JPH0525480B2 - - Google Patents
Info
- Publication number
- JPH0525480B2 JPH0525480B2 JP60211730A JP21173085A JPH0525480B2 JP H0525480 B2 JPH0525480 B2 JP H0525480B2 JP 60211730 A JP60211730 A JP 60211730A JP 21173085 A JP21173085 A JP 21173085A JP H0525480 B2 JPH0525480 B2 JP H0525480B2
- Authority
- JP
- Japan
- Prior art keywords
- methyl ester
- phenylalanine methyl
- acetyl
- aspartyl
- water
- 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.)
- Expired - Lifetime
Links
- 238000000034 method Methods 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- VSDUZFOSJDMAFZ-VIFPVBQESA-N methyl L-phenylalaninate Chemical compound COC(=O)[C@@H](N)CC1=CC=CC=C1 VSDUZFOSJDMAFZ-VIFPVBQESA-N 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 11
- 108090001109 Thermolysin Proteins 0.000 claims description 10
- OTCCIMWXFLJLIA-BYPYZUCNSA-N N-acetyl-L-aspartic acid Chemical compound CC(=O)N[C@H](C(O)=O)CC(O)=O OTCCIMWXFLJLIA-BYPYZUCNSA-N 0.000 claims description 9
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 7
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 5
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 229960005261 aspartic acid Drugs 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 125000003047 N-acetyl group Chemical class 0.000 claims description 2
- 230000003301 hydrolyzing effect Effects 0.000 claims description 2
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 claims 1
- 108010016626 Dipeptides Proteins 0.000 claims 1
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 150000002148 esters Chemical class 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 20
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 125000006239 protecting group Chemical group 0.000 description 6
- 239000002002 slurry Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 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 5
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-Phenylalanine Natural products OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 235000003704 aspartic acid Nutrition 0.000 description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 3
- 229960005190 phenylalanine Drugs 0.000 description 3
- 108090000526 Papain Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 229940055729 papain Drugs 0.000 description 2
- 235000019834 papain Nutrition 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- AAQFSZFQCXLMNT-ACMTZBLWSA-N (3s)-3-amino-4-[[(2s)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino]-4-oxobutanoic acid;hydrochloride Chemical compound Cl.OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 AAQFSZFQCXLMNT-ACMTZBLWSA-N 0.000 description 1
- GWKOSRIHVSBBIA-REOHCLBHSA-N (3s)-3-aminooxolane-2,5-dione Chemical compound N[C@H]1CC(=O)OC1=O GWKOSRIHVSBBIA-REOHCLBHSA-N 0.000 description 1
- OSEHTEQTVJQGDE-RYUDHWBXSA-N (3s)-3-formamido-4-[[(2s)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino]-4-oxobutanoic acid Chemical compound OC(=O)C[C@H](NC=O)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 OSEHTEQTVJQGDE-RYUDHWBXSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- YZQCXOFQZKCETR-UWVGGRQHSA-N Asp-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 YZQCXOFQZKCETR-UWVGGRQHSA-N 0.000 description 1
- -1 N-acetyl compound Chemical class 0.000 description 1
- MQUUQXIFCBBFDP-VKHMYHEASA-N N-formyl-L-aspartic acid Chemical compound OC(=O)C[C@@H](C(O)=O)NC=O MQUUQXIFCBBFDP-VKHMYHEASA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 150000005690 diesters Chemical class 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 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
- BXRNXXXXHLBUKK-UHFFFAOYSA-N piperazine-2,5-dione Chemical compound O=C1CNC(=O)CN1 BXRNXXXXHLBUKK-UHFFFAOYSA-N 0.000 description 1
- JPWDURLJLRNJAT-NSHDSACASA-N propan-2-yl (2s)-2-amino-3-phenylpropanoate Chemical compound CC(C)OC(=O)[C@@H](N)CC1=CC=CC=C1 JPWDURLJLRNJAT-NSHDSACASA-N 0.000 description 1
- IZNCNDSZTRKDQH-MERQFXBCSA-N propan-2-yl (2s)-2-amino-3-phenylpropanoate;hydrochloride Chemical compound Cl.CC(C)OC(=O)[C@@H](N)CC1=CC=CC=C1 IZNCNDSZTRKDQH-MERQFXBCSA-N 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、Ｎ−アセチル−Ｌ−アスパラギン酸
とＬ−フエニルアラニンアルキルエステルを水性
溶媒中でプロテアーゼを用いて反応させ、α−Ｌ
−アスパルチル−Ｌ−フエニルアラニンメチルエ
ステルを製造する方法に関する。Detailed Description of the Invention [Industrial Application Field] The present invention involves reacting N-acetyl-L-aspartic acid and L-phenylalanine alkyl ester using protease in an aqueous solvent to obtain α-L
- A method for producing aspartyl-L-phenylalanine methyl ester.
