JPH03183495A - Method for enzymically synthesizing amino acid oligomer having single molecular weight - Google Patents
Method for enzymically synthesizing amino acid oligomer having single molecular weightInfo
- Publication number
- JPH03183495A JPH03183495A JP24943889A JP24943889A JPH03183495A JP H03183495 A JPH03183495 A JP H03183495A JP 24943889 A JP24943889 A JP 24943889A JP 24943889 A JP24943889 A JP 24943889A JP H03183495 A JPH03183495 A JP H03183495A
- Authority
- JP
- Japan
- Prior art keywords
- amino acid
- molecular weight
- single molecular
- acid oligomer
- oligomer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 150000001413 amino acids Chemical class 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title description 18
- 230000002194 synthesizing effect Effects 0.000 title description 4
- 239000004365 Protease Substances 0.000 claims abstract description 13
- 108091005804 Peptidases Proteins 0.000 claims abstract description 9
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 8
- 150000003862 amino acid derivatives Chemical class 0.000 claims abstract description 8
- 239000003054 catalyst Substances 0.000 claims abstract description 3
- 238000005580 one pot reaction Methods 0.000 claims abstract description 3
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000001308 synthesis method Methods 0.000 claims description 4
- 239000000758 substrate Substances 0.000 abstract description 9
- 108090000317 Chymotrypsin Proteins 0.000 abstract description 2
- 239000000560 biocompatible material Substances 0.000 abstract description 2
- 239000007853 buffer solution Substances 0.000 abstract description 2
- 229960002376 chymotrypsin Drugs 0.000 abstract description 2
- 238000001914 filtration Methods 0.000 abstract description 2
- 238000006116 polymerization reaction Methods 0.000 abstract description 2
- 229940024606 amino acid Drugs 0.000 description 18
- 235000001014 amino acid Nutrition 0.000 description 18
- 238000003786 synthesis reaction Methods 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 9
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 229960003136 leucine Drugs 0.000 description 5
- 235000019419 proteases Nutrition 0.000 description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 4
- QVDXUKJJGUSGLS-LURJTMIESA-N methyl L-leucinate Chemical compound COC(=O)[C@@H](N)CC(C)C QVDXUKJJGUSGLS-LURJTMIESA-N 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 235000019454 L-leucine Nutrition 0.000 description 3
- 239000004395 L-leucine Substances 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- 108090000526 Papain Proteins 0.000 description 3
- 238000004992 fast atom bombardment mass spectroscopy Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- DODCBMODXGJOKD-RGMNGODLSA-N methyl (2s)-2-amino-4-methylpentanoate;hydrochloride Chemical compound Cl.COC(=O)[C@@H](N)CC(C)C DODCBMODXGJOKD-RGMNGODLSA-N 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 235000019834 papain Nutrition 0.000 description 3
- 229940055729 papain Drugs 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 230000006340 racemization Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 108010005843 Cysteine Proteases Proteins 0.000 description 2
- 102000005927 Cysteine Proteases Human genes 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- YGYAWVDWMABLBF-UHFFFAOYSA-N Phosgene Chemical compound ClC(Cl)=O YGYAWVDWMABLBF-UHFFFAOYSA-N 0.000 description 2
- 108010022999 Serine Proteases Proteins 0.000 description 2
- 102000012479 Serine Proteases Human genes 0.000 description 2
- 150000001370 alpha-amino acid derivatives Chemical class 0.000 description 2
- 235000008206 alpha-amino acids Nutrition 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- SWVMLNPDTIFDDY-FVGYRXGTSA-N methyl (2s)-2-amino-3-phenylpropanoate;hydrochloride Chemical compound Cl.COC(=O)[C@@H](N)CC1=CC=CC=C1 SWVMLNPDTIFDDY-FVGYRXGTSA-N 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 229960005190 phenylalanine Drugs 0.000 description 2
- 239000003505 polymerization initiator Substances 0.000 description 2
