl)amino)-3-hydroxypropanoate (compound 13)
Z-L-セリン(化合物12)(997.3 mg, 4.18 mmol, 1.0 eq)、ベンジルトリエチルアンモニウムクロリド(0.95 mg, 4.18 mmol, 1.0 eq)および炭酸カリウム(3.8 g, 27.2 mmol, 6.5 eq)をMeCN(13 mL)中で室温にて5時間激しく撹拌した。これに2-ブロモ-2-メチルプロパン(5.0 mL, 42.6 mmol, 10.2 eq)を加え、反応混合物を50℃まで加温し、急速に撹拌した。反応混合物は、2~3時間後に非常に高粘度となった。これにさらにMeCN(5 mL)を加えて撹拌を促進し、得られた反応混合物を24時間撹拌した。その後、反応混合物を室温まで冷却し、大部分のMeCNをロータリーエバポレーターにより除去した。そして、反応混合物をEtOAcおよび水の間で分割した。水層をEtOAcにて抽出した。EtOAc層を飽和食塩水にて洗浄し、Na2SO4上での乾燥、濾過および濃縮をおこなった。シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 40:1)で精製し、生成物である化合物13を無色固体(926.3 mg, 3.13 mmol, 75%)として得た; Rf 0.51 (hexane/EtOAc = 1:1); [α]D 20 -16.8 (c 1.1, EtOH); 1H NMR (300 MHz, CDCl3) δ7.36 (5H, m, Bn), 5.63 (1H, m, NH), 5.13 (2H, s, CH2Ph), 4.32 (1H, m, CH), 3.94 (2H, d, J = 3.6 Hz, CH2), 1.48 (9H, s, tBu)。 ZL-serine (compound 12) (997.3 mg, 4.18 mmol, 1.0 eq), benzyltriethylammonium chloride (0.95 mg, 4.18 mmol, 1.0 eq) and potassium carbonate (3.8 g, 27.2 mmol, 6.5 eq) were dissolved in MeCN ( 13 mL) and vigorously stirred at room temperature for 5 hours. To this was added 2-bromo-2-methylpropane (5.0 mL, 42.6 mmol, 10.2 eq) and the reaction mixture was warmed to 50° C. and stirred rapidly. The reaction mixture became very viscous after 2-3 hours. Additional MeCN (5 mL) was added to facilitate stirring and the resulting reaction mixture was stirred for 24 hours. The reaction mixture was then cooled to room temperature and most of the MeCN was removed by rotary evaporation. The reaction mixture was then partitioned between EtOAc and water. The aqueous layer was extracted with EtOAc. The EtOAc layer was washed with brine , dried over Na2SO4 , filtered and concentrated. Purification by silica gel column chromatography (hexane/EtOAc = 40:1) gave the product Compound 13 as a colorless solid (926.3 mg, 3.13 mmol, 75%); R f 0.51 (hexane/EtOAc = 1: 1); [α] D 20 -16.8 (c 1.1, EtOH); 1 H NMR (300 MHz, CDCl 3 ) δ 7.36 (5H, m, Bn), 5.63 (1H, m, NH), 5.13 (2H , s, CH2Ph ), 4.32 (1H, m, CH), 3.94 (2H, d, J = 3.6 Hz, CH2 ), 1.48 (9H, s, tBu).
(合成例3)
(S)-tert-Butyl 2-(((benzyloxy)carbonyl)amino)-3-iodopropanoate(化合物14)の合成
(Synthesis Example 3)
Synthesis of (S)-tert-Butyl 2-(((benzyloxy)carbonyl)amino)-3-iodopropanoate (compound 14)
トリフェニルホスフィン(1.55 g, 6.26 mmol, 2.0 eq)およびイミダゾール(405.1 mg, 6.26 mmol, 2.0 eq)を、窒素雰囲気下、0℃にて撹拌しながらCH2Cl2(9 mL)中に溶解した。ヨウ素粉末(1.51 g, 6.26 mmol, 2.0 eq)を加えた後、混合物を室温まで温め、10分間撹拌したのち、0℃まで冷却した。冷却した溶液に、(S)-tert-butyl 2-(((benzyloxy)carbonyl)amino)-3-hydroxypropanoate(化合物13)(878.6 mg, 3.13 mmol, 1.0 eq)のCH2Cl2(8 mL)溶液を加えた。1時間0℃にて撹拌後、反応混合物を減圧濃縮した。残渣を中性シリカゲルのショートカラムにて濾過し、hexane/Et2O = 1/1にて溶出し、ろ液を濃縮した。シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 10:1)で精製し、化合物14を黄色固体として得た。これをペンタンから再結晶化して無色結晶(993.3 mg, 2.45 mmol, 78%)を得た; Rf 0.68 (hexane/EtOAc = 2:1); [α]D 22 -8.1 (c 1.05, EtOH); 1H NMR (300 MHz, CDCl3) δ7.36 (5H, m, Bn), 5.60 (1H, m, NH), 5.13 (2H, s, CH2Ph), 4.41 (1H, m, CH), 3.59 (2H, d, J = 3.6 Hz, CH2), 1.50 (9H, s, tBu)。 Triphenylphosphine (1.55 g, 6.26 mmol, 2.0 eq) and imidazole (405.1 mg, 6.26 mmol, 2.0 eq) were dissolved in CH 2 Cl 2 (9 mL) with stirring at 0° C. under nitrogen atmosphere. . After adding iodine powder (1.51 g, 6.26 mmol, 2.0 eq), the mixture was allowed to warm to room temperature, stirred for 10 minutes and then cooled to 0°C. To the cooled solution was added (S)-tert-butyl 2-(((benzyloxy)carbonyl)amino)-3-hydroxypropanoate (compound 13) (878.6 mg, 3.13 mmol, 1.0 eq) in CH2Cl2 ( 8 mL). solution was added. After stirring for 1 hour at 0° C., the reaction mixture was concentrated under reduced pressure. The residue was filtered through a neutral silica gel short column, eluted with hexane/Et 2 O=1/1, and the filtrate was concentrated. Purification by silica gel column chromatography (hexane/EtOAc = 10:1) gave compound 14 as a yellow solid. This was recrystallized from pentane to give colorless crystals (993.3 mg, 2.45 mmol, 78%); R f 0.68 (hexane/EtOAc = 2:1); [α] D 22 -8.1 (c 1.05, EtOH). 1 H NMR (300 MHz, CDCl 3 ) δ 7.36 (5H, m, Bn), 5.60 (1H, m, NH), 5.13 (2H, s, CH 2 Ph), 4.41 (1H, m, CH) , 3.59 (2H, d, J = 3.6 Hz, CH2 ), 1.50 (9H, s, tBu).
(合成例4)
Bromo(trimethylsilyl)acetylene(化合物16)の合成
(Synthesis Example 4)
Synthesis of Bromo(trimethylsilyl)acetylene (compound 16)
トリメチルシリルアセチレン(化合物15)(10.8 mL, 75.0 mmol, 1.0 eq)のアセトン(250 mL)溶液にN-ブロモスクシンイミド(NBS)(14.7g, 82.5 mmol, 1.1 eq)およびAgNO3(1.28 g, 7.5 mmol, 0.1 eq)を室温にて加えた。室温にて2時間撹拌後、反応混合物を氷水で反応停止し、ペンタンで抽出し、飽和食塩水で洗浄し、Na2SO4上で乾燥、氷浴を用いて濃縮した。これを蒸留して精製し、化合物16を無色液体(4.77 g, 26.9 mmol, 36%)として得た; 1HNMR (300 MHz, CDCl3) δ 0.19 (9H, s, TMS)。 To a solution of trimethylsilylacetylene (compound 15) (10.8 mL, 75.0 mmol, 1.0 eq) in acetone (250 mL) was added N-bromosuccinimide (NBS) (14.7 g, 82.5 mmol, 1.1 eq) and AgNO 3 (1.28 g, 7.5 mmol). , 0.1 eq) was added at room temperature. After stirring for 2 hours at room temperature, the reaction mixture was quenched with ice water, extracted with pentane, washed with brine, dried over Na 2 SO 4 and concentrated using an ice bath. It was purified by distillation to give compound 16 as a colorless liquid (4.77 g, 26.9 mmol, 36%); 1 HNMR (300 MHz, CDCl 3 ) δ 0.19 (9H, s, TMS).
(合成例5)
(S)-tert-Butyl 2-(((benzyloxy)carbonyl)amino)-5-trimethylsilyl-4-pentynoate(化合物17)の合成
(Synthesis Example 5)
Synthesis of (S)-tert-Butyl 2-(((benzyloxy)carbonyl)amino)-5-trimethylsilyl-4-pentynoate (Compound 17)
CuCN(492.8 mg, 5.50 mmol, 1.0 eq)およびLiCl(466.5 mg, 11.0 mmol, 2.0 eq)を1つのフラスコ中で減圧下150℃にて2時間加熱し一緒に乾燥した。CuCNおよびLiClを乾燥している間に、亜鉛粉末(2.23 g, 34.1 mmol, 6.2 eq)を、ヒートガンを用いて5分間かけて減圧加熱により乾燥した。亜鉛粉末の入ったフラスコにDMF(2.7 mL)およびTMSCl(216 μL, 0.31 mmol)を室温にて加えた。30分間室温にて撹拌した後、この分散液にゆっくりとtert-Butyl 16-(S)-((benzyloxycarbonyl)amino)-15-iodopropanoate(化合物14)(2.23 g, 5.50 mmol, 1.0 eq)のDMF(2.7 mL, 0.55 mLにて洗浄 × 2)溶液を加えた。TLCにより化合物14-Znの生成を確認した後、DMF(10 mL)中にCuCNおよびLiClを含み、-20℃に冷却した別のフラスコに、化合物14-Znの溶液を30分かけて加えた。-20℃にて15分間撹拌後、新たに調製した(2-ブロモエチニル)トリメチルシラン(1.58 mL, 11.0 mmol, 2.0 eq)のDMF(1.67 mL)溶液を反応混合物に5分かけて滴下した。得られた混合物を室温までゆっくりと温め、17時間撹拌を続けた。混合物をEtOAcで希釈し、飽和NH4Cl溶液にて反応停止した。そして、水層をEtOAcで抽出した。有機層を合わせ、飽和食塩水で洗浄し、Na2SO4上で乾燥し、減圧濃縮した。シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 40:1)で精製して化合物17を黄色固体(1.00 g, 2.66 mmol, 49%)として得た; Rf 0.57 (hexane/EtOAc = 4:1); [α]D 25 +183.3 (c 0.10, CHCl3); IR (ATR, cm-1) 2962, 1730, 1506, 1456, 1349, 1251, 1530, 1220, 1159, 1049, 846, 759, 698; 1H NMR (300 MHz, CDCl3) δ 7.36 (5H, m, Bn), 5.60 (1H, d, J = 8.2 Hz, NH), 5.13 (2H, s, CH2Ph), 4.41 (1H, m, CH), 2.78 (2H, m, CH2), 1.50 (9H, s, tBu), 0.13 (9H, s, TMS); 13C NMR (125 MHz, CDCl3) δ 169.2, 155.5, 136.3, 128.6, 128.2, 128.1, 100.7, 88.2, 82.5, 67.0, 52.8, 28.0, 24.4, 0.1。 CuCN (492.8 mg, 5.50 mmol, 1.0 eq) and LiCl (466.5 mg, 11.0 mmol, 2.0 eq) were dried together in one flask under reduced pressure at 150° C. for 2 h. While CuCN and LiCl were drying, zinc powder (2.23 g, 34.1 mmol, 6.2 eq) was dried by heating under reduced pressure using a heat gun for 5 minutes. DMF (2.7 mL) and TMSCl (216 μL, 0.31 mmol) were added to the flask containing zinc powder at room temperature. After stirring for 30 minutes at room temperature, tert-Butyl 16-(S)-((benzyloxycarbonyl)amino)-15-iodopropanoate (compound 14) (2.23 g, 5.50 mmol, 1.0 eq) in DMF was slowly added to the dispersion. (2.7 mL, 0.55 mL wash x 2) solution was added. After confirming the formation of compound 14-Zn by TLC, the solution of compound 14-Zn was added over 30 minutes to another flask containing CuCN and LiCl in DMF (10 mL) cooled to -20°C. . After stirring for 15 minutes at −20° C., a freshly prepared solution of (2-bromoethynyl)trimethylsilane (1.58 mL, 11.0 mmol, 2.0 eq) in DMF (1.67 mL) was added dropwise to the reaction mixture over 5 minutes. The resulting mixture was slowly warmed to room temperature and continued to stir for 17 hours. The mixture was diluted with EtOAc and quenched with saturated NH 4 Cl solution. The aqueous layer was then extracted with EtOAc. The organic layers were combined, washed with saturated brine, dried over Na 2 SO 4 and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane/EtOAc = 40:1) gave compound 17 as a yellow solid (1.00 g, 2.66 mmol, 49%); R f 0.57 (hexane/EtOAc = 4:1); [α] D 25 +183.3 (c 0.10, CHCl 3 ); NMR (300 MHz, CDCl3 ) δ 7.36 (5H, m, Bn), 5.60 (1H, d, J = 8.2 Hz, NH), 5.13 (2H, s, CH2Ph ), 4.41 (1H, m, CH ), 2.78 (2H, m, CH2 ), 1.50 (9H, s, tBu), 0.13 ( 9H , s, TMS); , 128.1, 100.7, 88.2, 82.5, 67.0, 52.8, 28.0, 24.4, 0.1.
