JPH05115292A - Production of mannotriose - Google Patents
Production of mannotrioseInfo
- Publication number
- JPH05115292A JPH05115292A JP12318091A JP12318091A JPH05115292A JP H05115292 A JPH05115292 A JP H05115292A JP 12318091 A JP12318091 A JP 12318091A JP 12318091 A JP12318091 A JP 12318091A JP H05115292 A JPH05115292 A JP H05115292A
- Authority
- JP
- Japan
- Prior art keywords
- man
- alpha
- mannopyranoside
- nitrophenyl
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
- B23D—PLANING; SLOTTING; SHEARING; BROACHING; SAWING; FILING; SCRAPING; LIKE OPERATIONS FOR WORKING METAL BY REMOVING MATERIAL, NOT OTHERWISE PROVIDED FOR
- B23D45/00—Sawing machines or sawing devices with circular saw blades or with friction saw discs
- B23D45/06—Sawing machines or sawing devices with circular saw blades or with friction saw discs with a circular saw blade arranged underneath a stationary work-table
- B23D45/061—Sawing machines or sawing devices with circular saw blades or with friction saw discs with a circular saw blade arranged underneath a stationary work-table the saw blade being mounted on a carriage
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、マンノースを構成要素
とする各種結合形態のマンノトリオースを製造する方法
に関するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing mannotriose having various bonding forms containing mannose as a constituent.
【０００２】[0002]
【従来の技術】複合糖質を構成している要素であるオリ
ゴ糖の合成は、免疫化学的研究や生化学的な興味から広
く研究されている（文献１〜３）。マンノースを含む様
々な構成物も化学的方法で（文献４〜６）或いは酵素的
方法で（文献７、８）合成されている。酵素的方法によ
る例としては、Ｊｏｈａｎｓｓｏｎらはタチナタマメ由
来のα−Ｄ−マンノシダーゼを用いて、高濃度のＤ−マ
ンノース溶液からマンノオリゴ糖混合物を７０％の収率
で得た（文献９）。また、Ｎｉｌｓｓｏｎはタチナタマ
メ由来のα−Ｄ−マンノシダーゼの糖転移活性を利用し
て、ｐ−ニトロフェニルα−Ｄ−マンノピラノシドから
マンノビオースとして種々のｐ−ニトロフェニルグリコ
シドを得た（文献１０）。2. Description of the Related Art The synthesis of oligosaccharides, which are the constituents of glycoconjugates, has been widely studied from immunochemical studies and biochemical interests (References 1 to 3). Various constituents containing mannose have also been synthesized by chemical methods (References 4 to 6) or enzymatic methods (References 7 and 8). As an example of the enzymatic method, Johanns et al. Obtained a 70% yield of a mannooligosaccharide mixture from a highly concentrated D-mannose solution using α-D-mannosidase derived from jack bean (Reference 9). In addition, Nilsson obtained various p-nitrophenyl glycosides as mannobioses from p-nitrophenyl α-D-mannopyranoside by utilizing the transglycosylation activity of α-D-mannosidase derived from jack bean (Reference 10).
【０００３】[0003]
【発明が解決しようとする課題】しかしながら、化学的
方法でマンノースからマンノトリオースを合成するには
数多くの工程を要し、収率も低い。例えば、Ｌｉｐｔａ
ｋらはＭａｎ（α１−２）Ｍａｎ（α１−２）Ｍａｎを
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）から化学的
手法で１０もの多くの工程を経て合成したが、全体での
収率は２．４％に過ぎなかった。このように化学的合成
法はマンノトリース以上のマンノオリゴ糖の合成には実
用的ではない。However, synthesizing mannotriose from mannose by a chemical method requires many steps and the yield is low. For example, Lipta
k et al. described Man (α1-2) Man (α1-2) Man as benzyl 3-O-benzyl-4,6-O-benzylidene-α-D-mannopyranoside (benzyl 3-O-).
Benzyl-4,6-O-benzylidene-
It was synthesized from α-D-mannopyranoside) by many chemical steps including 10 steps, but the overall yield was only 2.4%. As described above, the chemical synthesis method is not practical for the synthesis of manno-oligosaccharides beyond mannotrise.
【０００４】一方酵素的方法では、α−Ｄ−マンノピラ
ノシル−（１→２）−α−Ｄ−マンノピラノースとα−
Ｄ−マンノピラノシル−（１→６）−α−Ｄ−マンノピ
ラノース（以下、それぞれＭａｎ（α１−２）Ｍａｎ及
びＭａｎ（α１−６）Ｍａｎと記す；その他のオリゴ糖
も、このような短縮表示で記すことができる）の二種類
のマンノオリゴ糖しか得られていない。On the other hand, in the enzymatic method, α-D-mannopyranosyl- (1 → 2) -α-D-mannopyranose and α-
D-mannopyranosyl- (1 → 6) -α-D-mannopyranose (hereinafter referred to as Man (α1-2) Man and Man (α1-6) Man, respectively); other oligosaccharides are also represented by such abbreviations. , Which can be described in the above).