- -1 promain Proteins 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- SAUDSWFPPKSVMK-LBPRGKRZSA-N (2s)-2-(n-phenylanilino)propanoic acid Chemical compound C=1C=CC=CC=1N([C@@H](C)C(O)=O)C1=CC=CC=C1 SAUDSWFPPKSVMK-LBPRGKRZSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 108010006303 Carboxypeptidases Proteins 0.000 description 1
- 102000005367 Carboxypeptidases Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108090000270 Ficain Proteins 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108010020346 Polyglutamic Acid Proteins 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 108090000787 Subtilisin Proteins 0.000 description 1
- 108090001109 Thermolysin Proteins 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012776 electronic material Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 150000002168 ethanoic acid esters Chemical class 0.000 description 1
- 235000019836 ficin Nutrition 0.000 description 1
- POTUGHMKJGOKRI-UHFFFAOYSA-N ficin Chemical compound FI=CI=N POTUGHMKJGOKRI-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
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- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical group [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- QVDXUKJJGUSGLS-UHFFFAOYSA-N methyl 2-amino-4-methylpentanoate Chemical compound COC(=O)C(N)CC(C)C QVDXUKJJGUSGLS-UHFFFAOYSA-N 0.000 description 1
- VSDUZFOSJDMAFZ-VIFPVBQESA-N methyl L-phenylalaninate Chemical compound COC(=O)[C@@H](N)CC1=CC=CC=C1 VSDUZFOSJDMAFZ-VIFPVBQESA-N 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
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- 239000003607 modifier Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229920001308 poly(aminoacid) Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920002643 polyglutamic acid Polymers 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 230000007065 protein hydrolysis Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000003890 succinate salts Chemical class 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
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- 238000005809 transesterification reaction Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野］
本発明はプロテアーゼを用いた単一の分子量を有するア
ミノ酸オリゴマーの酵素的合成方法に関する。アミノ酸
オリゴマーは、生体適合材料、機能性担体、膜材料、エ
レクトロニクス材料としての利用のばかドラッグデリバ
リ−システムの基材として重要である。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for enzymatically synthesizing amino acid oligomers having a single molecular weight using a protease. Amino acid oligomers are important as base materials for drug delivery systems for use as biocompatible materials, functional carriers, membrane materials, and electronic materials.
［従来の技術］
アミノ酸オリゴマーの製造方法としては化学的な方法、
微生物発酵法若しくは酵素的合成方法が知られている。[Prior art] Methods for producing amino acid oligomers include chemical methods,
Microbial fermentation methods or enzymatic synthesis methods are known.
このうち代表的な化学的合成法としては、α−アミノ酸
からＮ−カルボキシ−α−アくノ酸無水物（ＮＣＡと略
す）を合成し、適当な重合開始剤の存在下、重合させる
ＮＣＡ法が挙げられる。Among these, a typical chemical synthesis method is the NCA method, in which N-carboxy-α-acinoic anhydride (abbreviated as NCA) is synthesized from α-amino acid and polymerized in the presence of a suitable polymerization initiator. can be mentioned.
ＮＣＡ法以外の化学的合成法としては活性エステルによ
る方法、混合酸無水物、Ｎ、Ｎ’−ジシクロへキシルカ
ルボシイ主ドなどの縮合剤を用いる方法がある。微生物
発酵の例としてポリグルタミン酸、ポリリジンの合成が
、報告されている。又、酵素的合成方法としてはタンパ
ク質分解酵素（プロテアーゼ）によるタンパク質加水分
解反応の逆反応あるいはエステル交換反応を利用した合
成法が報告されている。Chemical synthesis methods other than the NCA method include methods using active esters and methods using condensing agents such as mixed acid anhydrides and N,N'-dicyclohexyl carboxylic acid. Synthesis of polyglutamic acid and polylysine has been reported as an example of microbial fermentation. Furthermore, as an enzymatic synthesis method, a synthesis method using a reverse reaction of a protein hydrolysis reaction using a protease or a transesterification reaction has been reported.
［発明が解決しようとする課題］
化学的合成法のうちＮＣＡ法は、アミノ酸オリゴマー若
しくはポリマーの合成に最も汎用的に用いられている方
法であるがその使用にあたっては幾つかの制限がある。[Problems to be Solved by the Invention] Among chemical synthesis methods, the NCA method is the method most commonly used for synthesizing amino acid oligomers or polymers, but there are some limitations in its use.
