JP3508563B2 - Pyrazine derivative-recognizing antibody and method for measuring 1,2-dicarbonyl derivative using the same - Google Patents

Pyrazine derivative-recognizing antibody and method for measuring 1,2-dicarbonyl derivative using the same

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Publication number
JP3508563B2
JP3508563B2 JP24912298A JP24912298A JP3508563B2 JP 3508563 B2 JP3508563 B2 JP 3508563B2 JP 24912298 A JP24912298 A JP 24912298A JP 24912298 A JP24912298 A JP 24912298A JP 3508563 B2 JP3508563 B2 JP 3508563B2
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JP
Japan
Prior art keywords
derivative
group
antibody
compound
pyrazine
Prior art date
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JP24912298A
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Japanese (ja)
Other versions
JPH11181000A (en
Inventor
好昭 内田
義裕 倉野
哲 伊藤
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Fujirebio Inc
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Fujirebio Inc
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Description

【発明の詳細な説明】Detailed Description of the Invention

【0001】[0001]

【発明の属する技術分野】本発明は、生体内におけるタ
ンパク質の糖化反応の中間体である1,2−ジカルボニ
ル誘導体の反応物であるピラジン誘導体を認識する抗
体、該抗体を誘起するための免疫原及び該抗体を用いた
1,2−ジカルボニル誘導体の免疫測定方法に関する。
TECHNICAL FIELD The present invention relates to an antibody which recognizes a pyrazine derivative which is a reaction product of a 1,2-dicarbonyl derivative which is an intermediate of a glycation reaction of a protein in vivo, and an immunity for inducing the antibody. The present invention relates to an immunoassay method for a 1,2-dicarbonyl derivative using a raw material and the antibody.

【0002】糖尿病はインスリンに起因する代謝異常の
疾患であり、慢性化しやすいため、種々の合併症を引き
起こす。この合併症は、例えば、眼疾患、腎疾患、神経
症、心血管系合併症、壊疽等があげられ、合併症の有無
や程度によって患者の予後は大きく左右される。
[0002] Diabetes is a metabolic disorder caused by insulin and is prone to be chronic, and thus causes various complications. The complications include, for example, eye diseases, renal diseases, neuroses, cardiovascular complications, gangrene, etc., and the prognosis of patients is greatly influenced by the presence or absence of complications and the degree thereof.

【0003】一方、糖尿病における蛋白質の糖化反応の
有力な原因とされる中間体に、デオキシグルコゾンがあ
る。デオキシグルコゾンは、蛋白質間のクロスリンカー
として作用し、糖化反応の最終反応物の形成を促進する
機能を持つ物質である。このデオキシグルコゾンが、高
血糖状態の持続しやすい糖尿病患者、特に腎症を合併し
ている患者の血中において増加していること(Biochem.
Biophys. Res. Commun., Vol196, p837-843, 1993)
や、糖尿病性動脈硬化を引き起こしている患者の血中に
おいて増加していること(DIABETES, Vol.45, SP3, S81
-83, 1996 )が示されてから、糖尿病合併症の病状を把
握する指標として注目された。また、メチルグルオキザ
ールも糖尿病性動脈硬化を引き起こしている患者の血中
において増加していること(DIABETES, Vol.45, SP3, S
81-83, 1996 )が示唆され、糖尿病における1,2−ジ
カルボニル誘導体の動向が注目されている。
On the other hand, deoxyglucosone is an intermediate that is considered to be a major cause of the glycation reaction of proteins in diabetes. Deoxyglucosone is a substance that functions as a cross-linker between proteins and has a function of promoting the formation of the final reaction product of the saccharification reaction. This deoxyglucosone is increased in the blood of diabetic patients with persistent hyperglycemia, especially those with nephropathy (Biochem.
Biophys. Res. Commun., Vol196, p837-843, 1993)
And in the blood of patients with diabetic arteriosclerosis (DIABETES, Vol.45, SP3, S81
-83, 1996), it attracted attention as an index for understanding the condition of diabetic complications. Methylgluoxal is also increased in the blood of patients with diabetic arteriosclerosis (DIABETES, Vol.45, SP3, S
81-83, 1996), and attention has been paid to the trend of 1,2-dicarbonyl derivatives in diabetes.

【0004】しかしながら、デオキシキグルコゾンを初
めとする1,2−ジカルボニル誘導体は、非常に不安定
な物質であるため、これまでは1,2−ジカルボニル誘
導体を誘導体、あるいは、安定な代謝物に変換した後抽
出し、GS/MSやHPLC等を用いたクロマトグラフ
ィー分析にて測定していた。これらの方法は、1,2−
ジカルボニル誘導体の抽出操作及びクロマトグラフィー
分析操作が煩雑で時間を要するため、更に簡便で多検体
測定が可能な測定手法が待ち望まれていた。
However, since 1,2-dicarbonyl derivatives such as deoxyquiglucosone are very unstable substances, the 1,2-dicarbonyl derivatives have hitherto been derivatives or stable metabolites. It was converted to a substance, extracted, and measured by chromatographic analysis using GS / MS, HPLC, or the like. These methods are 1,2-
Since the extraction operation and the chromatographic analysis operation of the dicarbonyl derivative are complicated and time-consuming, there has been a long-felt demand for a simpler measurement method capable of measuring multiple samples.

【0005】[0005]

【発明が解決しようとする課題】従って、本発明の目的
は、簡便で多検体測定が可能な1,2−ジカルボニル誘
導体の測定方法を提供することである。さらに、本発明
の目的は、該測定方法に用いられる抗体、及び該抗体の
産生を誘起するための免疫原を提供することである。
SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a method for measuring a 1,2-dicarbonyl derivative which is simple and capable of measuring many analytes. Further, it is an object of the present invention to provide an antibody used in the measuring method and an immunogen for inducing the production of the antibody.

【0006】[0006]

【課題を解決するための手段】本願発明者らは、鋭意研
究の結果、1,2−ジカルボニル誘導体をジアミノ誘導
体と反応させて安定なピラジン誘導体に変え、このピラ
ジン誘導体を認識する抗体を用いた免疫測定で該ピラジ
ン誘導体を測定することにより、検体中の1,2−ジカ
ルボニル誘導体を測定することができることを見出し、
かつ、上記の抗体を現実に提供し、本発明を完成した。
Means for Solving the Problems As a result of earnest research, the inventors of the present application have used an antibody that recognizes a pyrazine derivative by reacting a 1,2-dicarbonyl derivative with a diamino derivative to convert it into a stable pyrazine derivative. It was found that the 1,2-dicarbonyl derivative in a sample can be measured by measuring the pyrazine derivative by immunoassay,
The present invention was completed by actually providing the above-mentioned antibody.

【0007】すなわち、本発明は、下記一般式(I)で
示されるピラジン誘導体の少なくともR1 又はR2 を包
含する領域を認識する抗体を提供する。
That is, the present invention provides an antibody that recognizes a region containing at least R 1 or R 2 of a pyrazine derivative represented by the following general formula (I).

【0008】[0008]

【化1】 [Chemical 1]

【0009】(ただし、式中、R は水素、R トリ
ヒドロキシブチル基であり、Aはピラジン環と結合して
5員又は6員の芳香族炭化水素基、芳香族複素環基又は
脂環式炭化水素基を形成する基であり、Rは架橋性残
基であり、Aにより形成される環は、上記Rの他に、
5員環の場合には1又は2個、6員環の場合には1〜3
個のさらなる置換基によって置換されていてもよい)。
(Wherein, R 1 is hydrogen, R 2 is a trihydroxybutyl group, A is a 5- or 6-membered aromatic hydrocarbon group or aromatic heterocyclic group bonded to the pyrazine ring, or R 3 is a group that forms an alicyclic hydrocarbon group, R 3 is a crosslinkable residue, and the ring formed by A is, in addition to the above R 3 ,
1 or 2 in case of 5-membered ring, 1-3 in case of 6-membered ring
Optionally further substituted by 1).

【0010】また、本発明は、上記一般式(I)で示さ
れるピラジン誘導体又は該ピラジン誘導体のR3 に免疫
原性担体が結合したものから成る免疫原を提供する。
The present invention also provides an immunogen comprising a pyrazine derivative represented by the above general formula (I) or an R 3 of the pyrazine derivative bound to an immunogenic carrier.

【0011】さらに、本発明は、生体内におけるタンパ
ク質の糖化反応の中間体である1,2−ジカルボニル誘
導体を免疫測定する方法であって、検体中の1,2−ジ
カルボニル誘導体を、一般式(II)
Furthermore, the present invention is a method for immunoassaying a 1,2-dicarbonyl derivative which is an intermediate of a glycation reaction of a protein in a living body, wherein the 1,2-dicarbonyl derivative in a sample is generally used. Formula (II)

【化2】 (R3 及びAは、上記一般式(I)の場合と同義)で示
されるジアミノ誘導体と反応させて上記一般式(I)で
示されるピラジン誘導体を生成させ、該ピラジン誘導体
と上記本発明の抗体との抗原抗体反応を利用した免疫測
定により該ピラジン誘導体を測定し、それによって検体
中の1,2−ジカルボニル誘導体を測定することから成
る、1,2−ジカルボニル誘導体の免疫測定方法を提供
する。
[Chemical 2] (R 3 and A have the same meanings as in the case of the above general formula (I)) to produce a pyrazine derivative represented by the above general formula (I), and the pyrazine derivative and the above An immunoassay method for a 1,2-dicarbonyl derivative, which comprises measuring the pyrazine derivative by an immunoassay utilizing an antigen-antibody reaction with an antibody, and thereby measuring the 1,2-dicarbonyl derivative in a sample. provide.

【0012】[0012]

【発明の実施の形態】上述のように、本発明の抗体は、
上記一般式(I)で示されるピラジン誘導体を認識する
ものである。
BEST MODE FOR CARRYING OUT THE INVENTION As described above, the antibody of the present invention is
It recognizes the pyrazine derivative represented by the general formula (I).

【0013】一般式(I)中のR、Rの定義は上記
の通りであり、Rが水素でRがトリヒドロキシブチ
ル基である。
The definitions of R 1 and R 2 in the general formula (I) are as described above, wherein R 1 is hydrogen and R 2 is a trihydroxybutyl group.

【0014】一般式(I)中、Aの定義は上記の通りで
あり、Aにより形成される環がピリジン、ベンゼン、フ
ラン又はチオフェンであることが好ましい。また、Aに
より形成される環は、R3 の他に、5員環の場合には1
又は2個、6員環の場合には1〜3個のさらなる任意の
置換基によって置換されていてもよい。該置換基として
は、カルボキシ基、スルホニル基、アルコキシカルボニ
ル基、カルバモイル基、シアノ基、ホルミル基、ヒドロ
キシ基、メルカプト基、ニトロ基、ハロゲン原子;、カ
ルボキシ基、スルホニル基、アルコキシカルボニル基、
カルバモイル基、シアノ基、ホルミル基、ヒドロキシ
基、メルカプト基、ニトロ基、ハロゲン原子で置換され
ていてもよい芳香族炭化水素基、芳香族複素環基、また
は脂肪族炭化水素基(該脂肪族炭化水素基は炭素鎖が酸
素またはイオウ原子によって置換されていてもよい)等
を挙げることができる。
In the general formula (I), the definition of A is as described above, and the ring formed by A is preferably pyridine, benzene, furan or thiophene. The ring formed by A is 1 in the case of a 5-membered ring in addition to R 3.
Alternatively, in the case of a 2-membered or 6-membered ring, it may be substituted with 1 to 3 further optional substituents. As the substituent, a carboxy group, a sulfonyl group, an alkoxycarbonyl group, a carbamoyl group, a cyano group, a formyl group, a hydroxy group, a mercapto group, a nitro group, a halogen atom; a carboxy group, a sulfonyl group, an alkoxycarbonyl group,
A carbamoyl group, a cyano group, a formyl group, a hydroxy group, a mercapto group, a nitro group, an aromatic hydrocarbon group which may be substituted with a halogen atom, an aromatic heterocyclic group, or an aliphatic hydrocarbon group (the aliphatic hydrocarbon group). Examples of the hydrogen group include a carbon chain which may be substituted with oxygen or sulfur atom).

【0015】一般式(I)中、R3 は架橋性残基であ
る。本明細書において架橋性残基とは、ピラジン誘導体
と他の物質とを結合することのできる化学構造を意味す
る。R3 は、ピラジン部分を免疫原性担体や標識のよう
な他の化合物と結合することを目的とするのであるか
ら、この目的が達成されるならばその具体的な構造は如
何なるものでもよい。従って、架橋性残基による結合手
段としては、共有結合及び非共有結合のどちらをも包含
する。非共有結合としては、疎水結合、水素結合、イオ
ン結合、配位結合等を挙げることができ、具体的には、
長鎖の脂肪族炭化水素(炭素数は好ましくは8〜1
8)、カルボキシレートイオン、アンモニウムイオン等
を挙げることがきる。共有結合の場合は、R3 はスペー
サー部分と反応部分とから成ることが好ましい。反応部
分とは他の化合物と反応して共有結合を形成することの
できる官能基を意味し、例えば、カルボキシル基、水酸
基、スルフヒドリル基、アミノ基、マレイミド基、アル
デヒド基、ハロゲン原子等、及び他の化合物と結合する
ために活性化されたこれら官能基の誘導体を挙げること
ができる。スペーサー部分とは、ピラジン部分と反応部
分とを適当な距離に置くことのできる化学構造を意味
し、例えば、脂肪族炭化水素(炭素数は好ましくは2〜
6)、芳香族炭化水素、またはこれらが互いにエステル
結合、アミド結合、エーテル結合、チオエーテル結合、
ジスルフィド結合、シッフ塩基結合等により連結された
構造等を挙げることができるが、R3 は反応部分だけで
スペーサー部分を含まない構造も取り得る。
In the general formula (I), R 3 is a crosslinkable residue. In the present specification, the crosslinkable residue means a chemical structure capable of binding the pyrazine derivative and another substance. Since R 3 has the purpose of binding the pyrazine moiety to another compound such as an immunogenic carrier or a label, any specific structure thereof can be used as long as this purpose is achieved. Therefore, the means for binding by the crosslinkable residue includes both covalent bond and non-covalent bond. Examples of the non-covalent bond include a hydrophobic bond, a hydrogen bond, an ionic bond, a coordinate bond, and the like.
Long-chain aliphatic hydrocarbon (carbon number is preferably 8 to 1)
8), carboxylate ion, ammonium ion and the like. In the case of a covalent bond, R 3 preferably consists of a spacer moiety and a reactive moiety. The reactive portion means a functional group capable of reacting with another compound to form a covalent bond, and examples thereof include a carboxyl group, a hydroxyl group, a sulfhydryl group, an amino group, a maleimide group, an aldehyde group, a halogen atom, and the like. Mention may be made of derivatives of these functional groups which have been activated to bind to the compounds of The spacer portion means a chemical structure capable of placing the pyrazine portion and the reaction portion at an appropriate distance, and includes, for example, an aliphatic hydrocarbon (carbon number is preferably 2 to
6), aromatic hydrocarbons, or these are ester bonds, amide bonds, ether bonds, thioether bonds,
Examples thereof include a structure linked by a disulfide bond, a Schiff base bond, etc., but R 3 may have a structure that includes only a reactive portion and does not include a spacer portion.

