JP2024063770A - Novel compound and method for producing the same - Google Patents
Novel compound and method for producing the same Download PDFInfo
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- JP2024063770A JP2024063770A JP2023182303A JP2023182303A JP2024063770A JP 2024063770 A JP2024063770 A JP 2024063770A JP 2023182303 A JP2023182303 A JP 2023182303A JP 2023182303 A JP2023182303 A JP 2023182303A JP 2024063770 A JP2024063770 A JP 2024063770A
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- 125000000217 alkyl group Chemical group 0.000 claims abstract description 16
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Landscapes
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Abstract
Description
本発明は、Ｏ結合型糖鎖の定量解析に有益な新規化合物及びその製造方法に関する。 The present invention relates to a novel compound useful for quantitative analysis of O-linked glycans and a method for producing the same.
糖鎖は糖タンパク質の構造の安定化、体内動態、生物活性、免疫原性等に関与しており、糖タンパク質を有効成分とする医薬品において糖鎖構造は有効性・安全性に影響する重要な品質特性である。様々な糖タンパク質の構造・機能の解析や、糖タンパク質医薬品の品質管理のための様々な定性・定量分析法が開発されてきた（非特許文献１，２）。 Glycosylation is involved in the stabilization of glycoprotein structure, pharmacokinetics, biological activity, immunogenicity, etc., and the glycan structure is an important quality characteristic that affects the efficacy and safety of pharmaceuticals that use glycoproteins as active ingredients. Various qualitative and quantitative analytical methods have been developed to analyze the structure and function of various glycoproteins and to control the quality of glycoprotein pharmaceuticals (Non-Patent Documents 1, 2).
しかし、N結合型糖鎖の解析には多大な努力が払われている一方で、O結合型糖鎖（タンパク質のセリン又はスレオニン側鎖に結合した糖鎖）のハイスループット定量解析は十分に研究されているとは言い難い。これはタンパク質骨格からO結合型糖鎖を遊離させる普遍的な酵素が存在しないことに加え、従来の化学的な遊離法では副反応(Peeling反応)が強く、もとの糖鎖構造を正確に反映した解析結果を得ることが困難であり、また，煩雑なサンプル調製が必要であったからである（特許文献１，２，非特許文献３）。 However, while a great deal of effort has been devoted to the analysis of N-linked glycans, it cannot be said that high-throughput quantitative analysis of O-linked glycans (glycans bound to the serine or threonine side chains of proteins) has been sufficiently studied. This is because there is no universal enzyme that can release O-linked glycans from the protein backbone, and because conventional chemical release methods have a strong side reaction (Peeling reaction), making it difficult to obtain analysis results that accurately reflect the original glycan structure, and because complicated sample preparation is required (Patent Documents 1 and 2, Non-Patent Document 3).
O結合型糖鎖の解析に用いられている3-Methyl-1-phenyl-5-pyrazolone（以下MPPと略することがある。）は、Peeling反応の抑制と誘導体化を同時に行うことが可能であることが知られているが、検出はUVで行うこと、過剰な試薬を取り除くための洗浄操作が必要等のことから、検出感度や定量性に課題があった（特許文献３，非特許文献４，５）。 3-Methyl-1-phenyl-5-pyrazolone (hereinafter sometimes abbreviated as MPP), which is used in the analysis of O-linked glycans, is known to be capable of simultaneously suppressing the Peeling reaction and derivatizing the product. However, since detection is performed using UV light and a washing step is required to remove excess reagent, there are problems with detection sensitivity and quantitation (Patent Document 3, Non-Patent Documents 4 and 5).
本発明はかかる問題点に鑑みてなされたものであって、簡便な操作でO結合型糖鎖を高感度に検出できる誘導体化試薬等に利用できる新規化合物を提供することを目的とする。 The present invention was made in consideration of these problems, and aims to provide a novel compound that can be used as a derivatization reagent that can detect O-linked glycans with high sensitivity by simple operations.
本発明にかかる新規化合物は、下記化学式からなる。 The novel compound of the present invention has the following chemical formula:
式中、Ｘ１及びＸ２は、各々独立に、置換基（この置換基は、炭素数１から４のアルキル基、アルコキシ基、アルコキシアルキル基、エステル基、エステルアルキル基、又は、アミノ基、水酸基、チオール基である）若しくはヘテロ原子を有してもよい分岐可能な、Ｃ０～３アルキル基又はＣ０～３アルケニル基であり、
Ｚは、－(CH2)ｎ－（ここでnは１～１５の整数を表す）、又は、下記構造式(Ｙは、Ｃ又はＮである。)であり、
In the formula, X1 and X2 each independently represent a branched C0-3 alkyl group or C0-3 alkenyl group which may have a substituent (the substituent is an alkyl group, an alkoxy group, an alkoxyalkyl group, an ester group, an esteralkyl group, or an amino group, a hydroxyl group, or a thiol group, each having 1 to 4 carbon atoms) or a heteroatom;
Z is -( CH2 ) n- (wherein n is an integer from 1 to 15) or the following structural formula (Y is C or N):
Ｌ１は、－NHCO－、又は、－CONH－であり、
Ｌ２は、化学結合、又は、置換基（この置換基は、水酸基、カルボキシル基、アミノ基、アミド基、カルバメート基、又はケトン基である）若しくはヘテロ原子を有しても良い分岐可能な、Ｃ１～８アルキル基若しくはＣ２～８アルケニル基であり、
L３は、化学結合、O、－OCH２－、－CH2O－、－(CH2OCH２)ｎ－、－NH－、－NHCO－、－CONH－、若しくは、－NHCONH－（ここで、nは１～１５の整数を表す）であり、
Ａは、ベンゼン、ペンタレン、インデン、ナフタレン、アズレン、ヘプタレン、インダセン、アセナフタレン、フルオレン、ベンゾフルオレン、ジベンゾフルオレン、フェナレン、フェナントレン基、アントラセン、フルオランテン、ピレン、クリセン、ナフタセン、ピセン、ペリレン、ピロール、チオフェン、フラン、シロール、イミダゾール、ピラゾール、チアゾール、イソチアゾール、オキサゾール、イソオキサゾール、ピリジン、ピラジン、ピリミジン、ピリダジン、トリアジン、ベンゾフラン、ベンゾチオフェン、ジベンゾフラン、ジベンゾチオフェン、カルバゾール、ベンゾシロール、ジベンゾシロール、キノリン、イソキノリン、ベンゾイミダゾール、イミダゾピリジン又はイミダゾピリミジンである。
L1 is -NHCO- or -CONH-;
L2 is a chemical bond, or a branched C1-8 alkyl group or C2-8 alkenyl group which may have a substituent (the substituent is a hydroxyl group, a carboxyl group, an amino group, an amide group, a carbamate group, or a ketone group) or a heteroatom;
L3 is a chemical bond, O , -OCH2- , -CH2O- , -( CH2OCH2 ) n- , -NH-, -NHCO-, -CONH-, or -NHCONH- (wherein n is an integer of 1 to 15);
A is benzene, pentalene, indene, naphthalene, azulene, heptalene, indacene, acenaphthalene, fluorene, benzofluorene, dibenzofluorene, phenalene, phenanthrene group, anthracene, fluoranthene, pyrene, chrysene, naphthacene, picene, perylene, pyrrole, thiophene, furan, silole, imidazole, pyrazole, thiazole, isothiazole, oxazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, triazine, benzofuran, benzothiophene, dibenzofuran, dibenzothiophene, carbazole, benzosilole, dibenzosilole, quinoline, isoquinoline, benzimidazole, imidazopyridine or imidazopyrimidine.