すなわち、極めて毒性の高いホスゲンを使用するアミノ
基のホスゲン化工程が含まれることや側鎖にアミノ基、
カルボキシル基、水酸基、チオール基などの官能基を有
するアミノ酸誘導体を用いる場合には合成の前に適切な
保護基によりこれら官能基を保護し、合成終了後その脱
保護が必要であるなど合成工程が複雑になる。In other words, it involves a step of phosgenation of amino groups that uses extremely toxic phosgene, and that the side chain contains amino groups,
When using amino acid derivatives with functional groups such as carboxyl groups, hydroxyl groups, and thiol groups, it is necessary to protect these functional groups with appropriate protecting groups before synthesis, and to deprotect them after the synthesis is completed. It gets complicated.
更にホスゲンまたは重合開始剤や生成するオリゴマーを
溶解する適切な有ｍ溶媒を選択する必要がある。その他
、一般に化学的合成法では操作が多段階に互りさらに生
成物のラセミ化を伴うことが多く光学活性なポリマーを
得ることは困難である。Furthermore, it is necessary to select an appropriate solvent that dissolves phosgene or the polymerization initiator and the oligomers produced. In addition, chemical synthesis methods generally require multiple steps and often involve racemization of the product, making it difficult to obtain optically active polymers.
一方、微生物発酵によるアミノ酸オリゴマーポリマーの
合成では目的物を効率良く産生ずる微生物のスクリーニ
ング、改質に莫大な労力が必要であり、培地の成分に生
成物が左右されることや得られるオリゴマーは一般的に
分子量分布の極めて広いものが得られるなどの短所があ
る。On the other hand, the synthesis of amino acid oligomer polymers by microbial fermentation requires a huge amount of effort to screen and modify microorganisms that can efficiently produce the target product, and the product depends on the components of the culture medium and the oligomers obtained are common. However, it has disadvantages such as the fact that it can give an extremely wide molecular weight distribution.
これに対して、酵素によるペプチド合成では、反応を水
溶液系または有機溶媒を含む水溶液系において穏やかな
条件下で反応を行う事ができ、ラセミ化が起こらず高い
光学純度を有する生成物を得ることが可能であるとされ
ている。In contrast, in enzymatic peptide synthesis, the reaction can be carried out under mild conditions in an aqueous solution system or an aqueous solution system containing an organic solvent, and it is possible to obtain a product with high optical purity without racemization. is said to be possible.
しかし、これまでじ開示されたプロテアーゼによるペプ
チド合成反応は、同種または異種のアミノ酸あるいはそ
の誘導体間に加水分解反応の逆反応を利用し、ペプチド
結合を形成させるものであるが、生成物が各ｆ！溶媒に
溶けにくいため分析手段がなく単離、構造解析された例
は少ない。However, the peptide synthesis reactions using proteases that have been disclosed so far utilize the reverse reaction of the hydrolysis reaction between the same or different amino acids or their derivatives to form peptide bonds; ! Because it is difficult to dissolve in solvents, there are few cases where it has been isolated and structurally analyzed due to the lack of analytical methods.
前記のごとくアミノ酸オリゴマー若しくはポリマーの化
学合成では、極めて毒性の高いガスを使用することやア
くノ酸側鎖への保護基の導入、脱保護や生成物のラセミ
化など合成工程が煩雑であった。As mentioned above, the chemical synthesis of amino acid oligomers or polymers requires complicated synthesis steps such as the use of highly toxic gases, the introduction of protective groups into the acetic acid side chains, deprotection, and racemization of the product. Ta.
そこで、本発明者は、均一な重合度を有するアミノ酸オ
リゴマーを単一の操作で合成できる反応系を検討してき
た。アミノ酸誘導体を基質としてプロテアーゼを用いた
酵素反応の諸条件（ｐＨ，）Ｊｔ街液の種類、塩濃度、
反応温度０反応時間、基質／酵素の比率など）を極々検
討した結果、単一の操作で阜−の分子量を有するアミノ
酸オリゴマーを得る合成法を確立した。実施例に示すよ
うに本発明によりラセミ体など光学的に不純なアくノ酸
エステルを出発原料として光学活性なアミノ酸オリゴマ
ーを得ることが可能となった。また文献（）ｌｅｌｖｅ
ｔｉｃａ Ｃｈｉｍｉｃａ Ａｃｔａ、 ６２．４８８
（１９７９）及びＪ、 Ｃｈｅｗ、 Ｔｅｃｈ、 ＢＩ
ｏｔｅｃｈｎｏｌ、、 ３５Ｂ、 ２８２（＋９８５）
）に開示されているところではロイシンあるいはフェニ
ルアラニンのメチルエステルは単独ではオリゴマーを生
成しないとされている。しかし、本発明によりこれらの
アミノ酸のメチルエステルを基質としてそのホモオリゴ
ペプチドの製造が可能となった。Therefore, the present inventor has studied a reaction system that can synthesize amino acid oligomers having a uniform degree of polymerization in a single operation. Conditions for enzymatic reaction using protease with amino acid derivative as substrate (pH,) Type of Jt street solution, salt concentration,
As a result of careful consideration of the reaction temperature, reaction time, substrate/enzyme ratio, etc., we established a synthetic method for obtaining amino acid oligomers with a certain molecular weight in a single operation. As shown in the Examples, the present invention has made it possible to obtain optically active amino acid oligomers using optically impure acetic acid esters such as racemates as starting materials. Also, literature () level
tica Chimica Acta, 62.488
(1979) and J. Chew, Tech, BI.