(合成例6)
(S)-tert-Butyl 2-(((benzyloxy)carbonyl)amino)-4-pentynoate(化合物8)の合成
(Synthesis Example 6)
Synthesis of (S)-tert-Butyl 2-(((benzyloxy)carbonyl)amino)-4-pentynoate (compound 8)
化合物17(1.37 g, 3.70 mmol, 1.0 eq)のTHF(46.2 mL)およびEtOH(1.08 mL, 18.5 mmol, 5.0 eq)溶液を0℃に冷却し、これにテトラブチルアンモニウムフルオリド(TBAF)(THF中1.0 M溶液; 1.85 mL, 1.85 mmol, 0.5 eq)を加えた。0℃にて1.5時間撹拌後、反応混合物をEtOAcで希釈し、飽和NH4Cl溶液にて反応停止した。そして、水層をEtOAcで抽出した。有機層を合わせ、飽和食塩水で洗浄し、Na2SO4上で乾燥し、減圧濃縮した。シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 10:1)により精製し、化合物8を黄色固体(1.06 g, 3.49 mmol, 96%)として得た; Rf 0.42 (hexane/EtOAc = 4/1); [α]D 25 +78.7 (c 0.10, CHCl3); IR (ATR, cm-1) 3355, 3299, 1722, 1691, 1533, 1342, 1292, 1264, 1158, 1059, 996, 647; 1H NMR (300 MHz, CDCl3) δ 7.36 (5H, m, Bn), 5.60 (1H, d, J = 7.6 Hz, NH), 5.13 (2H, s, CH2Ph), 4.41 (1H, m, CH), 2.74 (2H, m, CH2), 2.00 (1H, t, J = 2.7 Hz, CH) 1.50 (9H, s, tBu); 13C NMR (125 MHz, CDCl3) δ169.4, 165.4, 155.5, 136.1, 128.6, 128.0, 100.7, 82.7, 71.5, 67.0, 52.6, 28.0, 22.9; ESI-HRMS (m/z) calcd for C17H21N1NaO4 [M+Na]+ 326.1368, found 326.1343。 A solution of compound 17 (1.37 g, 3.70 mmol, 1.0 eq) in THF (46.2 mL) and EtOH (1.08 mL, 18.5 mmol, 5.0 eq) was cooled to 0° C., to which tetrabutylammonium fluoride (TBAF) (THF) was added. medium 1.0 M solution; 1.85 mL, 1.85 mmol, 0.5 eq) was added. After stirring for 1.5 hours at 0° C., the reaction mixture was diluted with EtOAc and quenched with saturated NH 4 Cl solution. The aqueous layer was then extracted with EtOAc. The organic layers were combined, washed with saturated brine, dried over Na 2 SO 4 and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane/EtOAc = 10:1) gave Compound 8 as a yellow solid (1.06 g, 3.49 mmol, 96%); R f 0.42 (hexane/EtOAc = 4/1); α] D 25 +78.7 (c 0.10, CHCl 3 ); IR (ATR, cm −1 ) 3355, 3299, 1722, 1691, 1533, 1342, 1292, 1264, 1158, 1059, 996, 647; 300 MHz, CDCl3 ) δ 7.36 (5H, m, Bn), 5.60 (1H, d, J = 7.6 Hz, NH), 5.13 (2H, s, CH2Ph ), 4.41 (1H, m, CH), 2.74 (2H, m, CH2 ), 2.00 (1H, t, J = 2.7 Hz, CH) 1.50 (9H, s, tBu); 13C NMR (125 MHz, CDCl3 ) δ169.4, 165.4, 155.5, 136.1, 128.6, 128.0, 100.7, 82.7, 71.5, 67.0, 52.6, 28.0, 22.9; ESI - HRMS ( m/z) calcd for C17H21N1NaO4 [M+Na] + 326.1368 , found 326.1343.
(合成例7)
4-Methyl L-aspartate hydrochloride(化合物19)の合成
(Synthesis Example 7)
Synthesis of 4-Methyl L-aspartate hydrochloride (compound 19)
L-アスパラギン酸(化合物18)(4.95 g, 37.55 mmol, 1.0 eq)をメタノール(24.5 mL, 16.05 eq)に加え、-10℃に冷却した。塩化チオニル(3.66 mL, 1.34 eq)を混合物に滴下し、冷却槽を除去して溶液をゆっくりと室温まで加温した。25分間置いた後、冷却したジエチルエーテルを混合物に冷却および振とうしながら加え、生成物を白色固体として沈澱させた。この白色固体を直ちに濾過し、氷冷したジエチルエーテルで洗浄し、収集した。L-aspartic acid methyl ester hydrochloride(化合物19)を無色固体(6.60 g, 35.95 mmol, 97%)として得た; 1H NMR (300 MHz, CDCl3) δ 4.32 (1H, t, J = 5.1 Hz, CH), 3.76 (3H, s, Me), 3.07-3.01 (2H, m, CH2)。 L-aspartic acid (compound 18) (4.95 g, 37.55 mmol, 1.0 eq) was added to methanol (24.5 mL, 16.05 eq) and cooled to -10°C. Thionyl chloride (3.66 mL, 1.34 eq) was added dropwise to the mixture and the cooling bath was removed to slowly warm the solution to room temperature. After standing for 25 minutes, chilled diethyl ether was added to the mixture with cooling and shaking to precipitate the product as a white solid. The white solid was immediately filtered, washed with ice cold diethyl ether and collected. L-aspartic acid methyl ester hydrochloride (compound 19) was obtained as a colorless solid (6.60 g, 35.95 mmol, 97%); 1 H NMR (300 MHz, CDCl 3 ) δ 4.32 (1H, t, J = 5.1 Hz, CH), 3.76 (3H, s, Me), 3.07-3.01 (2H, m, CH2 ).
(合成例8)
(S)-20-((tert-Butoxycarbonyl)amino)-18-methoxy-21-oxobutanoic acid(化合物20)の合成
(Synthesis Example 8)
Synthesis of (S)-20-((tert-Butoxycarbonyl)amino)-18-methoxy-21-oxobutanoic acid (compound 20)
L-aspartic acid methyl ester hydrochloride(化合物19)(1.90 g, 10.4 mmol, 1.0 eq)のジオキサン(38.3 mL)溶液を撹拌し、0℃にて、これにNa2CO3(0.5 M, 41.4 mL, 2.0 eq)水溶液を加え、次いでBoc2O(2.62 mL, 11.4 mmol, 1.1 eq)を加えた。反応混合物を室温にて18時間撹拌した後、濃縮した。得られた溶液に1M HClを加えて0℃にてpH3.0に酸性化し、EtOAcにて抽出した。有機層を合わせて水で洗浄し、Na2SO4上で乾燥し、減圧濃縮した。シリカゲルカラムクロマトグラフィー(MeOH/CH2Cl2, 1/5)で精製し、化合物20を黄色油状物(2.27 g, 9.18 mmol, 91%)として得た; Rf 0.64 (MeOH/CH2Cl2 = 1:5); 1H NMR (300 MHz, CDCl3) δ 5.56 (1H, d, J = 8.64 Hz, NH), 4.62 (1H, m, H20), 3.72 (3H, s, OCH3), 3.04 (1H, dd, J = 17.2, 4.5 Hz, H19), 2.85 (1H, dd, J = 17.4, 4.9 Hz, H19), 1.43 (9H, s, tBu)。 A solution of L-aspartic acid methyl ester hydrochloride (compound 19) (1.90 g, 10.4 mmol, 1.0 eq) in dioxane (38.3 mL) was stirred and at 0° C. Na 2 CO 3 (0.5 M, 41.4 mL, 2.0 eq) aqueous solution was added, followed by Boc 2 O (2.62 mL, 11.4 mmol, 1.1 eq). The reaction mixture was stirred at room temperature for 18 hours and then concentrated. The resulting solution was acidified to pH 3.0 by adding 1M HCl at 0° C. and extracted with EtOAc. The combined organic layers were washed with water, dried over Na2SO4 and concentrated in vacuo. Purification by silica gel column chromatography ( MeOH/CH2Cl2, 1/5) gave compound 20 as a yellow oil (2.27 g, 9.18 mmol, 91%); Rf 0.64 ( MeOH/CH2Cl2 = 1:5); 1 H NMR (300 MHz, CDCl 3 ) δ 5.56 (1H, d, J = 8.64 Hz, NH), 4.62 (1H, m, H20), 3.72 (3H, s, OCH 3 ), 3.04. (1H, dd, J = 17.2, 4.5 Hz, H19), 2.85 (1H, dd, J = 17.4, 4.9 Hz, H19), 1.43 (9H, s, tBu).
