【0001】
以下で、DMFはジメチルホルムアミドの略号である。
【発明の属する技術分野】
本発明は、蛍光を有する糖鎖プローブに関するものである。
【0002】
【従来の技術】
生体内で重要な役割を果たしている糖鎖の機能を探索するために、糖鎖部分の構造が明確な糖鎖プローブを開発することが必要である。また、糖鎖自体には検出に有用な官能基が無いために、微量のサンプルを取り扱うには、例えば蛍光標識の様に、高感度で簡便な検出を可能にする官能基を持った糖鎖プローブが望まれる。
例えば、代表的な糖鎖の蛍光標識法として、2−アミノピリジンを還元アミノ化によって糖鎖の還元末端に結合させるピリジルアミノ化法は、糖鎖の構造解析や相互作用解析に多用されてきた(例えば、非特許文献1参照)。しかし、ピリジルアミノ化法には、還元末端の糖の環状構造を壊す、合成糖鎖の取り扱いには適していない等の問題が指摘されており、新たな蛍光糖鎖プローブの開発が望まれていた。
蛍光を有する化合物として、ポルフィリンやクロロフィルを挙げることができる。しかし、糖鎖を結合したポルフィリン誘導体の合成例は多いものの、癌治療剤や抗生物質(例えば、非特許文献2、3参照)、ゲル化剤(例えば、非特許文献4参照)等を目的としたものであり、糖鎖プローブとしての分子設計がなされていなかった。また、糖鎖を結合したクロロフィル誘導体の合成例は知られていなかった。
【0003】
【非特許文献1】
蛋白質核酸酵素、1992年、37巻、p.2054−2059
【非特許文献2】
テトラヘドロン・レターズ(Tetrahedron Letters)、2002年、43巻、p.603−605
【非特許文献3】
ケミカル・コミュニケーションズ(Chemical Communications)、2001年、p.81−82
【非特許文献4】
アンゲバンテ・ヘミー・インターナショナル・エディション(Angewandte Chemie Int. Ed.)、2002年、41巻、p.853−856
【0004】
【発明が解決しようとする課題】
本発明の課題は、糖鎖の還元末端が環状構造を持ち、合成糖鎖の取り扱いにも適した糖鎖プローブである、クロロフィル誘導体を提供することにある。
【0005】
【課題を解決するための手段】
上記課題を鋭意検討した結果、本発明者らは、糖鎖とクロロフィル誘導体とをアミノアルコールを介して結合させることにより、式(1)または式(2)で示されるクロロフィル誘導体を合成し、上記課題を解決できることを見出し、本発明を完成するに至った。
【化3】
(式中、R1は糖鎖を、R2はCH=CH2またはCHOまたはCH2OHを、nは2から6の整数を表す。)
【化4】
(式中、R1は糖鎖を、R2はCH=CH2またはCHOまたはCH2OHを、Mは金属原子または配位子をともなった金属原子を、nは2から6の整数を表す。)
即ち、本発明は、式(1)および式(2)で示されるクロロフィル誘導体を提供する。
【0006】
糖鎖プローブとしての分子設計として、任意の糖鎖を容易に結合できること、糖鎖とクロロフィル部分とを結ぶ構造に充分な柔軟性があり、糖鎖を認識する蛋白質等との相互作用にクロロフィル部分が影響を与えないこと等が必要である。任意の糖鎖を結合する手段として、一旦糖鎖にアルコール誘導体を結合させた後、官能基変換でクロロフィル部分と結合する合成法を用いることにより、合成由来の糖鎖にも天然由来の糖鎖にも対応でき、還元末端の環状構造を保ったままの糖鎖プローブを実現した。
【0007】
本発明のクロロフィル誘導体を糖鎖プローブとして用いる方法として、化合物をそのまま、あるいは、リポソームに組み込んでから、糖鎖を認識する蛋白質等との結合を測定する方法、バイオセンサー表面や電極表面に固定して相互作用を検出する手法等が挙げられる。原料のクロロフィル誘導体は、分子会合体を形成することが知られているので、糖鎖プローブとして用いる際に、いわゆるクラスター効果によって相互作用を増強することも期待される。
【0008】
【発明の実施の形態】
本発明の化合物の合成は、いかなる方法によっても構わない。