otechnol,, 35B, 282 (+985)
) discloses that leucine or phenylalanine methyl ester alone does not produce oligomers. However, the present invention has made it possible to produce homo-oligopeptides using methyl esters of these amino acids as substrates.
［問題点を解決するための手段］ 本発明は、下記（１）の構成を有する。[Means for solving problems] The present invention has the following configuration (1).
（１） アミノ酸誘導体を用いて単一の分子量を有する
α−アミノ酸オリゴマーを製造し、または２種以上のア
ミノ酸誘導体を用いて単一の分子量を有するコオリゴマ
ーを製造する方法において触媒としてプロテアーゼを用
い、ワンポットで合成することを特徴とするアミノ酸オ
リゴマーの酵素的合成方法。(1) Protease is used as a catalyst in a method for producing an α-amino acid oligomer having a single molecular weight using an amino acid derivative or a cooligomer having a single molecular weight using two or more amino acid derivatives. , a method for enzymatic synthesis of amino acid oligomers characterized by one-pot synthesis.
本発明の構成と効果につき以下に詳述する。The configuration and effects of the present invention will be explained in detail below.
本発明を概説すれば、セリンプロテアーゼ若しくはシス
ティンプロテアーゼで例示される酵素による単一の分子
量を有するアミノ酸オリゴマーの簡便１（酵素的製造法
であって、基質としてアミノ酸または、そのカルボキシ
ル末端をエステル化した種々の天然アミノ酸を用い、緩
衝液中でプロテアーゼの存在下反応させ、生成物を沈殿
物として反応系外に出すことにより均一な分子量を有す
るホモまたはコポリアミノ酸を合成する方法に関する。To summarize the present invention, it is a method for producing amino acid oligomers having a single molecular weight using an enzyme such as serine protease or cysteine protease. This invention relates to a method for synthesizing homo- or co-polyamino acids having uniform molecular weights by reacting various natural amino acids in the presence of protease in a buffer and discharging the product as a precipitate from the reaction system.
本発明に用いられる酵素としては、キモトリプシン、ト
リプシン、ズブチリシン、カルボキシペプチダーゼなど
のセリンプロテアーゼ、パパイン、プロメイン、フィシ
ンなどのシスティンプロテアーゼ、サーモライシンなど
の金属プロテアーゼである。これら酵素をそのまま、ま
たはポリエチレングリコールなどの修飾剤で修飾して用
いる事もできる。Enzymes used in the present invention include serine proteases such as chymotrypsin, trypsin, subtilisin, and carboxypeptidase, cysteine proteases such as papain, promain, and ficin, and metalloproteases such as thermolysin. These enzymes can be used as they are or modified with a modifier such as polyethylene glycol.
基質としては、２０ｆ！の天然ア主）酸およびその他の
合成アミノ酸または、そのエステルを用いる事ができる
。ただし、酵素のもつ基質特異性により基質を選ぶ必要
がある。エステルとしては、アルキル、アリール若しく
はフェニルエステルなどが使用出来る。As a substrate, 20f! Natural amino acids and other synthetic amino acids or esters thereof can be used. However, the substrate must be selected depending on the substrate specificity of the enzyme. As the ester, alkyl, aryl or phenyl esters can be used.