(合成例9)
(S)-21-tert-Butyl 18-methyl-20-((tert-butoxycarbonyl)amino)oxobutanedioate(化合物21)の合成
(Synthesis Example 9)
Synthesis of (S)-21-tert-Butyl 18-methyl-20-((tert-butoxycarbonyl)amino)oxobutanedioate (compound 21)
化合物20(1.16 mg, 4.69 mmol, 1.0 eq)、ベンジルトリエチルアンモニウムクロリド(1.06 mg, 4.69 mmol, 1.0 eq)および炭酸カリウム(4.22 g, 30.5 mmol, 6.5 eq)の化合物をMeCN(15.6 mL)中、室温中で5時間激しく撹拌した。2-ブロモ-2-メチルプロパン(5.37 mL, 47.8 mmol, 10.2 eq)を加え、反応混合物を50℃に温めて急速に撹拌した。反応混合物は2~3時間後に非常に高粘度となった。これにさらにMeCN(7 mL)を加えて撹拌を促進し、得られた反応混合物を24時間撹拌した。その後、反応混合物を室温まで冷却し、大部分のMeCNをロータリーエバポレーターにより除去した。そして、反応混合物をEtOAcおよび水の間で分割した。水層をEtOAcにて抽出した。EtOAc層を飽和食塩水にて洗浄し、Na2SO4上での乾燥、濾過および濃縮をおこなった。シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 8:1)で精製し、生成物である化合物21を無色固体(1.14 g, 3.76 mmol, 80%)として得た; Rf 0.58 (hexane/EtOAc = 2:1); 1H NMR (300 MHz, CDCl3) δ5.43 (1H, d, J = 8.8 Hz, NH), 4.48-4.42 (1H, m, H20), 3.69 (3H, s, OCH3), 2.95 (1H, dd, J = 16.6, 4.6 Hz, H19), 2.76 (1H, dd, J = 16.6, 5.0 Hz, H19), 1.45 (9H, s, tBu), 14.8 (9H, s, tBu)。 Compound 20 (1.16 mg, 4.69 mmol, 1.0 eq), benzyltriethylammonium chloride (1.06 mg, 4.69 mmol, 1.0 eq) and potassium carbonate (4.22 g, 30.5 mmol, 6.5 eq) were dissolved in MeCN (15.6 mL). Stir vigorously at room temperature for 5 hours. 2-Bromo-2-methylpropane (5.37 mL, 47.8 mmol, 10.2 eq) was added and the reaction mixture was warmed to 50° C. and stirred rapidly. The reaction mixture became very viscous after 2-3 hours. Additional MeCN (7 mL) was added to this to facilitate stirring and the resulting reaction mixture was stirred for 24 hours. The reaction mixture was then cooled to room temperature and most of the MeCN was removed by rotary evaporation. The reaction mixture was then partitioned between EtOAc and water. The aqueous layer was extracted with EtOAc. The EtOAc layer was washed with brine , dried over Na2SO4 , filtered and concentrated. Purification by silica gel column chromatography (hexane/EtOAc = 8:1) gave the product Compound 21 as a colorless solid (1.14 g, 3.76 mmol, 80%); R f 0.58 (hexane/EtOAc = 2: 1); 1 H NMR (300 MHz, CDCl 3 ) δ 5.43 (1H, d, J = 8.8 Hz, NH), 4.48-4.42 (1H, m, H20), 3.69 (3H, s, OCH 3 ), 2.95 (1H, dd, J = 16.6, 4.6 Hz, H19), 2.76 (1H, dd, J = 16.6, 5.0 Hz, H19), 1.45 (9H, s, tBu), 14.8 (9H, s, tBu).
(合成例10)
(S)-21-tert-Butyl 20-((tert-butoxycarbonyl)amino)-18-oxobutanoic acid(化合物22)の合成
(Synthesis Example 10)
Synthesis of (S)-21-tert-Butyl 20-((tert-butoxycarbonyl)amino)-18-oxobutanoic acid (compound 22)
化合物12(500.7 mg, 1.65 mmol, 1.0 eq)のMeOH(0.75 mL)溶液に、2M NaOH(0.85 mL)を加えた。室温にて3時間撹拌後、MeOHを減圧下で除去し、残った水層をエーテルで洗浄した。そして、水層に3M HClを加えてpH2に酸性化し、EtOAcにより抽出した。有機層を合わせて飽和食塩水で洗浄し、Na2SO4上で乾燥し、減圧濃縮した。シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 3/1)で精製して化合物22(437 mg, 1.51 mmol, 92%)を無色固体として得た; 1H NMR (300 MHz, CDCl3) δ5.40 (1H, d, NH), 4.47-4.43 (1H, m, H20), 3.00 (2H, dd, J = 18.5, 4.6 Hz, H19), 2.84 (2H, dd, J = 17.2, 5.02 Hz, H19), 1.48 (9H, s, tBu), 1.45 (9H, s, tBu)。 To a solution of compound 12 (500.7 mg, 1.65 mmol, 1.0 eq) in MeOH (0.75 mL) was added 2M NaOH (0.85 mL). After stirring for 3 hours at room temperature, MeOH was removed under reduced pressure and the remaining aqueous layer was washed with ether. The aqueous layer was then acidified to pH 2 by adding 3M HCl and extracted with EtOAc. The organic layers were combined, washed with saturated brine, dried over Na 2 SO 4 and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane/EtOAc = 3/1) gave Compound 22 (437 mg, 1.51 mmol, 92%) as a colorless solid; 1 H NMR (300 MHz, CDCl 3 ) δ5.40 ( 1H, d, NH), 4.47-4.43 (1H, m, H20), 3.00 (2H, dd, J = 18.5, 4.6 Hz, H19), 2.84 (2H, dd, J = 17.2, 5.02 Hz, H19), 1.48 (9H, s, tBu), 1.45 (9H, s, tBu).
(合成例11)
(S)-21-tert-Butyl 20-((tert-butoxycarbonyl)amino)-18-hydroxybutanoate(化合物23)の合成
(Synthesis Example 11)
Synthesis of (S)-21-tert-Butyl 20-((tert-butoxycarbonyl)amino)-18-hydroxybutanoate (compound 23)
化合物22(1.33 mg, 4.60 mmol, 1.0 eq)のTHF(95.8 mL)溶液を-15℃に冷却し、そこへN-メチルモルホリン(556 μL, 5.06 mmol, 1.1 eq)およびEtOCOCl(481.3 μL, 5.06 mmol, 1.1 eq)を加えた。-15℃で1時間撹拌した後、沈澱したN-メチルモルホリン塩酸塩を濾過して除去し、THF(73.2 mL)にて洗浄した。ろ液を0℃に冷却してこれにNaBH4(257 mg, 6.89 mmol, 1.5 eq)を加え、次いで水を滴下した。0℃にて0.5時間撹拌後、反応混合物に飽和NH4Cl溶液を加えて反応停止した。そして、水層をEtOAcで抽出した。有機層を合わせて飽和食塩水で洗浄し、Na2SO4上で乾燥し、減圧濃縮した。シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 3/1)で精製してアルコールである化合物23を白色固体(1.24 mg, 4.50 mmol, 98%)として得た; Rf 0.30 (hexane/EtOAc = 2:1); 1H NMR (300 MHz, CDCl3) δ 5.34 (1H, d, J = 6.6 Hz, NH), 4.38-4.31 (1H, m, H20), 3.71-3.63 (2H, m, H19), 2.19-2.04 (1H, m, H18), 1.67-1.58 (1H, m, H19), 1.47 (9H, s, tBu), 1.44 (9H, s, tBu)。 A solution of compound 22 (1.33 mg, 4.60 mmol, 1.0 eq) in THF (95.8 mL) was cooled to −15° C., and N-methylmorpholine (556 μL, 5.06 mmol, 1.1 eq) and EtOCOCl (481.3 μL, 5.06 μL) were added thereto. mmol, 1.1 eq) was added. After stirring for 1 hour at -15°C, the precipitated N-methylmorpholine hydrochloride was filtered off and washed with THF (73.2 mL). The filtrate was cooled to 0° C. and NaBH 4 (257 mg, 6.89 mmol, 1.5 eq) was added to it, followed by dropwise addition of water. After stirring for 0.5 h at 0° C., the reaction mixture was quenched with saturated NH 4 Cl solution. The aqueous layer was then extracted with EtOAc. The organic layers were combined, washed with saturated brine, dried over Na 2 SO 4 and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane/EtOAc = 3/1) gave alcohol Compound 23 as a white solid (1.24 mg, 4.50 mmol, 98%); R f 0.30 (hexane/EtOAc = 2:1). ); 1 H NMR (300 MHz, CDCl 3 ) δ 5.34 (1H, d, J = 6.6 Hz, NH), 4.38-4.31 (1H, m, H20), 3.71-3.63 (2H, m, H19), 2.19 -2.04 (1H, m, H18), 1.67-1.58 (1H, m, H19), 1.47 (9H, s, tBu), 1.44 (9H, s, tBu).
(合成例12)
(S)-21-tert-Butyl 20-((tert-butoxycarbonyl)amino)-18-iodobutanoate(化合物9)の合成
(Synthesis Example 12)
Synthesis of (S)-21-tert-Butyl 20-((tert-butoxycarbonyl)amino)-18-iodobutanoate (compound 9)
トリフェニルホスフィン(2.51 g, 9.0 mmol, 2.0 eq)およびイミダゾール(653.0 mg, 9.0 mmol, 2.0 eq)を、窒素雰囲気下、20℃で撹拌しながら、CH2Cl2(14.2 mL)に溶解した。ヨウ素粉末(2.43 g, 9.0 mmol, 2.0 eq)を加えた後、混合物を室温まで温め、10分間撹拌したのち、0℃まで冷却した。この冷却溶液に、化合物23すなわち(S)-21-tert-butyl 20-((tert-butoxycarbonyl)amino)-18-hydroxybutanoate(1.32 g, 4.5 mmol, 1.0 eq)のCH2Cl2(15 mL)溶液を加えた。0℃にて1時間撹拌後、反応混合物を減圧濃縮した。残渣を中性シリカゲルのショートカラムに通し、hexane/Et2O = 1/1にて溶出し、ろ液を濃縮した。シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 10:1)で精製し、化合物9を白色固体(1.69 mg, 4.38 mmol, 92%)として得た; Rf 0.68 (hexane/EtOAc = 2:1); 1H NMR (300 MHz, CDCl3)δ 5.06 (1H, s, NH), 4.22-4.17 (1H, m, H20), 3.20-3.14 (2H, m, H19), 2.44-2.32 (1H, m, H18), 2.21-2.09 (1H, m, H19), 1.45 (9H, s, tBu), 1.48 (9H, s, tBu)。 Triphenylphosphine (2.51 g, 9.0 mmol, 2.0 eq) and imidazole (653.0 mg, 9.0 mmol, 2.0 eq) were dissolved in CH 2 Cl 2 (14.2 mL) with stirring at 20° C. under a nitrogen atmosphere. After adding iodine powder (2.43 g, 9.0 mmol, 2.0 eq), the mixture was allowed to warm to room temperature, stirred for 10 minutes and then cooled to 0°C. To this cooled solution was added compound 23, i.e. (S)-21-tert-butyl 20-((tert-butoxycarbonyl)amino)-18 - hydroxybutanoate (1.32 g, 4.5 mmol, 1.0 eq) in CH2Cl2 ( 15 mL). solution was added. After stirring at 0° C. for 1 hour, the reaction mixture was concentrated under reduced pressure. The residue was passed through a short column of neutral silica gel, eluted with hexane/Et 2 O=1/1, and the filtrate was concentrated. Purification by silica gel column chromatography (hexane/EtOAc = 10:1) gave compound 9 as a white solid (1.69 mg, 4.38 mmol, 92%); R f 0.68 (hexane/EtOAc = 2: 1 ); H NMR (300 MHz, CDCl3 ) δ 5.06 (1H, s, NH), 4.22-4.17 (1H, m, H20), 3.20-3.14 (2H, m, H19), 2.44-2.32 (1H, m, H18 ), 2.21-2.09 (1H, m, H19), 1.45 (9H, s, tBu), 1.48 (9H, s, tBu).