本発明によれば簡便な操作でO結合型糖鎖を高感度に検出できる誘導体化試薬等に利用できる新規化合物が得られる。 The present invention provides a novel compound that can be used as a derivatization reagent that can detect O-linked glycans with high sensitivity using simple procedures.
以下、添付の図面を参照して本発明の実施形態について具体的に説明するが、当該実施形態は本発明の原理の理解を容易にするためのものであり、本発明の範囲は、下記の実施形態に限られるものではなく、当業者が以下の実施形態の構成を適宜置換した他の実施形態も、本発明の範囲に含まれる。 The following describes in detail an embodiment of the present invention with reference to the attached drawings. However, this embodiment is intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the following embodiment. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the following embodiment are also included in the scope of the present invention.
本発明者は、新規蛍光性糖鎖誘導体化試薬を開発するにあたり、下記式に示されるPMPが有する利点を活かしつつ、複数の課題を一気に克服する方法を考案した。 In developing a novel fluorescent glycan derivatization reagent, the inventors devised a method to overcome multiple problems at once while taking advantage of the advantages of PMP, as shown in the formula below.
本発明にかかる新規化合物は、下記化学式からなる。 The novel compound of the present invention has the following chemical formula:
式中、Ｘ１及びＸ２は、各々独立に、置換基（この置換基は、炭素数１から４のアルキル基、アルコキシ基、アルコキシアルキル基、エステル基、エステルアルキル基、又は、アミノ基、水酸基、チオール基である）若しくはヘテロ原子を有してもよい分岐可能な、Ｃ０～３アルキル基又はＣ０～３アルケニル基である。 In the formula, X1 and X2 each independently represent a branched C0-3 alkyl group or C0-3 alkenyl group which may have a substituent (the substituent is an alkyl group, an alkoxy group, an alkoxyalkyl group, an ester group, an esteralkyl group, or an amino group, a hydroxyl group, or a thiol group, each having 1 to 4 carbon atoms) or a heteroatom.
Ｚは、－(CH2)ｎ－（ここでnは１～１５の整数を表す）、又は、下記構造式(Ｙは、Ｃ又はＮである。)である。 Z is --(CH 2 ) n -- (wherein n is an integer of 1 to 15) or the following structural formula (Y is C or N):
Ｌ１は、－NHCO－、又は、－CONH－である。 L1 is -NHCO- or -CONH-.
Ｌ２は、化学結合、又は、置換基（この置換基は、水酸基、カルボキシル基、アミノ基、アミド基、カルバメート基、又はケトン基である）若しくはヘテロ原子を有しても良い分岐可能な、Ｃ１～８アルキル基若しくはＣ２～８アルケニル基である。 L2 is a chemical bond or a branched C1-8 alkyl or C2-8 alkenyl group which may have a substituent (the substituent is a hydroxyl group, a carboxyl group, an amino group, an amide group, a carbamate group, or a ketone group) or a heteroatom.
L３は、化学結合、O、－OCH２－、－CH2O－、－(CH2OCH２)ｎ－、－NH－、－NHCO－、－CONH－、若しくは、－NHCONH－（ここで、nは１～１５の整数を表す）である。 L3 is a chemical bond, O, -OCH 2 -, -CH 2 O-, -(CH 2 OCH 2 ) n -, -NH-, -NHCO-, -CONH-, or -NHCONH- (wherein n represents an integer of 1 to 15).
Ａは、ベンゼン、ペンタレン、インデン、ナフタレン、アズレン、ヘプタレン、インダセン、アセナフタレン、フルオレン、ベンゾフルオレン、ジベンゾフルオレン、フェナレン、フェナントレン基、アントラセン、フルオランテン、ピレン、クリセン、ナフタセン、ピセン、ペリレン、ピロール、チオフェン、フラン、シロール、イミダゾール、ピラゾール、チアゾール、イソチアゾール、オキサゾール、イソオキサゾール、ピリジン、ピラジン、ピリミジン、ピリダジン、トリアジン、ベンゾフラン、ベンゾチオフェン、ジベンゾフラン、ジベンゾチオフェン、カルバゾール、ベンゾシロール、ジベンゾシロール、キノリン、イソキノリン、ベンゾイミダゾール、イミダゾピリジン又はイミダゾピリミジンである。 A is benzene, pentalene, indene, naphthalene, azulene, heptalene, indacene, acenaphthalene, fluorene, benzofluorene, dibenzofluorene, phenalene, phenanthrene group, anthracene, fluoranthene, pyrene, chrysene, naphthacene, picene, perylene, pyrrole, thiophene, furan, silole, imidazole, pyrazole, thiazole, isothiazole, oxazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, triazine, benzofuran, benzothiophene, dibenzofuran, dibenzothiophene, carbazole, benzosilole, dibenzosilole, quinoline, isoquinoline, benzimidazole, imidazopyridine, or imidazopyrimidine.
また本発明にかかる新規化合物は、下記化学式からなる。 The novel compound according to the present invention has the following chemical formula:
式中、R1、R2、R3、R4、R5は、それぞれ独立に、置換基を有していても良い芳香族炭化水素基、置換基を有していても良いアルケニル基、置換基を有していても良いアルキニル基、置換基を有していても良いアルキル基、置換基を有していても良いシリル基、置換基を有していても良い複素環基、置換基を有していても良いアリールアミノ基、置換基を有していても良いアルコキシル基、置換基を有していてもよいアリールボリル基を表す。 In the formula, R1, R2, R3, R4, and R5 each independently represent an aromatic hydrocarbon group which may have a substituent, an alkenyl group which may have a substituent, an alkynyl group which may have a substituent, an alkyl group which may have a substituent, a silyl group which may have a substituent, a heterocyclic group which may have a substituent, an arylamino group which may have a substituent, an alkoxyl group which may have a substituent, or an arylboryl group which may have a substituent.