【0016】R3 で示される架橋性残基は、ピラジン誘
導体と他の物質とを直接結合させることも、2価性の反
応性架橋剤を介して間接的に結合させることもできる。
ここで、2価性の反応性架橋剤の具体例として、スクシ
ンイミジル3−(2−ピリジルチオ)プロピオネ−ト
(SPDP)、N−スクシンイミジル4−マレイミド酪
酸(GMBS)、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)、N,
N’−ジシクロヘキシルカルボジイミド(DCC)、3
−(2−ピリジルジチオ)プロピオニルヒドラジド(P
DPH)、4−(4−マレイミドメチル)ブタン酸ヒド
ラジド塩酸塩(MPBH)等を挙げることができる。
The crosslinkable residue represented by R 3 can be directly bonded to the pyrazine derivative and another substance, or indirectly bonded via a divalent reactive crosslinker.
Here, as specific examples of the divalent reactive crosslinking agent, succinimidyl 3- (2-pyridylthio) propionate (SPDP), N-succinimidyl 4-maleimidobutyric acid (GMBS), 1-ethyl-3- (3 -Dimethylaminopropyl) carbodiimide hydrochloride (EDC), N,
N'-dicyclohexylcarbodiimide (DCC), 3
-(2-pyridyldithio) propionyl hydrazide (P
DPH), 4- (4-maleimidomethyl) butanoic acid hydrazide hydrochloride (MPBH), and the like.

【0017】一般式(I)で示される化合物としての好
ましい構造は、R1 及びR2 のどちらか一方が水素、他
方が2,3,4−トリヒドロキシブチル基、Aがベンゼ
ン、R3 が4−カルボキサミドブタノイル−(2−メル
カプト)エチルアミド基である化合物N−(4−(3−
(2,3,4−トリヒドロキシブチル)キノキサリン−
6−カルボキサミド)ブタノイル)−2−メルカプトエ
チルアミンまたはN−(4−(2−(2,3,4−トリ
ヒドロキシブチル)キノキサリン−6−カルボキサミ
ド)ブタノイル)−2−メルカプトエチルアミンが好ま
しい。N−(4−(3−(2,3,4−トリヒドロキシ
ブチル)キノキサリン−6−カルボキサミド)ブタノイ
ル)−2−メルカプトエチルアミン及びN−(4−(2
−(2,3,4−トリヒドロキシブチル)キノキサリン
−6−カルボキサミド)ブタノイル)−2−メルカプト
エチルアミンの化学式を以下に示す。
A preferred structure of the compound represented by formula (I) is that one of R 1 and R 2 is hydrogen, the other is 2,3,4-trihydroxybutyl group, A is benzene, and R 3 is 4-carboxamidobutanoyl- (2-mercapto) ethylamide compound N- (4- (3-
(2,3,4-trihydroxybutyl) quinoxaline-
6-carboxamido) butanoyl) -2-mercaptoethylamine or N- (4- (2- (2,3,4-trihydroxybutyl) quinoxaline-6-carboxamido) butanoyl) -2-mercaptoethylamine is preferred. N- (4- (3- (2,3,4-trihydroxybutyl) quinoxaline-6-carboxamido) butanoyl) -2-mercaptoethylamine and N- (4- (2
The chemical formula of-(2,3,4-trihydroxybutyl) quinoxaline-6-carboxamido) butanoyl) -2-mercaptoethylamine is shown below.

【0018】[0018]

【化3】 [Chemical 3]

【0019】[0019]

【化4】 [Chemical 4]

【0020】本発明の抗体は、上記したピラジン誘導体
を認識するものであるが、特に、該ピラジン誘導体の少
なくともR1 又はR2 を包含する領域を認識するもので
ある。すなわち、より詳細に後述するように、本発明の
抗体は検体中の1,2−ジカルボニル誘導体を測定する
ために用いられるものである。この1,2−ジカルボニ
ル誘導体が後述するジアミノ誘導体と反応して上記一般
式(I)で示されるピラジン誘導体を形成し、形成され
たピラジン誘導体を本発明の抗体を用いて測定するので
ある。そして、1,2−ジカルボニル誘導体は、ピラジ
ン誘導体中のR1 及びR2 並びにこれらの置換基が結合
しているピラジン環の部分を構成する。従って、本発明
の抗体は、ピラジン誘導体の少なくともR1 又はR2
包含する領域を認識するものである必要があり、例え
ば、R3 のみを認識するような抗体は本発明の抗体では
ない。もっとも、R3 を含む、ピラジン誘導体全体を認
識するような抗体は、当然、本発明の抗体である。
The antibody of the present invention recognizes the above-mentioned pyrazine derivative, and particularly recognizes a region including at least R 1 or R 2 of the pyrazine derivative. That is, as described in more detail below, the antibody of the present invention is used for measuring a 1,2-dicarbonyl derivative in a sample. This 1,2-dicarbonyl derivative reacts with a diamino derivative described below to form a pyrazine derivative represented by the above general formula (I), and the formed pyrazine derivative is measured using the antibody of the present invention. The 1,2-dicarbonyl derivative constitutes the pyrazine ring portion to which R 1 and R 2 in the pyrazine derivative and their substituents are bonded. Therefore, the antibody of the present invention needs to recognize a region including at least R 1 or R 2 of the pyrazine derivative, and for example, an antibody that recognizes only R 3 is not the antibody of the present invention. However, an antibody that recognizes the entire pyrazine derivative including R 3 is, of course, the antibody of the present invention.

【0021】本発明の抗体は、ポリクローナル抗体でも
モノクローナル抗体でもよいが、均一な反応特異性を有
する抗体を再現性良く得るという観点からモノクローナ
ル抗体が好ましい。なお、ポリクローナル抗体の場合に
は、ピラジン誘導体の少なくともR1 又はR2 を包含す
る領域以外の領域を認識する抗体が含まれていても構わ
ない。
The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred from the viewpoint of obtaining an antibody having uniform reaction specificity with good reproducibility. In the case of a polyclonal antibody, an antibody that recognizes a region other than the region containing at least R 1 or R 2 of the pyrazine derivative may be included.

【0022】本発明の抗体は、一般式(I)で示される
ピラジン誘導体又は該ピラジン誘導体のR3 に免疫原性
担体が結合したものから成る免疫原を用いて常法により
調製することができ、後者の免疫原を用いることがより
好ましい。免疫原性担体としては、BSAやKLHのよ
うなタンパク質が好ましい。
The antibody of the present invention can be prepared by a conventional method using an immunogen comprising a pyrazine derivative represented by the general formula (I) or an R 3 of the pyrazine derivative bound to an immunogenic carrier. More preferably, the latter immunogen is used. Proteins such as BSA and KLH are preferred as immunogenic carriers.

【0023】ポリクローナル抗体の場合には、上記の免
疫原を動物に免疫し、抗血清から常法により抗体を生成
することにより本発明の抗体を得ることができる。ま
た、モノクローナル抗体の場合には、上記免疫原を免疫
した動物の脾臓細胞等のような抗体産生細胞と、ミエロ
ーマ細胞のような腫瘍細胞とを、ポリエチレングリコー
ル等のような融合剤で融合してハイブリドーマを作製す
る。次いでハイブリドーマをHAT培地のような選択培
地を用いて選択し、限界希釈法等の適当な方法でモノク
ロン化して培養する。この培養上清を酵素免疫測定法の
ような適当な免疫測定法で分析し、目的とする抗ピラジ
ン誘導体抗体を産生しているクローンを選択する。これ
らのモノクローナル抗体作製の手法は、公知の方法、例
えば、ケーラーとミルシュタイン(Nature 256 495 197
5 )、シェーラー(Nature 285 4461980 )等の方法に
より行うことができる。また上述の手法により作製した
モノクローナル抗体は、培養上清から、塩折、イオン交
換クロマトグラフィー、ゲルろ過クロマトグラフィー等
の分析・精製手段により回収することができる。さら
に、ピラジン誘導体の少なくともR又はRを包含す
る領域を認識する抗体は、下記実施例に詳述するよう
に、1,2−ジカルボニル誘導体の濃度を変えて後述す
る本発明の方法により1,2−ジカルボニル誘導体の測
定を行って検量線を書き、該検量線が1,2−ジカルボ
ニル誘導体の濃度に依存して変化するものを選択するこ
とにより得ることができる。
In the case of a polyclonal antibody, the antibody of the present invention can be obtained by immunizing an animal with the above immunogen and producing the antibody from antiserum by a conventional method. In the case of a monoclonal antibody, antibody-producing cells such as spleen cells of an animal immunized with the above immunogen and tumor cells such as myeloma cells are fused with a fusion agent such as polyethylene glycol. Create hybridomas. Next, the hybridomas are selected using a selective medium such as HAT medium, subjected to monocloning by an appropriate method such as limiting dilution method, and cultured. The culture supernatant is analyzed by an appropriate immunoassay such as enzyme immunoassay to select a clone producing the desired anti-pyrazine derivative antibody. These monoclonal antibody production methods are known methods, for example, Keller and Milstein (Nature 256 495 197).
5) and Scheller (Nature 285 4461980). Further, the monoclonal antibody produced by the above-mentioned method can be recovered from the culture supernatant by analysis / purification means such as salt folding, ion exchange chromatography, gel filtration chromatography and the like. Furthermore, an antibody recognizing a region containing at least R 1 or R 2 of a pyrazine derivative can be prepared by varying the concentration of a 1,2-dicarbonyl derivative according to the method of the present invention described below, as described in detail in the Examples below. It can be obtained by measuring the 1,2-dicarbonyl derivative, writing a calibration curve, and selecting one that changes depending on the concentration of the 1,2-dicarbonyl derivative.

【0024】次に、本発明の抗体を用いた1,2−ジカ
ルボニル誘導体の測定方法について説明する。
Next, a method for measuring a 1,2-dicarbonyl derivative using the antibody of the present invention will be described.

【0025】本発明の免疫測定方法の測定対象である
1,2−ジカルボニル誘導体は、デオキシグルコゾン
の、生体内におけるタンパク質の糖化反応の中間体であ
る1,2−ジカルボニル誘導体であり、例えば、2−デ
オキシグルコゾン、3−デオキシグルコゾン、4−デオ
キシグルコゾン等を挙げることができる。
The 1,2-dicarbonyl derivative to be measured by the immunoassay method of the present invention is a 1,2-dicarbonyl derivative which is an intermediate of protein saccharification reaction in vivo of deoxyglucosone, For example, 2-deoxyglucosone, 3-deoxyglucosone, 4-deoxyglucosone, etc. can be mentioned.

【0026】また、1,2−ジカルボニル誘導体を含む
検体としては、特に限定されないが、通常、血清、血
漿、血液、尿等の体液や生体組織である。
The sample containing the 1,2-dicarbonyl derivative is not particularly limited, but it is usually a body fluid such as serum, plasma, blood, urine or a biological tissue.

【0027】本発明の免疫測定方法では、先ず、検体中
の1,2−ジカルボニル誘導体を、上記一般式(II)で
示されるジアミノ誘導体と反応させて上記一般式(I)
で示されるピラジン誘導体を生成させる。この反応は、
好ましくは、4℃〜37℃、さらに好ましくは室温で、
好ましくは12〜20時間程度、単に混合するだけで行
うことができる。また、反応させるジアミノ誘導体の濃
度は、通常、1〜50μg/ml程度、好ましくは5〜
20μg/ml程度である。なお、1,2−ジカルボニ
ル誘導体とジアミノ誘導体との反応は、免疫反応の前に
行うことも、同時に行うこともできる。以下に、例とし
て、1,2−ジカルボニル誘導体の一種である3−デオ
キシグルコゾンとジアミノ誘導体の一種であるジアミノ
ベンゼン誘導体との反応式を示す。
In the immunoassay method of the present invention, first, the 1,2-dicarbonyl derivative in the sample is reacted with the diamino derivative represented by the general formula (II) to give the general formula (I).
The pyrazine derivative represented by is produced. This reaction is
Preferably at 4 ° C to 37 ° C, more preferably at room temperature,
It can be carried out preferably by simply mixing for about 12 to 20 hours. The concentration of the diamino derivative to be reacted is usually about 1 to 50 μg / ml, preferably 5 to
It is about 20 μg / ml. The reaction between the 1,2-dicarbonyl derivative and the diamino derivative can be performed before the immune reaction or simultaneously. As an example, a reaction formula of 3-deoxyglucosone, which is one of 1,2-dicarbonyl derivatives, and a diaminobenzene derivative, which is one of diamino derivatives, is shown below.