【０００５】[0005]
【課題を解決するための手段】そこで、本発明者らは複
合糖質のブロック合成の部分構造としてのマンノース含
有オリゴ糖の酵素的合成について探求したところ、アー
モンド由来のα−Ｄ−マンノシダーゼ（ＥＣ ３．２．
１．２４）を利用することにより、ｐ−ニトロフェニエ
ルα−Ｄ−マンノピラノシドをマンノース供与体、マン
ノビオースをマンノース受容体として、様々な結合（α
１→２、α１→３、α１→６）を有する種々のマンノト
リオースを得ることに成功した。本発明において複合糖
質の構成要素として有用であるマンノースがα１→２結
合、α→６結合或いはα１→３結合によって３個以上連
結されているマンノオリゴ糖を製造する道を開いたとい
うことができる。The inventors of the present invention have investigated the enzymatic synthesis of mannose-containing oligosaccharides as a partial structure for block synthesis of glycoconjugates. As a result, almond-derived α-D-mannosidase (EC 3.2.
1.24), p-nitrophenyl α-D-mannopyranoside is used as a mannose donor, and mannobiose is used as a mannose acceptor.
We succeeded in obtaining various mannotrioses having 1 → 2, α1 → 3, α1 → 6). In the present invention, it can be said that it opened the way for producing mannooligosaccharides in which three or more mannoses useful as constituents of glycoconjugates are linked by α1 → 2 bond, α → 6 bond or α1 → 3 bond. ..
【０００６】本発明においてはマンノース供与体として
ｐ−ニトロフェニルα−Ｄ−マンノピラノシド又はｏ−
ニトロフェニルα−Ｄ−マンノピラノシド又はα−Ｄ−
マンノピラノシルフルオリドを使用することが出来る。In the present invention, p-nitrophenyl α-D-mannopyranoside or o-is used as the mannose donor.
Nitrophenyl α-D-mannopyranoside or α-D-
Mannopyranosyl fluoride can be used.
【０００７】[0007]
【実施例】以下の実施例において、アーモンド由来のα
−Ｄ−マンノシダーゼは米国シグマ社から溶液の形で購
入し、それ以上精製することなくそのまま用いた（但し
実施例の一部では、このα−Ｄ−マンノシダーゼ溶液を
メンブレンフィルターで５倍濃縮したものを用いた）。
酵素反応はＣａｒｂｏｐａｃ ＰＡｌとパルスドアンペ
ロメトリック検出器が取り付けられた米国ＤｉｏｎｅＸ
社の液体クロマトグラフィー（以下ＨＰＬＣと記す）で
モニターした。溶出は１００ｍＭの水酸化ナトリウム溶
液を用いて同一組成のままで行った。１Ｈ−ＮＭＲ（４
００ＭＨｚ）及び１３Ｃ−ＮＭＲ（１００ＭＨｚ）スペ
クトルは、Ｖａｒｉａｎ ＸＬ−４００ ＮＭＲ分光計
で測定した。１３Ｃ−ＮＭＲの化学シフトの基準には、
内部アセトニトリル（１．３ｐｐｍ）のメチルシグナル
を用いた。EXAMPLES In the following examples, almond-derived α
-D-mannosidase was purchased in the form of a solution from Sigma, USA and used as is without further purification (however, in some of the examples, this α-D-mannosidase solution was concentrated 5 times with a membrane filter). Was used).
The enzymatic reaction is Carbopac PAl and a pulsed amperometric detector attached to the US DioneX
It was monitored by liquid chromatography (hereinafter referred to as HPLC) manufactured by the same company. Elution was performed with the same composition using a 100 mM sodium hydroxide solution. 1 H-NMR (4
00 MHz) and 13 C-NMR (100 MHz) spectra were measured with a Varian XL-400 NMR spectrometer. The standard of 13 C-NMR chemical shift is
The methyl signal of internal acetonitrile (1.3 ppm) was used.
