JP4624700B2 - Synthetic substrate of N-acetylglucosamine transferase V and method for measuring enzyme activity - Google Patents
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本発明は、N-アセチルグルコサミン転移酵素Vの酵素活性測定方法に関する。 The present invention relates to a method for measuring enzyme activity of N-acetylglucosamine transferase V.
癌の悪性化に伴い糖鎖が変化することは古くから知られており、癌と糖転移酵素の関係について多くの研究が行われてきた。糖転移酵素は現在約300種類存在すると言われているが、その中でもN-アセチルグルコサミン転移酵素V （以下GnT-Vと略する）は、発癌過程や癌転移に深く関与することが、これまでの研究で示唆されている。例えば、癌におけるGnT-Vの発現増加は、1982年に初めてマウスリンパ腫細胞中で確認されており（非特許文献１参照）、その後、種々の腫瘍細胞及びヒト癌組織中において発癌の初期過程からGnT-Vの発現増加が確認されている（非特許文献２−８参照）。また、GnT-Vノックアウトマウスの研究から、GnT-Vが癌の転移に必須の分子であることが証明されている（非特許文献９参照）。GnT-Vが癌転移を促進するメカニズムについては種々の研究が行われているが、最近の研究から、本来細胞内のゴルジ装置に存在するGnT-Vが可溶型GnT-Vとなって細胞外に流出し、本来の糖転移酵素活性の機能とは別に血管新生誘導活性を示すことが見出されており（非特許文献１０参照）、このことが癌転移メカニズムの一因であると推測されている。このようにGnT-Vは発癌過程や癌転移などに深く関与しているため、血液、尿および組織などの生体試料中におけるGnT-V活性の測定は、癌の悪性度、癌患者の予後を評価する上で重要な検査となる可能性がある。 It has been known for a long time that sugar chains change as cancers become malignant, and many studies have been conducted on the relationship between cancer and glycosyltransferases. There are currently about 300 types of glycosyltransferases. Among them, N-acetylglucosaminyltransferase V (hereinafter abbreviated as GnT-V) has been deeply involved in carcinogenic processes and cancer metastasis. Suggested in the study. For example, increased expression of GnT-V in cancer was confirmed in mouse lymphoma cells for the first time in 1982 (see Non-Patent Document 1), and thereafter from the initial stage of carcinogenesis in various tumor cells and human cancer tissues. Increased expression of GnT-V has been confirmed (see Non-Patent Documents 2-8). In addition, studies on GnT-V knockout mice have proved that GnT-V is an essential molecule for cancer metastasis (see Non-Patent Document 9). Various studies have been conducted on the mechanism by which GnT-V promotes cancer metastasis. However, recent research has shown that GnT-V, which is originally present in the Golgi apparatus in cells, becomes soluble GnT-V. It has been found that it flows out and exhibits angiogenesis-inducing activity separately from the function of the original glycosyltransferase activity (see Non-Patent Document 10), which is presumed to be a cause of cancer metastasis mechanism. Has been. GnT-V is thus deeply involved in the carcinogenesis process and cancer metastasis, so measurement of GnT-V activity in biological samples such as blood, urine, and tissues is important in determining the malignancy of cancer and the prognosis of cancer patients. It may be an important test for evaluation.
GnT-Vは糖転移酵素の１種であり、末端にＮ-アセチルグルコサミンβ1-2マンノースα1-6構造を有するアスパラギン結合型糖鎖のマンノースにN-アセチルグルコサミン (以下GlcNAcと略する)をβ1,6結合で転移する活性を有している。GnT-Vの酵素活性を測定する方法としては、糖鎖供与体であるウリジン5’-ニリン酸-N-アセチルグルコサミン（以下UDP-GlcNAcと略する）、あるいは糖鎖受容体のいずれかを放射性試薬あるいは蛍光試薬で標識し、GnT-Vにより生成した糖鎖を分離して定量する方法が知られている。 GnT-V is a type of glycosyltransferase, and N-acetylglucosamine (hereinafter abbreviated as GlcNAc) is added to the mannose of an asparagine-linked sugar chain having an N-acetylglucosamine β1-2 mannose α1-6 structure at the end. Therefore, it has the activity to transfer with 6 bonds. To measure the enzyme activity of GnT-V, either sugar chain donor uridine 5'-niphosphate-N-acetylglucosamine (hereinafter abbreviated as UDP-GlcNAc) or sugar chain acceptor is radioactive. A method is known in which a sugar chain generated by GnT-V is separated and quantified by labeling with a reagent or a fluorescent reagent.
糖鎖供与体の標識化合物を用いてGnT-Vの酵素活性を測定する方法としては、UDP-[3H]-GlcNAcあるいはUDP-[14C]-GlcNAcを用いる方法が知られている（非特許文献２、１１−１２参照）。例えば、UDP-[3H]-GlcNAcを用いる方法では、以下のようにしてGnT-V活性の測定が行われている（非特許文献１１参照）。まず、ウリジン5’-ニリン酸-N-グルコサミンとトリチウム標識無水酢酸を反応させ、UDP-[3H]-GlcNAcを合成する。また、糖鎖受容体として、GlcNAcβ1-2Manα1-6Manβ-O-(CH2)8-COOCH3を合成する。GnT-Vの測定対象となる細胞を緩衝液中に懸濁させ、超音波破砕機で処理する。この溶液中にUDP-[3H]-GlcNAcおよびGlcNAcβ1-2Manα1-6Manβ-O-(CH2)8-COOCH3を適量添加し、37℃で保温する。６時間後に溶液を遠心分離器にかけ、上静を逆相クロマトグラフィーカラムに通し、放射能が検出されなくなるまでカラムを水で洗い流す。この際、未反応のUDP-[3H]-GlcNAcは洗い流されて、カラムから流出してくる。その後、カラムにメタノールを通すと、GnT-V活性によりGlcNAcβ1-2Manα1-6Manβ-O-(CH2)8-COOCH3のマンノース6位に[3H]-GlcNAcが転移した化合物が流出してくるので、その放射能を液体シンチレーションカウンターで計測し、GnT-Vにより生成した化合物の量を算出することによりGnT-V酵素活性の測定が可能となる。ただしUDP-GlcNAcは、GnT-Vだけではなく他の糖転移酵素の糖鎖供与体にもなることが知られているため、この方法を用いた場合、他の糖転移酵素の活性も含めて測定している可能性があり、特異性が問題となっていた。 As a method for measuring the enzyme activity of GnT-V using a labeling compound of a sugar chain donor, a method using UDP- [ 3 H] -GlcNAc or UDP- [ 14 C] -GlcNAc is known (non- (See Patent Document 2 and 11-12). For example, in the method using UDP- [ 3 H] -GlcNAc, GnT-V activity is measured as follows (see Non-Patent Document 11). First, uridine 5′-niphosphate-N-glucosamine is reacted with tritium-labeled acetic anhydride to synthesize UDP- [ 3 H] -GlcNAc. In addition, GlcNAcβ1-2Manα1-6Manβ-O— (CH 2 ) 8 —COOCH 3 is synthesized as a sugar chain receptor. Cells to be measured for GnT-V are suspended in a buffer solution and processed with an ultrasonic crusher. Appropriate amounts of UDP- [ 3 H] -GlcNAc and GlcNAcβ1-2Manα1-6Manβ-O— (CH 2 ) 8 —COOCH 3 are added to this solution and kept at 37 ° C. After 6 hours, centrifuge the solution, pass the top through a reverse phase chromatography column, and flush the column with water until no radioactivity is detected. At this time, unreacted UDP- [ 3 H] -GlcNAc is washed away and flows out of the column. Subsequently, when methanol is passed through the column, a compound in which [ 3 H] -GlcNAc is transferred to the mannose 6 position of GlcNAcβ1-2Manα1-6Manβ-O- (CH 2 ) 8 -COOCH 3 by GnT-V activity flows out. Therefore, the GnT-V enzyme activity can be measured by measuring the radioactivity with a liquid scintillation counter and calculating the amount of the compound produced by GnT-V. However, since UDP-GlcNAc is known not only to be a GnT-V but also to other glycosyltransferases, it also includes the activities of other glycosyltransferases. There was a possibility of measurement, and specificity was a problem.
糖鎖受容体を標識した基質を用いてGnT-Vの酵素活性を測定する方法としては、チオグロブリン、トランスフェリン、または卵黄などのタンパク質を各種酵素処理したのち分離精製した糖鎖をI-125あるいは蛍光試薬で標識し、GnT-Vの基質として用いる方法が知られている（非特許文献１、１３−１５参照）。例えば蛍光試薬を用いる方法では、以下のようにしてGnT-V活性の測定が行われている（非特許文献１３参照）。まず、ヒトのトランスフェリンを蛋白分解酵素プロナーゼで処理したのちヒドラジン分解反応およびN-アセチル化反応を行い、その後2-アミノピリジンで蛍光標識する。得られた糖鎖をシアリダーゼおよびβ-ガラクトシダーゼで処理したのち逆相HPLCカラムで精製を行い、7糖を基本骨格とするGlcNAcβ1-2Manα1-3-(GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc-2-アミノピリジン（以下Gn，Gn-bi-PAと略する）を得る。GnT-V酵素活性の測定検体中にUDP-GlcNAおよびGn，Gn-bi-PAを適量加え、37℃で保温する。４時間後に100℃で2分間加熱して酵素反応を止めたのち、0.22μmのフィルターで濾過し、逆相HPLCで分析を行う。逆相HPLCの結果からGnT-Vにより生成した糖鎖を定量し、GnT-Vの酵素活性を算出する。このような方法によりGnT-Vの酵素活性測定が可能であるが、タンパク質などの生物由来物質を原料としてGnT-Vの基質を調製するには、各種酵素処理および分離精製の工程が必要であるため、操作が煩雑で、再現性にも問題がある。
本発明は、上記事情に鑑み、GnT-Vの糖鎖受容体となりうる放射性標識合成基質及びそれを用いた簡便かつ高感度なGnT-V酵素活性の測定方法を提供するものである。 In view of the above circumstances, the present invention provides a radiolabeled synthetic substrate that can be a GnT-V sugar chain receptor and a simple and highly sensitive method for measuring GnT-V enzyme activity using the same.
本発明者らは、上記課題を解決すべく、鋭意研究を重ねた結果、式１に示す放射性標識合成基質が生物由来のタンパク質を各種酵素処理、分離精製するといった煩雑な工程を必要とせず、再現性良く調製が可能で、かつ従来用いられてきた７糖を基本骨格とする蛍光標識体Gn,Gn-bi-PAよりもGnT-Vへ高い親和性を示し、これを用いることにより簡便かつ高感度でGnT-V酵素活性を測定できることを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention do not require a complicated process in which the radioactively labeled synthetic substrate shown in Formula 1 is subjected to various enzyme treatments, separation and purification of biologically derived proteins, It can be prepared with good reproducibility, and has a higher affinity for GnT-V than the conventional fluorescent label Gn, Gn-bi-PA with 7 saccharide as the basic skeleton. The inventors have found that GnT-V enzyme activity can be measured with high sensitivity, and have completed the present invention.
すなわち、本発明は、（１）式Ｉ： That is, the present invention provides (1) Formula I:
（式中、R1〜R3はそれぞれ独立に水素、ハロゲンまたはアセチル、nは2〜7の中から選択される整数、XはI-123、I-124、I-125およびI-131からなる群より選択される放射性同位元素）で示されるN-アセチルグルコサミン転移酵素Vの合成基質、
（２）R1〜R3が全て水素である（１）記載のN-アセチルグルコサミン転移酵素Vの合成基質、
（３）nが２である（２）記載のN-アセチルグルコサミン転移酵素Vの合成基質、
（４）ＸがI-125である（３）記載のN-アセチルグルコサミン転移酵素Vの合成基質、および、
（５）（１）から（４）の何れか１つに記載の合成基質を用いたN-アセチルグルコサミン転移酵素Vの酵素活性測定方法を提供するものである。
Wherein R 1 to R 3 are each independently hydrogen, halogen or acetyl, n is an integer selected from 2 to 7, X is from I-123, I-124, I-125 and I-131 A synthetic substrate of N-acetylglucosaminyltransferase V represented by a radioisotope selected from the group consisting of:
(2) The synthetic substrate of N-acetylglucosaminyltransferase V according to (1), wherein R 1 to R 3 are all hydrogen,
(3) The synthetic substrate for N-acetylglucosaminyltransferase V according to (2), wherein n is 2.