例えば、合成された糖鎖あるいは天然由来の糖鎖の、還元末端のヘミアセタール水酸基を含む全ての水酸基をアセチル基で保護し、三フッ化ホウ素・エーテル錯体を用いてブロモアルコールにグリコシド結合させ、引き続いて、ブロモ基をアジド基に変換し、糖鎖の水酸基のアセチル基を脱保護した後、アジド基を還元することによって、アミノアルコールが結合した糖鎖化合物へと誘導する。これに、別途調製した遊離のカルボキシル基を持ったクロロフィル誘導体(フォトケミストリー・アンド・フォトバイオロジー(Photochem. Photobio.)、1999年、69巻、p.448−456)を、カルボジイミド誘導体等の試薬を用いて、アミド縮合させることによって、目的とする式(1)で示される化合物が得られる。
式(1)で示される化合物のR1の糖鎖としては、ガラクトース、グルコース、マンノース、フコース、キシロース、N−アセチルガラクトサミン、N−アセチルグルコサミン等の単糖、ラクトース、セロビオース、キトビオース等天然型または合成で得られる二糖、オリゴ糖、多糖、糖蛋白質または糖脂質からヒドラジン分解やアルカリ分解あるいはエンド型酵素分解で得られる糖鎖等が挙げられる。還元末端のグリコシド結合の立体は、αでもβでも構わない。
さらに、式(1)で示される化合物に、金属イオンを配位させることにより、式(2)で示される化合物が得られる。金属原子としては、Mg、Zn、Cd、Co、Ni、Cu、Pd、Ag等が、配位子をともなう金属原子としては、MnOAc、FeCl等が挙げられる。好ましくは、金属原子として、Mg、Zn、Cdが挙げられる。
以下に、本発明をさらに詳細に説明するが、本発明は以下の記述に限定されるものではない。
【0009】
【実施例1】
(1−(2−アミノエチル)−β−D−ガラクトピラノシドの合成)
β−D−ガラクトースペンタアセタート(5.0 g)、三フッ化ホウ素・エーテル錯体(2.6 ml)、2−ブロモエタノール(2.8 ml)、モレキュラーシーブ4A(4.0 g)を、乾燥ジクロロメタン(30 ml)に加え、アルゴンガス気流下、室温で一晩撹拌した。不溶物をろ別し、ろ液を炭酸水素ナトリウム溶液および塩化ナトリウム溶液で洗浄後、硫酸マグネシウムで乾燥した。乾燥剤をろ別し、溶媒を留去した後、フラッシュカラムクロマトグラフィー(25%酢酸エチル/ヘキサン)で精製し、1−(2−ブロモエチル)−2,3,4,6−テトラ−O−アセチル−β−D−ガラクトピラノシド(4.6 g、79%)を得た。
【0010】
1−(2−ブロモエチル)−2,3,4,6−テトラ−O−アセチル−β−D−ガラクトピラノシド(1.0 g)のDMF(15 ml)溶液にアジ化ナトリウム(1.0 g)を加え、窒素気流下、70℃で2時間撹拌した後、酢酸エチルを加え、水で洗浄し、有機層を分離し、硫酸マグネシウムで乾燥した。乾燥剤をろ別し、溶媒を留去した後、フラッシュカラムクロマトグラフィー(50%酢酸エチル/ヘキサン)で精製し、1−(2−アジドエチル)−2,3,4,6−テトラ−O−アセチル−β−D−ガラクトピラノシド(0.76 g、81%)を得た。
【0011】
1−(2−アジドエチル)−2,3,4,6−テトラ−O−アセチル−β−D−ガラクトピラノシド(0.76 g)と28%ナトリウムメトキシドのメタノール溶液(1.0 ml)をメタノール(100 ml)に加え、室温で1時間撹拌した。これに、Amberlite IR−120 (plus)を加えて中和し、不溶物をろ別し、有機溶媒を留去した後、フラッシュカラムクロマトグラフィー(10%メタノール/クロロホルム)で精製し、1−(2−アジドエチル)−β−D−ガラクトピラノシド(0.41 g、89%)を得た。
1−(2−アジドエチル)−β−D−ガラクトピラノシド(0.41 g)と酸化白金(100 mg)をメタノール(150 ml)に加え、室温で2時間撹拌しながら水素添加を行った。不溶物をろ別し、有機溶媒を留去して、1−(2−アミノエチル)−β−D−ガラクトピラノシド(0.31 g、90%)を得た。
Mp 103−104℃. 1H−NMR (D2O) δ 2.87 (2H, t, J = 5 Hz), 3.47 (1H, dd, J = 8, 10 Hz), 3.59 (1H, dd, J = 3, 10 Hz), 3.64 (1H, t, J = 7 Hz), 3.66 (1H, dd, J = 7, 11 Hz), 3.70 (1H, dt, J = 11, 5 Hz), 3.73 (1H, dd, J = 7, 11 Hz), 3.86 (1H, d, J = 3Hz), 3.92 (1H, dt, J = 11, 5 Hz), 4.35 (1H, d, J = 8 Hz). IR (neat) 3396, 1577 cm−1. FAB−MS m/z 224 (MH+).