【０００８】[0008]
【実施例１（Ｍａｎ（α１−２）［Ｍａｎ
（α１−６）］Ｍａｎ及びＭａｎ（α１−２）Ｍａｎ
（α１−２）Ｍａｎの合成）】 まず、マンノトリオースを合成するための原料として、
Ｍａｎ（α１−２）ＭａｎとＭａｎ（α１−６）Ｍａｎ
を合成した。ｐ−ニトロフェニルα−Ｄ−マンノピラノ
シド（１２０ｍｇ）とＤ−マンノース（７２０ｍｇ）と
を０．１Ｍ燐酸緩衝液（ｐＨ６．０、２．８ｍｌ）とジ
メチルスルフオキシド（８００μｌ）の混合溶媒に溶か
し、これに５倍濃縮α−Ｄ−マンノシダーゼ（１４０μ
ｌ、２．７ユニット）を加えて３７℃で１６時間反応さ
せた。反応終了後反応液を熱湯浴で５分間加熱すること
によって酵素を変性させて濾別した。濾過液を活性炭素
カラム（５×５０ｃｍ）にかけ、０〜２０％のエタノー
ル水溶液濃度勾配で分画溶出した。各画分の糖濃度をフ
ェノール硫酸法（文献１１）で測定した後、二糖類を含
む各画分をＨＰＬＣにかけた。ＨＰＬＣの結果、２つの
ピークが見い出されたので、それぞれを１３Ｃ−ＮＭＲ
にかけて解析したところ、Ｍａｎ（α１−２）Ｍａｎと
Ｍａｎ（α１−６）Ｍａｎであることが判明した（これ
らの化学シフトを表１の中のそれぞれ１と２として示
す）。これらを９０％以上の純度で含んでいる画分を集
め、蒸留によって濃縮したところ、非晶性固体として１
７．６ｍｇ（収率１２．９％）のＭａｎ（α１−２）Ｍ
ａｎと１１．８ｍｇ（収率８．７％）のＭａｎ（α１−
６）Ｍａｎとが別々に得られた。これらのうち、Ｍａｎ
（α１−２）Ｍａｎは本実施例で以下に記すＭａｎ（α
１−２）［Ｍａｎ（α１−６）］Ｍａｎ及びＭａｎ（α
１−２）Ｍａｎ（α１−２）Ｍａｎの合成の原料に、Ｍ
ａｎ（α１−６）Ｍａｎは実施例２のＭａｎ（α１−
２）Ｍａｎ（α１−６）Ｍａｎ、Ｍａｎ（α１−６）Ｍ
ａｎ（α１−６）Ｍａｎ及びＭａｎ（α１−３）［Ｍａ
ｎ（α１−６）］Ｍａｎの合成の原料に用いた。Example 1 (Man (α1-2) [Man
(Α1-6)] Man and Man (α1-2) Man
(Α1-2) Man's Synthesis] First, as a raw material for synthesizing mannotriose,
Man (α1-2) Man and Man (α1-6) Man
Was synthesized. p-Nitrophenyl α-D-mannopyranoside (120 mg) and D-mannose (720 mg) were dissolved in a mixed solvent of 0.1 M phosphate buffer (pH 6.0, 2.8 ml) and dimethyl sulfoxide (800 μl), 5 times concentrated α-D-mannosidase (140μ
1, 2.7 units) was added and the mixture was reacted at 37 ° C. for 16 hours. After completion of the reaction, the reaction solution was heated in a hot water bath for 5 minutes to denature the enzyme and filtered. The filtrate was applied to an activated carbon column (5 × 50 cm), and fractionated and eluted with a 0-20% ethanol aqueous solution concentration gradient. After measuring the sugar concentration of each fraction by the phenol-sulfuric acid method (Reference 11), each fraction containing disaccharide was subjected to HPLC. HPLC results, because two peaks were found, each 13 C-NMR
It was found to be Man (α1-2) Man and Man (α1-6) Man when analyzed over time (these chemical shifts are shown as 1 and 2 in Table 1). Fractions containing 90% or more of these were collected and concentrated by distillation to give 1 as an amorphous solid.
7.6 mg (12.9% yield) of Man (α1-2) M
an and 11.8 mg (yield 8.7%) of Man (α1-
6) Man was obtained separately. Of these, Man
(Α1-2) Man is Man (α) described below in this embodiment.
1-2) [Man (α1-6)] Man and Man (α
1-2) As a raw material for the synthesis of Man (α1-2) Man, M
an (α1-6) Man is Man (α1−) of Example 2.
2) Man (α1-6) Man, Man (α1-6) M
an (α1-6) Man and Man (α1-3) [Ma
n (α1-6)] Man was used as a starting material for the synthesis.
