JPS587649B2 - NAD↑+-polyethylene glycol derivative - Google Patents
NAD↑+-polyethylene glycol derivativeInfo
- Publication number
- JPS587649B2 JPS587649B2 JP3789880A JP3789880A JPS587649B2 JP S587649 B2 JPS587649 B2 JP S587649B2 JP 3789880 A JP3789880 A JP 3789880A JP 3789880 A JP3789880 A JP 3789880A JP S587649 B2 JPS587649 B2 JP S587649B2
- Authority
- JP
- Japan
- Prior art keywords
- nad
- polyethylene glycol
- derivative
- glycol derivative
- molecular weight
- 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
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Description
【発明の詳細な説明】
本発明は、応用生化学、酵素工学、酵素レアクター等の
技術分野で用いられるＮＡＤ＋−ポリエチレングリコー
ル誘導体に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to NAD+-polyethylene glycol derivatives used in technical fields such as applied biochemistry, enzyme engineering, and enzyme reactors.
従来、補酵素、ＮＡＤ＋（＝コチンアミドーアデニンジ
ヌクレオチド）を高分子化して尚かつ補酵素活性を保有
させた高分子化ＮＡＤ＋とじては、次のようなものが合
成されている。Conventionally, the following polymerized NAD+, which is obtained by polymerizing the coenzyme NAD+ (=cotinamide adenine dinucleotide) and retains coenzyme activity, has been synthesized.
すなわち、Ｎ６−（６−ａｍｉｎｏｈｅｘｙｌ）一ｃａ
ｒｂａｍｏｙｌ一ＮＡＤ＋一ｄｅｘｔｒａｎ、Ｎ６−（
６−ａｍｉｎｏｈｅｘｙｌ）−ｃａｒｂａｍｏｙｌ−Ｎ
ＡＤ＋−ｓｅｐｈａｄｅｘ，８−（２−ｃａｒｂｏｘｙ
ｅｔｌｙｌｔｈｉｏ）−ＮＡＤ十一ｐｏｌｙｅｔｈｙｌ
ｃｎｅｉｍｉｎｅ、８−（２−ｃ’ａｒｂｏｘｙｅｔｌ
ｙｌｔｈｉｏ）−ＮＡＤ十−ｐｏｌｙｌｙｓｉｎｅ，Ｓ
ｕｃｃｉｎｙｌ−ＮＡＤ＋ｐｏｌｙｅｔ｝ｙｌｅｎｅｉ
ｍｉｎｅ、Ｓｕｃｃｉｎｙｌ−ＮＡＤ十−ｆｏｒｒｙｌ
ｐｏｌｙｅｔｈｙｌｅｎｅｉｍｉｎｅ、Ｎ６−（４−（
３−Ｈｙｄｒｏｘｙｂｕｔｙｌ））−ＮＡＤ＋−ｐｏｌ
ｙｅｔｈｙｌｅｎｅｉｍｉｎｅなどがある。That is, N6-(6-aminohexyl) - ca
rbamoyl-NAD+1-dextran, N6-(
6-aminohexyl)-carbamoyl-N
AD+-sephadex, 8-(2-carboxy
etlylthio)-NAD eleven polyethyl
cneimine, 8-(2-c'arboxyetl
ylthio)-NAD-polylysine,S
uccinyl-NAD+polyet}ylenei
mine, Succinyl-NAD-forryl
polyethyleneneimine, N6-(4-(
3-Hydroxybutyl))-NAD+-pol
There are such things as yetyleneimine.