α−Ｌ−アスパルチル−Ｌ−フエニルアラニン
メチルエステルはすぐれた甘味剤として知られて
おり、その製造法も種種知られている。例えば、
米国特許3786039、又は米国特許3933721において
はＮ−保護アスパラギン酸無水物を用いる化学合
成法が記載されているが、これらの方法において
は不要なβ−Ｌ−アスパルチル−Ｌ−フエニルア
ラニン誘導体を生成する為に必ずしも満足すべき
ものではなかつた。一方、上記の欠点を克服する
目的で、酵素を用いてペプチド結合を生成させる
方法も検討されており、例えば米国特許4086136
及び特開昭60−164495に記載されている方法があ
るが、前者は保護基として高価なベンジルオキシ
カルボニル基等を用いる事、後の方法においては
腐食性の強いギ酸を使用する事、又収率の点から
必ずしも満足すべきものではなかつた。
α-L-Aspartyl-L-phenylalanine methyl ester is known as an excellent sweetener, and various methods for producing it are also known. for example,
U.S. Patent No. 3786039 or U.S. Patent No. 3933721 describes a chemical synthesis method using N-protected aspartic acid anhydride, but these methods produce unnecessary β-L-aspartyl-L-phenylalanine derivatives. In order to do so, it was not necessarily something that I should be satisfied with. On the other hand, in order to overcome the above-mentioned drawbacks, methods of generating peptide bonds using enzymes are also being considered, such as U.S. Patent No. 4,086,136.
There is also a method described in JP-A No. 60-164495, but the former method uses an expensive benzyloxycarbonyl group as a protecting group, and the latter method uses highly corrosive formic acid, and The results were not necessarily satisfactory in terms of rate.
本発明は、上記Ｎ−保護アスパラギン酸を用い
酵素的にα−Ｌ−アスパルチル−Ｌ−フエニルア
ラニンメチルエステルを製造する際の従来法にお
ける問題点、即ち、使用する保護基が高価であ
る、或いは、腐食性が強い等を解決し、Ｎ−保護
アスパラギン酸とＬ−フエニルアラニンメチルエ
ステルとを酵素により効率よく結合及び保護基を
脱離することにより、α−Ｌ−アスパルチル−Ｌ
−フエニルアラニンメチルエステルを得ることを
目的とする。
The present invention solves problems in the conventional method of enzymatically producing α-L-aspartyl-L-phenylalanine methyl ester using the above-mentioned N-protected aspartic acid, namely, the protecting group used is expensive. Alternatively, α-L-aspartyl-L can be obtained by efficiently bonding N-protected aspartic acid and L-phenylalanine methyl ester and removing the protective group using an enzyme to solve the problem of strong corrosivity.
- Aim to obtain phenylalanine methyl ester.
上記問題点を解決するため、発明者等は鋭意検
討の結果驚くべき事にＮ−アセチル−Ｌ−アスパ
ラギン酸がＬ−フエニルアラニンアルキルエステ
ルと酵素により効率よく結合する事を見出した。
In order to solve the above problems, the inventors conducted intensive studies and surprisingly discovered that N-acetyl-L-aspartic acid is efficiently bonded to L-phenylalanine alkyl ester by an enzyme.
一方ペプチド合成におけるアミノ基の保護基と
してアセチル基は極めて安価であるにもかかわら
ず除去の際にジケトピペラジンが多量に副生され
る事が知られ（蛋白質化学第１巻、赤堀四朗編）、
効率のよい方法とは考えられず、α−Ｌ−アスパ
ルチル−Ｌ−フエニルアラニンメチルエステルの
製法においても米国特許3933721の本文中に保護
基としての記載はあるものの実施例はなく、実質
的に工業的な実施は不可能であると考えられて来
た。 On the other hand, although the acetyl group is extremely inexpensive as a protecting group for amino groups in peptide synthesis, it is known that a large amount of diketopiperazine is produced as a by-product when removed (Protein Chemistry Vol. 1, edited by Shiro Akahori). ,
It is not considered to be an efficient method, and although the text of U.S. Patent No. 3,933,721 describes a protecting group as a protecting group in the method for producing α-L-aspartyl-L-phenylalanine methyl ester, there are no examples, and it is essentially Industrial implementation has been considered impossible.