【０００９】ｐ−ニトロフェニルα−Ｄ−マンノピラノ
シド（３０ｍｇ）と上で得られたＭａｎ（α１−２）Ｍ
ａｎ（１０３ｍｇ）とを０．１Ｍ燐酸融衝液（ｐＨ６．
０、３５０μｌ）とジメチルスルフォキシド（１１５μ
ｌ）の混合溶媒に溶かし、α−Ｄ−マンノシダーゼ（７
８μｌ、０．３ユニット）を加え、３７℃で５時間反応
させた。反応終了後反応液を熱湯浴で５分間加熱するこ
とによって酵素を変性させて濾別した。得られた反応物
混合液のＨＰＬＣパターンを図１に示す。保持時間から
図１中のピークＡは二糖類のＭａｎ（α１−２）Ｍａｎ
であり、ピークＢとピークＣは三糖類である。この反応
混合液を活性炭素カラム（１．５×５０ｃｍ）にかけ、
０〜３０％エタノール水溶液を溶出液として濃度勾配分
画した。そしてフェノール硫酸法により各画分の糖濃度
を見積もるために４０５ｎｍにおける吸光度分光計で測
定した。吸光度の測定結果を図２に示す。ピークＢの画
分領域を取ってＨＰＬＣで調べたところ、図１のピーク
Ｂに対応する一種類の三糖類しか見い出されなかった。
この三糖類の１３Ｃ−ＮＭＲスペクトルを測定した。ス
ペクトルは図３のＡに、化学シフトは表１に示した。図
３Ａの１０３．０ｐｐｍのシグナルは、α１→２結合に
よって連結されている非還元末端のマンノシル残基のＣ
−１に対応するものであった。それ故、この三糖類の構
造はＭａｎα１−２［Ｍａｎα１−６］Ｍａｎと結論さ
れた。図２のピークＢ領域でこの三糖類を９０％以上の
純度で含む画分を集め、溶媒を蒸留で留去したところ、
非晶性固体として４．６ｍｇ（収率９．１％）の本物質
が得られた。P-Nitrophenyl α-D-mannopyranoside (30 mg) and the Man (α1-2) M obtained above.
an (103 mg) and 0.1 M phosphoric acid fusion solution (pH 6.
0,350 μl) and dimethyl sulfoxide (115 μl
l) dissolved in a mixed solvent of α-D-mannosidase (7
8 μl, 0.3 unit) was added, and the mixture was reacted at 37 ° C. for 5 hours. After completion of the reaction, the reaction solution was heated in a hot water bath for 5 minutes to denature the enzyme and filtered. The HPLC pattern of the resulting reaction mixture is shown in FIG. From the retention time, peak A in FIG. 1 indicates that the disaccharide is Man (α1-2) Man.
And peak B and peak C are trisaccharides. The reaction mixture was applied to an activated carbon column (1.5 x 50 cm),
A gradient gradient fractionation was performed using an aqueous solution of 0 to 30% ethanol as an eluent. Then, in order to estimate the sugar concentration of each fraction by the phenol-sulfuric acid method, it was measured by an absorption spectrometer at 405 nm. The measurement result of the absorbance is shown in FIG. When the fraction region of peak B was taken and examined by HPLC, only one type of trisaccharide corresponding to peak B in FIG. 1 was found.
The 13 C-NMR spectrum of this trisaccharide was measured. The spectra are shown in FIG. 3A and the chemical shifts are shown in Table 1. The signal at 103.0 ppm in FIG.
It corresponded to -1. Therefore, the structure of this trisaccharide was concluded to be Manα1-2 [Manα1-6] Man. Fractions containing this trisaccharide in a purity of 90% or more in the peak B region of FIG. 2 were collected and the solvent was distilled off.
4.6 mg (9.1% yield) of this substance was obtained as an amorphous solid.
【００１０】図２のピークＡ領域を集め、濃縮し、濃縮
溶液をＳｈｏｄｅｘ Ｒ１モニターを取り付けたＳｅｐ
ｈａｄｅｘ Ｇ−２５カラム（１．６×１００ｃｍ）に
かけ、更に蒸留水を用いて０．１８ｍｌ／分の速さで溶
出した。各画分をＨＰＬＣで調べ、三糖類を９０％以上
の純度で含む画分を集めた。溶媒を蒸留で留去したとこ
ろ、非晶性固体として４．９ｍｇ（収率９．７％）が得
られた。得られた三糖類の構造は、Ｌｉｐｔａｋら（文
献１２）によって報告された１３Ｃ−ＮＭＲの化学シフ
トのデータと本物質の化学シフト（表１参照）との比較
から、Ｍａｎ（α１−２）Ｍａｎ（α１−２）Ｍａｎで
あると同定された。The peak A region of FIG. 2 was collected and concentrated, and the concentrated solution was put on a Sepx R1 monitor.
It was applied to a hadex G-25 column (1.6 × 100 cm) and further eluted with distilled water at a rate of 0.18 ml / min. Each fraction was examined by HPLC, and the fractions containing trisaccharide in a purity of 90% or more were collected. When the solvent was distilled off, 4.9 mg (yield 9.7%) was obtained as an amorphous solid. The structure of the obtained trisaccharide was determined by comparing the chemical shift data of 13 C-NMR reported by Liptak et al. (Reference 12) and the chemical shift of this substance (see Table 1) with Man (α1-2). It was identified to be Man (α1-2) Man.