また本発明にかかる新規化合物は、下記化学式からなる。 The novel compound according to the present invention has the following chemical formula:
式中、nは１～１５の整数を表す。 In the formula, n represents an integer from 1 to 15.
また本発明にかかる新規化合物は、下記化学式からなる。なお前述の化学式においてn=3の場合であるためこの化合物をMPPP-C3と称することも可能である。 The novel compound according to the present invention has the following chemical formula. Note that since n=3 in the above chemical formula, this compound can also be called MPPP-C3.
また本発明にかかる新規化合物は、下記化学式からなる。 The novel compound according to the present invention has the following chemical formula:
式中、R1、R2、R3、R4、R5は、それぞれ独立に、置換基を有していても良い芳香族炭化水素基、置換基を有していても良いアルケニル基、置換基を有していても良いアルキニル基、置換基を有していても良いアルキル基、置換基を有していても良いシリル基、置換基を有していても良い複素環基、置換基を有していても良いアリールアミノ基、置換基を有していても良いアルコキシル基、置換基を有していてもよいアリールボリル基を表す。 In the formula, R1, R2, R3, R4, and R5 each independently represent an aromatic hydrocarbon group which may have a substituent, an alkenyl group which may have a substituent, an alkynyl group which may have a substituent, an alkyl group which may have a substituent, a silyl group which may have a substituent, a heterocyclic group which may have a substituent, an arylamino group which may have a substituent, an alkoxyl group which may have a substituent, or an arylboryl group which may have a substituent.
また本発明にかかる新規化合物は、下記化学式からなる。なおリンカー部分に-NMe2の官能基がついていることからこの化合物をMPPP-NMe2と称することも可能である。 The novel compound according to the present invention has the following chemical formula. Since the linker portion has a -NMe2 functional group, this compound can also be called MPPP-NMe2.
また本発明にかかる新規化合物は、下記化学式からなる。 The novel compound according to the present invention has the following chemical formula:
式中、R1、R2、R3、R4、R5は、それぞれ独立に、置換基を有していても良い芳香族炭化水素基、置換基を有していても良いアルケニル基、置換基を有していても良いアルキニル基、置換基を有していても良いアルキル基、置換基を有していても良いシリル基、置換基を有していても良い複素環基、置換基を有していても良いアリールアミノ基、置換基を有していても良いアルコキシル基、置換基を有していてもよいアリールボリル基を表し、nは１～１５の整数を表す。 In the formula, R1, R2, R3, R4, and R5 each independently represent an aromatic hydrocarbon group which may have a substituent, an alkenyl group which may have a substituent, an alkynyl group which may have a substituent, an alkyl group which may have a substituent, a silyl group which may have a substituent, a heterocyclic group which may have a substituent, an arylamino group which may have a substituent, an alkoxyl group which may have a substituent, or an arylboryl group which may have a substituent, and n represents an integer from 1 to 15.
また本発明にかかる新規化合物は、下記化学式からなる。式中、nは１～１５の整数を表す。 The novel compound according to the present invention has the following chemical formula: In the formula, n is an integer between 1 and 15.
また本発明にかかる新規化合物は、下記化学式からなる。 The novel compound according to the present invention has the following chemical formula:
式中、R1、R2、R3、R4、R5は、それぞれ独立に、置換基を有していても良い芳香族炭化水素基、置換基を有していても良いアルケニル基、置換基を有していても良いアルキニル基、置換基を有していても良いアルキル基、置換基を有していても良いシリル基、置換基を有していても良い複素環基、置換基を有していても良いアリールアミノ基、置換基を有していても良いアルコキシル基、置換基を有していてもよいアリールボリル基を表す。 In the formula, R1, R2, R3, R4, and R5 each independently represent an aromatic hydrocarbon group which may have a substituent, an alkenyl group which may have a substituent, an alkynyl group which may have a substituent, an alkyl group which may have a substituent, a silyl group which may have a substituent, a heterocyclic group which may have a substituent, an arylamino group which may have a substituent, an alkoxyl group which may have a substituent, or an arylboryl group which may have a substituent.
また本発明にかかる新規化合物は、下記化学式からなる。式中、nは１～１５の整数を表す。 The novel compound according to the present invention has the following chemical formula: In the formula, n is an integer from 1 to 15.
また本発明にかかる新規化合物は、下記化学式からなる。 The novel compound according to the present invention has the following chemical formula:
本発明にかかる新規化合物は、O結合型糖鎖と結合しO結合型糖鎖誘導体を生成するO結合型糖鎖誘導体化試薬として利用できる。 The novel compound of the present invention can be used as an O-linked glycan derivatization reagent that binds to O-linked glycans to generate O-linked glycan derivatives.
本明細書において「Ｏ結合型糖鎖」は、酸素（Ｏ）原子を介して結合された糖鎖又はなんらかの修飾を受けた（例えば、アセチル化、脱アセチル化）糖鎖をいう。代表的には、セリン又はスレオニンのＯＨ（水酸基）を介して結合することから、セリンスレオニン結合型糖鎖とも呼ばれる。 In this specification, "O-linked glycan" refers to a glycan linked via an oxygen (O) atom or a glycan that has been modified in some way (e.g., acetylated, deacetylated). Typically, it is linked via the OH (hydroxyl group) of serine or threonine, and is therefore also called a serine-threonine-linked glycan.
O結合型糖鎖としては、セリン又はスレオニン残基へのＮ－アセチルガラクトサミンの付加反応によって生じるＯ－Ｎ－アセチルガラクトサミン（Ｏ－ＧａｌＮＡｃ）型糖鎖、Ｏ－ＧｌｃＮＡｃ（Ｏ－Ｎ－アセチルグルコサミン）型糖鎖、フコースの付加によって生じるＯ－フコース型糖鎖（Ｎｏｔｃｈタンパク質のＥＧＦ様リピートのコンセンサス配列が－Ｃ－Ｘ－Ｘ－Ｇ－Ｇ－Ｓ／Ｔ－Ｃ－（Ｘは任意のタンパク質、フコースはＳ／Ｔに結合。）に付加するものが知られる。）、Ｏ－グルコース（Ｎｏｔｃｈタンパク質のＥＧＦ様リピートのコンセンサス配列が－Ｃ－Ｘ－Ｓ－Ｘ－Ｐ－Ｃ－（Ｘは任意のタンパク質、グルコースはＳ／Ｔに結合。）に付加するものが知られる）、Ｏ－マンノース型糖鎖、Ｏ－キシロース型糖鎖等があげられる。即ち、簡易に記載すれば、O結合型糖鎖は、例えば、GlcNAc、N-アセチルガラクトサミン(GalNAc)、フコース(Fuc)、マンノース(Man)、又は、キシロース(Xyl)であり，およびそれらの糖をタンパク質との結合点とした糖鎖である。 Examples of O-linked glycans include O-N-acetylgalactosamine (O-GalNAc) glycans and O-GlcNAc (O-N-acetylglucosamine) glycans produced by the addition of N-acetylgalactosamine to serine or threonine residues, O-fucose glycans produced by the addition of fucose (known to be added to the consensus sequence of the EGF-like repeats of Notch proteins -C-X-X-G-G-S/T-C- (X is any protein, and fucose is bound to S/T)), O-glucose (known to be added to the consensus sequence of the EGF-like repeats of Notch proteins -C-X-S-X-P-C- (X is any protein, and glucose is bound to S/T)), O-mannose glycans, and O-xylose glycans. In other words, simply stated, O-linked glycans are, for example, GlcNAc, N-acetylgalactosamine (GalNAc), fucose (Fuc), mannose (Man), or xylose (Xyl), and are glycans that use these sugars as the attachment point to a protein.