緩衝液のｐＨは、２−１０の領域で用いる事ができるが
、個々の酵素はそれぞれ至適ｐＨを有している０例えば
、パパインでは６−８、キモトリプシンでは７付近、ペ
プシンでは４付近である。ｍＷＩ液用の塩は、酢酸塩、
リン酸塩、コハク酸塩、炭酸塩を０．１Ｍ−８の領域で
用いることができるが好ましくは０．５−２Ｍである。The pH of the buffer solution can be used in the range of 2-10, but each individual enzyme has its own optimum pH. be. Salts for mWI solution include acetate,
Phosphates, succinates, carbonates can be used in the 0.1M-8 range, but preferably 0.5-2M.
本発明の方法において基質と酵素の使用比率は、 １０
０倍からｔｏ、ｏｏｏ倍であるが好ましくは１．０００
− ２，５００倍である。In the method of the present invention, the ratio of substrate to enzyme used is 10
0 times to to, ooo times, but preferably 1.000 times
- 2,500 times more.
反応温度は、０度から８０度（摂氏）で好ましくは、２
０度から４０度で、反応終了は生成物が完全に沈殿する
時点でもって終点とする。The reaction temperature is from 0 degrees to 80 degrees (Celsius), preferably 2
The reaction temperature is from 0 to 40 degrees, and the reaction is terminated when the product completely precipitates.
本発明により、ラセミ体アミノ酸エステルを出発原料と
して光学活性なアミノ酸オリゴマーを得ることが可能に
なった。The present invention has made it possible to obtain optically active amino acid oligomers using racemic amino acid esters as starting materials.
以下、実施例によって本発明を説明する。The present invention will be explained below with reference to Examples.
１［例１ （Ｌ−ロイシンのオリゴマーの合成）１
８２ＩＩｇのＬ−ロイシンメチルエステル塩酸塩の０．
５Ｍ炭酸ナトリウム緩衝液 （ｐＨａ、５）ｉｏ■ｌに
溶解し、あらかじめ還元剤ジチオスレイトール、および
エチレンジアミン四酢酸で活性化したパパイン（シグマ
社、タイプＩＩＩ ）をｌＯμＭとなるように加え、２
５℃にて緩やかに２日間振どう攪拌する。1 [Example 1 (Synthesis of L-leucine oligomer) 1
0.82IIg of L-leucine methyl ester hydrochloride.
Papain (Sigma, Type III), which had been dissolved in 5M sodium carbonate buffer (pH, 5) ioL and activated with the reducing agent dithiothreitol and ethylenediaminetetraacetic acid, was added to a concentration of 1OμM.
Stir gently for 2 days at 5°C.
反応液より析出した沈殿を濾取し、数回純水にて洗浄す
る。得られた結晶を自然乾燥し、９３−ｇのＬ−ロイシ
ンのオリゴマーを得た（収率５１％）、生成物をＦＴ−
ＮＭＲ，ＦＡＢ−ＭＳなどで分析したところ、スペクト
ルより明らかなとおり６量体オリゴペプチドであった。The precipitate deposited from the reaction solution is collected by filtration and washed several times with pure water. The obtained crystals were air-dried to obtain 93-g of L-leucine oligomer (yield 51%).
When analyzed by NMR, FAB-MS, etc., it was found to be a hexameric oligopeptide as clear from the spectrum.
’ＨＮＭＲ（ＣＦ、ＧＯＯＤ−ＴＭＳ）δ： ４．４８
（１）１．ｔ、−Ｃ）Ｉ−）Ｎ−末端メチン、４．８
５−４．７６ （ＩＨ，ａ、−ＣＨ−） Ｎ−末端以外
のメチン、前者と後者の比率は１：５゜
ＦＡＢ−ＭＳ
ｍ／ｅ ： ７１１（Ｍ＋Ｈ）、５８２，５６８，４２
７，３７１，３５４，３３７゜２８４．２５６，２４３
，２００，１４５．８６いずれも６量体のフラグメント
実施１１ｄ２（Ｌ−フェニルアラニンオリゴマーの合成
）
２１８１ｇのし一フェニルアラニンメチルエステル塩酸
塩を用いること以外、酵素、反応温度、反応時間、基質
／酵素の比率などの諸条件を実施例１と同じにして反応
した。'HNMR (CF, GOOD-TMS) δ: 4.48
(1)1. t, -C) I-) N-terminal methine, 4.8
5-4.76 (IH, a, -CH-) Methine other than the N-terminus, the ratio of the former to the latter is 1:5 °FAB-MS m/e: 711 (M + H), 582,568,42
7,371,354,337゜284.256,243
, 200, 145.86 Hexamer fragment implementation 11d2 (synthesis of L-phenylalanine oligomer) Enzyme, reaction temperature, reaction time, substrate/enzyme ratio, except for using 2181 g of phenylalanine methyl ester hydrochloride The reaction was carried out under the same conditions as in Example 1.