【0012】
【実施例2】
(式(4)で示されるクロロフィル誘導体の合成)
既に報告した方法(フォトケミストリー・アンド・フォトバイオロジー(Photochem. Photobio.)、1999年、69巻、p.448−456)によって合成した式(3)で示される化合物(26 mg)、1−(2−アミノエチル)−β−D−ガラクトピラノシド(27 mg)、1−ヒドロキシベンゾトリアゾール(20 mg)、水溶性カルボジイミド(31 mg)をDMF(2 ml)に加え、窒素気流下、室温で18時間撹拌した。クロロホルムを加え、水で洗浄し、有機層を分離し、有機溶媒を留去した後、フラッシュカラムクロマトグラフィー(10%メタノール/クロロホルム)で精製し、式(4)で示される化合物(20 mg、53%)を得た。
1H−NMR (1%CF3CO2D / CD3OD) δ 1.64 (3H, t, J = 7 Hz), 1.95 (3H, d, J = 7 Hz), 2.25−2.45 (2H, m), 2.61−2.90 (2H, m), 3.32−3.35 (1H, m), 3.39 (3H, s), 3.40−3.43 (1H, m), 3.43 (1H, dd, J = 3, 10 Hz), 3.46 (1H, t, J = 7 Hz), 3.49 (1H, dd, J = 3, 10 Hz), 3.57 (3H, s), 3.58−3.62 (1H, m), 3.62 (1H, dd, J = 7, 10 Hz), 3.67 (1H, dd, J = 7, 10 Hz), 3.75 (3H, s), 3.77 (1H, d, J = 3 Hz), 3.85 (1H, m), 3.93 (2H, q, J = 7 Hz), 4.20 (1H, d, J = 7 Hz), 4.53 (1H, d, J = 8 Hz), 4.82 (1H, q, J = 7 Hz), 5.26 (1H, d, J = 20 Hz), 5.50 (1H, d, J = 20 Hz), 6.36 (1H, d, J = 11 Hz), 6.41 (1H, d, J = 18 Hz), 8.23 (1H, dd, J = 11, 18 Hz), 9.36 (1H, s), 9.89 (1H, s), 10.26 (1H, s). IR (KBr) 3332, 1697 cm−1. TOF−MS m/z 758 [M+Na]+. Vis (MeOH) λmax = 666 nm (relative intensity: 46), 609 (12), 539 (13), 507 (14), 409 (100).
【化5】
【化6】
【0013】
【実施例3】
(式(6)で示されるクロロフィル誘導体の合成)
実施例2と同様に、既に報告した方法(フォトケミストリー・アンド・フォトバイオロジー(Photochem. Photobio.)、1999年、69巻、p.448−456)によって合成した式(5)で示される化合物から式(6)で示される化合物(21 mg、56%)を得た。
1H−NMR (1%CF3CO2D / CD3OD) δ 1.68 (3H, t, J = 7 Hz), 1.94 (3H, d, J = 7 Hz), 2.20−2.50 (2H, m), 2.61−2.90 (2H, m), 3.38−3.43 (1H, m), 3.45 (3H, m), 3.46−3.48 (1H, m), 3.49 (1H, dd, J = 3, 10 Hz), 3.53 (1H, t, J = 7 Hz), 3.55 (1H, dd, J = 3, 10 Hz), 3.63 (3H, s), 3.64−3.67 (1H, m), 3.68 (1H, dd, J = 7, 10 Hz), 3.74 (1H, dd, J = 7, 10 Hz), 3.78 (3H, s), 3.84 (1H, d, J = 3 Hz), 3.91 (1H, m), 4.02 (2H, q, J = 7 Hz), 4.20 (1H, d, J = 7 Hz), 4.54 (1H, d, J = 8 Hz), 4.87 (1H, q, J = 7 Hz), 5.30 (1H, d, J = 20 Hz), 5.60 (1H, d, J = 20 Hz), 9.40 (1H, s), 10.41 (1H, s), 10.47 (1H, s). IR (KBr) 3354, 1670 cm−1. TOF−MS m/z 759 [M+Na]+. Vis (CH2Cl2) λmax = 693 nm (relative intensity: 78), 632 (11), 554 (17), 522 (18), 430 (100).