しかしながら、驚くべき事に高濃度の酸溶液に
おいて、Ｎ−アセチル−Ｌ−アスパルチル−Ｌ−
フエニルアラニン及びそのモノ及びジエステルを
加水分解する事により、ペプチド結合を切断する
ことなくＮ−アセチル基のみを選択的に除去しう
る事を見出し、本発明を完成するに至つた。 However, surprisingly, in highly concentrated acid solutions, N-acetyl-L-aspartyl-L-
The present inventors have discovered that by hydrolyzing phenylalanine and its mono- and diesters, only the N-acetyl group can be selectively removed without cleaving the peptide bond, leading to the completion of the present invention.
本発明方法において原料として用いられるＮ−
アセチル−Ｌ−アスパラギン酸及びＬ−フエニル
アラニンメチルエステルは既知の方法により容易
に合成できる。 N- used as a raw material in the method of the present invention
Acetyl-L-aspartic acid and L-phenylalanine methyl ester can be easily synthesized by known methods.
本発明方法で用いられる酵素としては、蛋白分
解酵素であれば特に制限はなく、サーモライシ
ン、トリプシン、パパイン等あげられるが、中で
もサーモライシンが特に好適に用いられる。又、
酵素反応を行なう際の反応液のPHはサーモライシ
ンでは５〜8.5、トリプシンでは７〜８、パパイ
ンでは６〜７である。一方、酵素反応は通常、水
溶液中で行なわれるが、その際、反応に悪影響を
およぼさない有機溶媒が共存してもよい事は言う
までもない。本発明の酵素反応は温度10〜90℃、
酵素活性を維持する観点から、好ましくは20〜50
℃で行なう。反応は通常約30分から24時間で完結
するがこの反応時間に限定されるものではない。
本発明の方法において、両出発物質の使用濃度に
は特に制限はないが、本酵素反応では、生成した
Ｎ−アセチル−α−Ｌ−アスパルチル−Ｌ−フエ
ニルアラニンメチルエステルは未反応のＬ−フエ
ニルアラニンメチルエステルとの付加物を形成し
て析出してくるので、比較的高い方が望ましい。
両出発物質の使用比率も特に制限はないが、反応
の収率を向上させるためにはＮ−アセチル−Ｌ−
アスパラギン酸のＬ−フエニルアラニンメチルエ
ステルに対する比率が大きいほうがよい。 The enzyme used in the method of the present invention is not particularly limited as long as it is a proteolytic enzyme, and examples include thermolysin, trypsin, and papain, among which thermolysin is particularly preferably used. or,
The pH of the reaction solution used in enzymatic reactions is 5 to 8.5 for thermolysin, 7 to 8 for trypsin, and 6 to 7 for papain. On the other hand, enzymatic reactions are usually carried out in an aqueous solution, and it goes without saying that an organic solvent that does not adversely affect the reaction may coexist therein. The enzyme reaction of the present invention is carried out at a temperature of 10 to 90°C.
From the viewpoint of maintaining enzyme activity, preferably 20 to 50
Perform at ℃. The reaction is usually completed in about 30 minutes to 24 hours, but is not limited to this reaction time.
In the method of the present invention, there is no particular restriction on the concentration of both starting materials used, but in this enzymatic reaction, the produced N-acetyl-α-L-aspartyl-L-phenylalanine methyl ester is used as unreacted L- Since an adduct with phenylalanine methyl ester is formed and precipitated, a relatively high content is desirable.
There is no particular restriction on the ratio of the two starting materials used, but in order to improve the reaction yield, N-acetyl-L-
The larger the ratio of aspartic acid to L-phenylalanine methyl ester, the better.
