Glucosyl fluoride

S. Kitahata, C. F. Brewer, D. S. Genghof, T. Sawai, and E. J. Hehre, Scope and mechanism of carbohydrase action. Stereocomplementary hydrolytic and glu-cosyl-transferring actions of glucoamylase and glucodextranase with alpha- and beta-D-glucosyl fluoride, J. Biol. Chem., 256 (1981) 6017-6026. 

A small piece of Na was added to MeOH (5 mL) and the resulting solution was added to tetra-O-acetyl-a-D-glucopyranosyl fluoride (130 mg, 0.37 mmol) and 4,6-di-6>-acetyl-2-deoxy-2-fluoro-3-0-(tetra-6>-acetyl-/3-D-glucopyranosyl)-/ -D-glucosyl fluoride (44 mg, 0.07 mmol) in MeOH (5 mL) and the solution stirred (1 h) before being neutralized with Amberlite resin IR-120 (H+), fdtered and concentrated to afford a colourless residue. 

H. Kunz and W. Sager, Stereoselective glycosylation of alcohols and silyl ethers using glucosyl-fluorides and boron trifluoride etherate, Helv. Chim. Acta 68 283 (1985). 

Table I (104) shows the yields of products from maltose to malto-pentaose recovered from digests of 0.2 M crystalline a-D-glucosyl fluoride with crystalline a-amylase preparations from six different biological sources. The digests were incubated at 30 °C for 10 minutes, heat inactivated, and chromatographed for product isolation and analysis.

Maltose and maltotriose were synthesized from a-glucosyl fluoride by all the a-amylases. Maltotetraose and sizeable amounts of the pentaose... 

Table I. Maltosaccharide Synthesis from a-D-Glucosyl Fluoride...

Table II (104) shows the results of an experiment to learn the proportion of a-D-glucosyl fluoride that is hydrolyzed vs. that used for saccharide synthesis by the different crystalline a-amylases and by diluted saliva. The substrate was again 0.2 M and the enzymes at one-tenth to one-one hundredth the concentrations of the preceding experiment. These digests were analyzed directly for amounts of fluoride (104, 107) and D-glucose (glucose oxidase method) liberated during 2 hours at 30°C.

All the amylases caused release of more fluoride than D-glucose, both expressed as percent of substrate. The difference between them gives the percent substrate converted to saccharides. The main point is that with six of the seven a-amylases two to twenty times more a-D-glucosyl fluoride was converted to oligosaccharides than hydrolyzed to D-glucose. The great discrepancy was readily confirmed by direct visualization of the products by chromatography. In this experiment, preparations of so-... 

Table II. Saccharide Synthesis vs. a-D-Glucosyl Fluoride Hydrolysis by Different a-Amylases °...

The most important features are, first, that some of the a-amylases— i.e., those in digests Numbers 4, 5, 6, and 7 have catalyzed glycosylations from a-maltosyl fluoride at one-tenth to one-one hundredth the enzyme concentrations required for action on a-D-glucosyl fluoride. The Aspergillus oryzae a-amylase, which had the weakest synthetic action on a-D-glucosyl fluoride of the enzymes tested at 100 ftg/ml, has produced under conditions of this experiment an extended series of maltosaccharides at 1 fig/ml. 

The kinetic isotope effects Might/Mieavy, for the hydrolysis at pH 6.0 of a-glucosyl fluoride at 80 °C and /J-glucosyl fluoride at 50 °C, isotopically substituted as shown in structure 352, have been found381 to be as follows ... 

BEBOVIB-IV-TS structure for a-glucosyl fluoride hydrolysis involving the ring in a flattened 4C chair conformation and water and fluoride ion about the anomeric centre has been presented381,382. The conformations based on this structure reproduced well the a-D, j8-D, 5-D and [5-180] experimental KIE, but not the [1-13C] KIE (theor. KIE = 1.023 compared with 1.032 for exp. KIE). 

The qualitative TS structure for succinate-catalyzed hydrolysis of /1-glucosyl fluoride, shown as 353, has been proposed381. 

The manno thioglycoside 55 on treatment with DAST give the 2-deoxy-2-phenyl-thio-glucosyl fluoride as anomeric mixture 56. On glycosylation with the 6-hydroxy-glucoside 57 in dichloromethane and the presence of SnCl2, the ot,l- 6 disaccharide 58 is obtained exclusively (92%), which on reduction with Raney-Nickel yields the 2 -deoxy derivative 59. 

D-Glucosyl fluoride (500 mg.) and an equivalent amount of glycine in water (25 ml.) containing sodium bicarbonate was kept at room temperature for 1 day, and then for 3 days at 36-39°, at pH 7-8. The solution was now evaporated to dryness under diminished pressure, and a methanol extract was made. Addition of absolute ether precipitated the product (180 mg., 25% yield) as a colorless, amorphous material, [< ] +15.4° (in water). 

The fluoride LXXI is unaffected by aqueous solvents, and it undergoes alkaline hydrolysis, with difficulty, to give 2-acetamido-2-deoxy-D-glucose. 3,4,6-Tri-0-acetyl-2-deoxy-2 - (p - tolylsulfonamido)-a - d - glucosyl fluoride... 

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