(合成例13)
1-Benzyl-5-methyl-2-(S)-[(tert-butoxycarbonyl)-amino]-pentanedioate(化合物25)の合成
(Synthesis Example 13)
Synthesis of 1-Benzyl-5-methyl-2-(S)-[(tert-butoxycarbonyl)-amino]-pentanedioate (compound 25)
トリエチルアミン(0.185 mL, 1.33 mmol, 1.5 eq)を、2-(S)-[(tert-butoxycarbonyl)amino]-pentanedionic acid 1-benzyl ester(化合物24)(300.0 mg, 0.889 mmol, 1.0 eq)のCH2Cl2(4.03 mL)溶液に添加した。混合物を0℃に冷却し、その後、DMAP(10.9 mg, 88.9 μmol, 0.1 eq)およびMeOCOCl(82.3 μL, 1.07 mmol, 1.2 eq)を順次添加した。この溶液を室温まで温め、1時間撹拌した。反応混合物をCH2Cl2で希釈し、1M NaHCO3水溶液で洗浄した。水層をCH2Cl2で抽出した。有機層を合わせて飽和食塩水で洗浄し、Na2SO4上で乾燥し、減圧濃縮した。シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 4:1)にて精製し、1-benzyl-5-methyl-2-(S)-[(tert-butoxycarbonyl)-amino]-pentanedioate(化合物25)を無色固体(272.2 mg, 77.5 μmol, 87%)として得た; Rf 0.68 (hexane/EtOAc = 1:1); 1H NMR (300 MHz, CDCl3) δ7.40-7.32 (5H, m, Bn), 5.17 (2H, dd, J = 13.2, 12.3 Hz, Bn), 5.10 (1H, m, NH), 4.37 (1H, m, CH), 3.67 (3H, s, CO2CH3), 2.48-2.30 (2H, m, CH2), 2.27-2.12 (1H, m, CHCH2CH2), 2.01-1.89 (1H, m, CHCH2CH2), 1.42 (9H, s, Boc)。 Triethylamine (0.185 mL, 1.33 mmol, 1.5 eq) was added to 2-(S)-[(tert-butoxycarbonyl)amino]-pentanedionic acid 1-benzyl ester (compound 24) (300.0 mg, 0.889 mmol, 1.0 eq) in CH 2 Cl 2 (4.03 mL) was added to the solution. The mixture was cooled to 0° C., then DMAP (10.9 mg, 88.9 μmol, 0.1 eq) and MeOCOCl (82.3 μL, 1.07 mmol, 1.2 eq) were added sequentially. The solution was warmed to room temperature and stirred for 1 hour. The reaction mixture was diluted with CH 2 Cl 2 and washed with 1M aqueous NaHCO 3 solution. The aqueous layer was extracted with CH2Cl2 . The organic layers were combined, washed with saturated brine, dried over Na 2 SO 4 and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane/EtOAc = 4:1) gave 1-benzyl-5-methyl-2-(S)-[(tert-butoxycarbonyl)-amino]-pentanedioate (Compound 25) as a colorless solid. (272.2 mg, 77.5 μmol, 87%); R f 0.68 (hexane/EtOAc = 1:1); 1 H NMR (300 MHz, CDCl 3 ) δ7.40-7.32 (5H, m, Bn), 5.17 (2H, dd, J = 13.2, 12.3 Hz, Bn), 5.10 (1H, m, NH), 4.37 (1H, m, CH), 3.67 ( 3H , s, CO2CH3 ), 2.48-2.30 ( 2H, m, CH2 ), 2.27-2.12 ( 1H, m, CHCH2CH2 ), 2.01-1.89 (1H, m, CHCH2CH2 ), 1.42 (9H, s, Boc).
(合成例14)
2-(S)-[(tert-Butoxycarbonyl)-amino]-pentanedioic acid 1-tert-butyl ester 5-methyl ester(化合物26)の合成
(Synthesis Example 14)
Synthesis of 2-(S)-[(tert-Butoxycarbonyl)-amino]-pentanedioic acid 1-tert-butyl ester 5-methyl ester (compound 26)
1-benzyl-5-methyl-2-(S)-[(tert-butoxycarbonyl)-amino]-pentanedioate(化合物25)(303.5 mg, 0.864 mmol, 1.0 eq)のMeOH(1.4 mL)溶液を、10% Pd/C(9.19 mg, 8.64 μmol, 1.0 mol%)にて処理し、室温中、バルーン圧にて接触水素化した。室温中で3時間撹拌後、中性シリカゲルおよびセライトに通し、MeOHで溶出して不溶物を濾別した。そして、ろ液を減圧濃縮した。ろ液の濃縮物から5-methyl-2-(S)-[(tert-butoxycarbonyl)-amino]-pentanedioate(化合物25')(229.9 mg, 0.864 mmol, quant)を無色固体として得た; Rf 0.18 (hexane/EtOAc = 1:1); 1H NMR (300 MHz, CDCl3) δ5.10 (1H, m, NH), 4.37 (1H, m, CH), 3.66 (3H, s, CO2CH3), 2.50-2.44 (2H, m, CH2), 2.04-2.02 (1H, m, CHCH2CH2), 2.04-2.00 (1H, m, CHCH2CH2), 1.45 (9H, s, tBu)。 1-benzyl-5-methyl-2-(S)-[(tert-butoxycarbonyl)-amino]-pentanedioate (Compound 25) (303.5 mg, 0.864 mmol, 1.0 eq) in MeOH (1.4 mL) was diluted with 10% It was treated with Pd/C (9.19 mg, 8.64 μmol, 1.0 mol%) and catalytically hydrogenated at room temperature and balloon pressure. After stirring at room temperature for 3 hours, the mixture was passed through neutral silica gel and celite, and eluted with MeOH to filter off insoluble matter. The filtrate was then concentrated under reduced pressure. Concentration of the filtrate gave 5-methyl-2-(S)-[(tert-butoxycarbonyl)-amino]-pentanedioate (compound 25') (229.9 mg, 0.864 mmol, quant) as a colorless solid; R f 0.18 (hexane/EtOAc = 1: 1 ); 1H NMR (300 MHz, CDCl3 ) δ5.10 (1H, m, NH), 4.37 (1H, m, CH), 3.66 (3H, s, CO2CH 3 ), 2.50-2.44 (2H, m, CH2 ), 2.04-2.02 ( 1H, m, CHCH2CH2 ), 2.04-2.00 (1H, m, CHCH2CH2 ), 1.45 (9H, s, tBu ).
5-Methyl-2-(S)-[(tert-butoxycarbonyl)-amino]-pentanedioate(化合物25')(8.13 g, 31.5 mmol, 1.0 eq)のtBuOH(21 mL)溶液に、(Boc)2O(8.68 ml, 37.8 mmol, 1.2 eq)のtBuOH(42 mL)溶液およびDMAP(384.8 mg, 3.15 μmol, 0.1 eq)を添加した。反応混合物を3時間撹拌した。ショートカラムに通した後、シリカゲルクロマトグラフィー(hexane/EtOAc = 5:1)にて精製し、2-(S)-[(tert-butoxycarbonyl)-amino]-pentanedioic acid 1-tert-butyl ester 5-methyl ester(化合物26)を無色油状物(5.9 g, 18.6 mmol, 63%)として得た; Rf 0.43 (hexane/EtOAc = 2:1); 1H NMR (300 MHz, CDCl3) δ 5.10 (1H, m, NH), 4.12 (1H, dd, J = 9.6, 4.8 Hz, CH), 3.67 (3H, s, CO2CH3), 2.57-2.45 (1H, m, CHCH2CH2), 2.44-2.36 (1H, m, CH2), 2.27-2.15 (1H, m, CHCH2CH2), 1.50 (9H, s, tBu), 1.45 (9H, s, tBu)。 5-Methyl-2-(S)-[(tert-butoxycarbonyl)-amino]-pentanedioate (compound 25′) (8.13 g, 31.5 mmol, 1.0 eq) in tBuOH (21 mL) was added with (Boc) 2 O (8.68 ml, 37.8 mmol, 1.2 eq) in tBuOH (42 mL) and DMAP (384.8 mg, 3.15 μmol, 0.1 eq) were added. The reaction mixture was stirred for 3 hours. After passing through a short column, it was purified by silica gel chromatography (hexane/EtOAc = 5:1) to obtain 2-(S)-[(tert-butoxycarbonyl)-amino]-pentanedioic acid 1-tert-butyl ester 5- The methyl ester (compound 26) was obtained as a colorless oil (5.9 g, 18.6 mmol, 63%); R f 0.43 (hexane/EtOAc = 2:1); 1 H NMR (300 MHz, CDCl 3 ) δ 5.10 ( 1H, m, NH), 4.12 (1H, dd, J = 9.6, 4.8 Hz, CH), 3.67 ( 3H , s, CO2CH3 ), 2.57-2.45 (1H, m, CHCH2CH2 ), 2.44 -2.36 (1H, m, CH2 ), 2.27-2.15 (1H, m, CHCH2CH2 ), 1.50 (9H, s, tBu), 1.45 (9H, s, tBu).
(合成例15)
1-tert-Butyl-5-methyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-pentanedioate(化合物27)の合成
(Synthesis Example 15)
Synthesis of 1-tert-Butyl-5-methyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-pentanedioate (compound 27)
まず、DMAP(9.58 mg, 78.4 μmol, 0.2 eq)および2-(S)-[(tert-butoxycarbonyl)-amino]-pentanedioic acid 1-tert-butyl ester 5-methyl ester(化合物26)(124.4 g, 0.392 mmol, 1.0 eq)をMeCN(1.23 mL)に溶解した溶液に加えた。この混合物に、(Boc)2O(0.36 ml, 1.57 mmol, 4.0 eq)のMeCN(258 μL)溶液を室温にて加え、20時間撹拌した。シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 8:1)にて精製し、1-tert-butyl-5-methyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-pentanedioate(化合物27)を無色油状物(153 mg, 0.366 mmol, 94%)として得た; Rf 0.50 (hexane/EtOAc = 4:1); 1H NMR (300 MHz, CDCl3) δ 4.97 (1H, dd, J = 9.6, 4.8 Hz, CH), 3.67 (3H, s, CO2CH3), 2.57-2.45 (1H, m, CHCH2CH2), 2.44-2.36 (2H, m, CH2), 2.27-2.15 (1H, m, CHCH2CH2), 1.50 (18H, s, tBu), 1.45 (9H, s, tBu)。 First, DMAP (9.58 mg, 78.4 μmol, 0.2 eq) and 2-(S)-[(tert-butoxycarbonyl)-amino]-pentanedioic acid 1-tert-butyl ester 5-methyl ester (Compound 26) (124.4 g, 0.392 mmol, 1.0 eq) was added to a solution in MeCN (1.23 mL). A solution of (Boc) 2 O (0.36 ml, 1.57 mmol, 4.0 eq) in MeCN (258 μL) was added to the mixture at room temperature and stirred for 20 hours. Purification by silica gel column chromatography (hexane/EtOAc = 8:1) gave 1-tert-butyl-5-methyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-pentanedioate (Compound 27 ) as a colorless oil (153 mg, 0.366 mmol, 94%); R f 0.50 (hexane/EtOAc = 4:1); 1 H NMR (300 MHz, CDCl 3 ) δ 4.97 (1H, dd, J = 9.6, 4.8 Hz, CH), 3.67 ( 3H , s, CO2CH3 ), 2.57-2.45 (1H, m, CHCH2CH2 ), 2.44-2.36 (2H, m, CH2 ), 2.27-2.15 (1H, m , CHCH2CH2 ), 1.50 (18H, s, tBu), 1.45 (9H, s, tBu).