(4) The synthetic substrate for N-acetylglucosaminyltransferase V according to (3), wherein X is I-125, and
(5) Provided is a method for measuring the enzyme activity of N-acetylglucosaminyltransferase V using the synthetic substrate according to any one of (1) to (4).
本発明により、GnT-Vの糖鎖受容体となりうる放射性標識合成基質及びそれを用いた簡便かつ高感度なGnT-V酵素活性の測定方法が提供される。本発明のGnT-V合成基質は、生物由来物質を原料としないため、酵素処理や精製工程などの面倒な工程を必要としない。また、本発明のGnT-V合成基質は、従来用いられてきた７糖を基本骨格とする蛍光標識体Gn,Gn-bi-PAよりもGnT-Vへ高い親和性を示すため、高感度にGnT-Vの酵素活性を測定できる。これらのことは、GnT-V酵素活性検査の効率化および高性能化につながる。 INDUSTRIAL APPLICABILITY The present invention provides a radiolabeled synthetic substrate that can be a GnT-V sugar chain receptor and a simple and highly sensitive method for measuring GnT-V enzyme activity using the same. Since the GnT-V synthetic substrate of the present invention does not use biological materials as raw materials, it does not require troublesome processes such as enzyme treatment and purification processes. In addition, the GnT-V synthetic substrate of the present invention exhibits higher affinity for GnT-V than the conventional fluorescently labeled Gn, Gn-bi-PA having a heptasaccharide as a basic skeleton, and therefore has high sensitivity. The enzyme activity of GnT-V can be measured. These things lead to efficient and high-performance GnT-V enzyme activity tests.
以下、本発明の実施の形態について説明する。本発明のGnT-V合成基質は、上の一般式Iで示される化合物である。ここで、R1〜R3はそれぞれ独立に水素、ハロゲンまたはアセチルであるが、基質親和性を考慮した場合、R1〜R3は水素が好ましい。nは2〜7の中から選択される整数であるが、基質親和性を考慮した場合、ｎは２であることが好ましい。XはI-123、I-124、I-125、I-131からなる群より選択される放射性同位元素であるが、好ましくは適度な半減期及び放射線エネルギーを持つI-125を用いる。 Embodiments of the present invention will be described below. The GnT-V synthetic substrate of the present invention is a compound represented by the above general formula I. Here, R 1 to R 3 are each independently hydrogen, halogen, or acetyl, but considering substrate affinity, R 1 to R 3 are preferably hydrogen. n is an integer selected from 2 to 7. In consideration of substrate affinity, n is preferably 2. X is a radioisotope selected from the group consisting of I-123, I-124, I-125, and I-131, and preferably I-125 having an appropriate half-life and radiation energy is used.
本発明のGnT-V合成基質は、以下の方法により合成することができる。目的とするGnT-V基質をD-グルコサミン部位、D-マンノース部位、D-グルコース部位、p-ヨードフェニルアルキル部位の４つに分け、それぞれの誘導体を合成する。この時、p-ヨードフェニルアルキル部位のヨードの位置には、臭素を結合させておく。その後、D-グルコサミン誘導体とD-マンノース誘導体をβ1,2結合で、D-グルコース誘導体の４位にp-臭素フェニルアルキル誘導体をそれぞれ結合させ、次いでそれぞれの結合体を化学修飾し、臭素の位置にトリブチルスズを結合させて両者を結合させる。得られた3糖トリブチルスズフェニルアルキル誘導体に当該分野で良く知られているトリブチルスズ−ヨウ素置換反応を施し、保護基を脱離することにより目的とするGnT-V基質を合成できる。 The GnT-V synthesis substrate of the present invention can be synthesized by the following method. The target GnT-V substrate is divided into four parts: a D-glucosamine moiety, a D-mannose moiety, a D-glucose moiety, and a p-iodophenylalkyl moiety, and each derivative is synthesized. At this time, bromine is bonded to the iodo position of the p-iodophenylalkyl moiety. After that, D-glucosamine derivative and D-mannose derivative are linked by β1,2 bond, p-bromine phenylalkyl derivative is bonded to the 4-position of D-glucose derivative, respectively, and then each conjugate is chemically modified. Both are bonded by bonding tributyltin. By subjecting the resulting trisaccharide tributyltin phenylalkyl derivative to a tributyltin-iodine substitution reaction well known in the art and removing the protecting group, the target GnT-V substrate can be synthesized.
本発明の合成基質を用いるGnT-V酵素活性測定は、次の手順で行うことができる。まず、患者から尿、血液または組織などの生体試料を採取する。癌患者の場合には、癌摘出手術後の組織や生検用に採取した組織をGnT-V酵素活性測定用の試料として用いても良い。測定試料が組織の場合には、あらかじめ緩衝溶液中で超音波処理などを行い、懸濁液にしておく。測定対象となる溶液中に糖鎖供与体であるUDP-GlcNAc、TritonX-100、EDTAを含むMES緩衝液を加えた後、さらに本発明の放射性標識合成基質を糖鎖受容体として加え、37℃で保温する。4時間後に100℃で1分間加熱することにより反応を止め、逆相HPLCにて分析を行う。本発明の放射性標識合成基質からGnT-Vにより生成する糖鎖を標品としてあらかじめ合成しておき、逆相HPLCで事前に分析しておくことで、得られた逆相HPLCの結果からGnT-V酵素活性を算出することができる。 Measurement of GnT-V enzyme activity using the synthetic substrate of the present invention can be performed by the following procedure. First, a biological sample such as urine, blood or tissue is collected from a patient. In the case of a cancer patient, a tissue after cancer removal surgery or a tissue collected for biopsy may be used as a sample for measuring GnT-V enzyme activity. When the measurement sample is a tissue, it is subjected to ultrasonic treatment or the like in a buffer solution in advance to form a suspension. After adding a MES buffer containing UDP-GlcNAc, TritonX-100, and EDTA, which are sugar chain donors, to the solution to be measured, the radiolabeled synthetic substrate of the present invention is added as a sugar chain acceptor at 37 ° C. Keep warm. After 4 hours, the reaction is stopped by heating at 100 ° C. for 1 minute and analyzed by reverse phase HPLC. The sugar chain produced by GnT-V from the radiolabeled synthetic substrate of the present invention is pre-synthesized as a standard, and analyzed in advance by reverse-phase HPLC, so that the obtained GnT- V enzyme activity can be calculated.
以下に実施例を挙げて本発明を具体的に説明するが、これらの実施例は本発明を何ら限定するものではない。なお、化合物名は〔 〕内に英文名を併記した。 EXAMPLES The present invention will be specifically described below with reference to examples, but these examples do not limit the present invention. The compound names are shown in English in English.
2-(4-ヨードフェニル)エチル-O-(2-アセタミド-2-デオキシ-β-D-グルコピラノシル)-(1→2)-O-α-D-マンノピラノシル-(1→6)-β-D-グルコピラノシド〔2-(4-Iodophenyl)ethyl-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-O-α-D-mannopyranosyl-(1→6)-β-D-glucopyranoside (IPGMG)〕の合成
なお、合成の概略スキームを下記に図示した。該要約に示された化合物5, 8, 13, 21, 26または28、 ならびに、29, 30, 31および32の合成スキーム(Scheme)を各合成の記述の前に図示した。
2- (4-Iodophenyl) ethyl-O- (2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -O-α-D-mannopyranosyl- (1 → 6) -β- D-glucopyranoside (2- (4-Iodophenyl) ethyl-O- (2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -O-α-D-mannopyranosyl- (1 → 6) -β-D-glucopyranoside (IPGMG)] A schematic scheme of the synthesis is shown below. The synthetic schemes of compounds 5 , 8 , 13 , 21 , 26 or 28 , and 29 , 30 , 31 and 32 shown in the summary are illustrated prior to the description of each synthesis.
3,4,6-トリ-O-アセチル-2-デオキシ-2-フタルイミド-β-D-グルコピラノシル ブロミド〔3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl bromide〕(5)は、D-グルコサミン ヒドロクロリド〔D-glucosamine hydrochloride〕から文献既知 (Yakugakuzasshi 1957,77,726-.) の方法に従って5段階で合成した。またベンジル-α-D-マンノピラノシド〔benzyl-α-D-mannopyranoside〕(6)は、D-マンノース〔D-mannose〕から文献既知 (J. Chem. Soc..,Perkin. I 1981,377-.) の方法に従って合成した。更にベンジル 4,6-O-ベンジリデン -α- D-マンノピラノシド〔benzyl 4,6-O-benzylidene-α- D-mannopyranoside 〕(7)は、6から文献既知 (Carbohydr. Res. 1975,45,105-.) の方法に従って合成した。 3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl bromide [3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D -glucopyranosyl bromide] ( 5 ) was synthesized in 5 steps from D-glucosamine hydrochloride according to the method known in the literature (Yakugakuzasshi 1957, 77, 726-.). Also, benzyl-α-D-mannopyranoside (benzyl-α-D-mannopyranoside) ( 6 ) is known from D-mannose (J. Chem. Soc., Perkin. I 1981, 377-. ). Further, benzyl 4,6-O-benzylidene-α-D-mannopyranoside (benzyl 4,6-O-benzylidene-α-D-mannopyranoside) ( 7 ) is known from 6 (Carbohydr. Res. 1975, 45, 105-. ).
ベンジル 3-O-ベンジル-4,6-O-ベンジリデン-α-D-マンノピラノシド〔Benzyl 3-O-benzyl-4,6-O-benzylidene-α-D-mannopyranoside 〕(8)
窒素気流下、7 (2.7 g,7.53 mmol) のトルエン〔toluene〕 (100 ml) 溶液にテトラブチルアンモニウム ヨーダイド〔tetrabutylammonium iodide〕 (4.2 g,11.4 mmol),ジブチルスズオキシド〔dibutyltin oxide〕 (2.1 g,8.44 mmol) を加え生成する水を除去しながら3.5時間還流した。反応液に氷冷下、ベンジル ブロミド〔benzyl bromide〕 (1.2 ml,10.1 mmol) を加えた後、130 °Cで20時間撹拌した。反応終了後、反応液を酢酸エチル〔ethyl acetate〕で希釈し、有機層を5 % チオ硫酸ナトリウム水溶液、 飽和食塩水で洗浄後、乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン〔hexane〕:アセトン〔acetone 〕= 2 : 1) で精製し8 (2.85 g,85 %)を得た。
Benzyl 3-O-benzyl-4,6-O-benzylidene-α-D-mannopyranoside (Benzyl 3-O-benzyl-4,6-O-benzylidene-α-D-mannopyranoside) ( 8 )
Tetrabutylammonium iodide (4.2 g, 11.4 mmol), dibutyltin oxide (2.1 g, 8.44) in a toluene (100 ml) solution of 7 (2.7 g, 7.53 mmol) in a nitrogen stream mmol) and refluxed for 3.5 hours while removing the water formed. Benzyl bromide (1.2 ml, 10.1 mmol) was added to the reaction solution under ice cooling, followed by stirring at 130 ° C. for 20 hours. After completion of the reaction, the reaction solution is diluted with ethyl acetate, and the organic layer is washed with 5% aqueous sodium thiosulfate solution and saturated brine, dried (sodium sulfate), filtered, and the solvent is retained. The crude product obtained after purification was purified by silica gel column chromatography (hexane [hexane]: acetone [2: 1]) to obtain 8 (2.85 g, 85%).