【００１１】[0011]
【表１】 [Table 1]
【００１２】表１の横軸において１〜７はマンノオリゴ
糖は、それぞれＭａｎ（α１−２）Ｍａｎ、Ｍａｎ（α
１−６）Ｍａｎ、Ｍａｎ（α１−２）［Ｍａｎ（α１−
６）］Ｍａｎ、Ｍａｎ（α１−２）Ｍａｎ（α１−２）
Ｍａｎ、Ｍａｎ（α１−２）Ｍａｎ（α１−６）Ｍａ
ｎ、Ｍａｎ（α１−６）Ｍａｎ（α１−６）Ｍａｎ及び
Ｍａｎ（α１−３）［Ｍａｎ（α１−６）］Ｍａｎであ
る。また、表１の縦軸においてａ、ｂ及びｃはこれらの
各マンノオリゴ糖中のマンノシル残基の位置を表す。そ
の位置は以下の通りである。In the horizontal axis of Table 1, 1 to 7 are manno-oligosaccharides, Man (α1-2) Man and Man (α), respectively.
1-6) Man, Man (α1-2) [Man (α1-
6)] Man, Man (α1-2) Man (α1-2)
Man, Man (α1-2) Man (α1-6) Ma
n, Man (α1-6) Man (α1-6) Man and Man (α1-3) [Man (α1-6)] Man. Further, a, b and c on the vertical axis of Table 1 represent the positions of mannosyl residues in each of these manno oligosaccharides. The location is as follows.
【００１３】[0013]
【化１】 [Chemical 1]
【００１４】実施例２（Ｍａｎ（α１−２）Ｍａｎ（α
１−６）Ｍａｎ、Ｍａｎ（α１−６）Ｍａｎ（α１−
６）Ｍａｎ及びＭａｎ（α１−３）［Ｍａｎ（α１−
６）］Ｍａｎの合成） ｐ−ニトロフェニルα−Ｄ−マンノピラノシド（９６．
５ｍｇ）と実施例１で得られたＭａｎ（α１−６）Ｍａ
ｎ（３３０ｍｇ）とを、０．１Ｍ燐酸緩衝液（ｐＨ６．
０、１１２０μｌ）とジメチルスルフォキシド（３６８
μｌ）の混合溶媒に溶かし、α−Ｄ−マンノシダーゼ
（２６０μｌ、１．０ユニット）を加え、３７℃で５時
間反応させた。反応終了後反応液を熱湯浴で５分間加熱
することによって酵素を変性させて濾別した。濾液をＨ
ＰＬＣにかけたところ、三糖類領域には２つのピークが
観察された（図４）。次いで活性炭素カラム（２．５×
５０ｃｍ）にかけ、０〜３０％のエタノール水溶液濃度
勾配で溶出させた。すると三糖類の領域に２つのピーク
が現れた。１Ｈ−ＮＭＲと１３Ｃ−ＮＭＲスペクトルと
から、図４のピークＣは、Ｍａｎ（α１−２）Ｍａｎ
（α１−６）Ｍａｎ、図４のピークＢはＭａｎ（α１−
６）Ｍａｎ（α１−６）Ｍａｎとそれぞれ同定された。
Ｍａｎ（α１−６）Ｍａｎ（α１−６）Ｍａｎの１Ｈ−
ＮＭＲスペクトルのアノマー領域はＹｏｋｏｙａｍａ及
びＢａｌｌｏｕ（文献８）の報告しているスペクトルと
よく一致していた。それぞれの三糖類を９０％以上の純
度で含有している画分を集めた。溶媒を蒸留で留去した
ところ、非晶性固体として７．１ｍｇ（収率４．４％）
のＭａｎ（α１−２）Ｍａｎ（α１−６）と６．３ｍｇ
のＭａｎ（α１−６）Ｍａｎ（ａ１−６）Ｍａｎ（収率
３．９％）とが得られた。これらの２つの三糖類の他
に、調製用カラムをつけたＨＰＬＣによりＭａｎ（α１
−６）Ｍａｎ（α１−６）Ｍａｎを含んでいる画分の後
方に第三の三糖類が得られた。この三糖類を調製ＨＰＬ
Ｃで精製し、１３Ｃ−ＮＭＲスペクトルを測定した。
（図３のＢ）。文献１４及び文献１５に報告された対応
するグリコシドの化学シフトの比較から、その構造はＭ
ａｎ（α１−３）［Ｍａｎ（α１−６）］Ｍａｎである
と決定された。しかしながら得られた量は痕跡量で重量
は測定できなかった。Example 2 (Man (α1-2) Man (α
1-6) Man, Man (α1-6) Man (α1-
6) Man and Man (α1-3) [Man (α1-
6)] Synthesis of Man) p-Nitrophenyl α-D-mannopyranoside (96.
5 mg) and Man (α1-6) Ma obtained in Example 1
n (330 mg) and 0.1 M phosphate buffer (pH 6.
0, 1120 μl) and dimethyl sulfoxide (368
μl) in a mixed solvent, α-D-mannosidase (260 μl, 1.0 unit) was added, and the mixture was reacted at 37 ° C. for 5 hours. After completion of the reaction, the reaction solution was heated in a hot water bath for 5 minutes to denature the enzyme and filtered. The filtrate is H
When subjected to PLC, two peaks were observed in the trisaccharide region (Fig. 4). Then activated carbon column (2.5 x
50 cm) and eluted with a 0-30% ethanol aqueous solution concentration gradient. Then, two peaks appeared in the trisaccharide region. From the 1 H-NMR and 13 C-NMR spectra, the peak C in FIG. 4 is Man (α1-2) Man.