【化7】
【化8】
【0014】
【実施例4】
(式(7)で示されるクロロフィル誘導体の合成)
式(6)で示される化合物(15 mg)を少量の溶媒(10%メタノール/クロロホルム)に溶解し、ボラン−t−ブチルアミン錯体(8.7 mg)を加えて、0℃で4時間撹拌した。クロロホルムを加え、2%塩酸水溶液で反応を停止し、4%炭酸水素ナトリウム水溶液と水で洗浄し、有機層を分離して、有機溶媒を留去した後、フラッシュカラムクロマトグラフィー(15%メタノール/クロロホルム)で精製し、式(7)で示される化合物(11.5 mg、77%)を得た。
1H−NMR (DMSO−d6) δ 1.65 (3H, t, J = 7 Hz), 1.78 (3H, d, J = 7 Hz), 2.10−2.25 (2H, m), 2.30−2.70 (2H, m), 3.00−3.30 (8H, m), 3.38 (3H, s), 3.40−3.50 (3H, m), 3.62 (3H, s), 3.55−3.70 (2H, m), 3.72 (2H, q, J = 7 Hz), 4.02 (1H, d, J = 7 Hz), 4.31 (1H, d, J = 8 Hz), 4.56 (1H, q, J = 7 Hz), 5.10 (1H, d, J = 20 Hz), 5.23 (1H, d, J = 20 Hz), 5.74 (2H, s), 8.80 (1H, s), 9.56 (1H, s), 9.71 (1H, s). IR (neat) 3333, 1622 cm−1. TOF−MS m/z 761 [M+Na]+. Vis (CH2Cl2) λmax = 663 nm (relative intensity: 53), 607 (11), 536 (12), 505 (14), 398 (100).
【化9】
【0015】
【実施例5】
(式(8)で示されるクロロフィル誘導体の合成)
式(7)で示される化合物(11.5 mg)のクロロホルム溶液(5 ml)に、亜鉛二水和物を飽和したメタノール(0.2 ml)を加えて、室温で2時間撹拌し、さらに、4%炭酸水素ナトリウム水溶液(20 ml)を加え、室温で20分撹拌した。水層をクロロホルムで抽出し、有機層と合わせて、水で洗浄し、硫酸ナトリウムで乾燥した。脱水剤をろ別して、有機溶媒を留去して、式(8)で示される化合物(11.2 mg、91%)を得た。
1H−NMR (CD3OD) δ 1.69 (3H, t, J = 7 Hz), 1.82 (3H, d, J = 7 Hz), 2.08−2.15 (2H, m), 2.32−2.65 (2H, m), 3.12−3.20 (1H, m), 3.22 (3H, s), 3.25−3.40 (4H, m), 3.41 (3H, s), 3.43−3.45 (1H, m), 3.50 (1H, dd, J = 7, 10 Hz), 3.56 (1H, dd, J = 7, 10 Hz), 3.57 (3H, s), 3.61 (1H, d, J = 3 Hz), 3.69 (1H, m), 3.72 (2H, q, J = 7 Hz), 3.78 (1H, d, J = 7 Hz), 4.24 (1H, d, J = 8 Hz), 4.50 (1H, q, J = 7 Hz), 5.02 (1H, d, J = 20 Hz), 5.23 (1H, d, J = 20 Hz), 5.70 (2H, s), 8.42 (1H, s), 9.26 (1H, s), 9.47 (1H, s). IR (KBr) 3332, 1654 cm−1. TOF−MS m/z 824 [M+Na]+. Vis (CH2Cl2) λmax = 655 nm (relative intensity: 68), 608 (26), 425 (100).