【0028】[0028]

【化5】 [Chemical 5]

【0029】次に、本発明の抗体を用いた免疫測定方法
により、上記の反応により形成された、一般式(I)で
示されるピラジン誘導体を測定する。免疫測定方法自体
は、この分野において周知であり、免疫組織染色法、免
疫比濁法、ラジオイムノアッセイ、エンザイムイムノア
ッセイ等の該抗体とピラジン誘導体との免疫反応を応用
するいずれの測定方法をも採用することができる。
Next, the pyrazine derivative represented by the general formula (I) formed by the above reaction is measured by the immunoassay method using the antibody of the present invention. The immunoassay method itself is well known in this field, and any immunoassay method such as an immunohistological staining method, an immunoturbidimetric method, a radioimmunoassay, an enzyme immunoassay or the like that applies an immunoreaction of the antibody with a pyrazine derivative is adopted. be able to.

【0030】好ましい免疫測定方法の一例として、上記
ジアミノ誘導体のR3 が検出可能な標識により標識され
ており、免疫測定は、本発明の抗体を固相に結合させ、
該固相結合抗体と前記ピラジン誘導体とを反応させ、洗
浄後、固相に結合された標識量を測定することにより前
記ピラジン誘導体を測定することから成る方法を挙げる
ことができる。この場合の標識としては、ビオチン、酵
素、放射性同位体等のこの分野において常用されるいず
れの標識をも用いることができる。
As an example of a preferred immunoassay method, R 3 of the above diamino derivative is labeled with a detectable label, and in the immunoassay, the antibody of the present invention is bound to a solid phase,
An example is a method comprising reacting the solid phase-bound antibody with the pyrazine derivative, washing, and measuring the amount of the label bound to the solid phase to measure the pyrazine derivative. As the label in this case, any label commonly used in this field such as biotin, enzyme, radioisotope and the like can be used.

【0031】[0031]

【実施例】以下、本発明を実施例に基づきさらに具体的
に説明する。もっとも、本発明は、下記実施例に限定さ
れるものではない。
EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

【0032】参考例1 N−t−ブトキシカルボニル−
4−アミノ酪酸(化合物(1) )の合成
Reference Example 1 Nt-butoxycarbonyl-
Synthesis of 4-aminobutyric acid (compound (1))

【0033】4−アミノ酪酸5.16gを水20mlに
溶解し、N−メチルモルホリン8.4mlと、アセトン
50mlに溶解した2−t−ブトキシカルボニルオキシ
イミノ−2−フェニルアセトニトリル11.08gを加
え、室温で一夜撹拌した。溶媒を約半分溜去し、水及び
酢酸エチルを加えてよく振り混ぜ、分液した。水層を酢
酸エチルで洗い、有機相を合わせて、5%炭酸水素ナト
リウム水溶液で抽出した。水層を合わせて酢酸エチルを
加え、6N塩酸でpHを約3とし、分液した。水層を更
に2回酢酸エチルで抽出し、抽出液を合わせて水洗いし
た。これを無水硫酸マグネシウムで乾燥した。乾燥剤を
濾去した後、N,N−ジシクロヘキシルアミンを加え結
晶化した。結晶を濾取し、エーテルで洗って乾燥した。
N,N−ジシクロヘキシルアミン塩の収量は15.07
gで、収率は79%であった。
5.16 g of 4-aminobutyric acid was dissolved in 20 ml of water, and 8.4 ml of N-methylmorpholine and 11.08 g of 2-t-butoxycarbonyloxyimino-2-phenylacetonitrile dissolved in 50 ml of acetone were added. Stir overnight at room temperature. About half of the solvent was distilled off, water and ethyl acetate were added, and the mixture was shaken well and separated. The aqueous layer was washed with ethyl acetate, the organic phases were combined and extracted with a 5% aqueous sodium hydrogen carbonate solution. The aqueous layers were combined, ethyl acetate was added, the pH was adjusted to about 3 with 6N hydrochloric acid, and the layers were separated. The aqueous layer was extracted twice more with ethyl acetate, and the extracts were combined and washed with water. This was dried over anhydrous magnesium sulfate. After the desiccant was filtered off, N, N-dicyclohexylamine was added to crystallize. The crystals were collected by filtration, washed with ether and dried.
Yield of N, N-dicyclohexylamine salt is 15.07.
The yield was 79% in g.

【0034】N,N−ジシクロヘキシルアミン塩全量を
酢酸エチルと5%クエン酸水溶液に溶解し、分液した。
水層を酢酸エチルで抽出し、有機相を合わせて、5%ク
エン酸水溶液及び水で洗って、無水硫酸マグネシウムで
乾燥した。溶媒を溜去し、N−t−ブトキシカルボニル
−4−アミノ酪酸(以下、本明細書中では化合物(1)と
記載する)として7.57gの油状物を得た。化合物
(1) の化学式を以下に示す。式中Bocはt−ブトキシ
カルボニル基を示す。
The total amount of N, N-dicyclohexylamine salt was dissolved in ethyl acetate and a 5% aqueous citric acid solution, and the layers were separated.
The aqueous layer was extracted with ethyl acetate, the organic phases were combined, washed with a 5% aqueous citric acid solution and water, and dried over anhydrous magnesium sulfate. The solvent was distilled off to obtain 7.57 g of an oily substance as Nt-butoxycarbonyl-4-aminobutyric acid (hereinafter referred to as compound (1) in the present specification). Compound
The chemical formula of (1) is shown below. In the formula, Boc represents a t-butoxycarbonyl group.

【0035】[0035]

【化6】 [Chemical 6]

【0036】化合物〔1〕のNMR測定結果は以下の通
りであった。 1H−NMR(400MHz,CDCl3 ,ppm)δ
1.44(9H,s),1.82(2H,m,J=7.
0,7.0Hz),2.40(2H,t,J=7.0H
z),3.18(2H,m),4.72(1H,s).
The NMR measurement results of the compound [1] are as follows. 1 H-NMR (400 MHz, CDCl 3 , ppm) δ
1.44 (9H, s), 1.82 (2H, m, J = 7.
0, 7.0 Hz), 2.40 (2H, t, J = 7.0H
z), 3.18 (2H, m), 4.72 (1H, s).

【0037】参考例2 S−(2−ピリジルチオ)−2
−メルカプトエチルアミン塩酸塩(化合物(2) )の合成 2,2’−ジピリジルジスルフィド13.2gをメタノ
ール100mlに溶解し、激しく撹拌しながら、メタノ
ール30mlに溶解した2−メルカプトエチルアミン塩
酸塩3.4gを滴下した。反応液を1時間40分撹拌し
た後、溶媒を溜去した。残渣に酢酸エチルを加えて結晶
化し、濾取した。得られた粗結晶をメタノール−エーテ
ルから再結晶した。母液を濃縮し、エーテルを加えてさ
らに結晶化し、S−(2−ピリジルチオ)−2−メルカ
プトエチルアミン塩酸塩(以下、本明細書中では化合物
(2) と記載する)を得た。化合物(2) の収量は5.62
gで、収率は84%であった。化合物(2) の化学式を以
下に示す。
Reference Example 2 S- (2-pyridylthio) -2
-Synthesis of mercaptoethylamine hydrochloride (compound (2)) 13.2 g of 2,2'-dipyridyl disulfide was dissolved in 100 ml of methanol, and 3.4 g of 2-mercaptoethylamine hydrochloride dissolved in 30 ml of methanol was stirred with vigorous stirring. Dropped. The reaction solution was stirred for 1 hour and 40 minutes, and then the solvent was distilled off. Ethyl acetate was added to the residue to crystallize and collect by filtration. The obtained crude crystal was recrystallized from methanol-ether. The mother liquor was concentrated, ether was added for further crystallization, and S- (2-pyridylthio) -2-mercaptoethylamine hydrochloride (hereinafter, referred to as compound in the present specification).
(Described as (2)) was obtained. The yield of compound (2) is 5.62.
The yield was 84% in g. The chemical formula of compound (2) is shown below.

【0038】[0038]

【化7】 [Chemical 7]

【0039】化合物(2) のNMR測定結果は以下の通り
であった。 1H−NMR(400MHz,DMSO−D6 ,pp
m)δ3.09(4H,m),7.30(1H,m,J
=1.1,4.8,7.4Hz),7.75(1H,
m,J=0.9,1.1,8.0Hz),7.84(1
H,m,J=1.8,7.4,8.0Hz),8.10
(3H,s),8.52(1H,m,J=0.9,1.
8,4.8Hz).
The NMR measurement results of the compound (2) are as follows. 1 H-NMR (400 MHz, DMSO-D 6 , pp
m) δ 3.09 (4H, m), 7.30 (1H, m, J
= 1.1, 4.8, 7.4 Hz), 7.75 (1H,
m, J = 0.9, 1.1, 8.0 Hz), 7.84 (1
H, m, J = 1.8, 7.4, 8.0 Hz), 8.10
(3H, s), 8.52 (1H, m, J = 0.9, 1.
8, 4.8 Hz).

【0040】参考例3 N−(4−t−ブトキシカルボ
キサミドブタノイル)−S−(2−ピリジルチオ)−2
−メルカプトエチルアミン(化合物(3) )の合成 参考例1で合成した化合物(1) を0. 98g、参考例2
で合成した化合物(2)を1. 07g及び1−ヒドロキシ
ベンゾトリアゾール0. 65gをDMF20mlに懸濁
し、氷/食塩浴で冷却下、水溶性カルボジイミド0. 8
8mlを加え、室温で6時間撹拌した。次いで、水溶性
カルボジイミド塩酸塩0. 28gを追加し、一夜撹拌し
た。溶媒を溜去し、残渣を水と酢酸エチルに溶解し分液
した。酢酸エチル相を水、5%炭酸水素ナトリウム水溶
液、水で洗って、無水硫酸マグネシウムで乾燥した。溶
媒を溜去し、油状の残渣をシリカゲルカラムクロマトグ
ラフィー(100ml、酢酸エチル)で精製した。N−
(4−t−ブトキシカルボキサミドブタノイル)−S−
(2−ピリジルチオ)−2−メルカプトエチルアミン
(以下、本明細書中では化合物(3) と記載する)の収量
は1. 10gで、収率は56%であった。化合物(3) の
化学式を以下に示す。
Reference Example 3 N- (4-t-butoxycarboxamidobutanoyl) -S- (2-pyridylthio) -2
-Synthesis of mercaptoethylamine (Compound (3)) 0.98 g of the compound (1) synthesized in Reference Example 1, Reference Example 2
1.07 g of the compound (2) synthesized in 1. and 0.65 g of 1-hydroxybenzotriazole were suspended in 20 ml of DMF, and water-soluble carbodiimide 0.8 was added under cooling with an ice / salt bath.
8 ml was added, and the mixture was stirred at room temperature for 6 hours. Next, 0.28 g of water-soluble carbodiimide hydrochloride was added, and the mixture was stirred overnight. The solvent was evaporated, the residue was dissolved in water and ethyl acetate, and the layers were separated. The ethyl acetate phase was washed with water, 5% aqueous sodium hydrogen carbonate solution and water, and dried over anhydrous magnesium sulfate. The solvent was distilled off, and the oily residue was purified by silica gel column chromatography (100 ml, ethyl acetate). N-
(4-t-Butoxycarboxamidobutanoyl) -S-
The yield of (2-pyridylthio) -2-mercaptoethylamine (hereinafter referred to as compound (3) in the present specification) was 1.10 g, and the yield was 56%. The chemical formula of compound (3) is shown below.

【0041】[0041]

【化8】 [Chemical 8]

【0042】化合物(3) のNMR測定結果は以下の通り
であった。 1H−NMR(400MHz,CDCl3 ,ppm)δ
1.44(9H,s),1.82(2H,m,J=7.
0Hz),2.27(2H,t,J=7.0Hz),
2.94(2H,t,J=6.0Hz),3.18(2
H,m),3.57(2H,m,J=6.0,6.0H
z),4.86(1H,s),7.15(1H,m,J
=1.8,4.9,6.7Hz),7.39(1H,
s),7.54(1H,m,J=0.5,0.9,8.
1Hz),7.63(1H,m,J=1.8,4.9,
6.7Hz),8.50(1H,m,J=0.9,1.
8,4.9Hz).
The NMR measurement results of the compound (3) are as follows. 1 H-NMR (400 MHz, CDCl 3 , ppm) δ
1.44 (9H, s), 1.82 (2H, m, J = 7.
0Hz), 2.27 (2H, t, J = 7.0Hz),
2.94 (2H, t, J = 6.0Hz), 3.18 (2
H, m), 3.57 (2H, m, J = 6.0, 6.0H
z), 4.86 (1H, s), 7.15 (1H, m, J
= 1.8, 4.9, 6.7 Hz), 7.39 (1H,
s), 7.54 (1H, m, J = 0.5, 0.9, 8.
1 Hz), 7.63 (1 H, m, J = 1.8, 4.9,
6.7 Hz), 8.50 (1 H, m, J = 0.9, 1.
8, 4.9 Hz).