(Α1-6) Man, peak B in FIG. 4 is Man (α1-
6) Man (α1-6) Man, respectively.
Man (α1-6) Man (α1-6) Man 1 H-
The anomeric region of the NMR spectrum was in good agreement with the spectrum reported by Yokoyama and Ballou (Reference 8). Fractions containing each trisaccharide in a purity of 90% or higher were collected. When the solvent was distilled off, 7.1 mg (yield 4.4%) was obtained as an amorphous solid.
Man (α1-2) Man (α1-6) and 6.3 mg
Man (α1-6) Man (a1-6) Man (yield 3.9%) was obtained. In addition to these two trisaccharides, Man (α1
-6) A third trisaccharide was obtained behind the fraction containing Man (α1-6) Man. Prepare this trisaccharide HPL
Purified by C, 13 C-NMR spectrum was measured.
(B in FIG. 3). From the comparison of the chemical shifts of the corresponding glycosides reported in Refs. 14 and 15, the structure is M
was determined to be an (α1-3) [Man (α1-6)] Man. However, the amount obtained was a trace amount and the weight could not be measured.
【００１５】[0015]
【引用文献】１．Ａｎｄｅｒｓｏｎ，Ｆ．，Ｂｉｒｂｅ
ｒｇ，Ｗ．，Ｆｕｇｅｄｉ，Ｐ．，ＧＡｒｅｇｇ，Ｐ．
Ｊ．，Ｎａｓｈｅｄ，Ｍ．ａｎｄ Ｐｉｌｏｔｔｉ，
Ａ．，（１９８９）Ｔｒｅｎｄｓ ｉｎ Ｓｙｎｔｈｅ
ｔｉｃ Ｃａｒｂｏｈｙｄｒａｔｅ Ｃｈｅｍｉｓｔｒ
ｙ：１１７−１３０． ２．Ｃｏｔｅ，Ｇ・Ｌ．ａｎｄ Ｔａｏ，Ｂ．Ｙ．，
（１９９０）Ｇｌｙｃｏｃｏｎｊｕｇａｔｅ Ｊ．７：
１４５−１６２． ３．Ａｊｉｓａｋａ，Ｋ．ａｎｄ Ｆｕｊｉｍｏｔｏ，
Ｈ．，（１９９０）Ｃａｒｂｏｈｙｄｒ．Ｒｅｓ．１９
９：２２７−２３４． ４．Ｋｈａｎ，Ｓ．Ｈ．，Ｊａｉｎ，Ｒ．Ｋ．ａｎｄ
Ｍａｔｔａ，Ｋ．Ｌ．，（１９９０）Ｃａｒｂｏｈｙｄ
ｒ．Ｒｅｓ．２０７：５７−６９． ５．Ｏｇａｗａ，Ｔ．ａｎｄ Ｙａｍａｍｏｔｏ，
Ｈ．，（１９８２）Ｃａｒｂｏｈｙｄｒ．Ｒｅｓ．１０
４：２７１−２８３． ６．Ａｗａｄ，Ｌ．Ｆ．，Ａｓｈｒｙ，Ｓ．Ｈ．ａｎｄ
Ｓｃｈｕｅｒｃｈ，Ｃ．，（１９８３）Ｃａｒｂｏｈ
ｙｄｒ．Ｒｅｓ．１２２：６９−７９． ７．Ｊｏｈａｎｓｓｏｎ，Ｅ．，Ｈｅｄｂｙｓ，Ｌ．，
Ｍｏｓｂａｃｈ，Ｋ．ａｎｄ Ｐ．Ｏ．，（１９８９）
Ｅｎｚｙｍｅ Ｍｉｃｒｏｂ．Ｔｅｃｈｎｏｌ．１１：
３４７−３５２． ８・Ｙｏｋｏｙａｍａ，Ｋ．ａｎｄ Ｂａｌｌｏｕ
Ｃ．Ｅ．，（１９８９）Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２６
４：２１６２１−２１６２８． ９．Ｊｏｈａｎｓｓｏｎ，Ｅ．，Ｈｅｄｂｙｓ，Ｌ．ａ
ｎｄ Ｌａｒｓｓｏｎ，Ｐ．Ｏ．，（１９８６）Ｂｉｏ
ｔｅｃｈｎｏｌ．Ｌｅｔｔｅｒｓ ８：４２１−４２
４． １０．Ｎｉｌｓｓｏｎ，Ｋ．Ｇ．，（１９８７）Ｃａｒ
ｂｏｈｙｄｒ．Ｒｅｓ．１６７：９５−１０３． １１．Ｄｕｂｏｉｓ，Ｍ．，Ｇｉｌｌｅｓ，Ｋ．Ａ．，
Ｈａｍｉｌｔｏｎ Ｊ．Ｋ．，Ｒｅｂｅｒｓ，Ｐ．Ａ．
ａｎｄ Ｓｍｉｔｈ，Ｆ．，（１９５６）Ａｎａｌ．Ｃ
ｈｅｍ．２８：３５０−３５６． １２．Ｏｇａｗａ，Ｔ．ａｎｄ Ｓａｓａｊｉｍａ，
Ｋ．，（１９８１）Ｃａｒｂｏｈｙｄｒ．Ｒｅｓ．９
７：２０５−２２７． １３．Ｅｋｂｏｒｇ，Ｇ．ａｎｄ Ｇｌａｕｄｅｍａｎ
ｓ Ｃ．Ｐ．Ｊ．，（１９８５）Ｃａｒｂｏｈｙｄｒ．
Ｒｅｓ．１４２：２１３−２２１．[Citations] 1. Anderson, F.M. , Birbe
rg, W. Fugedi, P .; , GAreggg, P .;
J. Nashed, M .; and Pilotti,
A. , (1989) Trends in Synthe
tic Carbohydrate Chemistr
y: 117-130. 2. Cote, G.L. and Tao, B .; Y. ，
(1990) Glycoconjugate J. et al. 7:
145-162. 3. Ajisaka, K .; and Fujimoto,
H. , (1990) Carbohydr. Res. 19
9: 227-234. 4. Khan, S .; H. Jain, R .; K. and
Matta, K .; L. , (1990) Carbohyd
r. Res. 207: 57-69. 5. Ogawa, T .; and Yamamoto,
H. , (1982) Carbohydr. Res. 10
4: 271-283. 6. Awad, L .; F. , Ashry, S .; H. and
Schuerch, C.I. , (1983) Carboh
ydr. Res. 122: 69-79. 7. Johansson, E .; Hedbys, L .; ，
Mosbach, K .; and P.D. O. , (1989)
Enzyme Microb. Technol. 11:
347-352. 8. Yokoyama, K .; and Ballou
C. E. , (1989) J. Biol. Chem. 26
4: 21621-21628. 9. Johansson, E .; Hedbys, L .; a
nd Larsson, P.N. O. , (1986) Bio
technology. Letters 8: 421-42
4. 10. Nilsson, K .; G. , (1987) Car
bohydr. Res. 167: 95-103. 11. Dubois, M .; Gilles, K .; A. ，
Hamilton J. K. Rebers, P .; A.
and Smith, F.M. , (1956) Anal. C
hem. 28: 350-356. 12. Ogawa, T .; and Sasajima,
K. , (1981) Carbohydr. Res. 9
7: 205-227. 13. Ekburg, G .; and Glaudeman
s C. P. J. , (1985) Carbohydr.
Res. 142: 213-221.
【発明の効果】本発明で合成された各種結合形態のマン
ノトリオースは、ハイマンノース型の複合糖質の主要構
成要素である。これらのオリゴ糖はそのような複合糖質
をブロツク合成する上で役立つことが期待される。EFFECTS OF THE INVENTION Mannotriose of various binding forms synthesized according to the present invention is a main constituent of a high-mannose type glycoconjugate. It is expected that these oligosaccharides will be useful in the block synthesis of such glycoconjugates.
【図１】アーモンド由来α−Ｄ−マンノシダーゼの存在
下でのｐ−ニトロフェニルα−Ｄ−マンノピラノシドと
Ｍａｎ（α１−２）Ｍａｎとの反応物のＨＰＬＣであ
る。図中でピークＡはＭａｎ（α１−２）Ｍａｎ、ピー
クＢはＭａｎ（α１−２）［Ｍａｎ（α１−６）］Ｍａ
ｎ、ピークＣはＭａｎ（α１−２）Ｍａｎ（α１−２）
Ｍａｎである。FIG. 1 is an HPLC of the reaction of p-nitrophenyl α-D-mannopyranoside with Man (α1-2) Man in the presence of almond-derived α-D-mannosidase. In the figure, peak A is Man (α1-2) Man, peak B is Man (α1-2) [Man (α1-6)] Ma.
n, peak C is Man (α1-2) Man (α1-2)
It is Man.
【図２】アーモンド由来α−Ｄ−マンノシダーゼの存在
下でのｐ−ニトロフェニルα−Ｄ−マンノピラノシドと
Ｍａｎ（α１−２）Ｍａｎとの反応物を活性炭素カラム
クロマトグラフィーにより分画し、フェノール硫酸法に
より４０５ｎｍにおける吸光度を測定したものである。
図中、Ａは二糖類領域、Ｂは三糖類領域である。図中で
両方向矢印で示された範囲の画分を集め、マンノトリオ
ースを単離精製した。[Fig. 2] A reaction product of p-nitrophenyl α-D-mannopyranoside and Man (α1-2) Man in the presence of almond-derived α-D-mannosidase was fractionated by activated carbon column chromatography to obtain phenol sulfate. The absorbance at 405 nm was measured by the method.