【化10】
【0016】
【発明の効果】
本発明は、糖鎖の還元末端が環状構造を持ち、合成糖鎖の取り扱いにも適した糖鎖プローブである、クロロフィル誘導体を提供することにある。[0001]
In the following, DMF is an abbreviation for dimethylformamide.
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a sugar chain probe having fluorescence.
[0002]
[Prior art]
In order to search for the function of a sugar chain that plays an important role in a living body, it is necessary to develop a sugar chain probe having a clear sugar chain structure. In addition, since the sugar chain itself does not have a useful functional group for detection, it is necessary to handle a very small amount of sample, for example, a sugar chain having a functional group that enables high sensitivity and easy detection, such as a fluorescent label. Probes are desired.
For example, as a typical fluorescent labeling method for sugar chains, a pyridyl amination method in which 2-aminopyridine is bonded to the reducing end of a sugar chain by reductive amination has been widely used for structural analysis and interaction analysis of sugar chains ( For example, see Non-Patent Document 1). However, it has been pointed out that the pyridyl amination method breaks the cyclic structure of the sugar at the reducing end and is not suitable for handling synthetic sugar chains, and the development of a new fluorescent sugar chain probe has been desired. .
Examples of the compound having fluorescence include porphyrin and chlorophyll. However, although there are many examples of synthesizing a porphyrin derivative having a sugar chain attached, it is intended for use as a cancer therapeutic agent, an antibiotic (for example, see Non-Patent Documents 2 and 3), a gelling agent (for example, see Non-Patent Document 4), and the like. The molecular design as a sugar chain probe was not made. In addition, a synthesis example of a chlorophyll derivative having a sugar chain bonded thereto has not been known.
[0003]
[Non-patent document 1]
Protein Nucleic Acid Enzyme, 1992, 37, p. 2054-2059
[Non-patent document 2]
Tetrahedron Letters, 2002, 43, p. 603-605
[Non-Patent Document 3]
Chemical Communications, 2001, p. 81-82
[Non-patent document 4]
Angewandte Chemie Int. Ed., 2002, Vol. 41, p. 853-856
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide a chlorophyll derivative, which is a sugar chain probe that has a cyclic structure at the reducing end of the sugar chain and is suitable for handling synthetic sugar chains.
[0005]
[Means for Solving the Problems]
As a result of intensive studies on the above problems, the present inventors synthesized a chlorophyll derivative represented by the formula (1) or (2) by binding a sugar chain to a chlorophyll derivative via an amino alcohol, The inventors have found that the problem can be solved, and have completed the present invention.
Embedded image
(In the formula, R 1 represents a sugar chain, R 2 represents CHCHCH 2 or CHO or CH 2 OH, and n represents an integer of 2 to 6.)
Embedded image
(Wherein, R 1 represents a sugar chain, R 2 represents CH = CH 2 or CHO or CH 2 OH, M represents a metal atom or a metal atom with a ligand, and n represents an integer of 2 to 6. .)
That is, the present invention provides chlorophyll derivatives represented by the formulas (1) and (2).
[0006]
The molecular design of the sugar chain probe is such that any sugar chain can be easily bonded, the structure connecting the sugar chain and the chlorophyll portion has sufficient flexibility, and the interaction between the sugar chain-recognizing protein and the chlorophyll portion Need to have no effect. As a means for bonding an arbitrary sugar chain, a synthetic method in which an alcohol derivative is once bonded to a sugar chain and then bonded to a chlorophyll moiety by conversion of a functional group is used, so that a sugar chain derived from a synthetic source and a sugar chain derived from a natural source are used. Thus, a sugar chain probe with the cyclic structure at the reducing end maintained.
[0007]
As a method of using the chlorophyll derivative of the present invention as a sugar chain probe, a method for measuring the binding to a protein or the like recognizing a sugar chain after immobilizing the compound as it is or after incorporating it into a liposome, or immobilizing the compound on the surface of a biosensor or an electrode. For detecting the interaction. It is known that the raw material chlorophyll derivative forms a molecular aggregate, and therefore, when used as a sugar chain probe, it is expected that the interaction is enhanced by the so-called cluster effect.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
The synthesis of the compounds of the present invention may be by any method.