ところで上述の高分子化ＮＡＤ＋は、いずれも補酵素活
性が不安定であるか、低いか、特定の酵素に対してしか
補酵素活性を示さないものであったし、あるいは高い濃
度で使用したとき高粘度のため酵素レアクターに応用で
きないものであった本発明は、上述の諸点に鑑みて提案
されるもので、脱水素酵素反応またはその逆反応生産物
の全部に適当しうるＮＡＤ＋−ポリエチレングリコール
誘導体を提供することを目的とする。By the way, all of the above-mentioned polymerized NAD+ have unstable or low coenzyme activity, or only show coenzyme activity against specific enzymes, or when used at high concentrations. The present invention, which could not be applied to enzyme reactors due to its high viscosity, was proposed in view of the above-mentioned points. The purpose is to provide
このため本発明は、分子量３０００〜２００００のポリ
エチレングリコールの末端にアミノ基を導入した誘導体
と、Ｎ６−カルボキシアルキル−ＮＡＤ＋とからなり、
ポリエチレングリコール誘導体１分子に対してＮＡＤ十
残基１分子がペプチド結合した化学構造式、
で示されるＮＡＤ十一ポリエチレングリコール誘導体を
要旨とする。Therefore, the present invention consists of a derivative of polyethylene glycol having a molecular weight of 3,000 to 20,000 and having an amino group introduced at the end, and N6-carboxyalkyl-NAD+.
The substance is an NAD 11 polyethylene glycol derivative represented by the following chemical structural formula, in which 1 molecule of NAD 10 residues is peptide-bonded to 1 molecule of the polyethylene glycol derivative.
以下、本発明の実施例について説明すると、その具体例
としては次のようなものがある。Hereinafter, embodiments of the present invention will be described. Specific examples thereof include the following.
こには、ＮＡＤ＋のアデニン環の６位のアミン基をカル
ボキシアルキル化したＮＡＤ十誘導体と、分子量３００
０〜２００００のポリエチレングリコールの末端水酸基
をテミノ基に置換した誘導体とをペプチド結合させた誘
導体である。In this case, an NAD derivative with a molecular weight of 300 and a carboxyalkylated amine group at the 6-position of the adenine ring of NAD
It is a derivative obtained by bonding a peptide bond with a derivative in which the terminal hydroxyl group of polyethylene glycol of 0 to 20,000 is substituted with a temino group.
ｎは７０以上４５０以下のもの、ｍは１以上６以下のも
のである。n is 70 or more and 450 or less, and m is 1 or more and 6 or less.
ただしポリエチレングリコール誘導体１分子ＫＮＡＤ十
残基１分子が結合されている。However, one molecule of polyethylene glycol derivative and one molecule of 10 KNAD residues are bound.
次に、その合成例について説明すると、Ｎ６−ｃａｒｂ
ｏｙｅｔｈｙｌ−ＮＡＤ＋０．６？と、ポリエチレング
リコールの末端をアミン化した誘導体（以下、ＮＨ２−
ＰＥＧと略称する。Next, to explain the synthesis example, N6-carb
oyethyl-NAD+0.6? and a terminally aminated derivative of polyethylene glycol (hereinafter referred to as NH2-
It is abbreviated as PEG.
）（分子量３０００〜３７００）４．９６ｆとを水１５
ｍｌに溶かし、ｐＨを４．５以下に保ちながら少量ずつ
ｄｉｃｙｃｌｏｈｅｘｙｌ ｃａｒｂｏｄｉ ｉｍｉｄ
ｅを合計５１加えて反応させる。) (molecular weight 3000-3700) 4.96f and water 15
ml, and add dicyclohexyl carbodi imid little by little while keeping the pH below 4.5.
A total of 51 pieces of e were added and reacted.
室温で２４時間反応させた反応混液をヂクロ口メタンで
抽出し、カチオン交換、アニオン交換、カラムクロマト
グラフイにより精製し、薄層クロマトグラフにより単一
スポットを与えるＮＡＤ十一ポリエチレングリコール誘
導体を得た。The reaction mixture, which was reacted for 24 hours at room temperature, was extracted with dichloromethane and purified by cation exchange, anion exchange, and column chromatography to obtain an NAD 11 polyethylene glycol derivative that gave a single spot by thin layer chromatography. .
これは、セファデツクスＧ−７５によるクロマトグラフ
イにより高分子化されたＮＡＤ＋であり、その収率は４
６％であった。This is NAD+ that has been polymerized by chromatography using Sephadex G-75, and the yield is 4.
It was 6%.