本発明の方法においては、生成するＮ−アセチ
ル−α−Ｌ−アスパルチル−Ｌ−フエニルアラニ
ンメチルエステルはＬ−フエニルアラニンメチル
エステルとの１：１の付加物として得られるの
で、酸で中和することにより遊離のＮ−アセチル
−α−Ｌ−アスパルチル−Ｌ−フエニルアラニン
メチルエステルとした後、アセチル基を除去すれ
ば容易にAPMをえることができる。 In the method of the present invention, the N-acetyl-α-L-aspartyl-L-phenylalanine methyl ester produced is obtained as a 1:1 adduct with L-phenylalanine methyl ester, so it is neutralized with an acid. APM can be easily obtained by combining the two to obtain free N-acetyl-α-L-aspartyl-L-phenylalanine methyl ester, and then removing the acetyl group.
アセチル基を除去するにはＮ−アセチル化合物
を高濃度の強酸の水溶液もしくはメタノール水溶
液と接触させればよい。本発明の方法において用
いられる酸の濃度としては、1.5N以上、好まし
くは1.5〜6.0Nが選ばれる。強酸の種類としては
塩酸、硫酸、Ｐ−トルエンスルホン酸等があげら
れるが、なかでも塩酸が好適に用いられる。Ｎ−
アセチル−α−Ｌ−アスパルチル−Ｌ−フエニル
アラニンメチルエステルの場合は強酸の水溶液よ
りもメタノール水溶液の方がより好ましい。又、
メタノールと水の比率は特に制限はなく、任意に
選択される。 To remove the acetyl group, the N-acetyl compound may be brought into contact with a highly concentrated aqueous solution of a strong acid or an aqueous methanol solution. The concentration of the acid used in the method of the present invention is selected to be 1.5N or more, preferably 1.5 to 6.0N. Examples of strong acids include hydrochloric acid, sulfuric acid, and P-toluenesulfonic acid, among which hydrochloric acid is preferably used. N-
In the case of acetyl-α-L-aspartyl-L-phenylalanine methyl ester, an aqueous methanol solution is more preferable than an aqueous solution of a strong acid. or,
The ratio of methanol and water is not particularly limited and can be selected arbitrarily.
以下実施例により詳細に説明するが、本発明の
実施の態様はこれに限定されないことは勿論であ
る。 Examples will be described in detail below, but it goes without saying that the embodiments of the present invention are not limited thereto.
参考例 １
Ｎ−アセチル−Ｌ−アスパラギン酸8.8ｇ
（50mmole）を少量の水に懸濁し水酸化ナトリウ
ム水溶液でPH６に調整した。Ｌ−フエニルアラニ
ンメチルエステル塩酸塩10.6ｇ（50mmole）を添
加した後、PHを6.5にし、更に水を加えて液量を
50mlにした。サーモライシン0.4ｇを加え、25℃
で24時間反応した後、得られたスラリーを分離し
た。得られた白色固体はＮ−アセチル−α−Ｌ−
アスパルチル−Ｌ−フエニルアラニンメチルエス
テル及びＬ−フエニルアラニンメチルエステルの
１：１付加物であることを確認した。この固体中
のＮ−アセチル−α−Ｌ−アスパルチル−Ｌ−フ
エニルアラニンメチルエステルをHPLCにて定量
したところ、3.8ｇのＮ−アセチル−α−Ｌ−ア
スパルチル−Ｌ−フエニルアラニンメチルエステ
ルが含まれていた。これは収率が35％であること
を示している。Reference example 1 N-acetyl-L-aspartic acid 8.8g
(50 mmole) was suspended in a small amount of water and the pH was adjusted to 6 with an aqueous sodium hydroxide solution. After adding 10.6 g (50 mmole) of L-phenylalanine methyl ester hydrochloride, the pH was adjusted to 6.5, and water was added to reduce the liquid volume.
The volume was reduced to 50ml. Add 0.4g of thermolysin and heat at 25°C.
After reacting for 24 hours, the resulting slurry was separated. The white solid obtained is N-acetyl-α-L-
It was confirmed that it was a 1:1 adduct of aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester. When the amount of N-acetyl-α-L-aspartyl-L-phenylalanine methyl ester in this solid was determined by HPLC, 3.8 g of N-acetyl-α-L-aspartyl-L-phenylalanine methyl ester was determined. It was included. This indicates a yield of 35%.