【0043】参考例4 3,4−ジ−t−ブトキシカル
ボキサミド安息香酸(化合物(4) )の合成 3,4−ジアミノ安息香酸3.04gを1N水酸化ナト
リウム水溶液25mlに溶解し、ジ−t−ブチルジカー
ボネート9.6gをアセトン30mlに溶解して加え、
一夜撹拌した。次いで、ジ−t−ブチルジカーボネート
2.0gと1N水酸化ナトリウム水溶液5mlを追加
し、さらに3日間撹拌を続けた。アセトンを溜去し、残
渣に水と酢酸エチルを加えてよく振り混ぜた後、分液し
た。水層を酢酸エチルで抽出し、酢酸エチル相を合わせ
て、5%炭酸水素ナトリウム水溶液で2回抽出した。水
層をすべて合わせて、酢酸エチルを加え、6N塩酸でp
Hを約3とした後、分液した。水層を酢酸エチルで2回
抽出し、酢酸エチル相を合わせて水洗した。抽出液を濃
縮し、析出した結晶を濾取した。酢酸エチルで結晶を洗
い、五酸化二燐上で乾燥した。3,4−ジ−t−ブトキ
シカルボキサミド安息香酸(以下、本明細書中では化合
物(4) と記載する)の収量は5. 55gで、収率は79
%であった。化合物(4) の化学式を以下に示す。
Reference Example 4 Synthesis of 3,4-di-t-butoxycarboxamide benzoic acid (compound (4)) 3.04 g of 3,4-diaminobenzoic acid was dissolved in 25 ml of 1N aqueous sodium hydroxide solution to give di-t. -Butyl dicarbonate 9.6 g dissolved in acetone 30 ml and added,
Stir overnight. Next, 2.0 g of di-t-butyl dicarbonate and 5 ml of a 1N sodium hydroxide aqueous solution were added, and stirring was continued for another 3 days. Acetone was distilled off, water and ethyl acetate were added to the residue, and the mixture was shaken well and then separated. The aqueous layer was extracted with ethyl acetate, the ethyl acetate phases were combined and extracted twice with a 5% aqueous sodium hydrogen carbonate solution. Combine all the aqueous layers, add ethyl acetate, and p with 6N hydrochloric acid.
After H was set to about 3, the layers were separated. The aqueous layer was extracted twice with ethyl acetate, and the ethyl acetate phases were combined and washed with water. The extract was concentrated, and the precipitated crystals were collected by filtration. The crystals were washed with ethyl acetate and dried over phosphorous pentoxide. The yield of 3,4-di-t-butoxycarboxamide benzoic acid (hereinafter referred to as compound (4) in the present specification) was 5.55 g, and the yield was 79.
%Met. The chemical formula of compound (4) is shown below.

【0044】[0044]

【化9】 [Chemical 9]

【0045】化合物(4) のNMR測定結果は以下の通り
であった。 1H−NMR(400MHz,DMSO−D6 ,pp
m)δ1.48(9H,s),1.49(9H,s),
7.63(1H,d,J=8.6Hz),7.71(1
H,d,J=8.6Hz),8.09(1H,s),
8.68(1H,s),8.73(1H,s),12.
6−13.0(1H,broad s).
The NMR measurement results of the compound (4) are as follows. 1 H-NMR (400 MHz, DMSO-D 6 , pp
m) δ 1.48 (9H, s), 1.49 (9H, s),
7.63 (1H, d, J = 8.6Hz), 7.71 (1
H, d, J = 8.6 Hz), 8.09 (1H, s),
8.68 (1H, s), 8.73 (1H, s), 12.
6-13.0 (1H, breads).

【0046】参考例5 3,4−ジ−t−ブトキシカル
ボキサミド安息香酸N−コハク酸イミドエステル(化合
物(5) )の合成 参考例4で合成した化合物(4) 5. 50gとN−ヒドロ
キシコハク酸イミド2. 70gをDMF50mlに溶解
し、氷冷下、水溶性カルボジイミド塩酸塩4.50gを
加え、撹拌した。2時間後、室温に戻し一夜撹拌した。
溶媒を溜去し、残渣を水と酢酸エチルを加えて溶解し、
分液した。酢酸エチル相を水、5%炭酸水素ナトリウム
水溶液、5%クエン酸水溶液、水で洗って、無水硫酸マ
グネシウムで乾燥した。溶媒を溜去し、ヘキサンを加え
て結晶化し、濾取した。3,4−ジ−t−ブトキシカル
ボキサミド安息香酸N−コハク酸イミドエステル(以
下、本明細書中では化合物(5) と記載する)の収量は
6. 63gで、収率は95%であった。化合物(5) の化
学式を以下に示す。化合物(5) の化学式中、NSuはコ
ハク酸イミド基を示す。
Reference Example 5 Synthesis of 3,4-di-t-butoxycarboxamide benzoic acid N-succinimide ester (Compound (5)) 5.50 g of the compound (4) synthesized in Reference Example 4 and N-hydroxysuccine 2.70 g of acid imide was dissolved in 50 ml of DMF, 4.50 g of water-soluble carbodiimide hydrochloride was added under ice cooling, and the mixture was stirred. After 2 hours, the mixture was returned to room temperature and stirred overnight.
The solvent was evaporated, the residue was dissolved by adding water and ethyl acetate,
The layers were separated. The ethyl acetate phase was washed with water, 5% aqueous sodium hydrogen carbonate solution, 5% aqueous citric acid solution, water and dried over anhydrous magnesium sulfate. The solvent was distilled off, hexane was added to crystallize, and the crystals were collected by filtration. The yield of 3,4-di-t-butoxycarboxamide benzoic acid N-succinimide ester (hereinafter referred to as compound (5) in the present specification) was 6.63 g, and the yield was 95%. . The chemical formula of compound (5) is shown below. In the chemical formula of the compound (5), NSu represents a succinimide group.

【0047】[0047]

【化10】 [Chemical 10]

【0048】化合物(5) のNMR測定結果は以下の通り
であった。 1H−NMR(400MHz,DMSO−D6 ,pp
m)δ1.49(9H,s),1.50(9H,s),
2.88(4H,s),7.78(1H,d,J=8.
6Hz),7.95(1H,d,J=8.6Hz),
8.28(1H,s),8.89(1H,s),8.9
9(1H,s).
The NMR measurement results of the compound (5) are as follows. 1 H-NMR (400 MHz, DMSO-D 6 , pp
m) δ 1.49 (9H, s), 1.50 (9H, s),
2.88 (4H, s), 7.78 (1H, d, J = 8.
6Hz), 7.95 (1H, d, J = 8.6Hz),
8.28 (1H, s), 8.89 (1H, s), 8.9
9 (1H, s).

【0049】参考例6 N−(4−(3,4−ジアミノ
ベンズアミド)ブタノイル)−S−(2−ピリジルチ
オ)−2−メルカプトエチルアミン(化合物(7) )の合
成 参考例3に記載の方法で合成した化合物(3) 1. 54g
をクロロホルム5mlに溶解し、トリフルオロ酢酸5m
lを加え、室温で30分撹拌後、溶媒を溜去した。残渣
にクロロホルムを加えて溜去を3回繰り返した後、DM
F20mlに溶解した。これを氷冷しトリエチルアミン
で中和し、化合物(5) 1. 86gを加え、トリエチルア
ミンでpHを7〜8位に調節しながら一夜反応させた。
溶媒を溜去し、残渣を水と酢酸エチルに溶解し、分液し
た。酢酸エチル相を5%クエン酸水溶液、5%炭酸水素
ナトリウム水溶液、水で洗って、無水硫酸マグネシウム
で乾燥した。溶媒を溜去し、N−(4−(3,4−ジ−
t−ブトキシカルボキサミドベンズアミド)ブタノイ
ル)−S−(2−ピリジルチオ)−2−メルカプトエチ
ルアミン(以下、本明細書中では化合物(6) と記載す
る)を油状物として得た。化合物(6) の化学式を以下に
示す。
Reference Example 6 Synthesis of N- (4- (3,4-diaminobenzamido) butanoyl) -S- (2-pyridylthio) -2-mercaptoethylamine (Compound (7)) By the method described in Reference Example 3. Synthesized compound (3) 1.54 g
Is dissolved in 5 ml of chloroform, and 5 m of trifluoroacetic acid is dissolved.
1 was added, the mixture was stirred at room temperature for 30 minutes, and the solvent was distilled off. Chloroform was added to the residue and evaporation was repeated 3 times, then DM
It was dissolved in 20 ml of F. This was ice-cooled and neutralized with triethylamine, 1.86 g of compound (5) was added, and the reaction was carried out overnight while adjusting the pH to the 7-8 position with triethylamine.
The solvent was distilled off, the residue was dissolved in water and ethyl acetate, and the layers were separated. The ethyl acetate phase was washed with 5% aqueous citric acid solution, 5% aqueous sodium hydrogen carbonate solution and water, and dried over anhydrous magnesium sulfate. The solvent was distilled off, and N- (4- (3,4-di-
t-Butoxycarboxamidobenzamido) butanoyl) -S- (2-pyridylthio) -2-mercaptoethylamine (hereinafter referred to as compound (6) in the present specification) was obtained as an oil. The chemical formula of compound (6) is shown below.

【0050】[0050]

【化11】 [Chemical 11]

【0051】化合物(6) のNMR測定結果は以下の通り
であった。 1H−NMR(300MHz,CDCl3 ,ppm)δ
1.51(9H,s),1.52(9H,s),1.9
7(2H,m),2.34(2H,t,J=6.9H
z),2.90(2H,t,J=5.5Hz),3.4
6−3.57(4H,m),6.7−6.9(1H,b
road s),7.10−7.17(2H,m),
7.52−7.70(4H,m),7.82(1H,
s),8.47(1H,d,J=4.9Hz).
The NMR measurement results of the compound (6) are as follows. 1H-NMR (300 MHz, CDCl 3 , ppm) δ
1.51 (9H, s), 1.52 (9H, s), 1.9
7 (2H, m), 2.34 (2H, t, J = 6.9H
z), 2.90 (2H, t, J = 5.5Hz), 3.4.
6-3.57 (4H, m), 6.7-6.9 (1H, b
roads), 7.10-7.17 (2H, m),
7.52-7.70 (4H, m), 7.82 (1H,
s), 8.47 (1H, d, J = 4.9 Hz).

【0052】化合物(6) 全量をトリフルオロ酢酸10m
lに溶解し、室温で1時間撹拌した。トリフルオロ酢酸
を溜去し、水を加えてもう一度溜去した。残渣を水に溶
解し、ダイアイオン HP−20カラム45mlに吸着
させ、水250mlで洗った後、40%アセトニトリル
水溶液300mlで溶出した。溶出液を集めて濃縮し、
分離してきた油状物を少量のアセトニトリルを加えて溶
解し、凍結乾燥して、N−(4−(3,4−ジアミノベ
ンズアミド)ブタノイル)−S−(2−ピリジルチオ)
−2−メルカプトエチルアミン(以下、本明細書中では
化合物(7) と記載する)を得た。化合物(7) の収量は
1. 30gであった。化合物(7) の化学式を以下に示
す。
The total amount of the compound (6) is 10 m of trifluoroacetic acid.
It was dissolved in 1 and stirred at room temperature for 1 hour. Trifluoroacetic acid was distilled off, water was added, and the mixture was distilled off again. The residue was dissolved in water, adsorbed on 45 ml of Diaion HP-20 column, washed with 250 ml of water, and then eluted with 300 ml of 40% acetonitrile aqueous solution. Collect and concentrate the eluate,
The separated oily substance was dissolved by adding a small amount of acetonitrile and lyophilized to give N- (4- (3,4-diaminobenzamido) butanoyl) -S- (2-pyridylthio).
2-Mercaptoethylamine (hereinafter referred to as compound (7) in the present specification) was obtained. The yield of compound (7) was 1.30 g. The chemical formula of compound (7) is shown below.

【0053】[0053]

【化12】 [Chemical 12]

【0054】化合物(7) のNMR測定結果は以下の通り
であった。 1H−NMR(400MHz,DMSO−D6 ,pp
m)δ1.70(2H,m),2.10(2H,t,J
=7.3Hz),2.89(2H,t,J=6.8H
z),3.18(2H,m),3.33(2H,m),
5.42(4H,broad s),6.53(1H,
d,J=8.1Hz)7.05(1H,dd,J=1.
9,8.1Hz),7.15(1H,d,J=1.9H
z),7.23(1H,m,J=1.1,4.8,7.
3Hz),7.76(1H,m,J=0.9,1.1,
8.4Hz),7.82(1H,m,J=1.8,7.
3,8.4Hz),7.94(1H,broad
t),8.06(1H,broadt),8.45(1
H,m,J=0.9,1.8,4.8Hz).
The NMR measurement results of the compound (7) are as follows. 1 H-NMR (400 MHz, DMSO-D 6 , pp
m) δ 1.70 (2H, m), 2.10 (2H, t, J
= 7.3 Hz), 2.89 (2H, t, J = 6.8H)
z), 3.18 (2H, m), 3.33 (2H, m),
5.42 (4H, broads), 6.53 (1H,
d, J = 8.1 Hz) 7.05 (1H, dd, J = 1.
9, 8.1 Hz), 7.15 (1H, d, J = 1.9H)
z), 7.23 (1H, m, J = 1.1, 4.8, 7.
3Hz), 7.76 (1H, m, J = 0.9, 1.1,
8.4 Hz), 7.82 (1H, m, J = 1.8, 7.
3, 8.4Hz), 7.94 (1H, broad
t), 8.06 (1H, broadt), 8.45 (1
H, m, J = 0.9, 1.8, 4.8 Hz).