また分子量２００００のポリエチレングリコールを出発
物質として末端をアミン基に置換し、Ｎ６−ｃａｒｂｏ
ｘｙｅｔｈｙｌ−ＮＡＤ十と結合させた。In addition, using polyethylene glycol with a molecular weight of 20,000 as a starting material, the terminal was substituted with an amine group, and N6-carbo
It was combined with xyethyl-NAD.
このようにして得られる本発明のＮＡＤ＋−ポリエチレ
ングリコール誘導体は、高い補酵素活性を広汎な脱水素
酵素に対して有するのみでなく、長期間の連続反応にも
安定であり、しかもＮＡＤ＋相当で３ｍＭに濃度を上げ
ても粘度、溶解性などの物性が酵素反応の障害にならな
いという特性がある。The NAD+-polyethylene glycol derivative of the present invention obtained in this way not only has high coenzyme activity against a wide range of dehydrogenases, but is also stable for long-term continuous reactions, and is 3mM in NAD+ equivalent. It has the characteristic that physical properties such as viscosity and solubility do not interfere with enzyme reactions even when the concentration is increased.
そして、適当な分子量のものを合成するのが容易である
。Moreover, it is easy to synthesize a compound having an appropriate molecular weight.
また本発明のＮＡＤ＋ーポリエチレングリコール誘導体
が試験したすべての脱水素酵素に対して高い補酵素活性
をもっていることは、次表により示される。Furthermore, the following table shows that the NAD+-polyethylene glycol derivative of the present invention has high coenzyme activity against all dehydrogenases tested.
ＮＡＤ＋−ポリエチレングリ
コール誘導体の補酵素活性
従来の高分子化ＮＡＤ＋では、アルコール脱水素酵素と
乳酸脱水素酵素との双方に対して５０％以上という高活
性を有するものが得られていない。Coenzyme Activity of NAD+-Polyethylene Glycol Derivatives Conventional polymerized NAD+ has not been found to have high activity of 50% or more against both alcohol dehydrogenase and lactate dehydrogenase.
その原因は、ポリマー分子にＮＡＤ＋を密に結合させる
と、却って酵素反応を阻害するものが生成するからであ
る。The reason for this is that when NAD+ is tightly bound to polymer molecules, something that actually inhibits the enzymatic reaction is produced.
これに対し、本発明では、ポリマー１分子に対してＮＡ
Ｄ＋ｌ残基しか結合させていないので、高い補酵素活性
を広範囲の脱水素酵素に対して発現しうるのである。In contrast, in the present invention, NA per molecule of polymer is
Since only D+l residues are bound, high coenzyme activity can be expressed against a wide range of dehydrogenases.
本発明のＮＡＤ＋−ポリエチレングリコール誘導体（Ｐ
ＥＧ−ＮＡＤ＋）の利用の１つとして、２種の脱水素酵
素の共役による連続反応系を組むことができる。NAD+-polyethylene glycol derivative of the present invention (P
As one of the uses of EG-NAD+), it is possible to set up a continuous reaction system by coupling two types of dehydrogenases.
この共役反応系は、次のようなものである。This conjugate reaction system is as follows.
限外沢過膜（許容分子量１ＯＯＯ）を付した反応器中に
、乳酸脱水素酵素２５ｎＭ、アルコール脱水素酵素７５
ｎＭおよびＰＥＧ−ＮＡＤ＋３ｍＭを入れ、これに１０
ｍＭピルビン酸５００ｍＭエタノール溶液を滞留時間３
０分の速度で注入する。In a reactor equipped with an ultrafiltration membrane (acceptable molecular weight 100), 25 nM of lactate dehydrogenase and 75 nM of alcohol dehydrogenase were added.
Add nM and PEG-NAD + 3mM and add 10
Residence time of 500mM ethanol solution of pyruvic acid 3
Inject at a rate of 0 minutes.
そして、注入液量と同量の液量を沢過膜を通して取出し
連続反応させた。Then, the same amount of liquid as the injected liquid was taken out through the filtration membrane and subjected to continuous reaction.
