Three novel oligosaccharides synthesized using Thermoanaerobacter brockii kojibiose phosphorylase

<p>Abstract</p> <p>Background</p> <p>Recently synthesized novel oligosaccharides have been produced primarily by hydrolases and glycosyltransferases, while phosphorylases have also been subject of few studies. Indeed, phosphorylases are expected to give good results via their reversible reaction. The purpose of this study was to synthesis other novel oligosaccharides using kojibiose phosphorylase.</p> <p>Results</p> <p>Three novel oligosaccharides were synthesized by glucosyltransfer from β-D-glucose 1-phosphate (β-D-G1P) to xylosylfructoside [<it>O</it>-α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside] using <it>Thermoanaerobacter brockii </it>kojibiose phosphorylase. These oligosaccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methyl derivatives, MALDI-TOF MS and NMR measurements were used for structural characterisation. The ¹H and ¹³C NMR signals of each saccharide were assigned using 2D-NMR including COSY (correlated spectroscopy), HSQC (herteronuclear single quantum coherence), CH₂-selected E-HSQC (CH₂-selected Editing-HSQC), HSQC-TOCSY (HSQC-total correlation spectroscopy) and HMBC (heteronuclear multiple bond correlation).</p> <p>Conclusion</p> <p>The structure of three synthesized saccharides were determined, and these oligosaccharides have been identified as <it>O</it>-α-D-glucopyranosyl-(1→2)-<it>O</it>-α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside (saccharide <b>1</b>), <it>O</it>-α-D-glucopyranosyl-(1→2)-<it>O</it>-α-D-glucopyranosyl-(1→2)-<it>O</it>-α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside (saccharide <b>2</b>) and <it>O</it>-α-D-glucopyranosyl-(1→[2-<it>O</it>-α-D-glucopyranosyl-1]₂→2)-<it>O</it>-α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside (saccharide <b>3</b>).</p

Advanced NMR approaches for a detailed structure analysis of natural products.

Some new nuclear magnetic resonance (NMR) approaches to elucidate chemical structures, which have not been determined by routine NMR methods, are presented. Selective detection of methine (CH), methylene (CH₂), or methyl (CH₃) signals in each subspectrum by editing NMR methods was utilized to reduce the complexity in crowded spectra. It also increased the peak separation and enhanced the sensitivity by limiting the measuring area of the 2D spectra. Several 2D methods to measure ²'³J CH values, which are useful for stereochemical assignment are then introduced. To determine the structure of a highly hydrogen-deficient molecule, efficient correlation methods for long-range ¹³C–¹³C coupling and ¹H-15N HMBC are also described

Application of 1D BIRD or X-filtered DEPT long-range C-C relay for detection of proton and carbon via four bonds and measuring long-range C-13-C-13 coupling constants

We propose the C-13-detecting 1D DEPT long-range C-C relay to detect super long-range H-C connectivity via four bonds (H-1-C-13-X-X-C-13, X represents C-12 or heteronuclear). It is derived from the DEPT C-C relay which detects the H-C correlations via two bonds (H-1-C-13-C-13) by setting the delays for J(CC) in the C-C relay sequence to the (LR)J(CC). This sequence gives correlation signals split by small (LR)J(CC), which seriously suffers from residual center signal. The unwanted signal is due to long-range C-H couplings ((LR)J(CH)). The expected relayed magnetization transfer (1)J(CH) -> (LR)J(CC) occurs in the H-1-C-13-X-(X)-C-13 isotopomer, whereas the unwanted signal of (LR)J(CH) comes from H-1-C-12-(X)-C-13 isotopomers, whose population is 100 times larger than that of the H-1-C-13-X-(X)-C-13 isotopomer. The large dispersive line of this unwanted center signal would be a fatal problem in the case of detecting small (LR)J(CC) couplings. This central signal could be removed by an insertion of BIRD pulse or X-filter. DEPT spectrum editing solved a signal overlapping problem and enabled accurate determination of particular (LR)J(CC) values. We demonstrate here the examples of structure determination using connectivity between H-1 and C-13 via four bonds, and the application of long-range C-C coupling constants to discrimination of stereochemical assignments

Thermoanaerobacter brockii Kojibiose Phosphorylaseによって合成された新規三および四糖の分離および同定

Novel tri- and tetra-saccharides were synthesized by glucosyltransfer from β-D-glucose 1-phosphate (β-D-G1P) to palatinose using Thermoanaerobacter brockii kojibiose phosphorylase. There saccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methyl derivatives, MALDI-TOF MS and NMR measurements were used for structural confirmation of the saccharides. The ^1H and ^13C NMR signals of the saccharides were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY and HMBC. These oligosaccharides were identified as 2^G-α-D-glucopyranosyl-palatinose; O-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl-(1→6)-D-fructofuranose and 2^G(2-α-D-glucopyranosyl)_2-palatinose; O-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl-(1→6)-D-fructofuranose. パラチノースは抗う蝕性のようなさまざまな機能をもつことが知られている．しかしながら，パラチノースはゆっくりではあるが小腸で加水分解をうけるため，糖尿病のような疾患をもつ患者への使用は薦められない．さらに，この糖のような低分子のオリゴ糖は比較的高浸透圧になりやすいため生体にとってよくない影響を及ぼすことがある．本研究では，二糖であるパラチノースを用い，Thermoanaerobacter brockii kojibiose phosphorylaseのグルコシル転移作用を利用し，グルコース1リン酸とパラチノースから新規オリゴ糖を合成した．反応は糖1および糖2が効率よく生成した48時間で止めた (Fig. 1)．また，転移生成物である糖1は反応10時間で最大となった (Fig. 2)．活性炭-セライトカラムおよび調製用HPLCを用いて糖1および糖2を単離し，MALDI-TOF-MS分析およびメチル誘導体のガスクロマトグラフィー分析を行い構造の推定を行った (Fig. 3)．さらにCOSY，HSQC，HSQC-TOCSYおよびHMBC (Fig. 4) の各手法を用いた2次元NMR解析により糖1を2^G-α-D-glucopyranosyl-palatinose; O-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl- (1→6)-D-fructofuranose，糖2を2^G(2-α-D-glucopyranosyl)_2-palatinose; O-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl- (1→2)-O-α-D-glucopyranosyl-(1→6)-D-fructofuranoseと同定した (Table 1)．今後，これらの糖の栄養機能について明らかにする必要があ