参考例 ２
Ｎ−アセチル−Ｌ−アスパラギン酸8.8ｇ
（50mmole）を少量の水に懸濁し水酸化ナトリウ
ム水溶液でPH5.5に調整した。Ｌ−フエニルアラ
ニンメチルエステル塩酸塩2.1ｇ（10mmole）を
添加した後、PHを5.8にし、更に水を加えて液量
を25mlにした。サーモライシン0.5ｇを加え、35
℃で2.5時間反応させた後、得られたスラリーを
分離し、得られた固体を水で洗浄した後、乾燥し
た。このようにしてＮ−アセチル−α−Ｌ−アス
パルチル−Ｌ−フエニルアラニンメチルエステル
とＬ−フエニルアラニンメチルエステルの１：１
付加生成物2.3ｇ（4.6mmole）が得られた。これ
は収率92％であることをしめしている。Reference example 2 N-acetyl-L-aspartic acid 8.8g
(50 mmole) was suspended in a small amount of water, and the pH was adjusted to 5.5 with an aqueous sodium hydroxide solution. After adding 2.1 g (10 mmole) of L-phenylalanine methyl ester hydrochloride, the pH was adjusted to 5.8, and water was further added to bring the liquid volume to 25 ml. Add 0.5g of thermolysin, 35
After reacting at °C for 2.5 hours, the resulting slurry was separated, and the resulting solid was washed with water and then dried. In this way, N-acetyl-α-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester were mixed in a 1:1 ratio.
2.3 g (4.6 mmole) of addition product were obtained. This indicates a yield of 92%.
参考例 ３
Ｎ−アセチル−Ｌ−アスパラギン酸8.8ｇ
（50mmole）を少量の水に懸濁し水酸化ナトリウ
ム水溶液でPH5.5に調整した。Ｌ−フエニルアラ
ニンメチルエステル塩酸塩21.2ｇ（100mmole）
を添加した後、PHを6.6にし、更に水を加えて液
量を50mlにした。サーモライシン1.0ｇを加え、
35℃で2.5時間反応させた後、得られたスラリー
を分離し、この固体を水で洗浄した後、乾燥し
た。このようにしてＮ−アセチル−α−Ｌ−アス
パルチル−Ｌ−フエニルアラニンメチルエステル
とＬ−フエニルアラニンメチルエステルの１：１
付加生成物11.5ｇ（22.5mmole）が得られた。こ
れは収率45％であることをしめしている。Reference example 3 N-acetyl-L-aspartic acid 8.8g
(50 mmole) was suspended in a small amount of water, and the pH was adjusted to 5.5 with an aqueous sodium hydroxide solution. L-phenylalanine methyl ester hydrochloride 21.2g (100mmole)
After adding , the pH was adjusted to 6.6, and water was further added to bring the liquid volume to 50 ml. Add 1.0g of thermolysin,
After reacting at 35° C. for 2.5 hours, the resulting slurry was separated, and the solid was washed with water and then dried. In this way, N-acetyl-α-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester were mixed in a 1:1 ratio.
11.5 g (22.5 mmole) of addition product were obtained. This indicates a yield of 45%.
参考比較例（参考例３に対比）
Ｎ−ホルミル−Ｌ−アスパラギン酸8.1ｇ
（50mmole）を少量の水に懸濁し、水酸化ナトリ
ウム水溶液でPH5.5に調整した。Ｌ−フエニルア
ラニンメチルエステル塩酸塩21.2ｇ（100mmole）
を添加した後、PHを6.5にし、更にに水を加えて
液量を50mlにした。サーモライシン1.0ｇを加え、
35℃で2.5時間反応させた後、得られたスラリー
を分離し、この固体を水で洗浄した後、乾燥し
た。このようにしてＮ−ホルミル−α−Ｌ−アス
パルチル−Ｌ−フエニルアラニンメチルエステル
とＬ−フエニルアラニンメチルエステルの１：１
付加物9.5ｇ（19mmole）が得られた。これは収
率38％であることをしめしている。Reference comparative example (compared to reference example 3) N-formyl-L-aspartic acid 8.1g
(50 mmole) was suspended in a small amount of water, and the pH was adjusted to 5.5 with an aqueous sodium hydroxide solution. L-phenylalanine methyl ester hydrochloride 21.2g (100mmole)
After adding , the pH was adjusted to 6.5, and water was further added to bring the liquid volume to 50 ml. Add 1.0g of thermolysin,
After reacting at 35° C. for 2.5 hours, the resulting slurry was separated, and the solid was washed with water and then dried. In this way, a 1:1 ratio of N-formyl-α-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester was obtained.