この条件でピルビン酸の乳酸への転換率は１００％であ
り、エタノール濃度を１００ｍＭに低下しても転換率は
９５％であった。Under these conditions, the conversion rate of pyruvic acid to lactic acid was 100%, and even when the ethanol concentration was lowered to 100 mM, the conversion rate was 95%.
７日間の連続運転後、アルコール脱水素酵素を添加する
と、再び９５％の転換率が得られたが、これはＰＥＧ−
ＮＡＤ＋が安定に作動していたことを示している。After 7 days of continuous operation, when alcohol dehydrogenase was added, a conversion rate of 95% was again obtained, which was lower than that of PEG-
This indicates that NAD+ was operating stably.
従来の高分子化ＮＡＤ＋を用いた連続反応では、転換率
は３０％以下であり、連続運転も１日以内であったので
、これにより本発明のＮＡＤ＋−ポリエチレングリコー
ル誘導体の高性能が証明されている。In the conventional continuous reaction using polymerized NAD+, the conversion rate was 30% or less, and the continuous operation was within one day.This proves the high performance of the NAD+-polyethylene glycol derivative of the present invention. There is.
なお、同定資料としては、次のものがある。Identification materials include the following:
（！）Ｎ６−（２−ｃａｒｂｏｘｙｅｔｈｙｌ）−ＮＡ
Ｄ＋の同定
（ａ）Ｕｖ；吸収：最犬２６６ｎｍ（ε−１８６００Ｍ
−ＩＣｍ−１）はＮＡＤ＋のアデニン環６位の修飾を示
す。(!)N6-(2-carboxyethyl)-NA
Identification of D+ (a) Uv; absorption: maximum 266 nm (ε-18600M
-ICm-1) indicates modification of the 6th position of the adenine ring of NAD+.
（ｂ）元素分析：測定値 Ｃ、３５．５１％；Ｈ、４，
０９％；Ｎ，１１．７４％：
Ｃ２４Ｈ２９Ｎ７０１６ｐ２”Ｎａ３Ｈ２０として計算
値Ｃ，３５．１２％；Ｈ１４．７９％一Ｎ−１１．９５
％を得る。(b) Elemental analysis: Measured value C, 35.51%; H, 4,
09%; N, 11.74%: C24H29N7016p2" Calculated value as Na3H20 C, 35.12%; H14.79%-N-11.95
Get %.
（ｅ）赤外スペクトル分析；１６３０Ｃｍ―’に広い吸
収がありＣＯＯ−の存在を示す。(e) Infrared spectrum analysis: There is a broad absorption at 1630 Cm-', indicating the presence of COO-.
（ｄ）原子吸光スペクトル分析；ＮＡＤ＋誘導体１分子
につき３．１モルのＮａ原子の存在を示した。(d) Atomic absorption spectroscopy; showed the presence of 3.1 mol of Na atoms per molecule of NAD+ derivative.
（ｅ）ＮＭＲスペクトル分析；Ｄ２０中でＮＡＤ十のプ
ロトンシグナルの他にδ値
２．５６ｐｐｍ（２Ｈ，ｔ，ＣＨ２ＣＯＯ）と３．６５
ｐｐｍ（（２Ｈ，ｔ，Ｎ６−ＣＨ２）が観測できた。(e) NMR spectrum analysis; in addition to the proton signal of NAD10 in D20, the δ value was 2.56 ppm (2H, t, CH2COO) and 3.65
ppm ((2H, t, N6-CH2)) could be observed.
（ｉｉ）アミノポリエチレングリコールの同定（ａ）Ｍ
Ｊニトロベ゛ンセンスルホン酸法により求めたアミン基
の数はポリエチレングリコール１分子当り１．２〜１．
４である。(ii) Identification of aminopolyethylene glycol (a) M
The number of amine groups determined by the J nitrobenzene sulfonic acid method was 1.2 to 1.2 per molecule of polyethylene glycol.
It is 4.
（ｔ）ＳＰ−Ｓｅｐｈａｄｅｘに吸着され４５ｍＭＮａ
Ｃｌで溶出される。(t) 45mMNa adsorbed on SP-Sephadex
It is eluted with Cl.