1,2,3,4-テトラ-O-アセチル -6-O-tert-ブチルジメチルシリル-D-グルコピラノシド〔1,2,3,4-Tetra-O-acetyl-6-O-tert-butyldimethylsilyl-D-glucopyranoside 〕(9)
窒素気流下、D-グルコース〔D-glucose 〕(4.06 g,22.5 mmol) のN,N-ジメチルホルムアミド〔N,N-dimethylformamide 〕(DMF,40 ml) 溶液にtert-ブチルジメチルシリルクロリド〔tert-butyldimethylsilyl chloride 〕(3.73 g,24.8 mmol),イミダゾール〔imidazole 〕(3.07 g,45.0 mmol) 加え、室温で撹拌した。43時間後、反応液に無水酢酸〔acetic anhydride 〕(15 ml)，ピリジン〔pyridine 〕(15 ml)，4-ジメチルアミノピリジン〔4-dimethylaminopyridine 〕(cat.) を加え、更に室温で4時間撹拌した。反応終了後、反応液に氷冷下、メタノール〔methanol〕を加えた後、溶媒を留去した。残査を酢酸エチル〔ethyl acetate 〕に希釈した有機層を蒸留水，飽和食塩水で洗浄後、乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン〔hexane 〕: 酢酸エチル〔ethyl acetate 〕= 3 : 1) で精製し9 (5.05 g,48 %)を得た。
1,2,3,4-Tetra-O-acetyl-6-O-tert-butyldimethylsilyl-D-glucopyranoside [1,2,3,4-Tetra-O-acetyl-6-O-tert-butyldimethylsilyl- (D-glucopyranoside) (9)
Under a nitrogen stream, tert-butyldimethylsilyl chloride (tert-butyldimethylsilyl chloride) was added to a solution of D-glucose (D-glucose) (4.06 g, 22.5 mmol) in N, N-dimethylformamide (N, N-dimethylformamide) (DMF, 40 ml). butyldimethylsilyl chloride] (3.73 g, 24.8 mmol), imidazole [imidazole] (3.07 g, 45.0 mmol) was added, and the mixture was stirred at room temperature. After 43 hours, acetic anhydride (15 ml), pyridine (pyridine) (15 ml), 4-dimethylaminopyridine (cat.) Was added to the reaction solution, and the mixture was further stirred at room temperature for 4 hours. did. After completion of the reaction, methanol was added to the reaction solution under ice cooling, and then the solvent was distilled off. A crude product obtained by diluting the residue in ethyl acetate, washing the organic layer with distilled water and saturated brine, drying (sodium sulfate), filtering and distilling off the solvent. The product was purified by silica gel column chromatography (hexane: ethyl acetate = 3: 1) to obtain 9 (5.05 g, 48%).
1,2,3,4-テトラ-O-アセチル-D-グルコピラノシド〔1,2,3,4-Tetra-O-acetyl-D-glucopyranoside 〕(10)
9 (2.46 g,5.31 mmol) のメタノール〔methanol 〕(40 ml) 溶液にピリジニウムp-トルエンスルホナート〔pyridinium p-toluenesulfonate 〕(PPTS,134 mg,0.53 mmol) を加え、60 °Cで2時間撹拌した。反応終了後、溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン〔hexane 〕:酢酸エチル〔ethyl acetate 〕= 1 : 1) で精製し10 (1.38 g,81 %) を得た。
1,2,3,4-Tetra-O-acetyl-D-glucopyranoside [1,2,3,4-Tetra-O-acetyl-D-glucopyranoside] (10)
Add pyridinium p-toluenesulfonate (PPTS, 134 mg, 0.53 mmol) to a solution of 9 (2.46 g, 5.31 mmol) in methanol (40 ml) and stir at 60 ° C for 2 hours. did. After completion of the reaction, the crude product obtained by distilling off the solvent was purified by silica gel column chromatography (hexane [hexane]: ethyl acetate = 1: 1) to obtain 10 (1.38 g, 81%). Obtained.
1,2,3,4-テトラ-O-アセチル-6-O-アリルオキシカルボニル-D-グルコピラノシド〔1,2,3,4-Tetra-O-acetyl-6-O-allyloxycarbonyl-D-glucopyranoside〕(11)
窒素気流下、10 (1.38 g,4.31 mmol) のテトラヒドロフラン〔tetrahydrofuran 〕(THF,30 ml) 溶液に氷冷下、アリル クロロホルメート〔allyl chloroformate 〕(1.37 ml,12.9 mmol)，ピリジン〔pyridine 〕(1.39 ml,17.2 mmol) を加えた後、室温で2時間撹拌した。反応終了後、反応液を飽和塩化アンモニウム水溶液に注ぎ酢酸エチル〔ethyl acetate〕で抽出した。得られた有機層を飽和食塩水で洗浄し、乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン〔hexane 〕: 酢酸エチル〔ethyl acetate 〕= 2 : 1) で精製し11 (1.63 g,87 %)を得た。
1,2,3,4-Tetra-O-acetyl-6-O-allyloxycarbonyl-D-glucopyranoside (1,2,3,4-Tetra-O-acetyl-6-O-allyloxycarbonyl-D-glucopyranoside) (11)
Under a nitrogen stream, 10 (1.38 g, 4.31 mmol) in tetrahydrofuran (THF, 30 ml) solution under ice-cooling, allyl chloroformate (1.37 ml, 12.9 mmol), pyridine (pyridine) ( 1.39 ml, 17.2 mmol) was added, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the reaction solution was poured into a saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The obtained organic layer was washed with saturated brine, dried (sodium sulfate), filtered, and then the solvent was distilled off. The resulting crude product was subjected to silica gel column chromatography (hexane [hexane]: acetic acid). Purification with ethyl acetate = 2: 1) gave 11 (1.63 g, 87%).
2,3,4-トリ-O-アセチル-6-O-アリロキシカルボニル-D-グルコピラノシル トリクロロアセトイミダート〔2,3,4-Tri-O-acetyl-6-O-allyoxycarbonyl-D-glucopyranosyl trichloroacetoimidate 〕(13)
窒素気流下、11 (3.45 g,11.4 mmol) のTHF (50 ml) 溶液にピペリジン〔piperidine 〕(2.26 ml,22.8 mmol) を加え室温で20時間撹拌した。反応終了後、反応液を酢酸エチル〔ethyl acetate〕で希釈した後、有機層を10 % 塩酸〔hydrochloric acid 〕(HCl)，飽和食塩水で洗浄後、乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン〔hexane 〕: 酢酸エチル〔ethyl acetate 〕= 2 : 1) で精製し2,3,4-テトラ-O-アセチル-6-O-アリルオキシカルボニル-D-グルコピラノシド〔2,3,4-tetra-O-acetyl-6-O-allyloxycarbonyl-D-glucopyranoside 〕(12,2.41 g,68 %)を得た。この12 (2.41 g,6.15 mmol) のDCM (30 ml) 溶液にトリクロロアセトニトリル〔trichloroacetonitrile 〕(1.23 ml,12.3 mmol)，1,8-ジアザビシクロ[5.4.0]ウンデセ-7-エン〔1,8-diazabicyclo[5.4.0]undec-7-ene 〕(46 μl,0.31 mmol) を加え室温で2時間撹拌した。反応終了後、反応溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン〔hexane 〕: 酢酸エチル〔ethyl acetate 〕= 3 : 1) で精製し13 (2.73 g,82 %)を得た。
2,3,4-Tri-O-acetyl-6-O-allyloxycarbonyl-D-glucopyranosyl trichloroacetimidate [2,3,4-Tri-O-acetyl-6-O-allyoxycarbonyl-D-glucopyranosyl trichloroacetoimidate 〕(13)
Under a nitrogen stream, piperidine (2.26 ml, 22.8 mmol) was added to a THF (50 ml) solution of 11 (3.45 g, 11.4 mmol), and the mixture was stirred at room temperature for 20 hours. After completion of the reaction, the reaction solution is diluted with ethyl acetate, and the organic layer is washed with 10% hydrochloric acid (HCl) and saturated saline, dried (sodium sulfate), filtered. Then, the crude product obtained by distilling off the solvent was purified by silica gel column chromatography (hexane [hexane]: ethyl acetate = 2: 1) and purified 2,3,4-tetra-O-acetyl. -6-O-allyloxycarbonyl-D-glucopyranoside [2,3,4-tetra-O-acetyl-6-O-allyloxycarbonyl-D-glucopyranoside] ( 12 , 2.41 g, 68%) was obtained. To a solution of 12 (2.41 g, 6.15 mmol) in DCM (30 ml), trichloroacetonitrile (1.23 ml, 12.3 mmol), 1,8-diazabicyclo [5.4.0] undec-7-ene [1,8- diazabicyclo [5.4.0] undec-7-ene] (46 μl, 0.31 mmol) was added, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the crude product obtained by distilling off the reaction solvent was purified by silica gel column chromatography (hexane [hexane]: ethyl acetate = 3: 1) and 13 (2.73 g, 82%) Got.
ベンジル-O-(3,4,6-トリ-O-アセチル-2-デオキシ-2-フタルイミド-β-D-グルコピラノシル)-(1→2)-3-O-ベンジル-4,6-O-ベンジリデン-α-D-マンノピラノシド〔Benzyl-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→2)-3-O-benzyl-4,6-O-benzylidene-α-D-mannopyranoside 〕(14)
窒素気流下、8 (255 mg,0.57 mmol)，銀トリフルオロメタンスルホナート〔silver trifluoromethanesulfonate 〕(263 mg,1.02 mmol)，γ-コリジン〔collidine 〕(135 μl,1.02 mmol) のニトロメタン〔nitromethane 〕(1 ml) 溶液に -20 °C下、5 (426 mg,0.86 mmol) のニトロメタン〔nitromethane 〕(2 ml) 溶液を加え同温で10分撹拌した後、室温で更に1.5時間撹拌した。反応終了後、Celite（登録商標）を用いて反応液を濾過した (クロロホルム〔chloroform〕) 後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (トルエン〔toluene 〕: ジエチルエーテル〔diethyl ether 〕= 3 : 1) で精製し14 (280 mg,57 %) を得た。
Benzyl-O- (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimide-β-D-glucopyranosyl)-(1 → 2) -3-O-benzyl-4,6-O- Benzylidene-α-D-mannopyranoside (Benzyl-O- (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1 → 2) -3-O- benzyl-4,6-O-benzylidene-α-D-mannopyranoside] (14)
Under nitrogen flow, 8 (255 mg, 0.57 mmol), silver trifluoromethanesulfonate (263 mg, 1.02 mmol), γ-collidine (135 μl, 1.02 mmol) of nitromethane (1) ml) A solution of 5 (426 mg, 0.86 mmol) of nitromethane (2 ml) was added to the solution at −20 ° C., and the mixture was stirred at the same temperature for 10 minutes, and further stirred at room temperature for 1.5 hours. After completion of the reaction, the reaction solution was filtered using Celite (registered trademark) (chloroform), and then the solvent was distilled off. The resulting crude product was purified by silica gel column chromatography (toluene): diethyl ether [ Diethyl ether] = 3: 1) to obtain 14 (280 mg, 57%).