In the figure, A is a disaccharide region and B is a trisaccharide region. Fractions in the range indicated by the double-headed arrow in the figure were collected to isolate and purify mannotriose.
【図３】ＡはＭａｎ（α１−２）［Ｍａｎ（α１−
６）］Ｍａｎ、ＢはＭａｎ（α１−３）［Ｍａｎ（α１
−６）］Ｍａｎの１３Ｃ−ＮＭＲスペクトルである。FIG. 3A is Man (α1-2) [Man (α1-
6)] Man and B are Man (α1-3) [Man (α1
-6)] A 13 C-NMR spectrum of Man.
【図４】α−Ｄ−マンノシダーゼの存在下でのｐ−ニト
ロフェニルα−Ｄ−マンノピラノシドとＭａｎ（α１−
６）Ｍａｎとの反応物のＨＰＬＣである。ＡはＭａｎ
（α１−６）Ｍａｎ、ＢはＭａｎ（α１−６）Ｍａｎ
（α１−２）Ｍａｎ、ＣはＭａｎ（α１−２）Ｍａｎ
（α１−６）Ｍａｎにそれぞれ対応する。FIG. 4 p-Nitrophenyl α-D-mannopyranoside and Man (α1-α-D in the presence of α-D-mannosidase.
6) HPLC of the reaction with Man. A is Man
(Α1-6) Man, B is Man (α1-6) Man
(Α1-2) Man, C is Man (α1-2) Man
They correspond to (α1-6) Man, respectively.
Claims (3)
とをアーモンド由来のα−Ｄ−マンノシダーゼの存在下
で糖転移反応を行わせることを特徴とするマンノトリオ
ースの製造方法。1. A method for producing mannotriose, which comprises subjecting a D-mannose donor and mannobiose to a transglycosylation reaction in the presence of almond-derived α-D-mannosidase.
ェニルα−Ｄ−マンノピラノシド又はｏ−ニトロフェニ
ル−α−Ｄ−マンノピラノシド又はα−Ｄ−マンノピラ
ノシルフルオリドである請求項１のマンノトリオースの
製造方法。2. The D-mannose donor is p-nitrophenyl α-D-mannopyranoside or o-nitrophenyl-α-D-mannopyranoside or α-D-mannopyranosyl fluoride. Mannotriose manufacturing method.
〔Ｍａｎ（α１−６）〕Ｍａｎ、Ｍａｎ（α１−２）Ｍ
ａｎ（α１−２）Ｍａｎ、Ｍａｎ（α１−２）Ｍａｎ
（α１−６）Ｍａｎ、Ｍａｎ（α１−６）Ｍａｎ（α１
−６）Ｍａｎ、又はＭａｎ（α１−３）〔Ｍａｎ（α１
−６）〕Ｍａｎである請求項１又は請求項２のマンノト
リオースの製造方法。3. Mannotriose is Man (α1-2)
[Man (α1-6)] Man, Man (α1-2) M
an (α1-2) Man, Man (α1-2) Man
(Α1-6) Man, Man (α1-6) Man (α1
-6) Man, or Man (α1-3) [Man (α1
-6)] The method for producing mannotriose according to claim 1 or 2, which is Man.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12318091A JPH05115292A (en) | 1991-03-05 | 1991-03-05 | Production of mannotriose |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12318091A JPH05115292A (en) | 1991-03-05 | 1991-03-05 | Production of mannotriose |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH05115292A true JPH05115292A (en) | 1993-05-14 |
Family
ID=14854173
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP12318091A Pending JPH05115292A (en) | 1991-03-05 | 1991-03-05 | Production of mannotriose |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH05115292A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005044861A1 (en) * | 2003-10-31 | 2005-05-19 | Wyeth Holdings Corporation | Polysaccharides of helicobacter pylori |
| CN117269360A (en) * | 2023-09-22 | 2023-12-22 | 广东省科学院生物与医学工程研究所 | Method for detecting content of mannooligosaccharide in fermented dairy product |
-
1991
- 1991-03-05 JP JP12318091A patent/JPH05115292A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005044861A1 (en) * | 2003-10-31 | 2005-05-19 | Wyeth Holdings Corporation | Polysaccharides of helicobacter pylori |
| US7527949B2 (en) | 2003-10-31 | 2009-05-05 | Wyeth Holdings Corporation | Polysaccharides of Helicobacter pylori |
| CN117269360A (en) * | 2023-09-22 | 2023-12-22 | 广东省科学院生物与医学工程研究所 | Method for detecting content of mannooligosaccharide in fermented dairy product |
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