【0055】実施例1 N−(4−(3−(2,3,4
−トリヒドロキシブチル)キノキサリン−6−カルボキ
サミド)ブタノイル)−S−(2−ピリジルチオ)−2
−メルカプトエチルアミン(化合物(8−1))及びN
−(4−(2−(2,3,4−トリヒドロキシブチル)
キノキサリン−6−カルボキサミド)ブタノイル)−S
−(2−ピリジルチオ)−2−メルカプトエチルアミン
(化合物(8−2))の合成 参考例6で合成した化合物(7) 28. 95mgをメタノ
ール1mlに溶解し、文献(Carbohyd. Res., 17, p183
-192, 1971)に記載の方法に従って合成した3−デオキ
シ−D−エリスロヘキソース−2−ウロース(3−デオ
キシグルコゾン)(以下、本明細書中では3−DGと記
載する)36. 92mgを1mlのPBSに溶解して混
合した。アルゴン雰囲気下、室温で一夜撹拌した。反応
液を水で希釈しダイアイオンHP−20カラム5mlに
吸着させ、水洗し、40%アセトニトリル水溶液で溶出
した。溶出液を濃縮し、別のダイアイオンHP−20カ
ラム7mlに吸着させ、5%、10%、20%アセトニ
トリル水溶液で順に溶出し、20%アセトニトリル水溶
液で溶出された画分を集めて濃縮し、これを凍結乾燥し
て化合物(8) を得た。化合物(8) は、N−(4−(3−
(2,3,4−トリヒドロキシブチル)キノキサリン−
6−カルボキサミド)ブタノイル)−S−(2−ピリジ
ルチオ)−2−メルカプトエチルアミン(以下、本明細
書中では化合物(8−1)と記載する)とN−(4−
(2−(2,3,4−トリヒドロキシブチル)キノキサ
リン−6−カルボキサミド)ブタノイル)−S−(2−
ピリジルチオ)−2−メルカプトエチルアミン(以下、
本明細書中では化合物(8−2)と記載する)との混合
物である。化合物(8) の収量は、異性体の混合物として
26. 70mgであった。化合物(8−1)及び(8−
2)の化学式を以下に示す。
Example 1 N- (4- (3- (2,3,4
-Trihydroxybutyl) quinoxaline-6-carboxamido) butanoyl) -S- (2-pyridylthio) -2
-Mercaptoethylamine (Compound (8-1)) and N
-(4- (2- (2,3,4-trihydroxybutyl))
Quinoxaline-6-carboxamido) butanoyl) -S
Synthesis of-(2-pyridylthio) -2-mercaptoethylamine (Compound (8-2)) 28.95 mg of the compound (7) synthesized in Reference Example 6 was dissolved in 1 ml of methanol and the reaction was carried out in the literature (Carbohyd. Res., 17, 17). p183
-192, 1971), 3-deoxy-D-erythrohexose-2-ulose (3-deoxyglucosone) (hereinafter, referred to as 3-DG in the present specification) (36.92 mg) It was dissolved in 1 ml of PBS and mixed. The mixture was stirred overnight at room temperature under an argon atmosphere. The reaction solution was diluted with water, adsorbed on 5 ml of Diaion HP-20 column, washed with water, and eluted with 40% acetonitrile aqueous solution. The eluate was concentrated, adsorbed on another 7 ml of Diaion HP-20 column, eluted with 5%, 10%, 20% acetonitrile aqueous solution in order, and the fractions eluted with 20% acetonitrile aqueous solution were collected and concentrated, This was freeze-dried to obtain the compound (8). Compound (8) is N- (4- (3-
(2,3,4-trihydroxybutyl) quinoxaline-
6-carboxamido) butanoyl) -S- (2-pyridylthio) -2-mercaptoethylamine (hereinafter referred to as compound (8-1) in the present specification) and N- (4-
(2- (2,3,4-trihydroxybutyl) quinoxaline-6-carboxamido) butanoyl) -S- (2-
Pyridylthio) -2-mercaptoethylamine (hereinafter,
In the present specification, it is a mixture with the compound (8-2). The yield of compound (8) was 26.70 mg as a mixture of isomers. Compounds (8-1) and (8-
The chemical formula of 2) is shown below.

【0056】[0056]

【化13】 [Chemical 13]

【0057】[0057]

【化14】 [Chemical 14]

【0058】化合物(8) のNMR測定結果は以下の通り
であった。 1H−NMR(400MHz,DMSO−D6 ,pp
m)δ1.81(2H,m),2.18(2H,t,J
=7.3Hz),2.90(2H,t,J=6.8H
z),3.02(2H,m),3.34(4H,m),
3.45(2H,m),3.61(1H,m),3.9
3(1H,m),4.45(1H,broads),
4.78(2H,m),7.22(1H,m),7.7
6(1H,d,J=8.1Hz),7.81(1H,d
d,J=7.2,8.1Hz),8.08(2H,
m),8.17(0.4H,d,J=8.8Hz),
8.21(0.6H,d,J=8.8Hz),8.45
(1H,d,J=4.6Hz),8.54(0.4H,
s),8.56(0.6H,s),8.82(1H,b
roadd,J=5.1Hz),8.90(0.4H,
s),8.91(0.6H,s).
The NMR measurement results of the compound (8) are as follows. 1 H-NMR (400 MHz, DMSO-D 6 , pp
m) δ1.81 (2H, m), 2.18 (2H, t, J
= 7.3 Hz), 2.90 (2H, t, J = 6.8H)
z), 3.02 (2H, m), 3.34 (4H, m),
3.45 (2H, m), 3.61 (1H, m), 3.9
3 (1H, m), 4.45 (1H, broads),
4.78 (2H, m), 7.22 (1H, m), 7.7
6 (1H, d, J = 8.1Hz), 7.81 (1H, d
d, J = 7.2, 8.1 Hz), 8.08 (2H,
m), 8.17 (0.4H, d, J = 8.8Hz),
8.21 (0.6H, d, J = 8.8Hz), 8.45
(1H, d, J = 4.6Hz), 8.54 (0.4H,
s), 8.56 (0.6H, s), 8.82 (1H, b
roadd, J = 5.1Hz), 8.90 (0.4H,
s), 8.91 (0.6H, s).

【0059】実施例2 KLH結合ピラジン誘導体の合
成 免疫原として、化合物(8) と貝ヘモシアニン(以下、本
明細書ではKLHと記載する)(カルビオケム社製)と
の結合物を合成した。すなわち、KLH11.16mg
を0. 1Mリン酸緩衝液(pH7.5)893μlに溶
解し、DMF(ジメチルホルムアミド)223μlに溶
解したGMBS(4−マレイミド酢酸N−コハク酸イミ
ドエステル、同人化学研究所製)2. 23mgを加え、
室温で1時間撹拌した。1mMのEDTAを含む0. 1
Mリン酸緩衝液(pH7. 0)で平衡化したPD−10
カラム(ファルマシア社製)二本に、反応液を500μ
lずつ分注し、同緩衝液で溶出した。それぞれ、最初の
2. 5mlを棄て、続く2. 0mlを集めた。
Example 2 Synthesis of KLH-Binding Pyrazine Derivative As an immunogen, a conjugate of compound (8) and shell hemocyanin (hereinafter referred to as KLH in the present specification) (manufactured by Calbiochem) was synthesized. That is, KLH 11.16 mg
Was dissolved in 893 μl of 0.1 M phosphate buffer (pH 7.5), and 223 mg of GMBS (4-maleimidoacetic acid N-succinimide ester, manufactured by Dojindo Laboratories) dissolved in 223 μl of DMF (dimethylformamide). In addition,
Stirred at room temperature for 1 hour. 0.1 with 1 mM EDTA
PD-10 equilibrated with M phosphate buffer (pH 7.0)
Add 500μ of reaction solution to two columns (Pharmacia).
Aliquots of 1 were dispensed and eluted with the same buffer. The first 2.5 ml was discarded and the subsequent 2.0 ml was collected, respectively.

【0060】一方、実施例1で合成した化合物〔8〕
3. 46mgを20%アセトニトリル水溶液300μl
に溶解し、ジチオスレイトール11. 56mgを加えて
室温で30分放置した。この還元溶液150μlを、逆
相HPLC(YMC−PackAP−322、内径10
mmx長さ150mm)を用いて、1mMのEDTAを
含む0. 1Mリン酸緩衝液(pH7. 0)とアセトニト
リル10−20%の濃度勾配で溶出分離し、保持時間1
0分のピーク分画(A)および10. 8分のピーク分画
(B)を分取した。分画(A)及び(B)を、上記のマ
レイミド化KLH溶液にそれぞれ加え、室温で撹拌し
た。40分後に残りの還元溶液150μlを分取しそれ
ぞれの反応液に追加した。通算で3時間反応させ、透析
チューブに移し、PBSに対し一夜透析し、KLH結合
ピラジン誘導体を得た。収量は、分画(A)由来のKL
H結合ピラジン誘導体が約7ml(346mg/m
l)、分画(B)由来のKLH結合ピラジン誘導体が約
6ml(375mg/ml)であった。
On the other hand, the compound [8] synthesized in Example 1
3. 46 mg of 20% acetonitrile aqueous solution 300 μl
Was dissolved in the solution, 11.56 mg of dithiothreitol was added, and the mixture was allowed to stand at room temperature for 30 minutes. 150 μl of this reducing solution was applied to reverse phase HPLC (YMC-PackAP-322, inner diameter 10
mm x length 150 mm), elution separation was performed with a 0.1 M phosphate buffer (pH 7.0) containing 1 mM EDTA and a concentration gradient of 10-20% acetonitrile, and a retention time of 1
A 0 minute peak fraction (A) and a 10.8 minute peak fraction (B) were collected. Fractions (A) and (B) were added to the above maleimidated KLH solution, respectively, and stirred at room temperature. After 40 minutes, 150 μl of the remaining reducing solution was collected and added to each reaction solution. The mixture was reacted for 3 hours in total, transferred to a dialysis tube, and dialyzed overnight against PBS to obtain a KLH-bonded pyrazine derivative. The yield is KL derived from fraction (A)
About 7 ml of H-bonded pyrazine derivative (346 mg / m
1), the KLH-bonded pyrazine derivative derived from fraction (B) was about 6 ml (375 mg / ml).

【0061】実施例3 BSA結合ピラジン誘導体の合
成 実施例2と同様の方法で、牛血清アルブミン(以下、本
明細書ではBSAと記載する)12. 88mgと実施例
1で合成した化合物(8) 3. 3mgとを用い、免疫測定
用の抗原として化合物(8) とBSAとの結合物を合成し
た。収量は、逆相HPLCで分取した分画(A)由来の
BSA結合ピラジン誘導体が約8ml(590mg/m
l)、分画(B)由来のBSA結合ピラジン誘導体が約
7ml(665mg/ml)であった。
Example 3 Synthesis of BSA-Bound Pyrazine Derivative In the same manner as in Example 2, 12.88 mg of bovine serum albumin (hereinafter referred to as BSA in this specification) and the compound (8) synthesized in Example 1 were used. Using 3.3 mg, a conjugate of compound (8) and BSA was synthesized as an antigen for immunoassay. The yield was about 8 ml (590 mg / m 2) of the BSA-bonded pyrazine derivative derived from the fraction (A) collected by reverse phase HPLC.
1), the BSA-bonded pyrazine derivative derived from the fraction (B) was about 7 ml (665 mg / ml).

【0062】実施例4 ビオチン結合ピラジン誘導体
(化合物(9) )の合成 免疫測定用の抗原として、化合物(8) とビオチンとの結
合物(以下、本明細書では化合物(9) と記載する)を合
成した。すなわち、実施例1に記載の方法で合成した化
合物(8) 21. 35mgを1mMのEDTAを含む0.
1Mリン酸緩衝液(pH7. 0)2mlとメタノール
0. 2mlに溶解し、ジチオスレイトール40. 53m
gを加えて、アルゴン雰囲気下室温で撹拌した。この溶
液を逆相HPLC(アサヒパックODP−90、20m
mI.D.x300mmL.昭和電工社製)を用い、1
mMのEDTAを含む0. 1Mリン酸緩衝液(pH7.
0)とアセトニトリル10−20%の濃度勾配で溶出分
離し、保持時間約26分のピーク分画(A)と27分の
ピーク分画(B)を分取した。ビオチン−PE−マレイ
ミド(同人化学研究所製)を、分画(A)には14. 0
mg、分画(B)には11. 3mg加え、アルゴン雰囲
気下、室温で撹拌した。分画(A)の反応液をダイアイ
オンHP−20カラム7mlに、分画(B)の反応液を
ダイアイオンHP−20カラム5mlに吸着させ、それ
ぞれ10%アセトニトリル水溶液で洗った後、40%ア
セトニトリル水溶液で溶出し、凍結乾燥した。収量は、
分画(A)由来のビオチン結合ピラジン誘導体が11.
35mg、分画(B)由来のビオチン結合ピラジン誘導
体が4.15mgであった。化合物(9−1)及び(9
−2)の化学式を以下に示す。
Example 4 Synthesis of biotin-bonded pyrazine derivative (compound (9)) As an antigen for immunoassay, a conjugate of compound (8) and biotin (hereinafter referred to as compound (9) in the present specification) Was synthesized. That is, 21.35 mg of the compound (8) synthesized by the method described in Example 1 containing 0.1 mM of EDTA was added.
Dissolved in 2 ml of 1M phosphate buffer (pH 7.0) and 0.2 ml of methanol, and dithiothreitol 40.53m
g was added, and the mixture was stirred at room temperature under an argon atmosphere. Reverse phase HPLC (Asahi Pack ODP-90, 20 m
mI. D. x300 mmL. Showa Denko KK), 1
0.1 M phosphate buffer (pH 7.
0) and acetonitrile were eluted and separated with a concentration gradient of 10-20%, and a peak fraction (A) with a retention time of about 26 minutes and a peak fraction (B) with a retention time of 27 minutes were fractionated. Biotin-PE-maleimide (manufactured by Dojindo Laboratories) was used in the fraction (A) of 14.0.
mg, 11.3 mg was added to the fraction (B), and the mixture was stirred at room temperature under an argon atmosphere. The reaction solution of the fraction (A) was adsorbed on 7 ml of Diaion HP-20 column, and the reaction liquid of the fraction (B) was adsorbed on 5 ml of Diaion HP-20 column. It was eluted with an aqueous acetonitrile solution and freeze-dried. The yield is
The biotin-bonded pyrazine derivative derived from the fraction (A) was 11.
35 mg, and the biotin-bonded pyrazine derivative derived from the fraction (B) was 4.15 mg. Compounds (9-1) and (9
The chemical formula of -2) is shown below.