ベンジル-O-(2-アセタミド-3,4,6-トリ-O-アセチル-2-デオキシ-β-D-グルコピラノシル)-(1→2)-3-O-ベンジル -4,6-O-ベンジリデン-α-D-マンノピラノシド〔Benzyl-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-3-O-benzyl-4,6-O-benzylidene-α-D-mannopyranoside 〕(16)
14 (900 mg,1.04 mmol) のエタノール〔ethanol 〕(50 ml) 溶液にヒドラジン水和物〔hydrazine・hydrate 〕(80 % in water,1.5 ml) を加え4.5時間還流した。反応終了後、反応液を濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (クロロホルム〔chloroform 〕: メタノール〔methanol〕 = 3 : 1) で精製しベンジルO-(2-アミノ-2-デオキシ-β-D-グルコピラノシル)-(1→2)-3-O-ベンジル-4,6-O-ベンジリデン-α-D-マンノピラノシド〔benzylO-(2-amino-2-deoxy-β-D-glucopyranosyl)-(1→2)-3-O-benzyl-4,6-O-benzylidene-α-D-mannopyranoside 〕(15,500 mg,95 %) を得た。この15 (500 mg,0.82 mmol) のピリジン〔pyridine 〕(5 ml) 溶液に無水酢酸〔acetic anhydride 〕(2.5 ml) を加え室温で一晩撹拌した。反応終了後、反応液を氷水に注ぎ酢酸エチル〔ethyl acetate〕で抽出した。得られた有機層を蒸留水で洗浄後、乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (クロロホルム〔chloroform 〕:メタノール〔methanol 〕= 20 : 1) で精製し16 (625 mg,84 %) を得た。
Benzyl-O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -3-O-benzyl-4,6-O- Benzylidene-α-D-mannopyranoside (Benzyl-O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -3-O- benzyl-4,6-O-benzylidene-α-D-mannopyranoside] (16)
To a solution of 14 (900 mg, 1.04 mmol) in ethanol (50 ml) was added hydrazine hydrate (80% in water, 1.5 ml) and refluxed for 4.5 hours. After completion of the reaction, the reaction mixture was filtered and the solvent was distilled off to obtain a crude product, which was purified by silica gel column chromatography (chloroform: methanol = 3: 1) to obtain benzyl O- (2 -Amino-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -3-O-benzyl-4,6-O-benzylidene-α-D-mannopyranoside (benzylO- (2-amino-2-deoxy -β-D-glucopyranosyl) - was obtained (1 → 2) -3-O -benzyl-4,6-O-benzylidene-α-D-mannopyranoside ] (15, 500 mg, 95% ). Acetic anhydride (2.5 ml) was added to a solution of this 15 (500 mg, 0.82 mmol) in pyridine (pyridine) (5 ml) and stirred overnight at room temperature. After completion of the reaction, the reaction solution was poured into ice water and extracted with ethyl acetate. The obtained organic layer was washed with distilled water, dried (sodium sulfate), filtered, and the solvent was distilled off to obtain a crude product, which was subjected to silica gel column chromatography (chloroform: methanol [ methanol]] = 20: 1) to obtain 16 (625 mg, 84%).
ベンジル-O-(2-アセタミド-3,4,6-トリ-O-アセチル-2-デオキシ-β-D-グルコピラノシル)-(1→2)-4,6-ジ-O-アセチル-3-O-ベンジル-α-D-マンノピラノシド(18)〔Benzyl-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-4,6-di-O-acetyl-3-O-benzyl-α-D-mannopyranoside 〕(18)
16 (160 mg,0.21 mmol) に70 % 酢酸〔acetic acid〕水溶液 (2 ml) を加え55 °Cで6.5時間撹拌した。反応終了後、反応液を氷水に注ぎ炭酸水素ナトリウムで中和した後、クロロホルム〔chloroform〕で抽出した。得られた有機層を飽和炭酸水素ナトリウム水溶液で洗浄後、乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (クロロホルム〔chloroform 〕: メタノール〔methanol 〕= 10 : 1) で精製しベンジル -O-(2-アセタミド-3,4,6-トリ-O-アセチル-2-デオキシ-β-D-グルコピラノシル)-(1→2)-3-O-ベンジル-α-D-マンノピラノシド〔benzyl -O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-3-O-benzyl-α-D-mannopyranoside 〕(17,125 mg,91 %) を得た。この17 (138 mg,0.20 mmol) のピリジン〔pyridine 〕(1 ml) 溶液に無水酢酸〔acetic anhydride 〕(500 μl) を加え室温で一晩撹拌した。反応終了後、反応液を氷水に注ぎ炭酸水素ナトリウムで中和した後クロロホルム〔chloroform〕で抽出した。得られた有機層を蒸留水で洗浄後、乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (クロロホルム〔chloroform 〕: メタノール〔methanol 〕= 20 : 1) で精製し18 (137 mg,88 %) を得た。
Benzyl-O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -4,6-di-O-acetyl-3- O-Benzyl-α-D-mannopyranoside (18) (Benzyl-O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -4,6-di-O-acetyl-3-O-benzyl-α-D-mannopyranoside] (18)
To 16 (160 mg, 0.21 mmol) was added 70% acetic acid aqueous solution (2 ml), and the mixture was stirred at 55 ° C for 6.5 hours. After completion of the reaction, the reaction solution was poured into ice water, neutralized with sodium hydrogen carbonate, and extracted with chloroform. The obtained organic layer was washed with a saturated aqueous solution of sodium bicarbonate, dried (sodium sulfate), filtered, and the solvent was distilled off to obtain a crude product, which was subjected to silica gel column chromatography (chloroform). : Purified with methanol (methanol) = 10: 1) benzyl-O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -3-O-benzyl-α-D-mannopyranoside (benzyl-O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -3-O-benzyl-α-D-mannopyranoside] ( 17 , 125 mg, 91%). Acetic anhydride (500 μl) was added to a solution of 17 (138 mg, 0.20 mmol) in pyridine (pyridine) (1 ml), and the mixture was stirred overnight at room temperature. After completion of the reaction, the reaction solution was poured into ice water, neutralized with sodium hydrogen carbonate, and extracted with chloroform. The obtained organic layer was washed with distilled water, dried (sodium sulfate), filtered, and the solvent was distilled off to obtain a crude product, which was subjected to silica gel column chromatography (chloroform: methanol [ methanol]] = 20: 1) to obtain 18 (137 mg, 88%).
O-(2-アセタミド-3,4,6-トリ-O-アセチル-2-デオキシ-β-D-グルコピラノシル)-(1→2)-1,3,4,6-テトラ-O-アセチル-α-D-マンノピラノシド〔O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-1,3,4,6-tetra-O-acetyl-α-D-mannopyranoside 〕(20)
18 (1.13 g,1.46 mmol) のエタノール〔ethanol 〕(35 ml)-酢酸〔acetic acid 〕(12 ml) 溶液にパラジウム〔palladium〕（，10 % 活性炭〔activated carbon〕担持 (600 mg)） を加え水素気流下、50 °Cで一晩撹拌した。反応液を濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (クロロホルム〔chloroform 〕: メタノール〔methanol 〕= 15 : 2) で精製しO-(2-アセタミド-3,4,6-トリ-O-アセチル-2-デオキシ-β-D-グルコピラノシル)-(1→2)-4,6-ジ-O-アセチル-α-D-マンノピラノシド〔O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-4,6-di-O-acetyl-α-D-mannopyranoside 〕(19,740 mg,quant.) を得た。この19 (225 mg,0.38 mmol) のピリジン〔pyridine 〕(4 ml) 溶液に無水酢酸〔acetic anhydride 〕(2 ml) を加え室温で一晩撹拌した。反応終了後、溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (クロロホルム〔chloroform 〕: メタノール〔methanol 〕= 10 : 1) で精製し20 (257 mg,86 %) を得た。
O- (2-Acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -1,3,4,6-tetra-O-acetyl- α-D-Mannopyranoside (O- (2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -1,3,4,6- (tetra-O-acetyl-α-D-mannopyranoside) (20)
18 (1.13 g, 1.46 mmol) in ethanol (35 ml) -acetic acid (12 ml) solution was added palladium (palladium (10% activated carbon supported (600 mg)). The mixture was stirred overnight at 50 ° C. under a hydrogen stream. The reaction mixture was filtered and the solvent was distilled off. The resulting crude product was purified by silica gel column chromatography (chloroform: methanol = 15: 2) and purified by O- (2-acetamide-3, 4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -4,6-di-O-acetyl-α-D-mannopyranoside [O- (2-acetamido- 3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -4,6-di-O-acetyl-α-D-mannopyranoside] ( 19 , 740 mg , quant.). Acetic anhydride (2 ml) was added to a solution of 19 (225 mg, 0.38 mmol) in pyridine (pyridine) (4 ml), and the mixture was stirred overnight at room temperature. After completion of the reaction, the crude product obtained by distilling off the solvent was purified by silica gel column chromatography (chloroform: methanol = 10: 1) to obtain 20 (257 mg, 86%). .
O-(2-アセタミド-3,4,6-トリ-O-アセチル-2-デオキシ-β-D-グルコピラノシル)-(1→2)-3,4,6-トリ-O-アセチル-α-D-マンノピラノシル ブロミド〔O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl bromide 〕(21)
窒素気流下、20 (136 mg,0.20 mmol) の無水酢酸〔acetic anhydride 〕(1.5 ml) 溶液に臭化水素〔hydrogen bromide 〕(30 % 酢酸中〔in acetic acid〕,3 ml) を加え室温で撹拌した。4時間後、臭化水素〔hydrogen bromide 〕(30 % 酢酸中〔in acetic acid〕，1.5 ml) を更に追加し室温で一晩撹拌した。反応終了後、反応液を氷水に注ぎクロロホルム〔chloroform〕で抽出した。得られた有機層を蒸留水，飽和炭酸水素ナトリウム水溶液，蒸留水で洗浄後、乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して21 (137 mg,quant.) を得た。
O- (2-Acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -3,4,6-tri-O-acetyl-α- D-mannopyranosyl bromide (O- (2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -3,4,6-tri-O -acetyl-α-D-mannopyranosyl bromide] (21)
Under nitrogen flow, hydrogen bromide (30% in acetic acid, 3 ml) was added to 20 (136 mg, 0.20 mmol) acetic anhydride (1.5 ml) at room temperature. Stir. After 4 hours, hydrogen bromide (30% in acetic acid, 1.5 ml) was further added, and the mixture was stirred overnight at room temperature. After completion of the reaction, the reaction solution was poured into ice water and extracted with chloroform. The obtained organic layer was washed with distilled water, saturated aqueous sodium hydrogen carbonate solution and distilled water, dried (sodium sulfate), filtered and the solvent was distilled off to obtain 21 (137 mg, quant.). It was.
2-(4-ブロモフェニル)エチル2,3,4-トリ-O-アセチル-6-O-アリルオキシカルボニル-β-D-グルコピラノシド〔2-(4-Bromophenyl)ethyl 2,3,4-tri-O-acetyl-6-O-allyloxycarbonyl-β-D-glucopyranoside 〕(22)
2-(4-ブロモフェニル)エチルアルコール〔2-(4-Bromophenyl)ethyl alcohol 〕(197 μl,1.41 mmol)，13 (904 mg,1.69 mmol) のDCM (20 ml) 溶液に -20 °C下、ボロントリフルオリドジエチルエーテラート〔boron trifluoride diethyl etherate 〕(36 μl,0.28 mmol) を加え同温で2時間撹拌した。反応終了後、反応液を飽和炭酸水素ナトリウム水溶液に注ぎクロロホルム〔chloroform〕で抽出した。得られた有機層を乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (トルエン〔toluene 〕: ジエチルエーテル〔diethyl ether 〕= 3 : 1) で精製し22 (584 mg,72 %) を得た。
2- (4-Bromophenyl) ethyl 2,3,4-tri-O-acetyl-6-O-allyloxycarbonyl-β-D-glucopyranoside [2- (4-Bromophenyl) ethyl 2,3,4-tri -O-acetyl-6-O-allyloxycarbonyl-β-D-glucopyranoside) (22)
2- (4-Bromophenyl) ethyl alcohol [2- (4-Bromophenyl) ethyl alcohol] (197 μl, 1.41 mmol), 13 (904 mg, 1.69 mmol) in DCM (20 ml) at -20 ° C Boron trifluoride diethyl etherate (36 μl, 0.28 mmol) was added and stirred at the same temperature for 2 hours. After completion of the reaction, the reaction solution was poured into a saturated aqueous sodium hydrogen carbonate solution and extracted with chloroform. The obtained organic layer was dried (sodium sulfate), filtered, and then the solvent was distilled off. The resulting crude product was subjected to silica gel column chromatography (toluene: diethyl ether) = 3 : Purification by 1) gave 22 (584 mg, 72%).