(合成例16)
(S)-2-[Bis-(tert-butoxycarbonylamino)]-4-(formylbutanoic acid)-tert-butyl ester(化合物28)の合成
(Synthesis Example 16)
Synthesis of (S)-2-[Bis-(tert-butoxycarbonylamino)]-4-(formylbutanoic acid)-tert-butyl ester (compound 28)
水素化ジイソブチルアルミニウム(DIBAL-H, ヘキサン中 1M溶液, 1.68 mL, 1.68 mmol, 1.4 eq)を、-78℃に冷却した1-tert-butyl-5-methyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-pentanedioate(化合物27)(502.2 mg, 1.20 mmol, 1.0 eq)のEt2O(12 mL)溶液に、3分かけて滴下した。反応混合物を5分間撹拌し、水で反応停止した後、室温まで温めた。得られた白色濃厚液をセライトパウダーで濾過し、Et2Oで洗浄した。ろ液を濃縮し、シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 4:1)で精製して(S)-2-[bis-(tert-butoxycarbonylamino)]-4-(formylbutanoic acid)-tert-butyl ester(化合物28)を無色油状物(417.2 mg, 1.08 mmol, 90%)として得た; Rf 0.38 (hexane/EtOAc = 4:1); 1H NMR (300 MHz, CDCl3) δ 9.77 (1H, s, CHO), 4.92 (1H, dd, J = 9.4, 5.0 Hz, CH), 2.69-2.44 (3H, m, CHCH2CH2), 2.24-2.13 (1H, m, CHCH2CH2), 1.50 (18H, s, tBu), 1.45 (9H, s, tBu)。 Diisobutylaluminum hydride (DIBAL-H, 1 M solution in hexane, 1.68 mL, 1.68 mmol, 1.4 eq) was cooled to −78° C. to give 1-tert-butyl-5-methyl-2-(S)-[bis- A solution of (tert-butoxycarbonyl)-amino]-pentanedioate (Compound 27) (502.2 mg, 1.20 mmol, 1.0 eq) in Et 2 O (12 mL) was added dropwise over 3 minutes. The reaction mixture was stirred for 5 minutes, quenched with water and then warmed to room temperature. The resulting white thick liquid was filtered through celite powder and washed with Et2O . The filtrate was concentrated and purified by silica gel column chromatography (hexane/EtOAc = 4:1) to give (S)-2-[bis-(tert-butoxycarbonylamino)]-4-(formylbutanoic acid)-tert-butyl ester (Compound 28) was obtained as a colorless oil (417.2 mg, 1.08 mmol, 90%); R f 0.38 (hexane/EtOAc = 4:1); 1 H NMR (300 MHz, CDCl 3 ) δ 9.77 (1H, s, CHO), 4.92 (1H, dd, J = 9.4, 5.0 Hz, CH), 2.69-2.44 (3H, m, CHCH2CH2 ), 2.24-2.13 (1H, m, CHCH2CH2 ), 1.50 (18H, s, tBu), 1.45 (9H, s, tBu).
(合成例17)
Benzyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-5-hexenoat(化合物29)の合成
(Synthesis Example 17)
Synthesis of Benzyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-5-hexenoat (compound 29)
nBuLi(ヘキサン中 2.65M溶液, 0.215 mL, 0.569 mmol, 2.0 eq)を、メチルトリフェニルホスホニウムブロミド(223.9 mg, 0.67 mmol, 2.2 eq)のTHF(3.45 mL)分散液に、-78℃にて滴下した。混合物を0℃で1時間撹拌した後、得られたオレンジ色のイリド液に、 (S)-2-[bis-(tert-butoxycarbonylamino)]-4-(formylbutanoic acid)-tert-butyl ester(化合物28)(110.4 g, 0.285 mmol, 1.0 eq)のTHF(1.3 mL)液を-78℃にて添加した。0℃にて0.5時間撹拌後、反応混合物に飽和NH4Cl溶液を加えて反応停止した。混合物を水で希釈し、水層をEtOAcで抽出した。有機層を合わせてNa2SO4上で乾燥し、減圧濃縮した。ショートカラムの後、シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 20:1)にて精製し、benzyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-5-hexenoate(化合物29)を無色油状物(65.9 mg, 0.171 mmol, 60%)として得た; Rf 0.65 (hexane/EtOAc = 4:1); 1H NMR (300 MHz, CDCl3) δ 5.82-5.79 (1H, tt, J = 6.47 Hz, CH2=CH), 5.08-4.89 (2H, m, CH=CH2), 4.92 (1H, dd, J = 9.4, 5.0 Hz, CH), 2.29-2.20 (1H, m, CHCH2CH2), 2.17-2.10 (2H, m, CHCH2CH2), 2.09-1.96 (1H, m, CHCH2CH2), 1.50 (18H, s, tBu), 1.45 (9H, s, tBu)。 nBuLi (2.65 M solution in hexane, 0.215 mL, 0.569 mmol, 2.0 eq) was added to a dispersion of methyltriphenylphosphonium bromide (223.9 mg, 0.67 mmol, 2.2 eq) in THF (3.45 mL) at -78°C. Dripped. After the mixture was stirred at 0°C for 1 hour, (S)-2-[bis-(tert-butoxycarbonylamino)]-4-(formylbutanoic acid)-tert-butyl ester (compound 28) (110.4 g, 0.285 mmol, 1.0 eq) in THF (1.3 mL) was added at -78°C. After stirring for 0.5 h at 0° C., the reaction mixture was quenched with saturated NH 4 Cl solution. The mixture was diluted with water and the aqueous layer was extracted with EtOAc. The organic layers were combined, dried over Na 2 SO 4 and concentrated in vacuo. After a short column, benzyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-5-hexenoate (compound 29) was purified by silica gel column chromatography (hexane/EtOAc = 20:1). was obtained as a colorless oil (65.9 mg, 0.171 mmol, 60%); R f 0.65 (hexane/EtOAc = 4:1); 1 H NMR (300 MHz, CDCl 3 ) δ 5.82-5.79 (1H, tt, J = 6.47 Hz, CH 2 =CH), 5.08-4.89 (2H, m, CH=CH 2 ), 4.92 (1H, dd, J = 9.4, 5.0 Hz, CH), 2.29-2.20 (1H, m, CHCH 2CH2 ), 2.17-2.10 (2H, m, CHCH2CH2 ), 2.09-1.96 (1H, m , CHCH2CH2 ), 1.50 (18H, s, tBu), 1.45 (9H, s, tBu) .
(合成例18)
tert-Butyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-6-hydroxyhexanoate(化合物30)の合成
(Synthesis Example 18)
Synthesis of tert-Butyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-6-hydroxyhexanoate (compound 30)
Benzyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-5-hexenoate(化合物29)(135.9 mg, 0.35 mmol, 1.0 eq)のTHF(1.57 mL)液を0℃に冷却し、NaBH4(17.4 mg, 0.46 mmol, 1.3 eq)を加えた。10分間撹拌後、BF3・Et2O(58.1 μL, 0.459 mmol, 1.3 eq)をこの溶液に加えた。混合物を室温まで温めた後、21時間撹拌した。その後、溶液を0℃まで冷却し、そして1N NaOH(0.523 mL, 0.523 mmol, 1.5 eq)および30% H2O2(0.43 mL)を順次添加した。0℃にて1.5時間撹拌した後、反応混合物を水で希釈した。水層をEtOAcにて抽出した。有機層を合わせてNa2SO4上で乾燥し、減圧濃縮した。シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 2:1)にて精製し、tert-butyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-6-hydroxyhexanoate(化合物30)を無色油状物(77.7 mg, 0.193 mmol, 55%)として得た; Rf 0.18 (hexane/EtOAc = 4:1); 1H NMR (300 MHz, CDCl3) δ 4.73 (1H, dd, J = 9.6, 5.1 Hz, CH), 3.64 (2H, t, J = 6.6 Hz, CH2O), 2.19-2.12 (1H, m, CHCH2), 1.97-1.89 (1H, m, CHCH2), 1.69-1.51 (4H, m, CHCH2CH2CH2), 1.50 (18H, s, tBu), 1.45 (9H, s, tBu)。 Benzyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-5-hexenoate (Compound 29) (135.9 mg, 0.35 mmol, 1.0 eq) in THF (1.57 mL) was cooled to 0°C. , NaBH4 ( 17.4 mg, 0.46 mmol, 1.3 eq) was added. After stirring for 10 minutes, BF 3 .Et 2 O (58.1 μL, 0.459 mmol, 1.3 eq) was added to the solution. After the mixture was allowed to warm to room temperature, it was stirred for 21 hours. The solution was then cooled to 0° C. and 1N NaOH (0.523 mL, 0.523 mmol, 1.5 eq) and 30% H 2 O 2 (0.43 mL) were added sequentially. After stirring for 1.5 hours at 0° C., the reaction mixture was diluted with water. The aqueous layer was extracted with EtOAc. The organic layers were combined, dried over Na 2 SO 4 and concentrated in vacuo. After purification by silica gel column chromatography (hexane/EtOAc = 2:1), tert-butyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-6-hydroxyhexanoate (Compound 30) was obtained as a colorless oil. (77.7 mg, 0.193 mmol, 55%); R f 0.18 (hexane/EtOAc = 4:1); 1 H NMR (300 MHz, CDCl 3 ) δ 4.73 (1H, dd, J = 9.6, 5.1 Hz, CH), 3.64 (2H, t, J = 6.6 Hz, CH2O ), 2.19-2.12 (1H, m, CHCH2 ), 1.97-1.89 (1H, m, CHCH2 ), 1.69-1.51 (4H , m , CHCH2CH2CH2 ), 1.50 ( 18H, s, tBu), 1.45 (9H, s, tBu).
(合成例19)
tert-Butyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-6-iodohexanoate(化合物32)の合成
(Synthesis Example 19)
Synthesis of tert-Butyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-6-iodohexanoate (compound 32)
トリフェニルホスフィン(54.2 mg, 0.562 mmol, 2.0 eq)およびイミダゾール(16.5 mg, 0.562 mmol, 2.4 eq)を、窒素雰囲気下、0℃にて撹拌しながらCH2Cl2(0.1849 mL)に溶解した。ヨウ素粉末(52.4 mg, 0.562 mmol, 2.0 eq)を添加した後、混合物を室温まで温め、10分間撹拌したのち、0℃に冷却した。tert-butyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-6-hydroxyhexanoate(化合物30)(113.4 mg, 0.281 mmol, 1.0 eq)のCH2Cl2(0.4622 mL)溶液を冷却した溶液に加えた。0℃にて1時間撹拌した後、反応混合物を減圧濃縮した。残渣を中性シリカゲルのショートカラムで濾過し、hexane/Et2O = 1/1で溶出し、ろ液を濃縮した。シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 40:1)にて精製し、化合物32を無色油状物(45.2 mg, 0.025 mmol, 85%)として得た:Rf 0.70 (hexane/EtOAc = 4:1); 1H NMR (300 MHz, CDCl3) δ 4.73 (1H, dd, J = 9.6, 5.1 Hz, CH), 3.18 (2H, t, J = 7.0 Hz, CH2I), 2.10-1.97 (1H, m, CHCH2), 1.94-1.75 (5H, m, CHCH2, CHCH2CH2CH2), 1.50 (18H, s, tBu), 1.45 (9H, s, tBu)。 Triphenylphosphine (54.2 mg, 0.562 mmol, 2.0 eq) and imidazole (16.5 mg, 0.562 mmol, 2.4 eq) were dissolved in CH 2 Cl 2 (0.1849 mL) with stirring at 0° C. under a nitrogen atmosphere. After addition of iodine powder (52.4 mg, 0.562 mmol, 2.0 eq), the mixture was allowed to warm to room temperature, stirred for 10 minutes and then cooled to 0°C. A CH 2 Cl 2 (0.4622 mL) solution of tert-butyl-2-(S)-[bis-(tert-butoxycarbonyl)-amino]-6-hydroxyhexanoate (compound 30) (113.4 mg, 0.281 mmol, 1.0 eq) was Added to the cooled solution. After stirring at 0° C. for 1 hour, the reaction mixture was concentrated under reduced pressure. The residue was filtered through a neutral silica gel short column, eluted with hexane/Et 2 O=1/1, and the filtrate was concentrated. Purification by silica gel column chromatography (hexane/EtOAc = 40:1) gave Compound 32 as a colorless oil (45.2 mg, 0.025 mmol, 85%): R f 0.70 (hexane/EtOAc = 4:1). 1 H NMR (300 MHz, CDCl 3 ) δ 4.73 (1H, dd, J = 9.6, 5.1 Hz, CH), 3.18 (2H, t, J = 7.0 Hz, CH 2 I), 2.10-1.97 (1H, m, CHCH2 ), 1.94-1.75 (5H, m , CHCH2 , CHCH2CH2CH2 ), 1.50 (18H, s, tBu), 1.45 (9H, s, tBu).