【0063】[0063]

【化15】 [Chemical 15]

【0064】[0064]

【化16】 [Chemical 16]

【0065】実施例5 ビオチン結合ジアミノベンゼン
誘導体(化合物(10))の合成 参考例6に記載の方法で合成した化合物(7) 100mg
を1mMのEDTAを含む0. 1Mリン酸緩衝液(pH
7. 0)4mlとアセトニトリル1mlに溶解し、ジチ
オスレイトール115mgを加えて1時間撹拌した。ダ
イヤイオンHP−20カラム10mlに吸着させ、水5
0mlで洗った後、20%アセトニトリル水溶液50m
lで溶出した。溶出液にビオチン−PE−マレイミド4
2.6mgを加え5時間攪拌した。ダイヤイオンHP−
20カラム20mlに吸着させ、水50mlで洗った
後、40%アセトニトリル水溶液50mlで溶出した。
溶出液を凍結乾燥し、粗生成物53.8mgを得た。逆
相HPLCを用いて精製し、化合物(10)8.05mgを
得た。化合物(10)の化学式を以下に示す。
Example 5 Synthesis of biotin-conjugated diaminobenzene derivative (compound (10)) 100 mg of compound (7) synthesized by the method described in Reference Example 6
0.1M phosphate buffer containing 1 mM EDTA (pH
7.0) 4 ml and acetonitrile 1 ml, it melt | dissolved in dithiothreitol 115 mg, and it stirred for 1 hour. Adsorbed on 10 ml of Diaion HP-20 column, water 5
After washing with 0 ml, 20m acetonitrile aqueous solution 50m
Elute with 1. Biotin-PE-maleimide 4 in the eluate
2.6 mg was added and stirred for 5 hours. Diaion HP-
It was adsorbed on 20 columns of 20 ml, washed with 50 ml of water, and then eluted with 50 ml of 40% acetonitrile aqueous solution.
The eluate was freeze-dried to obtain 53.8 mg of a crude product. Purification using reverse phase HPLC gave 8.05 mg of compound (10). The chemical formula of compound (10) is shown below.

【0066】[0066]

【化17】 [Chemical 17]

【0067】参考例7 Nα−(9−フルオレニルメト
キシカルボニル)−Nε−(3,4−ジ−t−ブトキシ
カルボキサミドベンゾイル)−L−リジン(化合物(1
1))の合成 Nα−(9−フルオレニルメトキシカルボニル)−Nε
−t−ブトキシカルボニル−L−リジン0.54gをク
ロロフォルム5mlに溶解し、トリフルオロ酢酸5ml
を加えて、室温で1時間撹拌した。溶媒を溜去し、エー
テルを加えて沈殿させ、上澄みを除いて、DMF5ml
に溶解した。氷冷下、トリエチルアミン322μlと
3,4−ジ−t−ブトキシカルボキサミド安息香酸N−
コハク酸イミドエステル0.67gを加え、室温で一夜
撹拌した。溶媒を溜去し、残渣を酢酸エチル、希塩酸に
溶解し、分液した。酢酸エチル相を水洗し、無水硫酸マ
グネシウムで乾燥した。溶媒を溜去し、残渣にn−ヘキ
サンを加え、結晶を濾取した。化合物(11)の収量は0.
60gであった。化合物(11)の化学式を以下に示す。化
合物(11)の化学式中、Fmocは9−フルオレニルメト
キシカルボニル基を示す。
Reference Example 7 Nα- (9-fluorenylmethoxycarbonyl) -Nε- (3,4-di-t-butoxycarboxamidobenzoyl) -L-lysine (Compound (1
1)) Synthesis Nα- (9-fluorenylmethoxycarbonyl) -Nε
0.54 g of -t-butoxycarbonyl-L-lysine was dissolved in 5 ml of chloroform, and 5 ml of trifluoroacetic acid was dissolved.
Was added and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off, ether was added to cause precipitation, the supernatant was removed, and DMF 5 ml was added.
Dissolved in. Under ice cooling, 322 μl of triethylamine and 3,4-di-t-butoxycarboxamide benzoic acid N-
0.67 g of succinimide ester was added, and the mixture was stirred overnight at room temperature. The solvent was evaporated, the residue was dissolved in ethyl acetate and dilute hydrochloric acid, and the layers were separated. The ethyl acetate phase was washed with water and dried over anhydrous magnesium sulfate. The solvent was distilled off, n-hexane was added to the residue, and the crystals were collected by filtration. The yield of compound (11) is 0.
It was 60 g. The chemical formula of compound (11) is shown below. In the chemical formula of compound (11), Fmoc represents a 9-fluorenylmethoxycarbonyl group.

【0068】[0068]

【化18】 [Chemical 18]

【0069】参考例8 Nε−(3,4−ジ−t−ブト
キシカルボキサミドベンゾイル)−L−リジン(化合物
(12))の合成 参考例7で合成した化合物(11)0.60gをDMF7m
lに溶解し、ピぺリジン3mlを加え、室温で10分間
撹拌した。反応液を1.5%トリフルオロ酢酸水溶液1
50ml中に注ぎ、不溶物を濾去した。水相をダイアイ
オンHP−20カラム10mlに通して目的物を吸着さ
せ、水50mlで洗った後、40%アセトニトリル水溶
液100mlで溶出した。溶出液を濃縮し、凍結乾燥し
た。化合物(12)の収量は101mgであった。化合物(1
2)の化学式を以下に示す。
Reference Example 8 Nε- (3,4-di-t-butoxycarboxamidobenzoyl) -L-lysine (Compound
Synthesis of (12)) 0.60 g of the compound (11) synthesized in Reference Example 7 was added to DMF7m
It was dissolved in 1 l, 3 ml of piperidine was added, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was 1.5% trifluoroacetic acid aqueous solution
It was poured into 50 ml and the insoluble matter was filtered off. The aqueous phase was passed through 10 ml of Diaion HP-20 column to adsorb the target product, washed with 50 ml of water, and then eluted with 100 ml of 40% acetonitrile aqueous solution. The eluate was concentrated and freeze-dried. The yield of compound (12) was 101 mg. Compound (1
The chemical formula of 2) is shown below.

【0070】[0070]

【化19】 [Chemical 19]

【0071】実施例6 Nα−(5−(ビオチニルアミ
ノ)−ペンタノイル)−Nε−(3,4−ジアミノベン
ゾイル)−L−リジン(化合物(13))の合成 参考例8で合成した化合物〔12〕30.29mgを
0.1Mリン酸緩衝液(pH7.5)3mlに懸濁し、
激しく撹拌しながらビオチン−AC5−OSu(同人化
学社製)のDMF溶液(10mg/ml)4mlを4回
に分けて加え、室温で一夜撹拌した。溶媒を溜去し、残
渣を酢酸エチルと水に溶解し、6N塩酸で酸性にした。
析出した沈殿を濾取し、水洗し、五酸化二燐上で乾燥し
た。得られた固体を、トリフルオロ酢酸1mlに溶解し
室温で1時間撹拌した。溶媒を溜去し、残渣を水に溶解
し凍結乾燥した。化合物(13)の収量は21.14mgで
あった。化合物(13)の化学式を以下に示す。
Example 6 Synthesis of Nα- (5- (biotinylamino) -pentanoyl) -Nε- (3,4-diaminobenzoyl) -L-lysine (Compound (13)) The compound synthesized in Reference Example 8 [12] ] 30.29 mg was suspended in 3 ml of 0.1 M phosphate buffer (pH 7.5),
With vigorous stirring, 4 ml of a DMF solution (10 mg / ml) of biotin-AC5-OSu (manufactured by Dojindo Kagaku) was added in 4 portions, and the mixture was stirred at room temperature overnight. The solvent was evaporated, the residue was dissolved in ethyl acetate and water and acidified with 6N hydrochloric acid.
The deposited precipitate was collected by filtration, washed with water, and dried over diphosphorus pentoxide. The obtained solid was dissolved in 1 ml of trifluoroacetic acid and stirred at room temperature for 1 hour. The solvent was distilled off, the residue was dissolved in water and freeze-dried. The yield of compound (13) was 21.14 mg. The chemical formula of compound (13) is shown below.

【0072】[0072]

【化20】 [Chemical 20]

【0073】化合物〔13〕のNMR測定結果は以下の
通りであった。 1H−NMR(400MHz,DMSO−D6 ,pp
m)δ1.2−1.8(16H,m),2.05(2
H,t,J=7.4Hz),2.10(2H,t,J=
7.3Hz),2.58(1H,d,J=12.4H
z),2.82(1H,dd,J=5.1,12.4H
z),3.00(2H,m),3.09(1H,m),
3.19(2H,m),3.2−4.0(4H,bro
ad s),4.14(2H,m),4.31(1H,
m),6.36(1H,broad s),6.42
(1H,broad s),6.71(1H,d,J=
8.4Hz),7.34(1H,dd,J=0.7,
8.4Hz),7.44(1H,s),7.72(1
H,m),8.00(1H,m),8.10(1H,
m).
The NMR measurement results of the compound [13] are as follows. 1 H-NMR (400 MHz, DMSO-D 6 , pp
m) δ1.2-1.8 (16H, m), 2.05 (2
H, t, J = 7.4 Hz), 2.10 (2H, t, J =
7.3 Hz), 2.58 (1H, d, J = 12.4H
z), 2.82 (1H, dd, J = 5.1, 12.4H
z), 3.00 (2H, m), 3.09 (1H, m),
3.19 (2H, m), 3.2-4.0 (4H, bro
ads), 4.14 (2H, m), 4.31 (1H,
m), 6.36 (1H, broads), 6.42
(1H, broads), 6.71 (1H, d, J =
8.4 Hz), 7.34 (1H, dd, J = 0.7,
8.4 Hz), 7.44 (1 H, s), 7.72 (1
H, m), 8.00 (1H, m), 8.10 (1H,
m).

【0074】参考例9 Nα−ビオチニル−Nε−
(3,4−ジアミノベンゾイル)−L−リジン(化合物
(14))の合成 参考例8で合成した化合物(12)41.07mgを0.1
Mリン酸緩衝液(pH7.5)4mlに懸濁し、激しく
撹拌しながらビオチン−OSu(同人化学社製)のDM
F溶液(10mg/ml)3mlを加え、室温で一夜撹
拌した。溶媒を溜去し、残渣を酢酸エチルと水に溶解
し、6N塩酸で酸性にした。分液し、酢酸エチル相を水
洗した。溶媒を溜去し、残渣をトリフルオロ酢酸1ml
に溶解して室温で1時間撹拌した。溶媒を溜去し、残渣
を水、アセトニトリルに溶解し凍結乾燥した。化合物(1
4)の収量は47mgであった。化合物(14)の化学式を以
下に示す。
Reference Example 9 Nα-Biotinyl-Nε-
(3,4-diaminobenzoyl) -L-lysine (compound
(14)) Compound (12) 41.07 mg synthesized in Reference Example 8
Suspended in 4 ml of M phosphate buffer (pH 7.5), and DM with biotin-OSu (manufactured by Dojindo Co., Ltd.) with vigorous stirring.
3 ml of F solution (10 mg / ml) was added, and the mixture was stirred at room temperature overnight. The solvent was evaporated, the residue was dissolved in ethyl acetate and water and acidified with 6N hydrochloric acid. The layers were separated, and the ethyl acetate phase was washed with water. The solvent was distilled off, and the residue was trifluoroacetic acid 1 ml.
And was stirred at room temperature for 1 hour. The solvent was distilled off, the residue was dissolved in water and acetonitrile and freeze-dried. Compound (1
The yield of 4) was 47 mg. The chemical formula of compound (14) is shown below.

【0075】[0075]

【化21】 [Chemical 21]

【0076】実施例7 抗ピラジン誘導体モノクローナ
ル抗体の作製 抗ピラジン誘導体モノクローナル抗体は、実施例2で合
成した分画(A)由来のKLH結合ピラジン誘導体をB
ALB/Cマウスに免疫し、その脾臓リンパ球とミエロ
ーマ細胞を融合することにより作製した。すなわち、B
ALB/Cマウスにフロイント完全アジュバントでエマ
ルジョン化したKLH結合ピラジン誘導体の25〜10
0μgを用いて初回免疫を行い、2〜3週間後、フロイ
ント不完全アジュバントでエマルジョン化した同抗原2
5〜100μgで追加免疫を行った。抗体価の上昇は、
後述のスクリーニング法と同様の手法で、実施例3で合
成した分画(A)由来のBSA結合ピラジン誘導体を固
相抗原としたELISAで確認した。抗体価の上昇を確
認後、KLH結合ピラジン誘導体25〜100μgを静
脈内に投与し、その3〜4日後、マウスから脾臓を取り
出し脾細胞を調製した。前もってRPMI−1640培
地で培養していたマウスミエローマ細胞(P3U1)と
脾細胞を1:2〜1:5の比率で混合し、ポリエチレン
グリコール(ベーリンガー社製)を用い細胞融合を行っ
た。融合した細胞はHAT培地に浮遊した後、96ウエ
ル培養プレートに分注し37℃二酸化炭素インキュベー
ターで培養した。
Example 7 Preparation of anti-pyrazine derivative monoclonal antibody As the anti-pyrazine derivative monoclonal antibody, the KLH-bonded pyrazine derivative derived from the fraction (A) synthesized in Example 2 was used.
It was prepared by immunizing ALB / C mice and fusing their spleen lymphocytes with myeloma cells. That is, B
25-10 of KLH-conjugated pyrazine derivatives emulsified in ALB / C mice with Freund's complete adjuvant
Initial immunization with 0 μg, and 2-3 weeks later, the same antigen 2 emulsified with Freund's incomplete adjuvant
A booster immunization was performed with 5 to 100 μg. The increase in antibody titer is
By a method similar to the screening method described later, the BSA-bonded pyrazine derivative derived from the fraction (A) synthesized in Example 3 was confirmed by ELISA using a solid-phase antigen. After confirming the increase in antibody titer, 25 to 100 μg of KLH-bonded pyrazine derivative was intravenously administered, and 3 to 4 days after that, the spleen was taken out from the mouse to prepare splenocytes. Mouse myeloma cells (P3U1) that had been previously cultured in RPMI-1640 medium were mixed with splenocytes at a ratio of 1: 2 to 1: 5, and cell fusion was performed using polyethylene glycol (Boehringer). The fused cells were suspended in HAT medium, dispensed into a 96-well culture plate, and cultured in a 37 ° C. carbon dioxide incubator.