2-(4-ブロモフェニル)エチル 2,3,4-トリ-O-アセチル-β-D-グルコピラノシド〔2-(4-Bromophenyl)ethyl 2,3,4-tri-O-acetyl-β-D-glucopyranoside 〕(23)
窒素気流下、22 (2.12 g,3.70 mmol) のTHF (30 ml) 溶液にテトラキス（トリフェニルホスフィン） パラジウム(0)〔tetrakis(triphenylphosphine) palladium(0) 〕(214 mg,0.18 mmol)，トリフェニルホスフィン〔triphenylphosphine 〕(291 mg,1.11 mmol)，ギ酸〔formic acid 〕(279 μl,7.39 mmol) を加え室温で6時間撹拌した。反応終了後、反応液を酢酸エチル〔ethyl acetate〕で希釈した後に蒸留水と飽和食塩水で洗浄後、乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン〔hexane 〕: 酢酸エチル〔ethyl acetate 〕= 1 : 1) で精製し23 (1.70 g,94 %) を得た。
2- (4-Bromophenyl) ethyl 2,3,4-tri-O-acetyl-β-D-glucopyranoside [2- (4-Bromophenyl) ethyl 2,3,4-tri-O-acetyl-β-D -glucopyranoside] (23)
Tetrakis (triphenylphosphine) palladium (0) (214 mg, 0.18 mmol), triphenyl in a solution of 22 (2.12 g, 3.70 mmol) in THF (30 ml) under nitrogen flow Phosphine [triphenylphosphine] (291 mg, 1.11 mmol) and formic acid [279 μl, 7.39 mmol] were added, and the mixture was stirred at room temperature for 6 hours. After completion of the reaction, the reaction solution was diluted with ethyl acetate, washed with distilled water and saturated brine, dried (sodium sulfate), filtered, and the crude solvent obtained by distilling off the solvent. The product was purified by silica gel column chromatography (hexane [hexane]: ethyl acetate = 1: 1) to obtain 23 (1.70 g, 94%).
2-(4-ブロモフェニル)エチル2,3,4-トリ-O-アセチル-6-O-1'-エトキシエチル-β-D-グルコピラノシド〔2-(4-Bromophenyl)ethyl 2,3,4-tri-O-acetyl-6-O-1'-ethoxyethyl-β-D-glucopyranoside 〕(24)
窒素気流下、23 (173 mg,0.35 mmol) のDCM (3 ml) 溶液にエチルビニルエーテル〔ethyl vinyl ether〕 (51 μl,0.53 mmol)，PPTS (9 mg,0.035 mmol) を加え室温で1時間撹拌した。反応終了後、溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン〔hexane 〕: 酢酸エチル〔ethyl acetate〕 = 2 : 1) で精製し24 (197 mg,99 %) を得た。
2- (4-Bromophenyl) ethyl 2,3,4-tri-O-acetyl-6-O-1'-ethoxyethyl-β-D-glucopyranoside [2- (4-Bromophenyl) ethyl 2,3,4 -tri-O-acetyl-6-O-1'-ethoxyethyl-β-D-glucopyranoside] (24)
Under a nitrogen stream, ethyl vinyl ether (51 μl, 0.53 mmol) and PPTS (9 mg, 0.035 mmol) were added to a DCM (3 ml) solution of 23 (173 mg, 0.35 mmol) and stirred at room temperature for 1 hour. did. After completion of the reaction, the crude product obtained by distilling off the solvent was purified by silica gel column chromatography (hexane [hexane]: ethyl acetate = 2: 1) to obtain 24 (197 mg, 99%). Obtained.
2-(4-トリブチルスタンニルフェニル)エチル2,3,4-トリ-O-アセチル-6-O-1'-エトキシエチル-β-D-グルコピラノシド〔2-(4-Tributylstannylphenyl)ethyl 2,3,4-tri-O-acetyl-6-O-1'-ethoxyethyl-β-D-glucopyranoside 〕(25)
窒素気流下、24 (161 mg,0.29 mmol) のDMF (5 ml) 溶液にテトラキス（トリフェニルホスフィン） パラジウム(0)〔tetrakis(triphenylphosphine) palladium(0) 〕(33 mg,0.029 mmol)，トリフェニルホスフィン〔triphenylphosphine 〕(15 mg,0.057 mmol)，ビス（トリブチルスズ）〔bis(tributyltin) 〕(289μl,0.57 mmol) を加え100 °Cで4時間撹拌した。反応終了後、溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン〔hexane 〕: 酢酸エチル〔ethyl acetate 〕= 5 : 1 to 2 : 1) で精製し25 (103 mg,47 %) を得た。
2- (4-Tributylstannylphenyl) ethyl 2,3,4-tri-O-acetyl-6-O-1'-ethoxyethyl-β-D-glucopyranoside [2- (4-Tributylstannylphenyl) ethyl 2,3 , 4-tri-O-acetyl-6-O-1'-ethoxyethyl-β-D-glucopyranoside] (25)
Tetrakis (triphenylphosphine) palladium (0) (33 mg, 0.029 mmol), triphenyl in a DMF (5 ml) solution of 24 (161 mg, 0.29 mmol) in a nitrogen stream Phosphine [triphenylphosphine] (15 mg, 0.057 mmol) and bis (tributyltin) (289 μl, 0.57 mmol) were added and stirred at 100 ° C. for 4 hours. After completion of the reaction, the crude product obtained by distilling off the solvent was purified by silica gel column chromatography (hexane [hexane]: ethyl acetate = 5: 1 to 2: 1) and 25 (103 mg, 47%).
2-(4-トリブチルスタンニルフェニル)エチル2,3,4-トリ-O-アセチル-β-D-グルコピラノシド〔2-(4-Tributylstannylphenyl)ethyl 2,3,4-tri-O-acetyl-β-D-glucopyranoside 〕(26)
25 (310 mg,0.40 mmol) のメタノール〔methanol 〕(10 ml) 溶液にPPTS (10 mg,0.040 mmol) を加え室温で2時間撹拌した。反応終了後、溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン〔hexane 〕: 酢酸エチル〔ethyl acetate 〕= 3 : 1 to 2 : 1) で精製し26 (268 mg,95 %) を得た。
2- (4-Tributylstannylphenyl) ethyl 2,3,4-tri-O-acetyl-β-D-glucopyranoside (2- (4-Tributylstannylphenyl) ethyl 2,3,4-tri-O-acetyl-β -D-glucopyranoside) (26)
PPTS (10 mg, 0.040 mmol) was added to a solution of 25 (310 mg, 0.40 mmol) in methanol [10 ml] and stirred at room temperature for 2 hours. After completion of the reaction, the crude product obtained by distilling off the solvent was purified by silica gel column chromatography (hexane [hexane]: ethyl acetate = 3: 1 to 2: 1) to obtain 26 (268 mg, 95%).
2-(4-ヨードフェニル)エチル2,3,4-トリ-O-アセチル-6-O-1'-エトキシエチル-β-D-グルコピラノシド〔2-(4-Iodophenyl)ethyl 2,3,4-tri-O-acetyl-6-O-1'-ethoxyethyl-β-D-glucopyranoside 〕(27)
窒素気流下、25 (305 mg,0.40 mmol) のDCM (10 ml) 溶液に氷冷下、ヨウ素〔iodine 〕(201 mg,0.79 mmol) を加え同温で2時間撹拌した。反応終了後、反応液を飽和炭酸水素ナトリウム-チオ硫酸ナトリウム水溶液に注ぎクロロホルム〔chloroform〕で抽出した。得られた有機層を乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (トルエン〔toluene 〕: ジエチルエーテル〔diethyl ether 〕= 3 : 1) で精製し27 (226 mg,94 %) を得た。
2- (4-Iodophenyl) ethyl 2,3,4-tri-O-acetyl-6-O-1'-ethoxyethyl-β-D-glucopyranoside [2- (4-Iodophenyl) ethyl 2,3,4 -tri-O-acetyl-6-O-1'-ethoxyethyl-β-D-glucopyranoside] (27)
Under a nitrogen stream, iodine (iodine) (201 mg, 0.79 mmol) was added to a DCM (10 ml) solution of 25 (305 mg, 0.40 mmol) under ice cooling, and the mixture was stirred at the same temperature for 2 hours. After completion of the reaction, the reaction solution was poured into a saturated aqueous solution of sodium bicarbonate-sodium thiosulfate and extracted with chloroform. The obtained organic layer was dried (sodium sulfate), filtered, and then the solvent was distilled off. The resulting crude product was subjected to silica gel column chromatography (toluene: diethyl ether) = 3 : Purification by 1) gave 27 (226 mg, 94%).
2-(4-ヨードフェニル)エチル2,3,4-トリ-O-アセチル-β-D-グルコピラノシド〔2-(4-Iodophenyl)ethyl 2,3,4-tri-O-acetyl-β-D-glucopyranoside 〕(28)
27 (222 mg,0.41 mmol) のメタノール〔methanol 〕(10 ml) 溶液にPPTS (10 mg,0.036 mmol) を加え55 °Cで2時間撹拌した。反応終了後、溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン〔hexane 〕: 酢酸エチル〔ethyl acetate 〕= 1 : 1) で精製し28 (192 mg,98 %) を得た。
2- (4-Iodophenyl) ethyl 2,3,4-tri-O-acetyl-β-D -glucopyranoside) (28)
PPTS (10 mg, 0.036 mmol) was added to a methanol (methanol) (10 ml) solution of 27 (222 mg, 0.41 mmol), and the mixture was stirred at 55 ° C for 2 hours. After completion of the reaction, the crude product obtained by distilling off the solvent was purified by silica gel column chromatography (hexane [hexane]: ethyl acetate = 1: 1) to obtain 28 (192 mg, 98%). Obtained.
2-(4-トリブチルスタンニルフェニル)エチル-O-(2-アセタミド-3,4,6-トリ-O-アセチル-2-デオキシ-β-D-グルコピラノシル)-(1→2)-O-(3,4,6-トリ-O-アセチル-α-D-マンノピラノシル)-(1→6)-2,3,4-トリ-O-アセチル-β-D-グルコピラノシド〔2-(4-Tributylstannylphenyl)ethyl-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-D-glucopyranoside 〕(29)
窒素気流下、26 (30 mg,0.043 mmol) のDCM (1 ml)-トルエン〔toluene 〕(1 ml) 溶液にγ-コリジン〔collidine 〕(14 μl,0.10 mmol) を加え室温で20分撹拌した。反応液に過塩素酸銀〔silver perchlorate 〕(11 mg,0.052 mmol) を加え室温で20分撹拌した。反応液に21 (36 mg,0.052 mmol) のDCM (2 ml)-トルエン〔toluene 〕(2 ml) 溶液を加え室温で5時間撹拌した。反応終了後、反応液を濾過した (クロロホルム〔chloroform〕)。得られた濾液を1 % HClで洗浄後、乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (酢酸エチル〔ethyl acetate〕) で精製し29 (24 mg,43 %) を得た。
2- (4-Tributylstannylphenyl) ethyl-O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -O- (3,4,6-Tri-O-acetyl-α-D-mannopyranosyl)-(1 → 6) -2,3,4-tri-O-acetyl-β-D-glucopyranoside [2- (4-Tributylstannylphenyl ) ethyl-O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -O- (3,4,6-tri- O-acetyl-α-D-mannopyranosyl)-(1 → 6) -2,3,4-tri-O-acetyl-β-D-glucopyranoside] (29)
Under a nitrogen stream, γ-collidine (14 μl, 0.10 mmol) was added to a solution of DCM (1 ml) -toluene (1 ml) in 26 (30 mg, 0.043 mmol) and stirred at room temperature for 20 minutes. . Silver perchlorate (11 mg, 0.052 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 20 minutes. A solution of 21 (36 mg, 0.052 mmol) in DCM (2 ml) -toluene [toluene] (2 ml) was added to the reaction solution and stirred at room temperature for 5 hours. After completion of the reaction, the reaction solution was filtered (chloroform). The obtained filtrate was washed with 1% HCl, dried (sodium sulfate), filtered, and the crude product obtained by distilling off the solvent was subjected to silica gel column chromatography (ethyl acetate). To obtain 29 (24 mg, 43%).