本発明者はエキサイマー蛍光の利用を着想した（図１）。エキサイマー蛍光とはピレン等の平面性の高い蛍光物質が励起状態になったときにもう一つのピレン分子と相互作用することにより通常より長波長にシフトした蛍光のことである。 The inventor came up with the idea of using excimer fluorescence (Figure 1). Excimer fluorescence is fluorescence that is shifted to a longer wavelength than normal when a highly planar fluorescent substance such as pyrene enters an excited state and interacts with another pyrene molecule.
即ち、本発明者は、例えば、PMPにピレンを導入した新規化合物であるMPPPを着想した（図２）。MPPPはPMP構造を有していることからO結合型糖鎖誘導体化時のPeeling反応を抑制しつつ糖に付加する。さらに反応生成物にはピレンが２分子存在することからエキサイマー蛍光が検出されるため、未反応の試薬に由来するピレン単体の蛍光と区別することができる。即ち未反応の試薬を除去する操作を省略できる利点を有する。 That is, the inventor came up with the idea of MPPP, a novel compound in which pyrene has been introduced into PMP (Figure 2). Because MPPP has a PMP structure, it is added to sugars while suppressing the Peeling reaction during O-linked glycan derivatization. Furthermore, because the reaction product contains two pyrene molecules, excimer fluorescence is detected, which can be distinguished from the fluorescence of pyrene alone derived from unreacted reagents. In other words, this has the advantage of eliminating the need to remove unreacted reagents.
(1)新規糖鎖誘導体化試薬MPPPの合成
下記反応式に示されるように、化合物1（261.3 mg, 1 mmol (2HCl salt)）、2（144.8mg, 0.5mmol）、HBTU（568.5 mg, 1.50 mmol）、DIPEA（193.5 mg，1.5 mmol）をDMF（10 ml）に溶かし、室温で一晩撹拌した。溶媒を減圧濃縮し、酢酸エチルを加え、0.2 N HCl及び飽和食塩水で洗浄した。溶媒を減圧留去し、シリカゲルカラムクロマトグラフィー（CH3OH：CH2Cl2= 99：1）によって精製し、化合物3（MPPP(前述のようにMPPP-C3と称することも可能である。)）を黄色固体として得た（162.7 mg, 71%）。
(1) Synthesis of a new glycan derivatization reagent, MPPP As shown in the reaction scheme below, compound 1 (261.3 mg, 1 mmol (2HCl salt)), 2 (144.8 mg, 0.5 mmol), HBTU (568.5 mg, 1.50 mmol), and DIPEA (193.5 mg, 1.5 mmol) were dissolved in DMF (10 ml) and stirred at room temperature overnight. The solvent was concentrated under reduced pressure, ethyl acetate was added, and the mixture was washed with 0.2 N HCl and saturated saline. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography ( CH3OH : CH2Cl2 = 99 :1) to obtain compound 3 (MPPP (which can also be referred to as MPPP-C3 as mentioned above)) as a yellow solid (162.7 mg, 71%).
NMRスペクトルを図３，図４に示す。図３はMPPPの1H NMR スペクトル図である。図４はMPPPの13C NMR スペクトル図である。
1H NMR (600 MHz, CDCl3) δ 8.42 (d, J = 9.0 Hz, 1H), 8.18-8.20 (m, 2H), 8.12-8.15 (m, 2H), 8.05 (s, 2H), 8.06-8.03 (m, 1H), 7.91 (d, J = 7.2 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.04 (s, 1H), 3.492 (t, J = 7.8 Hz, 2H), 3.44 (s, 2H), 2.45 (t, J = 6.6 Hz, 2H), 2.35 (m, 2H), 2.21 (s, 3H). 13C-NMR (150 MHz, CDCl3) δ 170.78, 170.43, 156.42, 135.71, 134.88, 134.37, 131.48, 130.97, 130.08, 128.92, 127.51, 126.86, 125.98, 125.18, 125.05, 124.90, 123.43, 120.14, 119.54, 43.13, 36.85, 32.60, 27.20, 17.04 ESI-HRMS calcd C30H26N3O2
+, [M+H]+: 460.2020, found : 460.2020
The NMR spectra are shown in Figures 3 and 4. Figure 3 shows the 1 H NMR spectrum of MPPP. Figure 4 shows the 13 C NMR spectrum of MPPP.
1H NMR (600 MHz, CDCl3 ) δ 8.42 (d, J = 9.0 Hz, 1H), 8.18-8.20 (m, 2H), 8.12-8.15 (m, 2H), 8.05 (s, 2H), 8.06-8.03 (m, 1H), 7.91 (d, J = 7.2 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.04 (s, 1H), 3.492 (t, J = 7.8 Hz, 2H), 3.44 (s, 2H), 2.45 (t, J = 6.6 Hz, 2H), 2.35 (m, 2H), 2.21 (s, 3H). 13C -NMR (150 MHz, CDCl3 ) δ 170.78, 170.43, 156.42, 135.71, 134.88 , 134.37, 131.48, 130.97, 130.08, 128.92, 127.51, 126.86, 125.98, 125.18, 125.05, 124.90, 123.43, 120.14, 119.54, 43.13, 36.85, 32.60, 27.20, 17.04 ESI-HRMS calcd C30H26N3O2 + , [ M +H] + : 460.2020, found : 460.2020
(2)MPPPによる誘導体化及びそのHPLC解析
50 mM MPPP（DMSO溶液）50 μL、20 mM 糖水溶液 25 μL、60mM NaOH 25 μL（終濃度25 mM MPPP、5 mM GalNAc、12 mM NaOH、 50% DMSO）を混和し、70℃、30 minで加熱した後、氷浴で2 min冷却した。下記式に示されるようにMPPPによる糖の誘導体化が行われた。
(2) Derivatization with MPPP and its HPLC analysis
50 μL of 50 mM MPPP (DMSO solution), 25 μL of 20 mM sugar solution, and 25 μL of 60 mM NaOH (final concentrations: 25 mM MPPP, 5 mM GalNAc, 12 mM NaOH, 50% DMSO) were mixed and heated at 70°C for 30 min, and then cooled in an ice bath for 2 min. Sugar derivatization with MPPP was carried out as shown in the following formula.