(合成例20)
(S)-tert-Butyl-16-(((benzyloxy)carbonyl)amino)-13-(3,5-dibromopyridin-1-yl)pent-14-ynoate(化合物34)の合成
(Synthesis Example 20)
Synthesis of (S)-tert-Butyl-16-(((benzyloxy)carbonyl)amino)-13-(3,5-dibromopyridin-1-yl)pent-14-ynoate (Compound 34)
3,5-ジブロモ-4-ヨードピリジン(化合物10)(300.0 mg, 827 μmol, 1.0 eq)、(S)-tert-butyl 2-(((benzyloxy)carbonyl)amino)-4-pentynoate(化合物8)(377.1 mg, 1.24 μmol, 1.5 eq)、Pd2dba3(75.7 mg, 82.7 μmol, 10 mol%)、P(2-furyl)3(76.8 mg, 331 μmol, 40 mol%)およびCuI(63.0 mg, 331 μmol, 40 mol%)のDMF(41.3 mL)溶液を、凍結脱気(freeze/pump/thaw techniques)により脱気した。iPr2NEt(8.3 mL)を得られた溶液に加えた。40℃で17時間撹拌した後、反応混合物をEtOAcで希釈し、飽和NH4Cl溶液で反応停止した。その後、水層をEtOAcで抽出した。有機層を合わせて飽和食塩水で洗浄し、Na2SO4上で乾燥し、減圧濃縮した。シリカゲルカラムクロマトグラフィー(hexane/EtOAc = 10:1)にて精製し、化合物34(309.7 mg, 575 μmol, 70%)を得た; Rf 0.34 (hexane/EtOAc = 4:1); 1H NMR (300 MHz, CDCl3) δ 8.62 (2H, s, H2/6), 7.33 (5H, m, Bn), 5.76 (1H, d, J = 7.8 Hz, NH), 5.13 (2H, s, CH2Ph), 4.55 (1H, m, H16), 3.26-3.02 (2H, m, H15), 1.46 (9H, s, tBu)。 3,5-dibromo-4-iodopyridine (compound 10) (300.0 mg, 827 μmol, 1.0 eq), (S)-tert-butyl 2-(((benzyloxy)carbonyl)amino)-4-pentynoate (compound 8 ) (377.1 mg, 1.24 μmol, 1.5 eq), Pd 2 dba 3 (75.7 mg, 82.7 μmol, 10 mol%), P(2-furyl) 3 (76.8 mg, 331 μmol, 40 mol%) and CuI (63.0 mg, 331 μmol, 40 mol%) in DMF (41.3 mL) was degassed by freeze/pump/thaw techniques. iPr 2 NEt (8.3 mL) was added to the resulting solution. After stirring for 17 hours at 40° C., the reaction mixture was diluted with EtOAc and quenched with saturated NH 4 Cl solution. The aqueous layer was then extracted with EtOAc. The organic layers were combined, washed with saturated brine, dried over Na 2 SO 4 and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane/EtOAc = 10:1) gave Compound 34 (309.7 mg, 575 µmol, 70%); R f 0.34 (hexane/EtOAc = 4:1); 1 H NMR. (300 MHz, CDCl3 ) δ 8.62 (2H, s, H2/6), 7.33 (5H, m, Bn), 5.76 (1H, d, J = 7.8 Hz, NH), 5.13 (2H, s, CH2 Ph), 4.55 (1H, m, H16), 3.26-3.02 (2H, m, H15), 1.46 (9H, s, tBu).
(合成例21)
((20S,20'S)-tert-Butyl-18,18'-(4-((S)-17-(tert-butyloxy)-16-(((benzyloxy)carbonyl)amino)-17-oxopent-13-ynyl)pyridine-3,5-diyl)bis-20-(tert-butoxycarbonylamino)butanoate(化合物7)の合成
(Synthesis Example 21)
((20S,20'S)-tert-Butyl-18,18'-(4-((S)-17-(tert-butyloxy)-16-(((benzyloxy)carbonyl)amino)-17-oxopent-13- Synthesis of ynyl)pyridine-3,5-diyl)bis-20-(tert-butoxycarbonylamino)butanoate (compound 7)
亜鉛末(182.2 mg, 2.79 mmol)を、窒素雰囲気にした1.5mLのマイクロチューブ内に配置した。ドライDMF(84 μL)およびトリメチルシリルクロリド(56.3 μL, 0.44 mmol)を添加し、得られた混合物を室温にて15分間激しく撹拌した。撹拌を停止し、溶液をマイクロシリンジで取り出した。残った固体を、減圧下、ドライヤーを用いて乾燥した。活性化した亜鉛を室温に冷却し、(S)-21-tert-butyl 20-((tert-butoxycarbonyl)amino)-18-iodobutanoate(化合物9)(178.9 mg, 0.465 mmol, 5.0 eq)のドライDMF溶液(141 μLおよび94 μLのDMFでリンス)を活性化亜鉛に加えた。反応混合物を室温にて1時間撹拌し、その時以後、TLC分析(hexane/EtOAc = 5/1)にて出発物質が残っていないことを確認した。撹拌を停止し、遠心分離機を用いて亜鉛末を沈澱させた。105μLのDMFの入ったマイクロシリンジで溶液を活性化亜鉛から取り除き、氷冷したPd-PEPPSI-IPr(12.6 mg, 20 mol%)および化合物34(50.6 mg, 0.094 mmol, 1.0 eq)の入った10mLフラスコに加えた。60℃まで昇温したスターラーに移したのち、1.5時間撹拌を続け、反応混合物をEtOAcにて希釈し、飽和NH4Cl溶液で反応停止した。その後、水層をEtOAcで抽出した。有機層を合わせて飽和食塩水で洗浄し、Na2SO4上で乾燥し、減圧濃縮して粗生成物を黄色油状物として得た。フラッシュカラムクロマトグラフィー((hexane/EtOAc = 2:1 → 1:1)にて精製し、純粋な化合物7(45.5 mg, 50.8 μmol, 54%)を褐色油状物として得た; Rf 0.56 (hexane/EtOAc = 5:1); 1H NMR (300 MHz, CDCl3) δ 8.29 (2H, s, H2/6), 7.33 (5H, m, Bn), 5.55 (1H, m, NH16), 5.20-5.03 (3H, m, H16/20/20'), 4.55 (2H, m, NH21/21'), 4.34 (2H, m, CH2Ph), 3.26-3.02 (2H, m, H15), 2.73 (4H, m, H18/18'), 2.12-1.84 (4H, m, H19/19'), 1.48 (18H, s, Boc), 1.46 (27H, s, tBu); ESI-HRMS (m/z) calcd for C48H70N4NaO12 [M+Na]+ 917.4889, found 917.4858。 Zinc dust (182.2 mg, 2.79 mmol) was placed in a 1.5 mL microtube under a nitrogen atmosphere. Dry DMF (84 μL) and trimethylsilyl chloride (56.3 μL, 0.44 mmol) were added and the resulting mixture was vigorously stirred at room temperature for 15 minutes. Stirring was stopped and the solution was removed with a microsyringe. The remaining solid was dried using a dryer under reduced pressure. The activated zinc was cooled to room temperature and treated with (S)-21-tert-butyl 20-((tert-butoxycarbonyl)amino)-18-iodobutanoate (compound 9) (178.9 mg, 0.465 mmol, 5.0 eq) in dry DMF. The solution (141 μL and 94 μL DMF rinses) was added to the activated zinc. The reaction mixture was stirred at room temperature for 1 hour, after which time TLC analysis (hexane/EtOAc = 5/1) confirmed that no starting material remained. Stirring was stopped and the zinc dust was precipitated using a centrifuge. The solution was removed from the activated zinc with a microsyringe containing 105 μL of DMF and 10 mL containing ice-cold Pd-PEPPSI-IPr (12.6 mg, 20 mol%) and compound 34 (50.6 mg, 0.094 mmol, 1.0 eq). added to the flask. After transferring to a stirrer heated to 60° C. and stirring continued for 1.5 h, the reaction mixture was diluted with EtOAc and quenched with saturated NH 4 Cl solution. The aqueous layer was then extracted with EtOAc. The combined organic layers were washed with saturated brine, dried over Na 2 SO 4 and concentrated under reduced pressure to give the crude product as a yellow oil. Purification by flash column chromatography ((hexane/EtOAc = 2:1 → 1:1) gave pure compound 7 (45.5 mg, 50.8 µmol, 54%) as a brown oil; R f 0.56 (hexane /EtOAc = 5:1); 1 H NMR (300 MHz, CDCl 3 ) δ 8.29 (2H, s, H2/6), 7.33 (5H, m, Bn), 5.55 (1H, m, NH16), 5.20- 5.03 (3H, m, H16/20/20'), 4.55 (2H, m, NH21/21'), 4.34 (2H, m, CH2Ph ), 3.26-3.02 (2H, m, H15), 2.73 ( 4H, m, H18/18'), 2.12-1.84 (4H, m, H19/19'), 1.48 (18H, s, Boc), 1.46 (27H, s, tBu); ESI-HRMS (m/z) calcd for C48H70N4NaO12 [M+Na] + 917.4889 , found 917.4858 .