2-(4-ヨードフェニル)エチル-O-(2-アセタミド-3,4,6-トリ-O-アセチル-2-デオキシ-β-D-グルコピラノシル)-(1→2)-O-(3,4,6-トリ-O-アセチル-α-D-マンノピラノシル)-(1→6)-2,3,4-トリ-O-アセチル-β-D-グルコピラノシド〔2-(4-Iodophenyl)ethyl-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-D-glucopyranoside 〕(30)
窒素気流下、28 (214 mg,0.40 mmol) のDCM (10 ml)-トルエン〔toluene 〕(10 ml) 溶液に炭酸銀〔silver carbonate 〕(121 mg,0.44 mmol) を加え室温で1時間撹拌した。反応液に過塩素酸銀〔silver perchlorate 〕(12 mg,0.060 mmol) を加え室温で20分撹拌した。反応液に21 (334 mg,0.48 mmol) のDCM (10 ml)-トルエン〔toluene 〕(10 ml) 溶液を加え室温で15時間撹拌した。反応終了後、反応液を濾過した (クロロホルム〔chloroform〕) 後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン)〔hexane 〕: 酢酸エチル〔ethyl acetate 〕= 4 : 1 to 酢酸エチルのみ〔ethyl acetate only〕) で精製し30 (310 mg,67 %) を得た。
2- (4-Iodophenyl) ethyl-O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -O- (3 , 4,6-Tri-O-acetyl-α-D-mannopyranosyl)-(1 → 6) -2,3,4-tri-O-acetyl-β-D-glucopyranoside [2- (4-Iodophenyl) ethyl -O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -O- (3,4,6-tri-O- acetyl-α-D-mannopyranosyl)-(1 → 6) -2,3,4-tri-O-acetyl-β-D-glucopyranoside) (30)
Under nitrogen stream, 28 (214 mg, 0.40 mmol) in DCM (10 ml) -toluene (10 ml) solution was added with silver carbonate (121 mg, 0.44 mmol) and stirred at room temperature for 1 hour. . Silver perchlorate (12 mg, 0.060 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 20 minutes. A solution of 21 (334 mg, 0.48 mmol) in DCM (10 ml) -toluene (10 ml) was added to the reaction solution and stirred at room temperature for 15 hours. After completion of the reaction, the reaction solution was filtered (chloroform), and then the solvent was distilled off. The resulting crude product was subjected to silica gel column chromatography (hexane): ethyl acetate = 4: 1 to ethyl acetate only) to obtain 30 (310 mg, 67%).
2-(4-ヨードフェニル)エチル-O-(2-アセタミド-2-デオキシ-β-D-グルコピラノシル)-(1→2)-O-α-D-マンノピラノシル-(1→6)-β-D-グルコピラノシド〔2-(4-Iodophenyl)ethyl-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-O-α-D-mannopyranosyl-(1→6)-β-D-glucopyranoside 〕(32)
窒素気流下、30 (188 mg,0.16 mmol) のメタノール〔methanol 〕(10 ml) 溶液にナトリウムメトキシド〔sodium methoxide 〕(28 % メタノール中〔in methanol〕，332 μl,1.63 mmol) を加え室温で2時間撹拌した。反応終了後、反応液をAmberlyst（登録商標）15 (1.0 g) で中和した後、得られた粗生成物をリサイクル〔recycle〕型逆相HPLC (メタノール〔methanol 〕: 蒸留水〔distilled water 〕= 7 : 3) で精製し32 (74 mg,58 %) を得た。
2- (4-Iodophenyl) ethyl-O- (2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -O-α-D-mannopyranosyl- (1 → 6) -β- D-glucopyranoside (2- (4-Iodophenyl) ethyl-O- (2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1 → 2) -O-α-D-mannopyranosyl- (1 → 6) -β-D-glucopyranoside) (32)
Under nitrogen flow, sodium methoxide (28% in methanol, 332 μl, 1.63 mmol) was added to 30 (188 mg, 0.16 mmol) in methanol (10 ml) at room temperature. Stir for 2 hours. After completion of the reaction, the reaction solution was neutralized with Amberlyst (registered trademark) 15 (1.0 g), and the obtained crude product was recycled (recycle) reverse phase HPLC (methanol: distilled water). = 7: 3) to obtain 32 (74 mg, 58%).
32: 無色無定形； 1H-NMR (300 MHz,CD3OD) δ: 1.99 (s,3H),2.89-2.92 (m,2H),3.16-4.04 (m,21H),4.28 (d,J = 7.3 Hz,1H),4.46 (d,J = 7.7 Hz,1H),7.06-7.09 (m,2H),7.59-7.62 (m,2H)； MS FAB(+) m/z 776 [(M+H)+,10]； HRMS calcd for C28H43O16NI [(M+H)+] 776.1627,found: 776.1635. 32 : colorless amorphous; 1 H-NMR (300 MHz, CD 3 OD) δ: 1.99 (s, 3H), 2.89-2.92 (m, 2H), 3.16-4.04 (m, 21H), 4.28 (d, J = 7.3 Hz, 1H), 4.46 (d, J = 7.7 Hz, 1H), 7.06-7.09 (m, 2H), 7.59-7.62 (m, 2H); MS FAB (+) m / z 776 [(M + H) + , 10]; HRMS calcd for C 28 H 43 O 16 NI [(M + H) + ] 776.1627, found: 776.1635.
IPGGMG (GnT-Ｖにより生成する化合物)の合成
ベンジル-O-(3,4,6-トリ-O-アセチル-2-デオキシ-2-フタルイミド-β-D-グルコピラノシル)-(1→2)-3-O-ベンジル-α-D-マンノピラノシド〔Benzyl-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→2)-3-O-benzyl-α-D-mannopyranoside 〕(33)
14 (1.45 g,1.67 mmol) に70 % 酢酸〔acetic acid〕水溶液 (10 ml) を加え55 °Cで2.5時間撹拌した。反応終了後、反応液を氷水に注ぎ炭酸水素ナトリウムで中和した後、クロロホルム〔chloroform〕で抽出した。得られた有機層を飽和炭酸水素ナトリウム水溶液で洗浄後、乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (クロロホルム〔chloroform 〕: メタノール〔methanol 〕= 20 : 1) で精製し33 (1.07 g,83 %) を得た。
Synthesis of IPGGMG (compound produced by GnT-V)
Benzyl-O- (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimide-β-D-glucopyranosyl)-(1 → 2) -3-O-benzyl-α-D-mannopyranoside Benzyl-O- (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1 → 2) -3-O-benzyl-α-D-mannopyranoside) (33)
To 14 (1.45 g, 1.67 mmol) was added 70% acetic acid aqueous solution (10 ml), and the mixture was stirred at 55 ° C for 2.5 hours. After completion of the reaction, the reaction solution was poured into ice water, neutralized with sodium hydrogen carbonate, and extracted with chloroform. The obtained organic layer was washed with a saturated aqueous solution of sodium bicarbonate, dried (sodium sulfate), filtered, and the solvent was distilled off to obtain a crude product, which was subjected to silica gel column chromatography (chloroform). : Methanol [methanol] = 20: 1) to obtain 33 (1.07 g, 83%).
ベンジル-O-(3,4,6-トリ-O-アセチル-2-デオキシ-2-フタルイミド-β-D-グルコピラノシル)-(1→2)-3-O-ベンジル-6-O-tert-ブチルジメチルシリル-α-D-マンノピラノシド〔Benzyl-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→2)-3-O-benzyl-6-O-tert-butyldimethylsilyl-α-D-mannopyranoside 〕(34)
窒素気流下、33 (1.07 g,1.38 mmol) のDMF (10 ml) 溶液にtert-ブチルジメチルシリルクロリド〔tert-butyldimethylsilyl chloride 〕(311 mg,2.06 mmol)，イミダゾール〔imidazole 〕(281 mg,4.13 mmol) 加え、室温で16時間撹拌した。反応終了後、反応液を飽和塩化アンモニウム水溶液に注ぎ酢酸エチル〔ethyl acetate〕で抽出した。得られた有機層を蒸留水で洗浄し、乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン〔hexane 〕: 酢酸エチル〔ethyl acetate 〕= 2 : 1) で精製し34 (1.15 g,94 %)を得た。
Benzyl-O- (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimide-β-D-glucopyranosyl)-(1 → 2) -3-O-benzyl-6-O-tert- Butyldimethylsilyl-α-D-mannopyranoside (Benzyl-O- (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1 → 2) -3- O-benzyl-6-O-tert-butyldimethylsilyl-α-D-mannopyranoside) (34)
Under a nitrogen stream, tert-butyldimethylsilyl chloride (tert-butyldimethylsilyl chloride) (311 mg, 2.06 mmol), imidazole (imidazole) (281 mg, 4.13 mmol) was added to a solution of 33 (1.07 g, 1.38 mmol) in DMF (10 ml). ) And stirred at room temperature for 16 hours. After completion of the reaction, the reaction solution was poured into a saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The obtained organic layer was washed with distilled water, dried (sodium sulfate), filtered, and then the solvent was distilled off. The resulting crude product was subjected to silica gel column chromatography (hexane [hexane]: ethyl acetate). Purification with [ethyl acetate] = 2: 1) gave 34 (1.15 g, 94%).
ベンジル-O-(3,4,6-トリ-O-アセチル-2-デオキシ-2-フタルイミド-β-D-グルコピラノシル)-(1→2)-4-O-アセチル-3-O-ベンジル-6-O-tert-ブチルジメチルシリル-α-D-マンノピラノシド〔Benzyl-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→2)-4-O-acetyl-3-O-benzyl-6-O-tert-butyldimethylsilyl-α-D-mannopyranoside 〕(35)
34 (1.14 g,1.28 mmol) のピリジン〔pyridine〕 (5 ml) 溶液に無水酢酸〔acetic anhydride 〕(5 ml) を加え室温で2時間撹拌した。反応終了後、氷冷下、反応液にmethanolを加え溶媒を減圧で留去した。得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン〔hexane 〕: 酢酸エチル〔ethyl acetate 〕= 2 : 1) で精製し35 (1.18 g,99 %)を得た。
Benzyl-O- (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimide-β-D-glucopyranosyl)-(1 → 2) -4-O-acetyl-3-O-benzyl- 6-O-tert-butyldimethylsilyl-α-D-mannopyranoside (Benzyl-O- (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1 → 2) -4-O-acetyl-3-O-benzyl-6-O-tert-butyldimethylsilyl-α-D-mannopyranoside] (35)
Acetic anhydride (5 ml) was added to a solution of 34 (1.14 g, 1.28 mmol) in pyridine (pyridine) (5 ml), and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, methanol was added to the reaction solution under ice cooling, and the solvent was distilled off under reduced pressure. The obtained crude product was purified by silica gel column chromatography (hexane [hexane]: ethyl acetate = 2: 1) to obtain 35 (1.18 g, 99%).
ベンジル-O-(3,4,6-トリ-O-アセチル-2-デオキシ-2-フタルイミド-β-D-グルコピラノシル)-(1→2)-4-O-アセチル-3-O-ベンジル-α-D-マンノピラノシド〔Benzyl-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→2)-4-O-acetyl-3-O-benzyl-α-D-mannopyranoside 〕(36)
35 (1.17 g,1.25 mmol) のメタノール〔methanol〕 (10 ml) 溶液にPPTS (32 mg,0.13 mmol) を加え50 °Cで2時間撹拌した。反応終了後、溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (ヘキサン〔hexane 〕: 酢酸エチル〔ethyl acetate 〕= 1 : 1) で精製し36 (765 mg,75 %) を得た。
Benzyl-O- (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimide-β-D-glucopyranosyl)-(1 → 2) -4-O-acetyl-3-O-benzyl- α-D-Mannopyranoside (Benzyl-O- (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1 → 2) -4-O-acetyl- 3-O-benzyl-α-D-mannopyranoside) (36)
PPTS (32 mg, 0.13 mmol) was added to a solution of 35 (1.17 g, 1.25 mmol) in methanol (10 ml), and the mixture was stirred at 50 ° C. for 2 hours. After completion of the reaction, the crude product obtained by distilling off the solvent was purified by silica gel column chromatography (hexane [hexane]: ethyl acetate = 1: 1) to obtain 36 (765 mg, 75%). Obtained.