冷却後10 mLとり、90 mLのDMSOで10倍希釈し、蛍光検出器を装備したLCMSで測定した。LCMSの測定条件は下記であった。 After cooling, 10 mL was taken and diluted 10-fold with 90 mL of DMSO, and measured using an LCMS equipped with a fluorescence detector. The LCMS measurement conditions were as follows:
〈測定条件〉
カラム：ODS（CapcelPak MGII）3 μm 2.0 mm I. D. x 35 mm
流速：0.4 mL/ min
注入量：1 μL
カラム温度：40℃
検出波長：UV 254 nm
FL：Ex 340 nm / Em 390 nm又はEx 340 nm / Em 490 nm
移動相：A）0.1% HCOOH/H2O，移動相：B）0.1% HCOOH/CH3CN，
溶出条件：B 0% to 100%（0.01-10 min），100% hold（10-10.5 min），100% to 0%（10.5-10.51 min），controller（10.51-12 min）
<Measurement condition>
Column: ODS (CapcelPak MGII) 3 μm 2.0 mm ID x 35 mm
Flow rate: 0.4 mL/min
Injection volume: 1 μL
Column temperature: 40℃
Detection wavelength: UV 254 nm
FL: Ex 340 nm/Em 390 nm or Ex 340 nm/Em 490 nm
Mobile phase: A) 0.1% HCOOH/ H2O , Mobile phase: B) 0.1% HCOOH/ CH3CN ,
Elution conditions: B 0% to 100% (0.01-10 min), 100% hold (10-10.5 min), 100% to 0% (10.5-10.51 min), controller (10.51-12 min)
蛍光スペクトルは下記にて測定された。即ち、50 mMの化合物溶液を調製し、分光蛍光光度計F-7000（Hitachi）にて蛍光スペクトルを測定した。励起波長は340nmを用い、必要に応じ、得られたスペクトル全蛍光強度を積算し、規格化した。 Fluorescence spectra were measured as follows: A 50 mM compound solution was prepared, and the fluorescence spectrum was measured using a spectrofluorometer F-7000 (Hitachi). The excitation wavelength was 340 nm, and the total fluorescence intensity of the obtained spectrum was integrated and normalized as necessary.
定量NMRは下記にて測定された。即ち、HPLC解析に用いた溶液を回収し、シリカゲルカラムクロマトグラフィーにて精製した（クロロホルム：メタノール＝１：１）。得られた2.81mgを定量NMRにて定量した。 Quantitative NMR was measured as follows. That is, the solution used in HPLC analysis was collected and purified by silica gel column chromatography (chloroform:methanol = 1:1). The resulting 2.81 mg was quantified by quantitative NMR.
(3)実験結果
(3-1)HPLC解析
既存のPMPの反応条件（Honda, et al, Anal Chem, 1989, 180, 351-357）を参考に、MPPPとGalNAcの反応を検討した。反応終了後の溶液を、蛍光検出器を装備したLCMS（LC-Fl-MS）で直接解析した。図５はMPPPとGalNAcとの反応生成物の解析結果を示す図である。UVクロマトグラム上に２つのピーク（A，B）が確認される。MSの結果からそれぞれ、化合物3及び目的生成物（4）であることが確認できた。また3にはピレンのモノマー蛍光（ex340nm/em390nm）が、4にはエキサイマー蛍光（ex340nm/em490nm）が観察された。この結果は本研究のデザインコンセプトが十分機能していることを示している。
(3) Experimental results
(3-1) HPLC analysis The reaction of MPPP and GalNAc was examined based on the existing PMP reaction conditions (Honda, et al, Anal Chem, 1989, 180, 351-357). The solution after the reaction was directly analyzed by LCMS equipped with a fluorescence detector (LC-Fl-MS). Figure 5 shows the analysis results of the reaction product of MPPP and GalNAc. Two peaks (A, B) were confirmed on the UV chromatogram. The MS results confirmed that they were compound 3 and the target product (4), respectively. In addition, pyrene monomer fluorescence (ex340nm/em390nm) was observed in 3, and excimer fluorescence (ex340nm/em490nm) was observed in 4. This result indicates that the design concept of this study is fully functional.
続いて、Glc、Galactose（Gal），Gal-GalNAcに対する反応性を検討した。反応生成物をLC-Fl-MSにて解析した。図６はMPPPとGlc、Gal又はGal-GalNAcとの反応生成物の解析結果を示す図である。各糖もUVクロマトグラム上で２本のピークが検出され、MSクロマトグラムで同定した反応生成物のピークにエキサイマー蛍光が観察された（図６）。反応生成物の保持時間は、GalNAcが8.25min，Glcが8.3minとGalが8.2minであったことから、糖の構造異性体を識別可能であることが示された。また、二糖であるGal-GalNAcとの反応生成物は7.9min付近にピークが観察された。 Next, the reactivity with Glc, Galactose (Gal), and Gal-GalNAc was examined. The reaction products were analyzed by LC-Fl-MS. Figure 6 shows the analysis results of the reaction products of MPPP with Glc, Gal, or Gal-GalNAc. Two peaks were detected on the UV chromatogram for each sugar, and excimer fluorescence was observed at the peaks of the reaction products identified on the MS chromatogram (Figure 6). The retention times of the reaction products were 8.25 min for GalNAc, 8.3 min for Glc, and 8.2 min for Gal, indicating that structural isomers of sugars can be distinguished. In addition, a peak was observed around 7.9 min for the reaction product with Gal-GalNAc, which is a disaccharide.