【0077】スクリーニングは抗原固相ELISAで行
った。すなわち、実施例3で合成した分画(A)由来の
BSA結合ピラジン誘導体を96ウエルELISAプレ
ート(ファルマシア社製)に1μg/mlの濃度で50
μl/ウエルづつ注し、4℃一晩放置することにより吸
着させた。プレートを1%スキムミルクでブロッキング
した後、0.05%ツィーン20を含むリン酸緩衝液
(以下本明細書中では、洗浄緩衝液と記載する)で3回
洗浄し、細胞融合を行ったプレートの培養上清50μl
を加え、37℃1時間反応させた。同様に洗浄緩衝液で
3回洗後、パーオキシダーゼ(以下本明細書中では、P
ODと記載する)標識抗マウスイムノグロブリン抗体
(ダコ社製)を加え、さらに37℃1時間反応させた。
洗浄緩衝液で4回洗浄後、基質ABTSを加え発色の見
られるウエルを選択した。BSA結合ピラジン誘導体に
反応性を示したウエルは、さらにフリーのピラジン誘導
体による抑制試験を行い特異性を確認した。すなわち、
固相抗原と培養上清との反応時に、一定濃度(約10μ
M)のピラジン誘導体を共存させ、抑制の有無で特異性
を確認した。この場合、抑制を示すウエルが特異的なウ
エルである。特異性を確認したウエルの細胞は限界希釈
法によりクローニングを行いモノクローン化した。抗ピ
ラジン誘導体モノクローナル抗体を産生する細胞は、大
量に培養後、マウス腹腔に投与し、抗ピラジン誘導体モ
ノクローナル抗体を含む腹水を回収した。さらにプロテ
インA−セファロースを用い、腹水から抗体を精製しモ
ノクローナル抗体を得た。この抗ピラジン誘導体モノク
ローナル抗体を3DG−451抗体と命名し、3DG−
451抗体を産生するハイブリドーマは工業技術院生命
工学工業技術研究所微生物寄託センターに寄託され、そ
の受託番号はFERM P−16179である。
The screening was carried out by an antigen solid phase ELISA. That is, the BSA-bonded pyrazine derivative derived from the fraction (A) synthesized in Example 3 was added to a 96-well ELISA plate (Pharmacia) at a concentration of 1 μg / ml.
μl / well was poured and left overnight at 4 ° C. for adsorption. After blocking the plate with 1% skim milk, the plate was washed 3 times with a phosphate buffer containing 0.05% Tween 20 (hereinafter referred to as a wash buffer) to perform cell fusion. 50 μl of culture supernatant
Was added and reacted at 37 ° C. for 1 hour. Similarly, after washing three times with a washing buffer, peroxidase (hereinafter, P
Labeled anti-mouse immunoglobulin antibody (denoted as OD) (manufactured by Dako) was added, and the mixture was further reacted at 37 ° C. for 1 hour.
After washing four times with the washing buffer, the substrate ABTS was added to select wells in which color development was observed. Wells showing reactivity to the BSA-bonded pyrazine derivative were further subjected to an inhibition test with a free pyrazine derivative to confirm the specificity. That is,
During the reaction between the solid-phase antigen and the culture supernatant, a fixed concentration (about 10 μm
The pyrazine derivative of M) was allowed to coexist, and the specificity was confirmed by the presence or absence of inhibition. In this case, the well showing suppression is the specific well. The cells in the wells whose specificity was confirmed were cloned by the limiting dilution method to be monocloned. The cells producing the anti-pyrazine derivative monoclonal antibody were cultured in a large amount and then intraperitoneally administered to mice to collect ascites fluid containing the anti-pyrazine derivative monoclonal antibody. The antibody was purified from ascites using protein A-Sepharose to obtain a monoclonal antibody. This anti-pyrazine derivative monoclonal antibody was named 3DG-451 antibody, and was named 3DG-
The hybridoma producing the 451 antibody has been deposited at the Microorganism Depositary Center, Institute of Biotechnology, Institute of Biotechnology, AIST, and the deposit number is FERM P-16179.

【0078】実施例8 抗ピラジン誘導体モノクローナ
ル抗体の特異性の確認 抗ピラジン誘導体モノクローナル抗体の特異性は、BS
A結合ピラジン誘導体を固相抗原としたELISAの抑
制試験で確認した。すなわち、ヌンク社製ELISAプ
レート(マキシソーブ)に実施例3で合成した分画
(A)由来または分画(B)由来のBSA結合ピラジン
誘導体をそれぞれ1μg/mlの濃度で75μl/ウエ
ル分注し、4℃一夜放置し吸着させ、分画(A)由来B
SA結合ピラジン誘導体吸着プレートをプレート
(A)、分画(B)由来BSA結合ピラジン誘導体吸着
プレートをプレート(B)とした。プレート(A)及び
プレート(B)を1%スキムミルクで37℃、3時間放
置してブロッキングした後、インヒビターとして、1m
Mから5n希釈した実施例1で合成したピラジン誘導体
(化合物(8) )、10mMから5n希釈した参考例6で
合成したジアミノベンゼン誘導体(化合物(7) )、10
mMから5n希釈したキノキサリン(東京化成工業社
製)の溶液をそれぞれ40μlづつ各ウエルに入れた。
次に、1μg/mlの3DG−451抗体を40μl加
え、37℃で1時間反応させた。洗浄緩衝液で充分洗浄
した後、POD標識抗マウスイムノグロブリン抗体(ダ
コ社製)を加え、37℃で1時間反応させた。同様に洗
浄緩衝液で充分洗浄した後、基質ABTSを加え、室温
で20〜30分放置した後、分光光度計にて405nm
の吸収を測定した。図1及び図2に示すように、BSA
結合ピラジン誘導体と3DG−451抗体との反応は、
ピラジン誘導体(化合物(8) )では抑制されたが、ジア
ミノベンゼン誘導体(化合物(7) )及びキノキサリンで
は全く抑制されなかった。3DG−451抗体のジアミ
ノベンゼン誘導体(化合物(7) )及びキノキサリンに対
する交差反応性は0.1%以下であり、ピラジン誘導体
(化合物(8) )特異的であることが確認できた。
Example 8 Confirmation of specificity of anti-pyrazine derivative monoclonal antibody
It was confirmed by an ELISA inhibition test using an A-bonded pyrazine derivative as a solid phase antigen. That is, 75 μl / well of the BSA-bonded pyrazine derivative derived from the fraction (A) or the fraction (B) synthesized in Example 3 was dispensed onto a Nunc ELISA plate (maxisorb) at a concentration of 1 μg / ml, respectively. Fraction (A) -derived B
The SA-bonded pyrazine derivative adsorption plate was used as the plate (A), and the fraction (B) -derived BSA-bonded pyrazine derivative adsorption plate was used as the plate (B). Plate (A) and plate (B) were left in 1% skim milk at 37 ° C. for 3 hours for blocking, and then 1 m as an inhibitor.
The pyrazine derivative (compound (8)) synthesized in Example 1 diluted with M from 5 n, and the diaminobenzene derivative (compound (7)) synthesized in Reference Example 6 diluted from 5 mM to 5 n, 10
40 μl of a solution of quinoxaline (manufactured by Tokyo Chemical Industry Co., Ltd.) diluted with 5 n from mM was added to each well.
Next, 40 μl of 1 μg / ml 3DG-451 antibody was added and reacted at 37 ° C. for 1 hour. After thorough washing with a washing buffer, POD-labeled anti-mouse immunoglobulin antibody (manufactured by Dako) was added, and the mixture was reacted at 37 ° C for 1 hour. Similarly, after thoroughly washing with a washing buffer, the substrate ABTS was added, and the mixture was allowed to stand at room temperature for 20 to 30 minutes, and then with a spectrophotometer at 405 nm.
Was measured. As shown in FIGS. 1 and 2, BSA
The reaction between the bound pyrazine derivative and the 3DG-451 antibody is
It was suppressed by the pyrazine derivative (compound (8)) but not by the diaminobenzene derivative (compound (7)) and quinoxaline. The cross-reactivity of the 3DG-451 antibody with respect to the diaminobenzene derivative (compound (7)) and quinoxaline was 0.1% or less, and it was confirmed that it was specific to the pyrazine derivative (compound (8)).

【0079】実施例9 ビオチン結合ピラジン誘導体
(化合物(9) )の測定 実施例4で合成した化合物(9) をサンドイッチELIS
A法にて測定した。ヌンク社製ELISAプレート(マ
キシソーブ)に3DG−451抗体を10μg/mlの
濃度で75μl/ウェルづつ分注し、4℃一晩放置して
吸着させた。プレートを1%スキムミルクで37℃、3
時間放置してブロッキングした後、実施例4で合成した
ビオチン結合ピラジン誘導体(化合物(9) )を1%BS
A含有トリス緩衝液で40ng/mlから5n希釈し、
各希釈液を抗体吸着プレートに75μl/ウェルづつ入
れ、37℃、1時間反応させた。同様に、1000ng
/mlより5n希釈した実施例5で合成したビオチン結
合ジアミノベンゼン誘導体(化合物(10))も測定した。
反応後、洗浄緩衝液で充分洗浄し、アルカリフォスファ
ターゼ(以下本明細書中では、ALPと記載する)標識
アビジン(ダコ社製)を各ウェルに75μl入れ、さら
に37℃、1時間反応させた。洗浄緩衝液で充分洗浄
し、基質pNPPを75μl/ウェル入れ室温で30分
放置後、405nmの波長を測定した。結果を図3に示
す。図3に示すように固相として用いた抗体3DG−4
51はジアミノベンゼン誘導体(化合物(10))には全く
反応しないが、ピラジン誘導体(化合物(9) )はおよそ
10pg/mlまで測定可能であった。
Example 9 Measurement of biotin-bonded pyrazine derivative (compound (9)) The compound (9) synthesized in Example 4 was sandwiched by ELISA.
It was measured by Method A. The 3DG-451 antibody was dispensed at a concentration of 10 μg / ml in an amount of 75 μl / well onto a Nunc ELISA plate (maxisorb) and left at 4 ° C. overnight for adsorption. Plate with 1% skim milk at 37 ° C for 3
After standing for blocking for a while, the biotin-bonded pyrazine derivative (compound (9)) synthesized in Example 4 was added to 1% BS.
Dilute 5 ng from 40 ng / ml with Tris buffer containing A,
75 μl / well of each diluted solution was added to the antibody adsorption plate and reacted at 37 ° C. for 1 hour. Similarly, 1000 ng
The biotin-conjugated diaminobenzene derivative (compound (10)) synthesized in Example 5 diluted with 5n from the solution / ml was also measured.
After the reaction, the wells were thoroughly washed with a wash buffer, and 75 μl of alkaline phosphatase (hereinafter referred to as ALP) -labeled avidin (manufactured by Dako) was added to each well, and further reacted at 37 ° C. for 1 hour. After sufficiently washing with the washing buffer, 75 μl / well of the substrate pNPP was added and left at room temperature for 30 minutes, and then the wavelength of 405 nm was measured. The results are shown in Fig. 3. Antibody 3DG-4 used as a solid phase as shown in FIG.
51 did not react with the diaminobenzene derivative (compound (10)) at all, but the pyrazine derivative (compound (9)) was measurable up to about 10 pg / ml.

【0080】実施例10 3−デオキシグルコゾン(3
−DG)の測定−1 3−DGと実施例5で合成したビオチン結合ジアミノベ
ンゼン誘導体(化合物(10))とをPBS中室温で一晩反
応させ、その反応物を3DG−451固相ELISAで
測定した。すなわち、5μg/mlから5n希釈した3
−DGにビオチン結合ジアミノベンゼン誘導体溶液(化
合物(10))を終濃度10μg/mlになるように加え、
室温、一晩反応させた。ヌンク社製ELISAプレート
(マキシソーブ)に3DG−451抗体を10μg/m
lの濃度で75μl/ウェル入れ、4℃一晩放置して吸
着させた後、1%スキムミルクでブロッキングし、EL
ISAプレートを作製した。対照として、ピラジン誘導
体には特異性を持たないモノクローナル抗体PCX22
A4を吸着させたELISAプレートも作製した。EL
ISAの測定手法は実施例9で示した方法に従い、作製
した2種類のプレートを用いて、3−DGとビオチン結
合ジアミノベンゼン誘導体(化合物(10))との反応物で
あるビオチン結合ピラジン誘導体を測定した。結果を図
4に示す。図4に示すように、本測定法で3−DGが測
定できることを確認した。
Example 10 3-deoxyglucosone (3
-DG) Measurement-1 3-DG and the biotin-conjugated diaminobenzene derivative (compound (10)) synthesized in Example 5 were reacted in PBS overnight at room temperature, and the reaction product was subjected to 3DG-451 solid phase ELISA. It was measured. That is, 3 diluted from 5 μg / ml to 5 n
Add a biotin-conjugated diaminobenzene derivative solution (compound (10)) to DG to a final concentration of 10 μg / ml,
The reaction was carried out overnight at room temperature. NDG ELISA plate (maxisorb) with 3DG-451 antibody at 10 μg / m
75 μl / well was added at a concentration of 1 and left overnight at 4 ° C. for adsorption, blocking with 1% skim milk, and EL
An ISA plate was made. As a control, a monoclonal antibody PCX22 having no specificity for pyrazine derivatives
An ELISA plate having A4 adsorbed was also prepared. EL
The measurement method of ISA was according to the method shown in Example 9, and using the two kinds of prepared plates, the biotin-bonded pyrazine derivative which is a reaction product of 3-DG and the biotin-bonded diaminobenzene derivative (compound (10)) was used. It was measured. The results are shown in Fig. 4. As shown in FIG. 4, it was confirmed that 3-DG could be measured by this measurement method.