(合成例22)
3,5-Bis-{20,20'-(tert-butoxycarbonyl)-20,20'-(S)-[(tert-butoxycarbonyl)-amino]-propyl}-4-{16-(tert-butoxycarbonyl)-16-(S)-amino]-buthyl}-pyridine(化合物6)の合成
(Synthesis Example 22)
3,5-Bis-{20,20'-(tert-butoxycarbonyl)-20,20'-(S)-[(tert-butoxycarbonyl)-amino]-propyl}-4-{16-(tert-butoxycarbonyl) Synthesis of -16-(S)-amino]-butyl}-pyridine (compound 6)
化合物7(11.0 mg, 12.2 μmol, 1.0 eq)のMeOH(0.534 mL)溶液を10% Pd/C(13.07 mg, 12.2 μmol, 100.0 mol%)で処理し、室温中、バルーン圧にて接触水素化した。室温にて4時間撹拌した後、反応混合物を減圧濃縮した。フラッシュカラムクロマトグラフィー(hexane/EtOAc = 0/1 → CH2Cl2/MeOH =20/1)にて精製し、化合物6(5.3 mg, 6.93 μmol, 56%)を黄色油状物として得た; Rf 0.56 (hexane/EtOAc = 5:1); 1H NMR (300 MHz, CDCl3) δ 8.07 (2H, s, H2/6), 3.93 (3H, m, H16/20/20'), 2.65 (6H, m, H13/18/18'), 2.00-1.55 (8H, m, H14/15/19/19'), 1.48 (18H, s, Boc), 1.46 (27H, s, tBu); ESI-HRMS (m/z) calcd for C48H68N4NaO10 [M+Na]+ 787.4833, found 787.4809。 Compound 7 (11.0 mg, 12.2 μmol, 1.0 eq) in MeOH (0.534 mL) was treated with 10% Pd/C (13.07 mg, 12.2 μmol, 100.0 mol%) and subjected to catalytic hydrogenation at room temperature under balloon pressure. did. After stirring at room temperature for 4 hours, the reaction mixture was concentrated under reduced pressure. Purification by flash column chromatography (hexane/EtOAc = 0/1 → CH 2 Cl 2 /MeOH = 20/1) gave compound 6 (5.3 mg, 6.93 µmol, 56%) as a yellow oil; f 0.56 (hexane/EtOAc = 5:1); 1 H NMR (300 MHz, CDCl 3 ) δ 8.07 (2H, s, H2/6), 3.93 (3H, m, H16/20/20'), 2.65 ( 6H, m, H13/18/18'), 2.00-1.55 (8H, m, H14/15/19/19'), 1.48 (18H, s, Boc), 1.46 (27H, s, tBu); ESI- HRMS (m/z) calcd for C48H68N4NaO10 [M+Na] + 787.4833 , found 787.4809 .
(合成例23)
N-Succinimidyl 4-maleimidobutyrate(化合物41)の合成
(Synthesis Example 23)
Synthesis of N-Succinimidyl 4-maleimidobutyrate (Compound 41)
4-アミノ酪酸(化合物38)(200 mg, 2.04 mmol, 1.0 eq)を、無水マレイン酸(化合物39)(10 mg, 2.04 mmol, 1.0 eq)のDMF(2 mL)溶液に加え、混合物を2時間撹拌した。得られた溶液を氷浴中で冷却し、N-ヒドロキシスクシンイミド(288.7 mg, 2.51 mmol, 1.23 eq)およびDCC(883.7 mg, 4.28 mmol, 2.1 eq)を順次加えた。5分後、氷浴を除去し、溶液を一晩激しく撹拌した。形成された白色沈澱を濾別し、DMFで洗浄し、ろ液を氷上に注いだ。水層をCH2Cl2で抽出した。CH2Cl2層をNa2SO4上で乾燥し、減圧濃縮した。シリカゲルカラムクロマトグラフィーにより、生成物として化合物41を無色固体(157.1 mg, 0.56 mmol, 28%)として得た; Rf 0.23 (hexane/EtOAc = 1:1) ; 1H NMR (300 MHz, CDCl3) δ6.71 (2H, s, CH=CH), 3.65(2H, t, J = 7.0 Hz, NCH2), 2.83(4H, s, COCH2CO), 2.65(2H, t, J = 7.7 Hz, CH2CO), 2.05(2H, m, CH2)。 4-aminobutyric acid (compound 38) (200 mg, 2.04 mmol, 1.0 eq) was added to a solution of maleic anhydride (compound 39) (10 mg, 2.04 mmol, 1.0 eq) in DMF (2 mL) and the mixture was Stirred for an hour. The resulting solution was cooled in an ice bath and N-hydroxysuccinimide (288.7 mg, 2.51 mmol, 1.23 eq) and DCC (883.7 mg, 4.28 mmol, 2.1 eq) were added sequentially. After 5 minutes, the ice bath was removed and the solution was vigorously stirred overnight. The white precipitate formed was filtered off, washed with DMF and the filtrate was poured onto ice. The aqueous layer was extracted with CH2Cl2 . The CH2Cl2 layer was dried over Na2SO4 and concentrated under reduced pressure. Silica gel column chromatography gave Compound 41 as a product as a colorless solid (157.1 mg, 0.56 mmol, 28%); Rf 0.23 (hexane/EtOAc = 1:1); 1 H NMR (300 MHz, CDCl 3 ). δ6.71 (2H, s, CH=CH), 3.65(2H, t, J = 7.0 Hz, NCH2 ), 2.83(4H, s, COCH2CO ), 2.65(2H, t, J = 7.7 Hz, CH2CO), 2.05(2H, m , CH2 ).
(合成例24)
3,5-bis-{20,20'-(tert-butoxycarbonyl)-20,20'-(S)-[(tert-butoxycarbonyl)-amino]-propyl}-4-{16-(tert-butoxycarbonyl)-16-(S)-[(25-maleimido-22-oxobuthyl)-amino]-buthyl}-pyridine(化合物5)の合成
(Synthesis Example 24)
3,5-bis-{20,20'-(tert-butoxycarbonyl)-20,20'-(S)-[(tert-butoxycarbonyl)-amino]-propyl}-4-{16-(tert-butoxycarbonyl) Synthesis of -16-(S)-[(25-maleimido-22-oxobutyl)-amino]-butyl}-pyridine (Compound 5)
化合物6(5.3 mg, 6.93 μmol, 1.0 eq)のDMF(100 μL)溶液に、化合物41(2.04 mg, 7.27 mmol, 1.05 eq)およびNMM(0.5 μL)を加えた。反応混合物を4時間撹拌した後、氷水中に注いだ。溶液をpH3に酸性化し、CH2Cl2で3回抽出した。有機層を合わせて水および飽和食塩水で洗浄し、Na2SO4上で乾燥し、減圧濃縮した。シリカゲルカラムクロマトグラフィーにて精製し、生成物である化合物5を無色油状物(5.2 mg, 5.59 mmol, 81%)として得た; Rf 0.14 (hexane/EtOAc = 1:1); 1H NMR (300 MHz, CDCl3) δ8.19 (2H, s, H2/6), 6.75 (2H, s, H27/28), 6.65 (1H, m. NH16) 5.45 (2H, m, NH20/20'), 4.50 (1H, m, H16), 4.26 (2H, m, H20/20'), 3.60 (2H, m, H25), 2.62 (8H, m, H13/18/18'/23), 2.00-1.55 (10H, m, H14/15/19/19'/24), 1.48 (18H, s, Boc), 1.46 (27H, s, tBu); ESI-HRMS (m/z) calcd for C48H76N5O13 [M+H]+ 930.5440, found 930.5390。 Compound 41 (2.04 mg, 7.27 mmol, 1.05 eq) and NMM (0.5 μL) were added to a DMF (100 μL) solution of compound 6 (5.3 mg, 6.93 μmol, 1.0 eq). The reaction mixture was stirred for 4 hours and then poured into ice water. The solution was acidified to pH 3 and extracted with CH 2 Cl 2 three times. The organic layers were combined, washed with water and saturated brine, dried over Na 2 SO 4 and concentrated under reduced pressure. Purification by silica gel column chromatography gave the product compound 5 as a colorless oil (5.2 mg, 5.59 mmol, 81%); R f 0.14 (hexane/EtOAc = 1:1); 1 H NMR ( 300 MHz, CDCl3 ) δ8.19 (2H, s, H2/6), 6.75 (2H, s, H27/28), 6.65 (1H, m. NH16) 5.45 (2H, m, NH20/20'), 4.50 (1H, m, H16), 4.26 (2H, m, H20/20'), 3.60 (2H, m, H25), 2.62 (8H, m, H13/18/18'/23), 2.00-1.55 ( 10H, m, H14/15/19/19'/24), 1.48 (18H, s, Boc), 1.46 (27H, s, tBu); ESI-HRMS ( m/z) calcd for C48H76N5 O13[M + H] +930.5440 , found 930.5390.
(合成例25)
3,5-bis-{20,20'-(tert-butoxycarbonyl)-20,20'-(S)-[(tert-butoxycarbonyl)-amino]-propyl}-4-{16-(tert-butoxycarbonyl)-16-(S)-[(25-maleimido-22-oxobuthyl)-amino]-buthyl}-1-{11-(tert-butoxycarbonyl)-1-(S)-[bis-(tert-tutoxycarbonyl)-amino]-pentyl}-pyridinium(化合物43)の合成
(Synthesis Example 25)
3,5-bis-{20,20'-(tert-butoxycarbonyl)-20,20'-(S)-[(tert-butoxycarbonyl)-amino]-propyl}-4-{16-(tert-butoxycarbonyl) -16-(S)-[(25-maleimido-22-oxobuthyl)-amino]-buthyl}-1-{11-(tert-butoxycarbonyl)-1-(S)-[bis-(tert-tutoxycarbonyl)- Synthesis of amino]-pentyl}-pyridinium (compound 43)
化合物5(6.1 mg, 6.55 μmol, 1.0 eq)および化合物32(6.73 mg, 13.1 μmol, 2.0 eq)の混合物をCH3NO2(0.18 mL)中、70℃にて8時間加熱した。反応混合物を減圧濃縮した。シリカゲルカラムクロマトグラフィー(Hexane/EtOAC = 1:1 → CH2Cl2/MeOH = 10:1)にて精製し、化合物43(5.9 mg, 4.08 μmol, 62%)を黄色油状物として得た; Rf 0.50 (CH2Cl2/MeOH = 10:1); IR (ATR, cm-1) 3036, 1709, 1593, 1367, 1248, 1155, 733, 477, 464, 453; 1H NMR (300 MHz, CDCl3) δ8.91 (2H, s, H2/6), 6.75 (2H, s, H27/28), 6.65 (1H, m. NH16) 5.65 (2H, d, J = 8.2 Hz, NH20/20'), 4.77 (1H, m, H11), 4.65 (2H, m, H7), 4.52 (1H, m, H16), 4.16 (2H, m, H20/20'), 3.57 (2H, m, H25), 3.00-2.85 (6H, m, H13/18/18'), 2.77 (2H, m, H23), 2.25 (2H, t, J = 7.2 Hz, H8), 2.20-1.98 (10H, m, H10/14/15/19/19'), 1.97-1.75 (4H, m, H9/24), 1.50 (27H, s, Boc), 1.40 (27H, s, tBu); 13C NMR (125 MHz, CDCl3) δ171.1, 170.8, 169.5, 152.5, 141.6, 140.7, 134.2, 83.1, 82.7, 82.4, 81.4, 80.0, 58.2, 36.9, 33.0, 32.7, 31.1, 28.3, 28.0, 27.9, 26.5, 23.1; ESI-HRMS (m/z) calcd for C68H111N6O19 [M]+ 1315.7904, found 1315.7874。 A mixture of compound 5 (6.1 mg, 6.55 μmol, 1.0 eq) and compound 32 (6.73 mg, 13.1 μmol, 2.0 eq) was heated in CH 3 NO 2 (0.18 mL) at 70° C. for 8 hours. The reaction mixture was concentrated under reduced pressure. Purification by silica gel column chromatography (Hexane/EtOAC = 1 : 1 → CH2Cl2/MeOH = 10:1) gave compound 43 (5.9 mg, 4.08 µmol, 62%) as a yellow oil; f 0.50 ( CH2Cl2/MeOH = 10: 1 ); IR (ATR, cm - 1 ) 3036, 1709, 1593, 1367, 1248, 1155, 733, 477, 464, 453; CDCl3 ) δ8.91 (2H, s, H2/6), 6.75 (2H, s, H27/28), 6.65 (1H, m. NH16) 5.65 (2H, d, J = 8.2 Hz, NH20/20' ), 4.77 (1H, m, H11), 4.65 (2H, m, H7), 4.52 (1H, m, H16), 4.16 (2H, m, H20/20'), 3.57 (2H, m, H25), 3.00-2.85 (6H, m, H13/18/18'), 2.77 (2H, m, H23), 2.25 (2H, t, J = 7.2 Hz, H8), 2.20-1.98 (10H, m, H10/14 /15/19/19'), 1.97-1.75 (4H, m, H9/24), 1.50 (27H, s, Boc), 1.40 (27H, s, tBu); 13C NMR (125 MHz, CDCl3 ) δ171.1, 170.8, 169.5, 152.5, 141.6, 140.7, 134.2, 83.1, 82.7, 82.4, 81.4, 80.0, 58.2, 36.9, 33.0, 32.7, 31.1, 28.3, 28.2, 36.9, SI2.0, 26.9 m/z) calcd for C68H111N6O19 [M] + 1315.7904 , found 1315.7874 .