さらに反応条件の検討を行った（図７）。GalNAcとの反応生成物の蛍光ピーク面積を用いてグラフ化した。NaOH濃度の検討は5, 10, 15, 20, 25 mMとし、5 mM GalNAc、25 mM MPPP（いずれも終濃度）、100 μL（50% DMSO）とした。MPPP濃度の検討は20, 25, 30, 35, 40, 45 mMとし、5 mM GalNAc、15 mM NaOH（いずれも終濃度）、100 μl（50% DMSO）とした。反応温度、反応時間については5 mM GalNAc、25mM MPPP、15mM NaOH、total 100 μL（50% DMSO）で行った。各条件で反応後，NaOH濃度は15mM付近が最大となった。反応時間は時間を追って生成物が増加した．MPPP濃度は過剰であれば良いことがわかった。反応の温度は70℃が最大となり，80℃は70℃と大きな違いはなかった。 The reaction conditions were further examined (Figure 7). The fluorescence peak area of the reaction product with GalNAc was used to make a graph. The NaOH concentrations examined were 5, 10, 15, 20, and 25 mM, with 5 mM GalNAc, 25 mM MPPP (final concentrations), and 100 μL (50% DMSO). The MPPP concentrations examined were 20, 25, 30, 35, 40, and 45 mM, with 5 mM GalNAc, 15 mM NaOH (final concentrations), and 100 μL (50% DMSO). The reaction temperature and reaction time were 5 mM GalNAc, 25 mM MPPP, 15 mM NaOH, and a total of 100 μL (50% DMSO). After the reaction under each condition, the NaOH concentration was maximum at around 15 mM. The reaction time increased with increasing product. It was found that an excessive MPPP concentration is sufficient. The reaction temperature reached its maximum at 70°C, with 80°C not significantly different from 70°C.
(3-2)蛍光スペクトルの環境依存性
MPPP単体の溶液状態での蛍光特性を検討するため、化合物のH2Oあるいはアセトニトリルにおける蛍光スペクトルを測定した。一般的にピレンのエキサイマー蛍光は溶媒の環境に強く依存する。アセトニトリルは水に比べて極性が低いため、ピレン同士の相互作用が起こらずエキサイマーの蛍光は観察されない。一方で、H2O中ではピレン同士の疎水性相互作用が起こりやすく、分子間でエキサイマー蛍光が観察されると考えた。
(3-2) Environmental Dependence of Fluorescence Spectrum
To investigate the fluorescence properties of MPPP alone in solution, we measured the fluorescence spectrum of the compound in H 2 O or acetonitrile. In general, pyrene excimer fluorescence is strongly dependent on the solvent environment. Since acetonitrile has a lower polarity than water, interactions between pyrenes do not occur and excimer fluorescence is not observed. On the other hand, in H 2 O, hydrophobic interactions between pyrenes are likely to occur, and we hypothesized that excimer fluorescence would be observed between molecules.
図８は、溶媒環境が異なる場合におけるMPPP単体の蛍光スペクトルを示す図である。測定の結果、MPPPのアセトニトリル中のスペクトルには380，400，420nmに極大蛍光が観察された（図８ 実線）。これはピレンのモノマー蛍光の特徴と一致する。H2O中のスペクトルは480 nmに極大蛍光が観察された（図８点線）。これはピレンのエキサイマー蛍光の特徴と一致する。スペクトルの形状が全く異なることから、アセトニトリル中では100%単体で存在し、一方でH2O中では分子間で相互作用しエキサイマー蛍光を発すると考えられる。 Figure 8 shows the fluorescence spectra of MPPP alone in different solvent environments. As a result of the measurements, maximum fluorescence was observed at 380, 400, and 420 nm in the spectrum of MPPP in acetonitrile (solid line in Figure 8). This coincides with the characteristics of pyrene monomer fluorescence. In the spectrum in H 2 O, maximum fluorescence was observed at 480 nm (dotted line in Figure 8). This coincides with the characteristics of pyrene excimer fluorescence. Since the spectral shapes are completely different, it is believed that MPPP exists as a 100% simple substance in acetonitrile, whereas in H 2 O it interacts with other molecules and emits excimer fluorescence.
(3-3)単離した反応生成物の環境依存的蛍光スペクトルの変化
LCのクロマトグラム（図５，図６）において糖との反応生成物にエキサイマー蛍光が観察されたことから、GalNAcとの反応生成物を単離し、蛍光スペクトルを測定した。溶媒にはアセトニトリルを用いた。測定の結果（図９）、MPPP単体には480nmの極大蛍光が観察されなかったのに対し、反応生成物には480nmの極大蛍光が観察された。アセトニトリル中では分子間エキサイマーは検出されない（図８）ことから、ここで検出された480nmの蛍光は分子内エキサイマー蛍光であることがわかった。また、Glucoseとの反応生成物も同様の結果を得た（図１０）。
(3-3) Changes in fluorescence spectra of isolated reaction products depending on the environment
Since excimer fluorescence was observed in the reaction product with sugar in the LC chromatograms (Figs. 5 and 6), the reaction product with GalNAc was isolated and its fluorescence spectrum was measured. Acetonitrile was used as the solvent. As a result of the measurement (Fig. 9), no maximum fluorescence at 480 nm was observed in MPPP alone, whereas a maximum fluorescence at 480 nm was observed in the reaction product. Since intermolecular excimers were not detected in acetonitrile (Fig. 8), it was found that the fluorescence at 480 nm detected here was intramolecular excimer fluorescence. The same result was obtained for the reaction product with glucose (Fig. 10).
(3-4)検量線
ガラクトースとの反応生成物をシリカゲルカラムクロマトグラフィーにて精製した。生成物を定量NMRにて定量し、溶液濃度2.89 mMに調整した。この溶液を下記の測定条件にてLCに供し、検量線を作成した（図１１）。
(3-4) Calibration curve The reaction product with galactose was purified by silica gel column chromatography. The product was quantified by quantitative NMR and the solution concentration was adjusted to 2.89 mM. This solution was subjected to LC under the following measurement conditions, and a calibration curve was created (Figure 11).
〈測定条件〉
カラム：AQUITY UPLC BEH C18, 130A, 1.7um, 2.1 mm x 50 mm
流速：0.3 mL/ min
カラム温度：60℃
検出波長：UV 254 nm
FL： Ex 340 nm / Em 490 nm
移動相：A）0.1% HCOOH/H2O，移動相：B）0.1% HCOOH/CH3CN，
溶出条件：A/B 40:60 (0 min) - 40/60 (1 min) - 10:90 (6 min)
<Measurement condition>
Column: AQUITY UPLC BEH C18, 130A, 1.7um, 2.1mm x 50mm
Flow rate: 0.3 mL/min
Column temperature: 60℃
Detection wavelength: UV 254 nm
FL: Ex 340 nm/Em 490 nm
Mobile phase: A) 0.1% HCOOH/ H2O , Mobile phase: B) 0.1% HCOOH/ CH3CN ,
Elution conditions: A/B 40:60 (0 min) - 40/60 (1 min) - 10:90 (6 min)
その結果、蛍光による検出限界は5.13 fmolであった。PMPのUVによる定量限界が1 pmol（Honda, et al, Anal Chem, 1989, 180, 351-357）であることから、MPPPはPMPに比べて194倍の感度が向上した。 As a result, the detection limit by fluorescence was 5.13 fmol. Since the quantification limit by UV for PMP is 1 pmol (Honda, et al, Anal Chem, 1989, 180, 351-357), the sensitivity of MPPP is 194 times higher than that of PMP.