８８ｍｇのし一フェニルアラニジメチルエステルオリコ
マーヲ得た（収率４Ｇ、７％） 、 ＦＴ−ＮＭＲ及び
ＦＡＢ−ＭＳによる分析の結果得られたものは５量体オ
リゴマーであった。88 mg of phenylalanidimethyl ester oligomer was obtained (yield: 4G, 7%). Analysis by FT-NMR and FAB-MS revealed that it was a pentameric oligomer.
’ＨＮＭＲ（ＣＦ、ＧＯＯＤ−ＴＭＳ）δ： ４．６３
（ＩＨ，ｔ、−Ｃ：Ｈ−）Ｎ−末端メチン、４．８３
−５．００ （１）１．ｍ、−Ｃ）ｌ−） Ｎ−末端以
外のメチン、前者と後者の比率は１：４゜
ＦＡＢ−ＭＳ
＋＊／ｅ ： ７５４ （（Ｍ−Ｃ）Ｉｓ）＋２Ｈ）
、７０８，６２１，２９５，２６７゜１４９．１２１，
１１９．５７いずれも５量体のフラグメント
実施例３ （Ｌ−ロイシン及びし−フェニルアラニン
のコオリゴペプチドの製造法）
Ｌ−ロイシンメチルエステル塩酸塩、Ｌ−フェニルアラ
ニンメチルエステル塩酸塩を下表のモル比率で加えたｐ
ｎ ａ、ｓの炭酸ナトリウムｌＩ衝液にパパインを各１
１μＭ加え、実施例１と同様に２５℃にて振とう攪拌す
る。得られた生成物は、ＦＴ−ＮＭＲの結果ロイシン、
フェニルアラニンを当初の混合モル比で含むオリゴコベ
ブチドであった。'HNMR (CF, GOOD-TMS) δ: 4.63
(IH,t, -C:H-)N-terminal methine, 4.83
-5.00 (1)1. m, -C)l-) Methine other than the N-terminus, the ratio of the former to the latter is 1:4゜FAB-MS +*/e: 754 ((M-C)Is)+2H)
, 708,621,295,267°149.121,
119.57 Both pentamer fragments Example 3 (Production method of cooligopeptide of L-leucine and di-phenylalanine) L-leucine methyl ester hydrochloride and L-phenylalanine methyl ester hydrochloride were mixed in the molar ratios shown in the table below. p added in
Add 1 portion each of papain to sodium carbonate lI solution of n a and s.
1 μM was added, and the mixture was shaken and stirred at 25° C. in the same manner as in Example 1. As a result of FT-NMR, the obtained product showed leucine,
It was an oligocobebutide containing phenylalanine at the original mixed molar ratio.
’ＨＮＭＲ（ＣＦｓＣＯＱＤ−ＴＭＳ）δ： ７．４２
−７．Ｈｌ （５）１．＊、フェニル）、１．０５−
０．９８（２Ｘ ３Ｆｌ、ｍ、−Ｃ８４）表
実施例４（Ｄ、Ｌ−ロイシンメチルエステル（ラセ果体
）よりＬ−ロイシンメチルエステ
ルオリゴマーの合成）
３Ｂ４＋ｅｇの［１，Ｌ−ロイシンメチルエステル塩酸
塩を用いること以外反応の諸条件を実施例１と同じにし
て反応した。 ７Ｇ園Ｈのし一ロイシンメチルエステル
オリゴマーを得た。オリゴマーの ’ ＨＮＭＲより６
量体であることが明らかである。収率２ｏ、８％。'HNMR (CFsCOQD-TMS) δ: 7.42
-7. Hl (5)1. *, phenyl), 1.05-
0.98 (2X 3Fl, m, -C84) Table Example 4 (Synthesis of L-leucine methyl ester oligomer from D, L-leucine methyl ester (race fruit)) 3B4+eg [1, L-leucine methyl ester hydrochloride The reaction was carried out under the same conditions as in Example 1 except for using a salt. A leucine methyl ester oligomer was obtained from 7GenH Noshiichi. From 'HNMR of oligomer 6
It is clear that it is a mercury. Yield 2o, 8%.