（１ｉ）ＮＡＤ＋−ポリエチレングリコール誘導体の同
定
（ａ）シリカゲル薄層クロマトグラフイ；溶媒系（イソ
酪酸：水：２８％アンモニア水−６６：３３：１、７）
または（ジクロロメタン：メタノール＝５：１）で単一
スポットを与える。(1i) Identification of NAD+-polyethylene glycol derivative (a) Silica gel thin layer chromatography; solvent system (isobutyric acid: water: 28% ammonia water - 66:33:1, 7)
or (dichloromethane:methanol=5:1) to give a single spot.
（ｈ｝透析膜Ｓｐｅｃｔｒａｐｏｒ Ｎｏ．６（透過最
大分子量＝１０００）２４時間透析で漏出せず。(h) Dialysis membrane Spectrapor No. 6 (maximum permeation molecular weight = 1000) No leakage after 24 hours of dialysis.
（ｃ）標品の２６６ｎｍ吸光度分析と、トリニトロさン
センスルホン酸によるアミノ基の測定からＮＡＤ＋ｌ残
基当りのアミノ基数は１．２〜１．４である。(c) The number of amino groups per NAD+1 residue is 1.2 to 1.4 from 266 nm absorbance analysis of the standard sample and measurement of amino groups using trinitrosense sulfonic acid.
（ｄ）ＳＰ−Ｓｅｐｈａｄｅｘ Ｃ−２５にもＤＥＡＥ
−Ｓｅｐｈａｄｅｘ Ａ−２５にも非吸着である。(d) DEAE for SP-Sephadex C-25
- It is also non-adsorbed to Sephadex A-25.
（ｅ）このものは酵母アルコール脱水素酵素、エタノー
ルの存在下で３４０ｎｍの吸光が増大する。(e) This substance is yeast alcohol dehydrogenase, and its absorbance at 340 nm increases in the presence of ethanol.
以上合成反応原料の化学構造と、生産物のアミン基数、
カルボキシール基の不存在および分子量とから化学構造
式が正しいものと考えられる。The chemical structure of the synthesis reaction raw materials, the number of amine groups in the product,
The chemical structural formula is believed to be correct based on the absence of carboxyl groups and the molecular weight.
Claims (1)
ールの末端にアミン基を導入した誘導体と、Ｎ６−カル
ボキシアルキルーＮＡＤ＋とからなり、ポリエチレング
リコール誘導体１分子に対してＮＡＤ＋残基１分子がペ
プチド結合した化学構造式、 で示される、ＮＡＤ＋−ポリエチレングリコール誘導体
。[Scope of Claims] 1 Consists of a derivative of polyethylene glycol having a molecular weight of 3,000 to 20,000 with an amine group introduced at the end, and N6-carboxyalkyl-NAD+, with one molecule of NAD+ residue per molecule of polyethylene glycol derivative being a peptide. A NAD+-polyethylene glycol derivative represented by the bonded chemical structural formula:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3789880A JPS587649B2 (en) | 1980-03-25 | 1980-03-25 | NAD↑+-polyethylene glycol derivative |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3789880A JPS587649B2 (en) | 1980-03-25 | 1980-03-25 | NAD↑+-polyethylene glycol derivative |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS56133334A JPS56133334A (en) | 1981-10-19 |
| JPS587649B2 true JPS587649B2 (en) | 1983-02-10 |
Family
ID=12510354
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3789880A Expired JPS587649B2 (en) | 1980-03-25 | 1980-03-25 | NAD↑+-polyethylene glycol derivative |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS587649B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0644927B2 (en) * | 1987-03-14 | 1994-06-15 | 日本油脂株式会社 | Sustained release active ingredient release agent |
| GB9814212D0 (en) | 1998-07-01 | 1998-09-02 | Dow Corning Gmbh | Polymer composition |
-
1980
- 1980-03-25 JP JP3789880A patent/JPS587649B2/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| JPS56133334A (en) | 1981-10-19 |
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