9.5 g (19 mmole) of adduct was obtained. This indicates a yield of 38%.
参考例 ４
Ｎ−アセチル−Ｌ−アスパラギン酸8.8ｇ
（50mmole）を少量の水に懸濁し水酸化ナトリウ
ム水溶液でPH5.5に調整した。Ｌ−フエニルアラ
ニンイソプロピルエステル塩酸塩2.4ｇ
（10mmole）を添加した後、PHを5.8にし、更に水
を加えて液量を50mlにした。サーモライシン0.5
ｇを加え、35℃で2.5時間反応させた後、得られ
たスラリーを分離し、得られた固体を水で洗浄し
た後、乾燥した。このようにしてＮ−アセチル−
α−Ｌ−アスパルチル−Ｌ−フエニルアラニンイ
ソプロピルエステルとＬ−フエニルアラニンイソ
プロピルエステルの１：１付加生成物2.7ｇ
（4.7mmole）が得られた。これは収率93％である
ことをしめしている。Reference example 4 N-acetyl-L-aspartic acid 8.8g
(50 mmole) was suspended in a small amount of water, and the pH was adjusted to 5.5 with an aqueous sodium hydroxide solution. L-phenylalanine isopropyl ester hydrochloride 2.4g
After adding (10 mmole), the pH was adjusted to 5.8, and water was added to make the liquid volume 50 ml. Thermolysin 0.5
After reacting at 35° C. for 2.5 hours, the resulting slurry was separated, and the resulting solid was washed with water and then dried. In this way, N-acetyl-
2.7 g of 1:1 addition product of α-L-aspartyl-L-phenylalanine isopropyl ester and L-phenylalanine isopropyl ester
(4.7 mmole) was obtained. This indicates a yield of 93%.
実施例 １
Ｎ−アセチル−Ｌ−アスパラギン酸44ｇ
（250mmole）を少量の水に懸濁し水酸化ナトリ
ウム水溶液でPH5.5に調整した。Ｌ−フエニルア
ラニンメチルエステル塩酸塩10.5ｇ（50mmole）
を添加した後、PHを5.8にし、更に水を加えて液
量を250mlにした。サーモライシン2.5ｇを加え、
35℃で2.5時間反応させた後、得られたスラリー
を分離し、この固体を水で洗浄した後、乾燥し
た。このようにしてＮ−アセチル−α−Ｌ−アス
パルチル−Ｌ−フエニルアラニンメチルエステル
とＬ−フエニルアラニンメチルエステルの１：１
付加生成物11.5ｇ（22.5mmole）が得られた。こ
れは収率92％にあたる。この固体を水に分散させ
PHを1.5に調整し、酢酸エチルで抽出した。酢酸
エチル層を減圧下に濃縮し、Ｎ−アセチル−α−
Ｌ−アスパルチル−Ｌ−フエニルアラニンメチル
エステル10ｇを得た。これを水５ml、メタノール
３mlの混合溶媒に加え、濃塩酸２mlを添加して、
60℃で20分、その後、更に濃塩酸６mlを添加して
25℃で４日間、反応させた。析出したα−Ｌ−ア
スパルチル−Ｌ−フエニルアラニンメチルエステ
ル塩酸塩を分離し、この結晶中のα−Ｌ−アスパ
ルチル−Ｌ−フエニルアラニンメチルエステルを
HPLCで定量したところ、結晶中には5.1ｇ
（17.3mmole）のα−Ｌ−アスパルチル−Ｌ−フ
エニルアラニンメチルエステルが含まれていた。Example 1 44g N-acetyl-L-aspartic acid
(250 mmole) was suspended in a small amount of water and the pH was adjusted to 5.5 with an aqueous sodium hydroxide solution. L-phenylalanine methyl ester hydrochloride 10.5g (50mmole)
After adding , the pH was adjusted to 5.8, and water was further added to bring the liquid volume to 250 ml. Add 2.5g of thermolysin,
After reacting at 35° C. for 2.5 hours, the resulting slurry was separated, and the solid was washed with water and then dried. In this way, N-acetyl-α-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester were mixed in a 1:1 ratio.