(合成例26)
4-(16-(S)-[(25-maleimido-22-oxobuthyl)-amino]-16-carboxy-buthyl)-1-(11)-(S)-amino-11-carboxy-pentyl)-3,5-bis-(20,20'-(S)-amino-20,20'-carboxy-propyl)-pyridinium(化合物2)の合成
(Synthesis Example 26)
4-(16-(S)-[(25-maleimido-22-oxobutyl)-amino]-16-carboxy-butyl)-1-(11)-(S)-amino-11-carboxy-pentyl)-3 Synthesis of ,5-bis-(20,20'-(S)-amino-20,20'-carboxy-propyl)-pyridinium (Compound 2)
TFAおよび蒸留水の混合物(1.25 mL, TFA/water = 95:5)を化合物43((8.0mg, 5.54 μmol, 1.0 eq)に室温で加え、2時間撹拌した。溶媒をロータリーエバポレーターにて除去した。C18シリカゲルカラムクロマトグラフィー(0.1% TFA in distilled water→0.1% TFA in distilled water/MeOH=9:1)にて精製し、化合物2を無色油状物(4.4 mg, 5.54 μmol, quant)として得た; Rf 0.4 [MeOH (0.1% TFA)/H2O (0.1% TFA) = 1:9]; 1H NMR (300 MHz, D2O) δ8.55 (2H, s, H2/6), 6.80 (2H, s, H27/28), 4.50 (2H, t. J = 6.4 Hz, H7) 4.37 (1H, m, H16), 4.02 (2H, t, J = 5.6 Hz, H20/20'), 3.89 (1H, t, J = 4.8 Hz, H11), 3.51 (2H, t, J = 6.4 Hz, H25), 3.05 (2H, m, H13), 2.93 (4H, m, H18/18'), 2.32 (2H, t, J = 6.4 Hz, H23), 2.22 (4H, m, H19/19'), 2.03 (2H,m, H8), 1.99-1.80 (6H, m, H10/15/24), 1.61 (2H, m, H14), 1.55-1.34 (2H, m, H9). ESI-HRMS (m/z) calcd for C32H47N6O11 [M]+ 691.3303, found 691.3319。 A mixture of TFA and distilled water (1.25 mL, TFA/water = 95:5) was added to compound 43 ((8.0 mg, 5.54 µmol, 1.0 eq) at room temperature and stirred for 2 hours. The solvent was removed with a rotary evaporator. Purification by C18 silica gel column chromatography (0.1% TFA in distilled water→0.1% TFA in distilled water/MeOH=9:1) gave Compound 2 as a colorless oil (4.4 mg, 5.54 μmol, quant). R f 0.4 [MeOH (0.1% TFA)/ H2O (0.1% TFA) = 1:9]; 1 H NMR (300 MHz, D2O) δ8.55 (2H, s, H2/6), 6.80 (2H, s, H27/28), 4.50 (2H, t. J = 6.4 Hz, H7) 4.37 (1H, m, H16), 4.02 (2H, t. J = 5.6 Hz, H20/20'), 3.89 (1H, t, J = 4.8 Hz, H11), 3.51 (2H, t, J = 6.4 Hz, H25), 3.05 (2H, m, H13), 2.93 (4H, m, H18/18'), 2.32 (2H, t, J = 6.4 Hz, H23), 2.22 (4H, m, H19/19'), 2.03 (2H, m, H8), 1.99-1.80 (6H, m, H10/15/24), 1.61 (2H, m, H14), 1.55-1.34 (2H, m, H9). ESI-HRMS (m/z) calcd for C32H47N6O11 [M] + 691.3303 , found 691.3319 .
(合成例27)
Thiolated BSA(化合物3)の合成
(Synthesis Example 27)
Synthesis of Thiolated BSA (compound 3)
BSA(10.2 mg, 0.15 μmol, 1.0 eq)およびtraut's試薬(0.8 mg, 6.0 μmol, 40 eq)のPBS(pH 8.4)(1 mL、10mM EDTAを含む。)中の混合物を32℃にて2時間インキュベートした。この溶液を、プレパックゲル脱塩カラムに適用し、1mMのEDTAを含むPBSにて各種を溶出した。タンパク質を含む画分をプールし、スルフヒドリル基濃度をエルマン分析により測定した。 A mixture of BSA (10.2 mg, 0.15 µmol, 1.0 eq) and Traut's reagent (0.8 mg, 6.0 µmol, 40 eq) in PBS (pH 8.4) (1 mL, containing 10 mM EDTA) was incubated at 32°C for 2 hours. incubated. This solution was applied to a prepacked gel desalting column and each species was eluted with PBS containing 1 mM EDTA. Fractions containing protein were pooled and sulfhydryl group concentrations were determined by Ellman's analysis.
(実施例1)
Desmosine BSA conjugate(化合物1)の合成
(Example 1)
Synthesis of Desmosine BSA conjugate (compound 1)
化合物2(2.2 mg, 2.70 μmol)をチオール化BSA(化合物3)のPBS(1 mM EDTAを含む。)溶液(0.5 mL)に溶解し、室温にて2時間撹拌した。NAP 5カラムを用いて反応混合物を精製し、未反応物を除去した。試料が完全に分離床に入った後、PBSにて溶出し、初めの1.3mLを回収した。
得られた複合体をMALDI-TOFMSにて分析したところ、BSA1分子あたり、平均5分子の化合物2が導入されていた。
Compound 2 (2.2 mg, 2.70 μmol) was dissolved in a solution (0.5 mL) of thiolated BSA (compound 3) in PBS (containing 1 mM EDTA) and stirred at room temperature for 2 hours. The reaction mixture was purified using a NAP 5 column to remove unreacted material. After the sample completely entered the separation bed, it was eluted with PBS and the first 1.3 mL was collected.
When the resulting complex was analyzed by MALDI-TOFMS, an average of 5 molecules of compound 2 were introduced per 1 molecule of BSA.
(合成例28)
Thiolated KLH(化合物3')の合成
(Synthesis Example 28)
Synthesis of Thiolated KLH (compound 3')
KLH(10.2 mg)およびtraut's試薬(0.8 mg, 6.0 μmol)のPBS(pH 8.4)(1 mL、10mM EDTAを含む。)中の混合物を32℃にて2時間インキュベートした。この溶液を、プレパックゲル脱塩カラムに適用し、1mMのEDTAを含むPBSにて各種を溶出した。タンパク質を含む画分をプールした。 A mixture of KLH (10.2 mg) and Traut's reagent (0.8 mg, 6.0 μmol) in PBS (pH 8.4) (1 mL, containing 10 mM EDTA) was incubated at 32° C. for 2 hours. This solution was applied to a prepacked gel desalting column and each species was eluted with PBS containing 1 mM EDTA. Fractions containing protein were pooled.
(実施例2)
Desmosine KLH conjugate(化合物1')の合成
(Example 2)
Synthesis of Desmosine KLH conjugate (compound 1')
化合物2(2.2 mg, 2.70 μmol)を、チオール化KLH(化合物3')溶液(0.5 mL, 1 mM EDTAを含むPBS)に溶解し、室温にて2時間撹拌した。NAP 5カラムを用いて反応混合物を精製し、未反応物を除去した。試料が完全に分離床に入った後、PBSにて溶出し、初めの1.3mLを回収した。
得られた複合体をMALDI-TOFMSにて分析したが、KLHの分子量が大きく、結合数を求めることができなかったため、上記式ではXと示す。
Compound 2 (2.2 mg, 2.70 μmol) was dissolved in thiolated KLH (compound 3′) solution (0.5 mL, PBS containing 1 mM EDTA) and stirred at room temperature for 2 hours. The reaction mixture was purified using a NAP 5 column to remove unreacted material. After the sample completely entered the separation bed, it was eluted with PBS and the first 1.3 mL was collected.
The resulting complex was analyzed by MALDI-TOFMS, but since the molecular weight of KLH was too large to determine the number of bonds, X is shown in the above formula.
本明細書において、以下の略記を用いた。
Me:メチル
Et:エチル
iPr:イソプロピル
Ac:アセチル
Boc:t-ブトキシカルボニル
Cbz:ベンジルオキシカルボニル
tBu:t-ブチル
DMF:N,N-ジメチルホルムアミド
THF:テトラヒドロフラン
NMM:N-メチルモルホリン
TMSCl:クロロトリメチルシラン
PEPPSI(商標):Pd-pyridine-enhanced precatalyst preparation stabilization and initiation
PBS:Phosphate Buffered Saline
rt:室温
h:時間
The following abbreviations have been used in this specification.
Me: methyl Et: ethyl iPr: isopropyl Ac: acetyl Boc: t-butoxycarbonyl Cbz: benzyloxycarbonyl tBu: t-butyl DMF: N,N-dimethylformamide THF: tetrahydrofuran NMM: N-methylmorpholine TMSCl: chlorotrimethylsilane PEPPSI™: Pd-pyridine-enhanced precatalyst preparation stabilization and initiation
PBS: Phosphate Buffered Saline
rt: room temperature h: time
Claims (5)
当該複合体が下記一般式(1)に示される、複合体。
A complex represented by the following general formula (1).
デスモシンの4位の側鎖に、反応性基が選択的に導入されたデスモシン類誘導体を準備する工程と、
前記デスモシン類誘導体とタンパク質またはその誘導体とを接触させて、前記複合体を形成する工程と、
を含む、複合体の製造方法。 A method for producing the composite according to any one of claims 1 to 3,
preparing a desmosine derivative in which a reactive group is selectively introduced into the 4-position side chain of desmosine;
contacting the desmosine derivative with a protein or a derivative thereof to form the complex;
A method of manufacturing a composite, comprising:
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JP2015178957A (en) | 2014-03-18 | 2015-10-08 | 学校法人上智学院 | Chemical compounds, standard substances for quantitative analysis using the same, and quantification method for desmosines |
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JP2015178957A (en) | 2014-03-18 | 2015-10-08 | 学校法人上智学院 | Chemical compounds, standard substances for quantitative analysis using the same, and quantification method for desmosines |
Non-Patent Citations (2)
Title |
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Connective Tissue Research,1980年,7 (4),263-267 |
日本胸部疾患学会雑誌,1989年,27 (12),1502-1508 |
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