For example, in a synthesized sugar chain or a naturally-derived sugar chain, all hydroxyl groups including a hemiacetal hydroxyl group at a reducing end are protected with an acetyl group, and a glycoside bond is formed with bromo alcohol using a boron trifluoride ether complex, Subsequently, the bromo group is converted into an azide group, the acetyl group of the hydroxyl group of the sugar chain is deprotected, and then the azide group is reduced, thereby leading to a sugar chain compound to which an amino alcohol is bound. Then, a separately prepared chlorophyll derivative having a free carboxyl group (Photochemistry and Photobiology (Photochem. Photobio.), 1999, Vol. 69, p. 448-456) was added to a reagent such as a carbodiimide derivative. By subjecting the compound to amide condensation, the desired compound represented by the formula (1) is obtained.
Examples of the sugar chain of R 1 in the compound represented by the formula (1) include monosaccharides such as galactose, glucose, mannose, fucose, xylose, N-acetylgalactosamine and N-acetylglucosamine, lactose, cellobiose, and chitobiose. Examples include sugar chains obtained by hydrazinolysis, alkali degradation, or endo-enzymatic degradation from disaccharides, oligosaccharides, polysaccharides, glycoproteins, or glycolipids obtained by synthesis. The configuration of the glycoside bond at the reducing end may be either α or β.
Further, by coordinating a metal ion to the compound represented by the formula (1), a compound represented by the formula (2) is obtained. Examples of the metal atom include Mg, Zn, Cd, Co, Ni, Cu, Pd, and Ag. Examples of the metal atom having a ligand include MnOAc and FeCl. Preferably, examples of the metal atom include Mg, Zn, and Cd.
Hereinafter, the present invention will be described in more detail, but the present invention is not limited to the following description.
[0009]
Embodiment 1
(Synthesis of 1- (2-aminoethyl) -β-D-galactopyranoside)
β-D-galactose pentaacetate (5.0 g), boron trifluoride / ether complex (2.6 ml), 2-bromoethanol (2.8 ml), and molecular sieve 4A (4.0 g) , Dry dichloromethane (30 ml), and the mixture was stirred at room temperature overnight under an argon gas stream. The insolubles were filtered off, and the filtrate was washed with a sodium hydrogen carbonate solution and a sodium chloride solution, and then dried over magnesium sulfate. After the desiccant was filtered off and the solvent was distilled off, the residue was purified by flash column chromatography (25% ethyl acetate / hexane) to give 1- (2-bromoethyl) -2,3,4,6-tetra-O-. Acetyl-β-D-galactopyranoside (4.6 g, 79%) was obtained.
[0010]
To a solution of 1- (2-bromoethyl) -2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (1.0 g) in DMF (15 ml) was added sodium azide (1. After stirring at 70 ° C. for 2 hours under a nitrogen stream, ethyl acetate was added, the mixture was washed with water, the organic layer was separated, and dried over magnesium sulfate. After the desiccant was filtered off and the solvent was distilled off, the residue was purified by flash column chromatography (50% ethyl acetate / hexane) to give 1- (2-azidoethyl) -2,3,4,6-tetra-O-. Acetyl-β-D-galactopyranoside (0.76 g, 81%) was obtained.
[0011]
1- (2-azidoethyl) -2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (0.76 g) and 28% sodium methoxide in methanol (1.0 ml) ) Was added to methanol (100 ml) and stirred at room temperature for 1 hour. To this, Amberlite IR-120 (plus) was added for neutralization, insolubles were filtered off, the organic solvent was distilled off, and the residue was purified by flash column chromatography (10% methanol / chloroform). 2-Azidoethyl) -β-D-galactopyranoside (0.41 g, 89%) was obtained.
1- (2-Azidoethyl) -β-D-galactopyranoside (0.41 g) and platinum oxide (100 mg) were added to methanol (150 ml), and hydrogenation was performed while stirring at room temperature for 2 hours. . The insolubles were removed by filtration, and the organic solvent was distilled off to obtain 1- (2-aminoethyl) -β-D-galactopyranoside (0.31 g, 90%).