11.5 g (22.5 mmole) of addition product were obtained. This corresponds to a yield of 92%. Disperse this solid in water
The pH was adjusted to 1.5 and extracted with ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure to give N-acetyl-α-
10 g of L-aspartyl-L-phenylalanine methyl ester was obtained. Add this to a mixed solvent of 5 ml of water and 3 ml of methanol, add 2 ml of concentrated hydrochloric acid,
20 minutes at 60℃, then add 6ml of concentrated hydrochloric acid.
The reaction was carried out at 25°C for 4 days. The precipitated α-L-aspartyl-L-phenylalanine methyl ester hydrochloride was separated, and α-L-aspartyl-L-phenylalanine methyl ester in the crystals was separated.
When quantified by HPLC, 5.1g was found in the crystals.
(17.3 mmole) of α-L-aspartyl-L-phenylalanine methyl ester.
Claims (1)
ンメチルエステルの製造方法において、Ｌ−アス
パラギン酸とＬ−フエニルアラニンメチルエステ
ルとの間のペプチド結合生成を媒介することが公
知の酵素の存在下で、Ｎ−アセチル−Ｌ−アスパ
ラギン酸とＬ−フエニルアラニンメチルエステル
を反応させ、次にこのＮ−アセチルジペプチドエ
ステルを加水分解してアセチル基を除去すること
を特徴とする、α−Ｌ−アスパルチル−Ｌ−フエ
ニルアラニンメチルエステルの製造方法。 ２ 酵素はサーモライシンである、特許請求の範
囲第１項記載の方法。 ３ アセチル基を除去する条件が、1.5N以上の
強酸の水溶液もしくはメタノール水溶液である特
許請求の範囲第２項の方法。[Claims] 1. In the method for producing α-L-aspartyl-L-phenylalanine methyl ester, it is known to mediate the formation of a peptide bond between L-aspartic acid and L-phenylalanine methyl ester. It is characterized by reacting N-acetyl-L-aspartic acid and L-phenylalanine methyl ester in the presence of an enzyme, and then hydrolyzing this N-acetyl dipeptide ester to remove the acetyl group. , a method for producing α-L-aspartyl-L-phenylalanine methyl ester. 2. The method according to claim 1, wherein the enzyme is thermolysin. 3. The method according to claim 2, wherein the conditions for removing the acetyl group are an aqueous solution of a strong acid of 1.5N or more or an aqueous methanol solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP21173085A JPS6274296A (en) | 1985-09-25 | 1985-09-25 | Enzymic linking of n-acetyl-l-aspartic acid to l-phenylalanine alkyl ester |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP21173085A JPS6274296A (en) | 1985-09-25 | 1985-09-25 | Enzymic linking of n-acetyl-l-aspartic acid to l-phenylalanine alkyl ester |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6274296A JPS6274296A (en) | 1987-04-06 |
| JPH0525480B2 true JPH0525480B2 (en) | 1993-04-13 |
Family
ID=16610644
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP21173085A Granted JPS6274296A (en) | 1985-09-25 | 1985-09-25 | Enzymic linking of n-acetyl-l-aspartic acid to l-phenylalanine alkyl ester |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6274296A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5144073A (en) * | 1988-08-31 | 1992-09-01 | Hubbs John C | Process for preparation of dipeptides |
| US4992552A (en) * | 1988-08-31 | 1991-02-12 | Eastman Kodak Company | Process for preparation of amino acids |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3933781A (en) * | 1973-11-05 | 1976-01-20 | Monsanto Company | Process for the preparation of α-L-aspartyl-L-phenylalanine alkyl esters |
| JPS5392729A (en) * | 1977-01-27 | 1978-08-15 | Toyo Soda Mfg Co Ltd | Adduct of dipeptide derivatives and amino acid derivatives and process for their preparation |
-
1985
- 1985-09-25 JP JP21173085A patent/JPS6274296A/en active Granted
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3933781A (en) * | 1973-11-05 | 1976-01-20 | Monsanto Company | Process for the preparation of α-L-aspartyl-L-phenylalanine alkyl esters |
| JPS5392729A (en) * | 1977-01-27 | 1978-08-15 | Toyo Soda Mfg Co Ltd | Adduct of dipeptide derivatives and amino acid derivatives and process for their preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6274296A (en) | 1987-04-06 |
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