【0081】実施例11 3−DGの測定−2 3−DGと実施例6で合成したビオチン化ジアミノベン
ゼン誘導体(化合物(13))または参考例9で合成したビ
オチン化ジアミノベンゼン誘導体(化合物(14))とをそ
れぞれPBS中室温で一晩反応させ、その反応物を3D
G−451固相ELISAで測定した。すなわち実施例
10と同様に、200ng/mlから3n希釈した3−
DGに化合物(13)または化合物(14)を終濃度10μg/
mlになるように加え、室温で一晩反応させた。ELI
SA測定方法は実施例10と同様に行った。結果を図5
に示す。図5に示すように、化合物(14)のビオチン化ジ
アミノベンゼン誘導体は反応を示さなかったが、化合物
(13)のビオチン化ジアミノベンゼン誘導体を用いた場
合、3−DGが100pg/ml程度より測定可能であ
った。
Example 11 Measurement of 3-DG-2 Biotinylated diaminobenzene derivative (compound (13)) synthesized in Example 6 with 3-DG or biotinylated diaminobenzene derivative synthesized in Reference Example 9 (compound (14 )) And each in PBS overnight at room temperature and the reaction product
It was measured by G-451 solid phase ELISA. That is, as in Example 10, 3-n diluted from 200 ng / ml to 3 n was used.
Compound (13) or compound (14) was added to DG at a final concentration of 10 μg /
It was added so that the amount became ml, and the reaction was carried out at room temperature overnight. ELI
The SA measurement method was the same as in Example 10. The results are shown in Figure 5.
Shown in. As shown in FIG. 5, the biotinylated diaminobenzene derivative of compound (14) showed no reaction, but the compound
When the biotinylated diaminobenzene derivative of (13) was used, 3-DG was measurable from about 100 pg / ml.

【0082】[0082]

【発明の効果】本発明により、合併症を伴う糖尿病の指
標となり得る1,2−ジカルボニル誘導体を測定し得る
抗体を提供し、該抗体を用いた測定法によって1,2−
ジカルボニル誘導体を簡便に測定できるようになった。
INDUSTRIAL APPLICABILITY According to the present invention, there is provided an antibody capable of measuring a 1,2-dicarbonyl derivative which can be an index of diabetes associated with complications, and a 1,2-dicarbonyl derivative by a measuring method using the antibody.
It has become possible to easily measure dicarbonyl derivatives.

【図面の簡単な説明】[Brief description of drawings]

【図1】分画(A)由来BSA結合ピラジン誘導体を用
いた抗ピラジン誘導体モノクローナル抗体の特異性を示
す図である。
FIG. 1 is a diagram showing the specificity of an anti-pyrazine derivative monoclonal antibody using a BSA-bonded pyrazine derivative derived from fraction (A).

【図2】分画(B)由来BSA結合ピラジン誘導体を用
いた抗ピラジン誘導体モノクローナル抗体の特異性を示
す図である。
FIG. 2 is a diagram showing the specificity of an anti-pyrazine derivative monoclonal antibody using a BSA-bonded pyrazine derivative derived from fraction (B).

【図3】抗ピラジン誘導体モノクローナル抗体によるビ
オチン結合ピラジン誘導体の測定結果を示す図である。
FIG. 3 is a diagram showing the measurement results of a biotin-bonded pyrazine derivative with an anti-pyrazine derivative monoclonal antibody.

【図4】化合物(10)を用いた抗ピラジン誘導体モノクロ
ーナル抗体による3−デオキシグルコゾンの測定結果を
示す図である。
FIG. 4 shows the measurement results of 3-deoxyglucosone using an anti-pyrazine derivative monoclonal antibody containing compound (10).

【図5】化合物(13)及び化合物(14)を用いた抗ピラジン
誘導体モノクローナル抗体による3−デオキシグルコゾ
ンの測定結果を示す図である。
FIG. 5 shows the measurement results of 3-deoxyglucosone using an anti-pyrazine derivative monoclonal antibody using Compound (13) and Compound (14).

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 FI C12N 15/02 C12N 15/00 C C12P 21/08 5/00 B (56)参考文献 FEBS Lett.,1997,Vo l.407,No.3,p.297−302 J.Clin.Invest., 1992,Vol.89,No.4,p.1102 −1112 (58)調査した分野(Int.Cl.7,DB名) C12P 1/00 - 41/00 BIOSIS/WPI(DIALOG) CA(STN) REGISTRY(STN) JSTPlus(JOIS)─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. 7 Identification code FI C12N 15/02 C12N 15/00 C C12P 21/08 5/00 B (56) Reference FEBS Lett. , 1997, Vol. 407, No. 3, p. 297-302 J. Clin. Invest. , 1992, Vol. 89, No. 4, p. 1102 -1112 (58) Fields surveyed (Int.Cl. 7 , DB name) C12P 1/00-41/00 BIOSIS / WPI (DIALOG) CA (STN) REGISTRY (STN) JSTPlus (JOIS)

Claims (14)

(57)【特許請求の範囲】(57) [Claims] 【請求項1】 下記一般式(I)で示されるピラジン誘
導体の少なくともR又はRを包含する領域を認識す
る抗体。 【化1】 (ただし、式中、R は水素、R トリヒドロキシブ
チル基であり、Aはピラジン環と結合して5員又は6員
の芳香族炭化水素基、芳香族複素環基又は脂環式炭化水
素基を形成する基であり、Rは架橋性残基であり、A
により形成される環は、上記Rの他に、5員環の場合
には1又は2個、6員環の場合には1〜3個のさらなる
置換基によって置換されていてもよい)。
1. An antibody which recognizes a region including at least R 1 or R 2 of a pyrazine derivative represented by the following general formula (I). [Chemical 1] (In the formula, R 1 is hydrogen, R 2 is a trihydroxybutyl group, A is a 5- or 6-membered aromatic hydrocarbon group, aromatic heterocyclic group or alicyclic group bonded to a pyrazine ring. R 3 is a group that forms a hydrocarbon group, R 3 is a crosslinkable residue, and
The ring formed by is optionally substituted with 1 or 2 additional substituents in the case of a 5-membered ring and 1 to 3 additional substituents in the case of a 6-membered ring in addition to the above R 3 ).
【請求項2】 Aにより形成される環はピリジン、ベン
ゼン、フラン又はチオフェンである請求項1に記載の抗
体。
2. The antibody according to claim 1, wherein the ring formed by A is pyridine, benzene, furan or thiophene.
【請求項3】 Rは反応部分とスペーサー部分を有す
る請求項1又は2に記載の抗体。
3. The antibody according to claim 1 or 2 , wherein R 3 has a reactive portion and a spacer portion.
【請求項4】 前記反応部分はカルボキシル基、水酸
基、スルフヒドリル基、アミノ基、マレイミド基、アル
デヒド基若しくはハロゲン原子又はこれらの誘導体であ
って他の化合物と反応して共有結合を形成できるもので
ある請求項記載の抗体。
4. The reactive moiety is a carboxyl group, a hydroxyl group, a sulfhydryl group, an amino group, a maleimide group, an aldehyde group or a halogen atom or a derivative thereof, which can react with another compound to form a covalent bond. The antibody according to claim 3 .
【請求項5】 前記スペーサー部分は、脂肪族炭化水素
基若しくは芳香族炭化水素基又はこれらが互いにエステ
ル結合、アミド結合、エーテル結合、チオエーテル結
合、ジスルフィド結合若しくはシッフ塩基結合により結
合されたものである請求項又は記載の抗体。
5. The spacer portion is an aliphatic hydrocarbon group or an aromatic hydrocarbon group or a group in which these are bonded to each other through an ester bond, an amide bond, an ether bond, a thioether bond, a disulfide bond or a Schiff base bond. The antibody according to claim 3 or 4 .
【請求項6】 Aが形成する環がベンゼンであり、R
が4−カルボキサミドブタノイル−(2−メルカプト)
エチルアミド基である請求項1記載の抗体。
6. The ring formed by A is benzene, and R 3
Is 4-carboxamidobutanoyl- (2-mercapto)
The antibody according to claim 1, which is an ethylamide group.
【請求項7】 モノクローナル抗体である請求項1ない
のいずれか1項に記載の抗体。
7. The antibody according to any one of claims 1 <br/> to 6 which is a monoclonal antibody.
【請求項8】 FERM P−16179の受託番号で
寄託されたハイブリドーマが産生するモノクローナル抗
体3DG−451である請求項記載の抗体。
8. The antibody according to claim 7, which is the monoclonal antibody 3DG-451 produced by the hybridoma deposited under the accession number of FERM P-16179.
【請求項9】 下記一般式(I)で示されるピラジン誘
導体のRに免疫原性担体が結合したものから成る免疫
原。 【化1】 (ただし、式中、R は水素、R トリヒドロキシブ
チル基であり、Aはピラジン環と結合して5員又は6員
の芳香族炭化水素基、芳香族複素環基又は脂環式炭化水
素基を形成する基であり、Rは架橋性残基であり、A
により形成される環は、上記Rの他に、5員環の場合
には1又は2個、6員環の場合には1〜3個のさらなる
置換基によって置換されていてもよい)。
9. An immunogen comprising a pyrazine derivative represented by the following general formula (I) in which R 3 is bound to an immunogenic carrier. [Chemical 1] (In the formula, R 1 is hydrogen, R 2 is a trihydroxybutyl group, A is a 5- or 6-membered aromatic hydrocarbon group, aromatic heterocyclic group or alicyclic group bonded to a pyrazine ring. R 3 is a group that forms a hydrocarbon group, R 3 is a crosslinkable residue, and
The ring formed by is optionally substituted with 1 or 2 additional substituents in the case of a 5-membered ring and 1 to 3 additional substituents in the case of a 6-membered ring in addition to the above R 3 ).
【請求項10】 前記免疫原性担体がタンパク質である
請求項記載の免疫原。
10. The immunogen according to claim 9 , wherein the immunogenic carrier is a protein.
【請求項11】 生体内におけるタンパク質の糖化反応
の中間体である1,2−ジカルボニル誘導体を免疫測定
する方法であって、検体中の1,2−ジカルボニル誘導
体を、一般式(II) 【化2】 (R及びAは、請求項1に記載される一般式(I)の
場合と同義)で示されるジアミノ誘導体と反応させて請
求項1に記載される一般式(I)で示されるピラジン誘
導体を生成させ、該ピラジン誘導体と請求項1ないし
のいずれか1項に記載の抗体との抗原抗体反応を利用し
た免疫測定により該ピラジン誘導体を測定し、それによ
って検体中の1,2−ジカルボニル誘導体を測定するこ
とから成る、1,2−ジカルボニル誘導体の免疫測定方
法。
11. A method for immunoassaying a 1,2-dicarbonyl derivative which is an intermediate of a glycation reaction of a protein in a living body, wherein the 1,2-dicarbonyl derivative in a sample is represented by the general formula (II): [Chemical 2] (R 3 and A have the same meanings as in the case of the general formula (I) described in claim 1) and are reacted with a diamino derivative represented by the general formula (I) described in claim 1. to generate, claims 1 and said pyrazine derivative 8
The method of claim 1, wherein the pyrazine derivative is measured by an immunoassay utilizing an antigen-antibody reaction with the antibody, and thereby the 1,2-dicarbonyl derivative in the sample is measured. Method for immunoassay of dicarbonyl derivative.
【請求項12】 上記ジアミノ誘導体のRが検出可能
な標識により標識されており、前記免疫測定は、前記抗
体を固相に結合させ、該固相結合抗体と前記ピラジン誘
導体とを反応させ、洗浄後、固相に結合された標識量を
測定することにより前記ピラジン誘導体を測定すること
から成る、請求項11記載の方法。
12. The diamino derivative R 3 is labeled with a detectable label, and in the immunoassay, the antibody is bound to a solid phase, and the solid phase-bound antibody is allowed to react with the pyrazine derivative. 12. The method according to claim 11 , comprising measuring the pyrazine derivative by measuring the amount of label bound to the solid phase after washing.
【請求項13】 前記標識はビオチンである請求項12
記載の方法。
Wherein said label claim 12 biotin
The method described.
【請求項14】 前記1,2−ジカルボニル誘導体は、
デオキシグルコゾンである、請求項11ないし13のい
ずれか1項に記載の方法。
14. The 1,2-dicarbonyl derivative is
The method according to any one of claims 11 to 13 , which is deoxyglucosone.
JP24912298A 1997-08-21 1998-08-19 Pyrazine derivative-recognizing antibody and method for measuring 1,2-dicarbonyl derivative using the same Expired - Fee Related JP3508563B2 (en)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FEBS Lett.,1997,Vol.407,No.3,p.297−302
J.Clin.Invest.,1992,Vol.89,No.4,p.1102−1112

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