Mp 103-104 ° C. 1 H-NMR (D 2 O) δ 2.87 (2H, t, J = 5 Hz), 3.47 (1H, dd, J = 8, 10 Hz), 3.59 (1H, dd, J = 3.10 Hz), 3.64 (1H, t, J = 7 Hz), 3.66 (1H, dd, J = 7, 11 Hz), 3.70 (1H, dt, J = 11.5 Hz) ), 3.73 (1H, dd, J = 7, 11 Hz), 3.86 (1H, d, J = 3 Hz), 3.92 (1H, dt, J = 11, 5 Hz), 4.35. (1H, d, J = 8 Hz). IR (neat) 3396, 1577 cm -1 . FAB-MS m / z 224 (MH <+> ).
[0012]
Embodiment 2
(Synthesis of chlorophyll derivative represented by formula (4))
The compound represented by the formula (3) (26 mg) synthesized by the method already reported (Photochemistry and Photobiology (Photochem. Photobio.), 1999, Vol. 69, p. 448-456), 1- (2-Aminoethyl) -β-D-galactopyranoside (27 mg), 1-hydroxybenzotriazole (20 mg) and water-soluble carbodiimide (31 mg) were added to DMF (2 ml), and the mixture was added under a nitrogen stream. Stir at room temperature for 18 hours. Chloroform was added thereto, washed with water, the organic layer was separated, the organic solvent was distilled off, and the residue was purified by flash column chromatography (10% methanol / chloroform) to obtain a compound represented by the formula (4) (20 mg, 53%).
1 H-NMR (1% CF 3 CO 2 D / CD 3 OD) δ 1.64 (3H, t, J = 7 Hz), 1.95 (3H, d, J = 7 Hz), 2.25- 2.45 (2H, m), 2.61-2.90 (2H, m), 3.32-3.35 (1H, m), 3.39 (3H, s), 3.40-3. 43 (1H, m), 3.43 (1H, dd, J = 3, 10 Hz), 3.46 (1H, t, J = 7 Hz), 3.49 (1H, dd, J = 3, 10) Hz), 3.57 (3H, s), 3.58-3.62 (1H, m), 3.62 (1H, dd, J = 7, 10 Hz), 3.67 (1H, dd, J) = 7, 10 Hz), 3.75 (3H, s), 3.77 (1H, d, J = 3 Hz), 3.85 (1H, m), .93 (2H, q, J = 7 Hz), 4.20 (1H, d, J = 7 Hz), 4.53 (1H, d, J = 8 Hz), 4.82 (1H, q, J) = 7 Hz), 5.26 (1H, d, J = 20 Hz), 5.50 (1H, d, J = 20 Hz), 6.36 (1H, d, J = 11 Hz), 6.41. (1H, d, J = 18 Hz), 8.23 (1H, dd, J = 11, 18 Hz), 9.36 (1H, s), 9.89 (1H, s), 10.26 (1H) , S). IR (KBr) 3332, 1697 cm -1 . TOF-MS m / z 758 [M + Na] + . Vis (MeOH) λ max = 666 nm (relative intensity: 46), 609 (12), 539 (13), 507 (14), 409 (100).
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[0013]
Embodiment 3
(Synthesis of chlorophyll derivative represented by formula (6))
Similarly to Example 2, a compound represented by the formula (5) synthesized by a method already reported (Photochemistry and Photobiology (Photochem. Photobio.), 1999, Vol. 69, p. 448-456). From the above, a compound represented by the formula (6) (21 mg, 56%) was obtained.
1 H-NMR (1% CF 3 CO 2 D / CD 3 OD) δ 1.68 (3H, t, J = 7 Hz), 1.94 (3H, d, J = 7 Hz), 2.20− 2.50 (2H, m), 2.61-2.90 (2H, m), 3.38-3.43 (1H, m), 3.45 (3H, m), 3.46-3. 48 (1H, m), 3.49 (1H, dd, J = 3, 10 Hz), 3.53 (1H, t, J = 7 Hz), 3.55 (1H, dd, J = 3, 10) Hz), 3.63 (3H, s), 3.64-3.67 (1H, m), 3.68 (1H, dd, J = 7, 10 Hz), 3.74 (1H, dd, J) = 7, 10 Hz), 3.78 (3H, s), 3.84 (1H, d, J = 3 Hz), 3.91 (1H, m), .02 (2H, q, J = 7 Hz), 4.20 (1H, d, J = 7 Hz), 4.54 (1H, d, J = 8 Hz), 4.87 (1H, q, J) = 7 Hz), 5.30 (1H, d, J = 20 Hz), 5.60 (1H, d, J = 20 Hz), 9.40 (1H, s), 10.41 (1H, s) , 10.47 (1H, s). IR (KBr) 3354, 1670 cm -1 . TOF-MS m / z 759 [M + Na] + . Vis (CH 2 Cl 2 ) λ max = 693 nm (relative intensity: 78), 632 (11), 554 (17), 522 (18), 430 (100).
