Major change in regiospecificity for the exo-1,3-ß-glucanase from Candida albicans following its conversion to a glycosynthase.

The exo-1,3-ß-glucanase (Exg) from Candida albicans is involved in cell wall ß-d-glucan metabolism and morphogenesis through its hydrolase and transglycosidase activities. Previous work has shown that both these activities strongly favor ß-1,3-linkages. The E292S Exg variant displayed modest glycosynthase activity using α-d-glucopyranosyl fluoride (α-GlcF) as the donor and pNP-ß-d-glucopyranoside (pNPGlc) as the acceptor but surprisingly showed a marked preference for synthesizing ß-1,6-linked over ß-1,3- and ß-1,4-linked disaccharide products. With pNPXyl as the acceptor, the preference became ß-1,4 over ß-1,3. The crystal structure of the glycosynthase bound to both of its substrates, α-GlcF and pNPGlc, is the first such ternary complex structure to be determined. The results revealed that the donor bound in the -1 subsite, as expected, while the acceptor was oriented in the +1 subsite to facilitate ß-1,6-linkage, thereby supporting the results from solution studies. A second crystal structure containing the major product of glycosynthesis, pNP-gentiobiose, showed that the -1 subsite allows another docking position for the terminal sugar; i.e., one position is set up for catalysis, whereas the other is an intermediate stage prior to the displacement of water from the active site by the incoming sugar hydroxyls. The +1 subsite, an aromatic "clamp", permits several different sugar positions and orientations, including a 180° flip that explains the observed variable regiospecificity. The p-nitrophenyl group on the acceptor most likely influences the unexpectedly observed ß-1,6-specificity through its interaction with F229. These results demonstrate that tailoring the specificity of a particular glycosynthase depends not only on the chemical structure of the acceptor but also on understanding the structural basis of the promiscuity of the native enzyme.

A single source femtosecond-millisecond broadband spectrometer.

Time-resolved measurement of population dynamics extending over femtosecond to millisecond time scales typically requires a combination of transient absorption techniques involving different laser systems and detection schemes. The spectrometer design presented here facilitates transient absorption measurements over 12 decades with a single ultrafast laser system by picking pump and probe pulses independently from the laser oscillator pulse train. Unamplified pulses seed a photonic crystal fiber to a supercontinuum probe source for spectrally resolved measurements. The utility of the system is demonstrated by measuring triplet state dynamics following photoexcitation of vitamin B(6) in aqueous solution.

Total synthesis of (+)-hatomarubigin B.

The first total synthesis of (+)-hatomarubigin 3 is described. The tetra-O-acetyl diborate promoted Diels-Alder reaction of 5-hydroxy-8-(2',3',4',6'-tetra-O-acetyl-beta-D-glucopyranosyloxy)-1,4-naphthoquinone 8 and (E, 1R*,5R*)-3-(2'-methoxyvinyl)cyclohex-2-enol (+/-)-7 gave a mixture of four cycloadducts from which (1S,3S,6S,6aR,12aR,12bS)-1,8-dihydroxy-6-dimethoxy-1-hydroxy-3-methyl-11-(2',3',4',6'-tetra-O-acetyl-beta-D-glucopyranosyloxy)-1,2,3,4,6,6a,12a,12b-octahydrobenz[a]anthracene-7,12-dione 12 was isolated in 51% yield. Selective methylation and acetylation of 12 gave (1S,3S,6S,6aR,12aR,12bS)-1-acetoxy-6,8-dimethoxy-3-methyl-11-(2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyloxy)-1,2,3,4,6,6a,12a,12b-octahydrobenz[a]anthracene-7,12-dione 10a. Sequential aromatization, photooxidation and hydrolysis of the glucosyl unit gave (+)-3 (98% ee) in an 8% overall yield from 8.

Oligosaccharides related to xyloglucan: synthesis and X-ray crystal structure of methyl alpha-L-fucopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-alpha-D-x ylopyranoside and the synthesis of methyl alpha-L-fucopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-beta-D-xy lopyranoside.

Trisaccharides, methyl alpha-L-fucopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-alpha-D-xy lopyranoside and methyl alpha-L-fucopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-beta-D-xyl opyranoside, which are related to the side chain of xyloglucan have been synthesised. The beta-galactopyranosyl linkage of each was constructed using silver trifluoromethanesulfonate-promoted glycosylations of 2-O-acetyl-3,4,6-tri-O-benzyl-beta-D-galactopyranosyl chloride and the corresponding anomer of methyl 3,4-tri-O-benzyl-D-xylopyranoside. The resulting disaccharides were deacetylated and fucosylated using assisted halide reactions with tri-O-benzyl-alpha-L-fucopyranosyl bromide. Hydrogenolytic debenzylation of the resulting protected trisaccharides gave the methyl glycosides of the fucose-containing xyloglucan side chain. The structure of methyl alpha-L-fucopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-alpha-D-xy lopyranoside as the monohydrate was confirmed by an X-ray crystallographic study.

Oligosaccharides related to xyloglucan: synthesis and X-ray crystal structure of methyl 2-0-(alpha-L-fucopyranosyl)-beta-D-galactopyranoside.

The disaccharides methyl 2-O-(alpha-L-fucopyranosyl)-beta-D-galactopyranoside and methyl 2-O-(alpha-L-fucopyranosyl)-alpha-D-galactopyranoside have been synthesised using the assisted halide reaction of tri-O-benzyl-alpha-L-fucopyranosyl bromide with methyl 3,4,6-tri-O-benzyl-beta-D-galactopyranoside and methyl 3,4,6-tri-O-benzyl-alpha-D-galactopyranoside to construct the interresidue glycosidic linkages. A crystal structure of methyl 2-O-(alpha-L-fucopyranosyl)-beta-D-galactopyranoside was determined using Mo-K alpha X-ray data at 183 K. The space group is P1 (No. 1) with the unit cell containing two molecules of the disaccharide with unique conformations and a water molecule. The structure was refined to R = 0.0566 for 2969 reflections. The L-fucopyranosyl and D-galactopyranosyl residues have the nominal 1C4 and 4C1 conformations, respectively. The interresidue torsion angles are comparable with those generated in a recent molecular modelling study.