第１図に実施例１で得たオリゴマーと実施例４で得たオ
リゴマーの ’ ｌ（ＮＭＲを示す。FIG. 1 shows NMR of the oligomer obtained in Example 1 and the oligomer obtained in Example 4.
第１図（＾）は実施例！で得られたし一ロイシンメチル
エステル６量体の ’ ＨＮＭＲスペクトルを第１図（
Ｂ）は実施例４で得られたＬ−ロイシンメチルエステル
６量体の’ＨＮＭＲスペクトルを示す。
以上
特許
出
願人
チッソ株式会社Figure 1 (^) is an example! Figure 1 shows the 'HNMR spectrum of the monoleucine methyl ester hexamer obtained in
B) shows the 'HNMR spectrum of the L-leucine methyl ester hexamer obtained in Example 4. Patent applicant: Chisso Corporation
Claims (1)
−アミノ酸オリゴマーを製造し、または２種以上のアミ
ノ酸誘導体を用いて単一の分子量を有するコオリゴマー
を製造する方法において触媒としてプロテアーゼを用い
、ワンポットで合成することを特徴とするアミノ酸オリ
ゴマーの酵素的合成方法。(1) α with a single molecular weight using amino acid derivatives
- A method for producing an amino acid oligomer or a cooligomer having a single molecular weight using two or more types of amino acid derivatives, which is characterized in that it is synthesized in one pot using a protease as a catalyst. Synthesis method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1249438A JP2525253B2 (en) | 1989-09-26 | 1989-09-26 | Enzymatic method for the synthesis of amino acid oligomers with a single molecular weight |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1249438A JP2525253B2 (en) | 1989-09-26 | 1989-09-26 | Enzymatic method for the synthesis of amino acid oligomers with a single molecular weight |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03183495A true JPH03183495A (en) | 1991-08-09 |
| JP2525253B2 JP2525253B2 (en) | 1996-08-14 |
Family
ID=17192972
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1249438A Expired - Lifetime JP2525253B2 (en) | 1989-09-26 | 1989-09-26 | Enzymatic method for the synthesis of amino acid oligomers with a single molecular weight |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2525253B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001032906A3 (en) * | 1999-10-29 | 2002-02-14 | Novus Int Inc | Oligomers and oligomeric segments of alpha-hydroxy carboxylic acids and alpha-amino acids |
| US6939693B2 (en) | 1999-10-29 | 2005-09-06 | Novus International, Inc. | Enantioselective oligomerization of α-hydroxy carboxylic acids and α-amino acids |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS587278A (en) * | 1981-07-03 | 1983-01-17 | 松下電工株式会社 | Blade of electric razor |
| JPS6246559A (en) * | 1985-08-26 | 1987-02-28 | Tech Res & Dev Inst Of Japan Def Agency | Manufacture of image pickup device |
| JPS6455192A (en) * | 1987-08-27 | 1989-03-02 | Kyowa Hakko Kogyo Kk | Production of acidic amino acid oligopeptide |
-
1989
- 1989-09-26 JP JP1249438A patent/JP2525253B2/en not_active Expired - Lifetime
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS587278A (en) * | 1981-07-03 | 1983-01-17 | 松下電工株式会社 | Blade of electric razor |
| JPS6246559A (en) * | 1985-08-26 | 1987-02-28 | Tech Res & Dev Inst Of Japan Def Agency | Manufacture of image pickup device |
| JPS6455192A (en) * | 1987-08-27 | 1989-03-02 | Kyowa Hakko Kogyo Kk | Production of acidic amino acid oligopeptide |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001032906A3 (en) * | 1999-10-29 | 2002-02-14 | Novus Int Inc | Oligomers and oligomeric segments of alpha-hydroxy carboxylic acids and alpha-amino acids |
| US6605590B1 (en) | 1999-10-29 | 2003-08-12 | Novus International, Inc. | Oligomers and oligomeric segments of alpha-hydroxy carboxylic acids and alpha-amino acids |
| US6939693B2 (en) | 1999-10-29 | 2005-09-06 | Novus International, Inc. | Enantioselective oligomerization of α-hydroxy carboxylic acids and α-amino acids |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2525253B2 (en) | 1996-08-14 |
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