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[0014]
Embodiment 4
(Synthesis of chlorophyll derivative represented by formula (7))
The compound represented by the formula (6) (15 mg) was dissolved in a small amount of a solvent (10% methanol / chloroform), a borane-t-butylamine complex (8.7 mg) was added, and the mixture was stirred at 0 ° C for 4 hours. . Chloroform was added, the reaction was stopped with a 2% aqueous hydrochloric acid solution, washed with a 4% aqueous sodium hydrogen carbonate solution and water, the organic layer was separated, and the organic solvent was distilled off. Then, flash column chromatography (15% methanol / Purification with chloroform) gave the compound of the formula (7) (11.5 mg, 77%).
1 H-NMR (DMSO-d6) δ 1.65 (3H, t, J = 7 Hz), 1.78 (3H, d, J = 7 Hz), 2.10-2.25 (2H, m) , 2.30-2.70 (2H, m), 3.00-3.30 (8H, m), 3.38 (3H, s), 3.40-3.50 (3H, m), 3 .62 (3H, s), 3.55-3.70 (2H, m), 3.72 (2H, q, J = 7 Hz), 4.02 (1H, d, J = 7 Hz), 4 .31 (1H, d, J = 8 Hz), 4.56 (1H, q, J = 7 Hz), 5.10 (1H, d, J = 20 Hz), 5.23 (1H, d, J) = 20 Hz), 5.74 (2H, s), 8.80 (1H, s), 9.56 (1H, s), 9.71 (1H, s). IR (neat) 3333, 1622 cm -1 . TOF-MS m / z 761 [M + Na] + . Vis (CH 2 Cl 2 ) λ max = 663 nm (relative intensity: 53), 607 (11), 536 (12), 505 (14), 398 (100).
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[0015]
Embodiment 5
(Synthesis of chlorophyll derivative represented by formula (8))
To a chloroform solution (5 ml) of the compound represented by the formula (7) (11.5 mg), methanol (0.2 ml) saturated with zinc dihydrate was added, and the mixture was stirred at room temperature for 2 hours. 4% aqueous sodium hydrogen carbonate solution (20 ml) was added, and the mixture was stirred at room temperature for 20 minutes. The aqueous layer was extracted with chloroform, combined with the organic layer, washed with water, and dried over sodium sulfate. The dehydrating agent was filtered off, and the organic solvent was distilled off to obtain a compound represented by the formula (8) (11.2 mg, 91%).
1 H-NMR (CD 3 OD) δ 1.69 (3H, t, J = 7 Hz), 1.82 (3H, d, J = 7 Hz), 2.08-2.15 (2H, m) , 2.32-2.65 (2H, m), 3.12-3.20 (1H, m), 3.22 (3H, s), 3.25-3.40 (4H, m), 3 .41 (3H, s), 3.43-3.35 (1H, m), 3.50 (1H, dd, J = 7, 10 Hz), 3.56 (1H, dd, J = 7, 10) Hz), 3.57 (3H, s), 3.61 (1H, d, J = 3 Hz), 3.69 (1H, m), 3.72 (2H, q, J = 7 Hz), 3 .78 (1H, d, J = 7 Hz), 4.24 (1H, d, J = 8 Hz), 4.50 (1H, q, J = 7 Hz), 5. 02 (1H, d, J = 20 Hz), 5.23 (1H, d, J = 20 Hz), 5.70 (2H, s), 8.42 (1H, s), 9.26 (1H, s) s), 9.47 (1H, s). IR (KBr) 3332, 1654 cm -1 . TOF-MS m / z 824 [M + Na] + . Vis (CH 2 Cl 2 ) λ max = 655 nm (relative intensity: 68), 608 (26), 425 (100).
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[0016]
【The invention's effect】
An object of the present invention is to provide a chlorophyll derivative, which is a sugar chain probe which has a cyclic structure at the reducing end of the sugar chain and is suitable for handling synthetic sugar chains.