(4)新規糖鎖誘導体化試薬MPPP-NMe2の合成
下記反応式にて、化合物（MPPP-NMe2）を固体として得た。
化合物1（43.0 mg，0.16 mmol（2HCl salt），5（74.0 mg，0.23 mmol），HBTU（208.6 mg，0.55 mmol），DIPEA（141.5 mg，1.1.1 mol）をDMF（5ml）に溶かし、室温で一晩撹拌した。溶媒を減圧濃縮し、酢酸エチルを加え、NaHCO3及び飽和食塩水で洗浄した。溶媒を減圧留去し、シリカゲルカラムクロマトグラフィー（CH2Cl2：MeOH＝94：6）によって精製し、化合物6（MPPP-NMe2）を黄色固体として得た（49.2 mg，63％）。
(4) Synthesis of a new glycan derivatization reagent, MPPP-NMe2 The compound (MPPP-NMe2) was obtained as a solid according to the following reaction scheme.
Compound 1 (43.0 mg, 0.16 mmol (2HCl salt), 5 (74.0 mg, 0.23 mmol), HBTU (208.6 mg, 0.55 mmol), and DIPEA (141.5 mg, 1.1.1 mol) were dissolved in DMF (5 mL) and stirred at room temperature overnight. The solvent was concentrated under reduced pressure, ethyl acetate was added, and the mixture was washed with NaHCO3 and saturated brine. The solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography ( CH2Cl2 :MeOH = 94:6) to give compound 6 (MPPP-NMe2) as a yellow solid (49.2 mg, 63%).
1H NMR (600 MHz, CDCl3)δ9.10 (s, 1H), 8.41 (d, J = 9.3 Hz, 1H), 8.15 - 8.17 (m, 2H), 8.12 (d, J = 9.3 Hz, 1H), 8.10 (d, J = 7.8 Hz, 1H), 8.04 (d, J = 7.8 Hz, 1H), 8.02 (s, 2H), 7.98 (t, J = 7.6 Hz, 1H), 7.80 (d, J = 8.9 Hz, 2H), 7.55 (d, J = 8.9 Hz, 2H), 4.17 (dd, J= 14.5 and 7.4 Hz, 1H), 3.87 (dd, J = 7.4 and 5.9 Hz, 1H), 3.71 (dd, J= 14.5 and 7.4 Hz, 1H), 3.41 (s, 2H), 2.04 (s, 6H), 2.19 (s, 3H) 1H NMR (600 MHz, CDCl3 ) δ 9.10 (s, 1H), 8.41 (d, J = 9.3 Hz, 1H), 8.15 - 8.17 (m, 2H), 8.12 (d, J = 9.3 Hz, 1H), 8.10 (d, J = 7.8 Hz, 1H), 8.04 (d, J = 7.8 Hz, 1H), 8.02 (s, 2H), 7.98 (t, J = 7.6 Hz, 1H), 7.80 (d, J = 8.9 Hz, 2H), 7.55 (d, J = 8.9 Hz, 2H), 4.17 (dd, J = 14.5 and 7.4 Hz, 1H), 3.87 (dd, J = 7.4 and 5.9 Hz, 1H), 3.71 (dd, J= 14.5 and 7.4 Hz, 1H), 3.41 (s, 2H), 2.04 (s, 6H), 2.19 (s, 3H)
(5)実験結果
MPPP-NMe2とGalNAcの反応を検討した。反応終了後の溶液を、蛍光検出器を装備したLCMS（LC-Fl-MS）で直接解析した。図１２はMPPP-NMe2とGalNAcとの反応生成物の解析結果を示す図である。UVクロマトグラム上に２つのピーク（C，D）が確認される。MSの結果からCはMPPP-NMe2、DはGalNAcとの反応生成物であることが確認できた。またCにはピレンのモノマー蛍光（ex340nm/em390nm）が、Dにはエキサイマー蛍光（ex340nm/em490nm）が観察された。
(5) Experimental results
The reaction of MPPP-NMe2 with GalNAc was investigated. The solution after the reaction was directly analyzed by LCMS equipped with a fluorescence detector (LC-Fl-MS). Figure 12 shows the analysis results of the reaction product of MPPP-NMe2 with GalNAc. Two peaks (C, D) are confirmed on the UV chromatogram. From the MS results, it was confirmed that C is the reaction product of MPPP-NMe2 and D is the reaction product with GalNAc. Furthermore, pyrene monomer fluorescence (ex340nm/em390nm) was observed in C, and excimer fluorescence (ex340nm/em490nm) was observed in D.
(6)新規糖鎖誘導体化試薬MPPP(noPh)の合成
化合物7（30.7 mg, 0.159 mmol（HCl salt）），8（47.5 mg, 0.177 mmol（HCl salt）），HBTU（242.9 mg, 0.640 mmol），DIPEA（168.5 mg, 1.30 mol）をDMF（5 ml）に溶かし、室温で100分間撹拌した。溶媒を減圧濃縮し、酢酸エチルを加え、0.2N HCl及び飽和食塩水で洗浄した。溶媒を減圧留去し、シリカゲルカラムクロマトグラフィー（CH2Cl2: MeOH）によって精製し、化合物9（MPPP(noPh)）を黄褐色固体として得た（21.3 mg, 36%）。
(6) Synthesis of a new glycan derivatization reagent, MPPP(noPh) Compound 7 (30.7 mg, 0.159 mmol (HCl salt)), 8 (47.5 mg, 0.177 mmol (HCl salt)), HBTU (242.9 mg, 0.640 mmol), and DIPEA (168.5 mg, 1.30 mol) were dissolved in DMF (5 ml) and stirred at room temperature for 100 minutes. The solvent was concentrated under reduced pressure, ethyl acetate was added, and the mixture was washed with 0.2N HCl and saturated saline. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography ( CH2Cl2 :MeOH) to give compound 9 (MPPP(noPh)) as a yellow-brown solid ( 21.3 mg, 36%).
1H NMR (600 MHz, CHLOROFORM-D) δ 8.24 - 8.18 (m, 3H), 8.18 - 8.12 (m, 2H), 8.06 (q, J = 8.9 Hz, 2H), 8.02 (t, J = 7.6 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 6.28 (s, 1H), 5.19 (d, J = 5.4 Hz, 2H), 4.38 (s, 2H), 3.13 (s, J = 0.8 Hz, 2H), 2.00 (s, J = 0.9 Hz, 3H).
13C NMR (151 MHz, CHLOROFORM-D) δ 172.87, 166.97, 156.97, 131.42, 131.33, 130.82, 130.51, 129.13, 128.44, 127.76, 127.44, 127.29, 126.27, 125.59, 125.53, 125.11, 124.91, 124.76, 122.81, 47.85, 42.23, 41.22, 17.09.