ベンジル-O-(3,4,6-トリ-O-アセチル-2-デオキシ-2-フタルイミド-β-D-グルコピラノシル)-(1→2)-[O-3,4,6-トリ-O-アセチル-2-デオキシ-2-フタルイミド-β-D-グルコピラノシル-(1→6)]-4-O-アセチル-3-ベンジル-α-D-マンノピラノシド〔Benzyl-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→2)-[O-3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→6)]-4-O-acetyl-3-benzyl-α-D-mannopyranoside 〕(37)
上記スキーム（Scheme 7）に従って合成した。
窒素気流下、36 (564 mg,0.69 mmol)，銀トリフルオロメタンスルホナート〔silver trifluoromethanesulfonate〕 (318 mg,1.24 mmol)，γ-コリジン〔collidine 〕(164μl,1.24 mmol) のニトロメタン〔nitromethane 〕(5 ml) 溶液に -20 °C下、5 (514 mg,1.03 mmol) のニトロメタン〔nitromethane 〕(5 ml) 溶液を加え同温で30分撹拌した後、室温で更に2時間撹拌した。反応終了後、Celite（登録商標）を用いて反応液を濾過した後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (1st； ヘキサン〔hexane 〕: 酢酸エチル〔ethyl acetate 〕= 1 : 1,2nd； クロロホルム〔chloroform 〕: メタノール〔methanol 〕= 20 : 1) で精製し37 (337 mg,40 %) を得た。
Benzyl-O- (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimide-β-D-glucopyranosyl)-(1 → 2)-[O-3,4,6-tri-O -Acetyl-2-deoxy-2-phthalimide-β-D-glucopyranosyl- (1 → 6)]-4-O-acetyl-3-benzyl-α-D-mannopyranoside [Benzyl-O- (3,4,6 -tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1 → 2)-[O-3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido -β-D-glucopyranosyl- (1 → 6)]-4-O-acetyl-3-benzyl-α-D-mannopyranoside] (37)
Synthesized according to the above scheme (Scheme 7).
Under nitrogen flow, 36 (564 mg, 0.69 mmol), silver trifluoromethanesulfonate (318 mg, 1.24 mmol), γ-collidine (164 μl, 1.24 mmol) of nitromethane (5 ml) ) A solution of 5 (514 mg, 1.03 mmol) of nitromethane (5 ml) was added to the solution at −20 ° C., and the mixture was stirred at the same temperature for 30 minutes, and further stirred at room temperature for 2 hours. After completion of the reaction, the reaction mixture was filtered using Celite (registered trademark), and then the solvent was distilled off. The resulting crude product was subjected to silica gel column chromatography (1 st ; hexane [hexane]: ethyl acetate [ethyl acetate]). = 1: 1,2 nd ; Chloroform [chloroform]: Methanol [methanol] = 20: 1) Purification was performed to obtain 37 (337 mg, 40%).
ベンジル-O-(2-アセタミド-3,4,6-トリ-O-アセチル-2-デオキシ-β-D-グルコピラノシル)-(1→2)-[O-2-アセタミド-3,4,6-トリ-O-アセチル-2-デオキシ-β-D-グルコピラノシル-(1→6)]-4-O-アセチル-3-O-ベンジル-α-D-マンノピラノシド〔Benzyl-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-[O-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl-(1→6)]-4-O-acetyl-3-O-benzyl-α-D-mannopyranoside 〕(38)
37 (157 mg,0.13 mmol) のエタノール〔ethanol 〕(10 ml) 溶液にヒドラジン水和物〔hydrazine・hydrate 〕(80 % in water,200 μl) を加え4時間還流した。反応終了後、溶媒を留去して得られた粗生成物のピリジン〔pyridine 〕(2 ml) 溶液に無水酢酸〔acetic anhydride 〕(2 ml) を加え室温で一晩撹拌した。反応終了後、反応液にメタノール〔methanol〕を加え溶媒を減圧で留去した。得られた粗生成物をシリカゲルカラムクロマトグラフィー (クロロホルム〔chloroform 〕: メタノール〔methanol 〕= 20 : 1) で精製し38 (125 mg,93 %) を得た。
Benzyl-O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2)-[O-2-acetamido-3,4,6 -Tri-O-acetyl-2-deoxy-β-D-glucopyranosyl- (1 → 6)]-4-O-acetyl-3-O-benzyl-α-D-mannopyranoside [Benzyl-O- (2-acetamido -3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2)-[O-2-acetamido-3,4,6-tri-O-acetyl-2 -deoxy-β-D-glucopyranosyl- (1 → 6)]-4-O-acetyl-3-O-benzyl-α-D-mannopyranoside] (38)
To a solution of 37 (157 mg, 0.13 mmol) in ethanol (10 ml) was added hydrazine hydrate (80% in water, 200 μl) and refluxed for 4 hours. After completion of the reaction, acetic anhydride (acetic anhydride) (2 ml) was added to a pyridine (pyridine) (2 ml) solution of the crude product obtained by distilling off the solvent and stirred overnight at room temperature. After completion of the reaction, methanol was added to the reaction solution and the solvent was distilled off under reduced pressure. The obtained crude product was purified by silica gel column chromatography (chloroform: methanol = 20: 1) to obtain 38 (125 mg, 93%).
O-(2-アセタミド-3,4,6-トリ-O-アセチル-2-デオキシ-β-D-グルコピラノシル)-(1→2)-[O-2-アセタミド-3,4,6-トリ-O-アセチル-2-デオキシ-β-D-グルコピラノシル-(1→6)]-1,3,4-トリ-O-アセチル-α-D-マンノピラノシド〔O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-[O-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl-(1→6)]-1,3,4-tri-O-acetyl-α-D-mannopyranoside 〕(39)
38 (118 mg,0.11 mmol) のエタノール〔ethanol 〕(10 ml) 溶液にパラジウム〔palladium〕,の10 % 活性炭担持物〔on activated carbon 〕(100 mg)，ギ酸アンモニウム〔ammonium formate 〕(140 mg,過剰〔excess〕) を加え一晩還流した。反応終了後、Celite（登録商標）を用いて反応液を濾過した後に溶媒を留去して得られた粗生成物のピリジン〔pyridine 〕(2 ml) 溶液に無水酢酸〔acetic anhydride 〕(2 ml) を加え室温で一晩撹拌した。反応終了後、氷冷下、反応液にメタノール〔methanol〕を加え溶媒を減圧で留去した。得られた粗生成物をシリカゲルカラムクロマトグラフィー (クロロホルム〔chloroform 〕: メタノール〔methanol 〕= 20 : 1) で精製し39 (78 mg,72 %) を得た。
O- (2-Acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2)-[O-2-acetamide-3,4,6-tri -O-acetyl-2-deoxy-β-D-glucopyranosyl- (1 → 6)]-1,3,4-tri-O-acetyl-α-D-mannopyranoside [O- (2-Acetamido-3,4 , 6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2)-(O-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β -D-glucopyranosyl- (1 → 6)]-1,3,4-tri-O-acetyl-α-D-mannopyranoside] (39)
38 (118 mg, 0.11 mmol) in ethanol (10 ml) in palladium (palladium), 10% activated carbon (100 mg), ammonium formate (140 mg, 140 mg, Excess) was added and refluxed overnight. After completion of the reaction, the reaction solution was filtered using Celite (registered trademark), and then the solvent was distilled off. The resulting crude product in pyridine (pyridine) (2 ml) solution was added with acetic anhydride (2 ml). ) Was added and stirred at room temperature overnight. After completion of the reaction, methanol was added to the reaction solution under ice cooling, and the solvent was distilled off under reduced pressure. The obtained crude product was purified by silica gel column chromatography (chloroform: methanol = 20: 1) to obtain 39 (78 mg, 72%).
2-(4-ヨードフェニル)エチル-O-(2-アセタミド-3,4,6-トリ-O-アセチル-2-デオキシ-β-D-グルコピラノシル)-(1→2)-[O-2-アセタミド-3,4,6-トリ-O-アセチル-2-デオキシ-β-D-グルコピラノシル-(1→6)]-O-(3,4-ジ-O-アセチル-α-D-マンノピラノシル)-(1→6)-2,3,4-トリ-O-アセチル-β-D-グルコピラノシド〔2-(4-Iodophenyl)ethyl-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-[O-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl-(1→6)]-O-(3,4-di-O-acetyl-α-D-mannopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-D-glucopyranoside 〕(41)
窒素気流下、39 (70 mg,0.073 mmol) の無水酢酸〔acetic anhydride 〕(1.5 ml) 溶液に臭化水素〔hydrogen bromide 〕(30 % in acetic acid,3 ml) を加え室温で一晩撹拌した。反応終了後、反応液を氷水に注ぎクロロホルム〔chloroform〕で抽出した。得られた有機層を蒸留水，飽和炭酸水素ナトリウム水溶液で洗浄後、乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して2-(4-ヨードフェニル)エチルO-(2-アセタミド-3,4,6-トリ-O- アセチル-2-デオキシ-β-D-グルコピラノシル)-(1→2)-[O-2-アセタミド-3,4,6-トリ-O-アセチル -2-デオキシ-β-D-グルコピラノシル-(1→6)]-O-(3,4-ジ-O-アセチル-α-D-マンノピラノシル)-(1→6)-2,3,4-トリ-O-アセチル-β-D-グルコピラノシル ブロミド〔2-(4-iodophenyl)ethyl O-(2-acetamido-3,4,6-tri-O- acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-[O-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl-(1→6)]-O-(3,4-di-O-acetyl-α-D-mannopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-D-glucopyranosyl bromide 〕(40) の粗生成物 (67 mg)を得た。窒素気流下、28 (30 mg,0.055 mmol) のDCM (1 ml)-トルエン〔toluene 〕(1 ml) 溶液に炭酸銀〔silver carbonate 〕(17 mg,0.06 mmol) を加え室温で1時間撹拌した。反応液に過塩素酸銀〔silver perchlorate〕 (2 mg,0.0082 mmol) を加え室温で30分間撹拌した。反応液に40 (65 mg,0.066 mmol) のDCM (2 ml)-トルエン〔toluene 〕(2 ml) 溶液を加え室温で15時間撹拌した。反応終了後、反応液を濾過した (クロロホルム〔chloroform〕) 後に溶媒を留去して得られた粗生成物をシリカゲルカラムクロマトグラフィー (クロロホルム〔chloroform 〕: メタノール〔methanol 〕= 20 : 1) で精製し41 (5 mg,7 %) を得た。
2- (4-Iodophenyl) ethyl-O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2)-[O-2 -Acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl- (1 → 6)]-O- (3,4-di-O-acetyl-α-D-mannopyranosyl )-(1 → 6) -2,3,4-Tri-O-acetyl-β-D-glucopyranoside [2- (4-Iodophenyl) ethyl-O- (2-acetamido-3,4,6-tri- O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2)-[O-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl- (1 → 6)]-O- (3,4-di-O-acetyl-α-D-mannopyranosyl)-(1 → 6) -2,3,4-tri-O-acetyl-β-D-glucopyranoside (41)
Under a nitrogen stream, hydrogen bromide (30% in acetic acid, 3 ml) was added to a solution of acetic anhydride (acetic anhydride) (1.5 ml) in 39 (70 mg, 0.073 mmol) and stirred overnight at room temperature. . After completion of the reaction, the reaction solution was poured into ice water and extracted with chloroform. The obtained organic layer was washed with distilled water and saturated aqueous sodium hydrogen carbonate solution, dried (sodium sulfate), filtered, and then the solvent was distilled off to give 2- (4-iodophenyl) ethyl O- (2 -Acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1 → 2)-[O-2-acetamide-3,4,6-tri-O-acetyl -2-deoxy-β-D-glucopyranosyl- (1 → 6)]-O- (3,4-di-O-acetyl-α-D-mannopyranosyl)-(1 → 6) -2,3,4- Tri-O-acetyl-β-D-glucopyranosyl bromide (2- (4-iodophenyl) ethyl O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl) -(1 → 2)-[O-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl- (1 → 6)]-O- (3,4- di-O-acetyl-α-D-mannopyranosyl)-(1 → 6) -2,3,4-tri-O-acetyl-β-D-glucopyranosyl bromide) ( 40 ) Obtained. Under a nitrogen stream, silver carbonate (17 mg, 0.06 mmol) was added to a DCM (1 ml) -toluene (1 ml) solution of 28 (30 mg, 0.055 mmol) and stirred at room temperature for 1 hour. . Silver perchlorate (2 mg, 0.0082 mmol) was added to the reaction solution and stirred at room temperature for 30 minutes. A solution of 40 (65 mg, 0.066 mmol) in DCM (2 ml) -toluene [toluene] (2 ml) was added to the reaction solution, and the mixture was stirred at room temperature for 15 hours. After completion of the reaction, the reaction solution was filtered (chloroform), and then the solvent was distilled off. The resulting crude product was purified by silica gel column chromatography (chloroform: methanol = 20: 1). 41 (5 mg, 7%) was obtained.