1H NMR (600 MHz, CHLOROFORM-D) δ 8.24 - 8.18 (m, 3H), 8.18 - 8.12 (m, 2H), 8.06 (q, J = 8.9 Hz, 2H), 8.02 (t, J = 7.6 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 6.28 (s, 1H), 5.19 (d, J = 5.4 Hz, 2H), 4.38 (s, 2H), 3.13 (s, J = 0.8 Hz, 2H), 2.00 (s, J = 0.9 Hz, 3H).
13C NMR (151 MHz, CHLOROFORM-D) δ 172.87, 166.97, 156.97, 131.42, 131.33, 130.82, 130.51, 129.13, 128.44, 127.76, 127.44, 127.29, 126.27, 125.59, 125.53, 125.11, 124.91, 124.76, 122.81, 47.85, 42.23, 41.22, 17.09.
(７)実験結果
MPPP(noPh)とGalNAcの反応を検討した。反応終了後の溶液を、蛍光検出器を装備したLCMS（LC-Fl-MS）で直接解析した。図１３はMPPP(noPh)とGalNAcとの反応生成物の解析結果を示す図である。UVクロマトグラム上に２つのピーク（E，F）が確認される。MSの結果からEはMPPP(noPh)、FはGalNAcとの反応生成物であることが確認できた。またEにはピレンのモノマー蛍光（ex340nm/em390nm）が、Fにはエキサイマー蛍光（ex340nm/em490nm）が観察された。
(7) Experimental results
The reaction of MPPP(noPh) with GalNAc was investigated. The solution after the reaction was directly analyzed by LCMS (LC-Fl-MS) equipped with a fluorescence detector. Figure 13 shows the analysis results of the reaction product of MPPP(noPh) with GalNAc. Two peaks (E, F) are confirmed on the UV chromatogram. From the MS results, it was confirmed that E is MPPP(noPh) and F is the reaction product with GalNAc. Furthermore, pyrene monomer fluorescence (ex340nm/em390nm) was observed in E, and excimer fluorescence (ex340nm/em490nm) was observed in F.
O結合型糖鎖の検出試薬等に利用できる。 It can be used as a detection reagent for O-linked glycans, etc.
Claims (16)
Ｘ１及びＸ２は、各々独立に、置換基（この置換基は、炭素数１から４のアルキル基、アルコキシ基、アルコキシアルキル基、エステル基、エステルアルキル基、又は、アミノ基、水酸基、チオール基である）若しくはヘテロ原子を有してもよい分岐可能な、Ｃ０～３アルキル基又はＣ０～３アルケニル基であり、
Ｚは、－(CH2)ｎ－（ここでnは１～１５の整数を表す）、又は、下記構造式(Ｙは、Ｃ又はＮである。)であり、
Ｌ２は、化学結合、又は、置換基（この置換基は、水酸基、カルボキシル基、アミノ基、アミド基、カルバメート基、又はケトン基である）若しくはヘテロ原子を有しても良い分岐可能な、Ｃ１～８アルキル基若しくはＣ２～８アルケニル基であり、
L３は、化学結合、O、－OCH２－、－CH2O－、－(CH2OCH２)ｎ－、－NH－、－NHCO－、－CONH－、若しくは、－NHCONH－（ここで、nは１～１５の整数を表す）であり、
Ａは、ベンゼン、ペンタレン、インデン、ナフタレン、アズレン、ヘプタレン、インダセン、アセナフタレン、フルオレン、ベンゾフルオレン、ジベンゾフルオレン、フェナレン、フェナントレン基、アントラセン、フルオランテン、ピレン、クリセン、ナフタセン、ピセン、ペリレン、ピロール、チオフェン、フラン、シロール、イミダゾール、ピラゾール、チアゾール、イソチアゾール、オキサゾール、イソオキサゾール、ピリジン、ピラジン、ピリミジン、ピリダジン、トリアジン、ベンゾフラン、ベンゾチオフェン、ジベンゾフラン、ジベンゾチオフェン、カルバゾール、ベンゾシロール、ジベンゾシロール、キノリン、イソキノリン、ベンゾイミダゾール、イミダゾピリジン又はイミダゾピリミジンである)。 A new compound consisting of the following chemical formula (
X1 and X2 each independently represent a branched C0-3 alkyl group or C0-3 alkenyl group which may have a substituent (the substituent is an alkyl group, an alkoxy group, an alkoxyalkyl group, an ester group, an esteralkyl group, or an amino group, a hydroxyl group, or a thiol group, each having 1 to 4 carbon atoms) or a heteroatom;
Z is -( CH2 ) n- (wherein n is an integer from 1 to 15) or the following structural formula (Y is C or N):
L2 is a chemical bond, or a branched C1-8 alkyl group or C2-8 alkenyl group which may have a substituent (the substituent is a hydroxyl group, a carboxyl group, an amino group, an amide group, a carbamate group, or a ketone group) or a heteroatom;
L3 is a chemical bond, O , -OCH2- , -CH2O- , -( CH2OCH2 ) n- , -NH-, -NHCO-, -CONH-, or -NHCONH- (wherein n is an integer of 1 to 15);
A is benzene, pentalene, indene, naphthalene, azulene, heptalene, indacene, acenaphthalene, fluorene, benzofluorene, dibenzofluorene, phenalene, phenanthrene group, anthracene, fluoranthene, pyrene, chrysene, naphthacene, picene, perylene, pyrrole, thiophene, furan, silole, imidazole, pyrazole, thiazole, isothiazole, oxazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, triazine, benzofuran, benzothiophene, dibenzofuran, dibenzothiophene, carbazole, benzosilole, dibenzosilole, quinoline, isoquinoline, benzimidazole, imidazopyridine or imidazopyrimidine).
式中、R1、R2、R3、R4、R5は、それぞれ独立に、置換基を有していても良い芳香族炭化水素基、置換基を有していても良いアルケニル基、置換基を有していても良いアルキニル基、置換基を有していても良いアルキル基、置換基を有していても良いシリル基、置換基を有していても良い複素環基、置換基を有していても良いアリールアミノ基、置換基を有していても良いアルコキシル基、置換基を有していてもよいアリールボリル基を表す)。
In the formula, R1, R2, R3, R4, and R5 each independently represent an aromatic hydrocarbon group which may have a substituent, an alkenyl group which may have a substituent, an alkynyl group which may have a substituent, an alkyl group which may have a substituent, a silyl group which may have a substituent, a heterocyclic group which may have a substituent, an arylamino group which may have a substituent, an alkoxyl group which may have a substituent, or an arylboryl group which may have a substituent.
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