41: 無色結晶； 1H-NMR (400 MHz,CDCl3)δ: 1.87 (s,3H),1.94 (s,3H),1.96 (s,3H),1.97 (s,3H),1.99 (s,3H),2.00 (s,3H),2.03 (s,3H),2.04 (s,3H),2.05 (s,3H),2.06 (s,3H),2.08 (s,3H),2.09 (s,3H),2.09 (s,3H),2.82-2.96 (m,3H),3.06-3.09 (m,1H),3.55-3.58 (m,1H),3.63-3.73 (m,5H),3.77-3.82 (m,1H),3.98-4.01 (m,1H),4.09-4.32 (m,8H),4.45 (d,J = 8.1 Hz,1H),4.82 (d,J = 1.5 Hz,1H),4.88-4.98 (m,3H),5.02-5.10 (m,3H),5.17 (t,J = 9.5 Hz,1H),5.35 (t,J = 9.6 Hz,1H),5.38 (d,J = 8.2 Hz,1H),5.92 (dd,J = 10.8,9.2 Hz,1H),6.43 (d,J = 9.9 Hz,1H),6.95-6.97 (m,2H),7.30 (d,J = 7.0 Hz,1H),7.56-7.59 (m,2H)； MS FAB(+) m/z 1463 [(M+Na)+,5]； HRMS calcd for C58H77N2O32NaI [(M+Na)+] 1463.3402,found: 1463.3394. 41 : colorless crystals; 1 H-NMR (400 MHz, CDCl 3 ) δ: 1.87 (s, 3H), 1.94 (s, 3H), 1.96 (s, 3H), 1.97 (s, 3H), 1.99 (s, 3H), 2.00 (s, 3H), 2.03 (s, 3H), 2.04 (s, 3H), 2.05 (s, 3H), 2.06 (s, 3H), 2.08 (s, 3H), 2.09 (s, 3H ), 2.09 (s, 3H), 2.82-2.96 (m, 3H), 3.06-3.09 (m, 1H), 3.55-3.58 (m, 1H), 3.63-3.73 (m, 5H), 3.77-3.82 (m , 1H), 3.98-4.01 (m, 1H), 4.09-4.32 (m, 8H), 4.45 (d, J = 8.1 Hz, 1H), 4.82 (d, J = 1.5 Hz, 1H), 4.88-4.98 ( m, 3H), 5.02-5.10 (m, 3H), 5.17 (t, J = 9.5 Hz, 1H), 5.35 (t, J = 9.6 Hz, 1H), 5.38 (d, J = 8.2 Hz, 1H), 5.92 (dd, J = 10.8,9.2 Hz, 1H), 6.43 (d, J = 9.9 Hz, 1H), 6.95-6.97 (m, 2H), 7.30 (d, J = 7.0 Hz, 1H), 7.56-7.59 (m, 2H); MS FAB (+) m / z 1463 [(M + Na) + , 5]; HRMS calcd for C 58 H 77 N 2 O 32 NaI [(M + Na) + ] 1463.3402, found: 1463.3394.
1-ヨード-4-(トリメチルシリル)ベンゼン〔1-Iodo-4-(trimethylsilyl)benzene 〕(42)
窒素気流下、1,4-ジヨードベンゼン〔1,4-diiodobenzene 〕(10.0 g,30.4 mmol) のTHF (150 ml) 溶液に -78 °C下、n-ブチル リチウム〔n-butyl lithium 〕(1.57 M ヘキサン溶液中〔in hexane solution〕,20.4 ml,32.0 mmol) を滴下し、同温で30分間撹拌した。反応液にクロロトリメチルシラン〔chlorotrimethylsilane 〕(4.64 ml,36.5 mmol) を滴下し、同温で2.5時間撹拌した。反応終了後、反応液を飽和塩化アンモニウム水溶液に注ぎ、酢酸エチル〔ethyl acetate〕で抽出した。得られた有機層を飽和チオ硫酸ナトリウム水溶液，飽和食塩水で洗浄後、乾燥 (硫酸ナトリウム〔sodium sulfate〕)、濾過した後に溶媒を留去して得られた粗生成物を減圧蒸留 (86 °C / 100 Pa) で精製し42 (7.29 g,87 %) を得た。
1-Iodo-4- (trimethylsilyl) benzene (1-Iodo-4- (trimethylsilyl) benzene) (42)
Under a nitrogen stream, a solution of 1,4-diiodobenzene (1,4-diiodobenzene) (10.0 g, 30.4 mmol) in THF (150 ml) at -78 ° C under n-butyl lithium (n-butyl lithium) ( 1.57 M in hexane solution, 20.4 ml, 32.0 mmol) was added dropwise and stirred at the same temperature for 30 minutes. Chlorotrimethylsilane (4.64 ml, 36.5 mmol) was added dropwise to the reaction solution, followed by stirring at the same temperature for 2.5 hours. After completion of the reaction, the reaction solution was poured into a saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The obtained organic layer was washed with a saturated aqueous solution of sodium thiosulfate and saturated brine, dried (sodium sulfate), filtered, and the solvent was distilled off to obtain a crude product obtained by distillation under reduced pressure (86 ° C). C / 100 Pa) to obtain 42 (7.29 g, 87%).
42: 無色油状； 1H-NMR (300 MHz,CDCl3) δ: 0.25 (s,9H),7.22-7.25 (m,2H),7.67-7.70 (m,2H). 42 : colorless oil; 1 H-NMR (300 MHz, CDCl 3 ) δ: 0.25 (s, 9H), 7.22-7.25 (m, 2H), 7.67-7.70 (m, 2H).
放射性ヨウ素標識IPGMGの合成Synthesis of radioiodine labeled IPGMG
有機スズ前駆体のメタノール溶液中に、10μL 5%H2O2、10μL 0.1N HClを加えた後、Na125Iを添加、室温で30分間反応させた。その後、飽和炭酸水素ナトリウム水溶液で中和し、酢酸エチルで抽出、酢酸エチルを減圧留去した後、ナトリウムメチラートによりアセチル基を脱保護し、Amberlystで中和した。その後、逆相HPLCにて分離精製を行った。（カラム；Cosmosil 5C18-AR-300,4.6×150 mm、溶出液；アセトニトリル：水=17：83、流速；1 ml/min） After adding 10 μL 5% H 2 O 2 and 10 μL 0.1N HCl in a methanol solution of the organotin precursor, Na 125 I was added and reacted at room temperature for 30 minutes. Thereafter, the mixture was neutralized with a saturated aqueous sodium hydrogen carbonate solution, extracted with ethyl acetate, and the ethyl acetate was distilled off under reduced pressure. Then, the acetyl group was deprotected with sodium methylate and neutralized with Amberlyst. Thereafter, separation and purification were performed by reverse phase HPLC. (Column: Cosmosil 5C18-AR-300, 4.6 × 150 mm, eluent: acetonitrile: water = 17: 83, flow rate: 1 ml / min)
IPGMGのGnT-Vによる反応生成物の測定
GnT-Vを高発現しているWidr cellを可溶化したもの、MES buffer (pH6.25)，UDP-GlcNAc，Triton X-100，EDTAを含む溶液にIPGMGを加え、37℃で保温した。4時間後、100℃で1分間加熱することにより反応を止めた。別途合成した1-IPGGMG (Compound 42) を標品とし、これらを、逆相HPLCにて分析した。（カラム；TSK-GEL ODS-80TM,4.6×150 mm、溶出液；アセトニトリル：水=15：85、流速；1 ml/min）逆相HPLCによる分析の結果、GnT-Vによる反応生成物が、標品であるIPGGMGと同じ保持時間を示した。また、LC-MSを用いた分析によっても、生成物がIPGGMGであることが確認された。この結果を図１に示す。
Measurement of reaction products by GnT-V of IPGMG
IPGMG was added to a solution containing solubilized Widr cells that highly express GnT-V, MES buffer (pH 6.25), UDP-GlcNAc, Triton X-100, and EDTA, and incubated at 37 ° C. After 4 hours, the reaction was stopped by heating at 100 ° C. for 1 minute. Separately synthesized 1-IPGGMG (Compound 42) was used as a standard, and these were analyzed by reverse phase HPLC. (Column: TSK-GEL ODS-80TM, 4.6 × 150 mm, eluent: acetonitrile: water = 15: 85, flow rate: 1 ml / min) As a result of analysis by reverse phase HPLC, the reaction product by GnT-V was The retention time was the same as the standard IPGGMG. Moreover, it was confirmed by LC-MS analysis that the product was IPGGMG. The result is shown in FIG.
GnT-VによるIPGMGの基質認識性
GnT-Vを高発現しているWidr cellを可溶化したもの、MES buffer (pH6.25)，UDP-GlcNAc，Triton X-100，EDTAを含む溶液に [125I]IPGMGを加え、37℃で保温した。4時間後、100℃の水浴で1分間加熱することにより反応を止めた。4℃、12000rpm,10min で遠心し、上清をHPLCで分析することによりKm、Vmaxを求めた。（カラム；Cosmosil 5C18-AR-300,4.6×150 mm、溶出液；アセトニトリル：水=15：85、流速；1 ml/min）Double reciprocal plotにより、 Kmは23.7μM、Vmaxは158.7 pmol/hrμg proteinと算出された。現在汎用されているGnT-Vの蛍光標識基質であるGn，Gn-bi-PAのKm値270μM（非特許文献１３参照）と比較すると、10倍以上もGnT-Vへの親和性が高いことが示された。この結果を図２及び図３に示す。
Substrate recognition of IPGMG by GnT-V
[ 125 I] IPGMG is added to a solution containing solubilized Widr cells expressing MnT-V, MES buffer (pH 6.25), UDP-GlcNAc, Triton X-100, and EDTA at 37 ° C. Keep warm. After 4 hours, the reaction was stopped by heating in a 100 ° C. water bath for 1 minute. Centrifugation was performed at 4 ° C., 12000 rpm, 10 min, and the supernatant was analyzed by HPLC to obtain Km and Vmax. (Column: Cosmosil 5C18-AR-300, 4.6 × 150 mm, eluent: acetonitrile: water = 15: 85, flow rate: 1 ml / min) According to double reciprocal plot, Km is 23.7 μM, Vmax is 158.7 pmol / hrμg protein And calculated. Compared with GnT-V, a commonly used fluorescent labeling substrate for GnT-V, with a Km value of 270 μM (see Non-Patent Document 13), the affinity for GnT-V is 10 times higher. It has been shown. The results are shown in FIGS.
IPGMGを用いたGnT-V酵素活性測定の検量線作成
種々の濃度のGnT-Vを含有するサンプルに関して[125I]IPGMGを用いてGnT-V酵素活性を測定し、検量線を作成した。それぞれのサンプル中に含まれるタンパク質の量は、Lowry法により定量した。得られた検量線は良好な直線性を示し、Gn，Gn-bi-PAを用いた場合の検量線と比較すると検量線の傾きが大きく、より高感度にGnT-V酵素活性を測定できることが示された。この結果を図４及び表１に示す。
Preparation of calibration curve for measuring GnT-V enzyme activity using IPGMG GnT-V enzyme activity was measured using [ 125 I] IPGMG for samples containing various concentrations of GnT-V, and a calibration curve was prepared. The amount of protein contained in each sample was quantified by the Lowry method. The obtained calibration curve shows good linearity, and the slope of the calibration curve is large compared to the calibration curve when Gn, Gn-bi-PA is used, and GnT-V enzyme activity can be measured with higher sensitivity. Indicated. The results are shown in FIG.
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