Maltosyl fluoride

Glycosides may also be prepared by enzyme-catalyzed condensation reactions utilizing a glycosyl fluoride. Thus 6-0-a-maltosylcyclodextrins were prepared enzymically from a-maltosyl fluoride (obtained from the corresponding heptaacetate by Zemplen deacetylation) and cyclodex-trins. " ... 

Hehre and coworkers showed that beta amylase from sweet potatoes, an inverting, a-specific exo-(l 4)-glucanase, catalyzes the hydrolysis of jS-maltosyl fluoride with complex kinetics which indicated the participation of two substrate molecules in the release of fluoride ion. Furthermore, the reaction was strongly accelerated by the addition of methyl ) -maltoside. Hydrolysis of a-maltosyl fluoride, on the other hand, obeyed Michaelis-Menten kinetics. The main product with both a- and yj-maltosyl fluoride was )S-maltose. The results with )3-maltosyl fluoride were interpreted by the assumption of a glycosylation reaction preceding hydrolysis by which a malto-tetraoside is formed by the replacement of fluoride ion by a second substrate molecule or added methyl -maltoside (see Scheme 5). 

Scheme 5.—Hydrolysis of a- and /J-Maltosyl Fluoride by Beta Amylase (R = o-d-G1uco-syl, R = H or a- Maltosyl).

A more attractive chemo-enrymatic approach for the production of he-mithiocyclodextrins has been developed. More than ten years ago, cyclodextrin glucosyltransferase (CGTase) was shown to use a-maltosyl fluoride for the synthesis of a-, p-, and y-cyclodextrins [47] and has been used for the chemoenzymatic synthesis of regioselectively substituted cyclodextrins [48]. For this purpose, 4-thio-a-maltosyl fluoride (58) was easily prepared from (45). Incubation of (58) in the presence of pure CGTases afforded hemithiocyclodex-trins (57a), (57b) and (57c) isolated in 14,16 and 15% yields respectively [49]. 

Maltosaccharide Synthesis from Dilute Solutions of a-Maltosyl Fluoride... 

As detailed under Experimental, we have obtained this compound via the known hepta-O-acetyl a-maltosyl fluoride (108), and although the preparations we have made and studied so far are only partly pure, we believe the results obtained with them are valid. A final chromatographic step is used to prepare the compound, but one by-product... 

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. 

In this case the predominance of glycosylation of C4-carbinols over substrate hydrolysis is in a mixture where the molar concentration of water was more than 3000 times that of the a-maltosyl fluoride. In other experiments the substrate concentration could be further reduced at least 5-fold without altering the fact of a substantially greater extent of maltosidic linkage synthesis than hydrolysis. We now are referring to a very dilute 4 mM, solution of substrate with which, if the hydrolase concept were valid, hydrolysis ought to have been predominant. 

Figure 3. Maltosaccharide synthesis from a-maltosyl fluoride by a-amylases. Mixtures (100 pliters) contained 1.4 mg substrate (a-malto-syl fluoride, 1.8 fimoles) and a-amylase in 0.05 M pH 5.5 acetate buffer (or buffer alone). After incubation (30°C, 30 min) 15 pliters of each mixture was chromatographed.

C = control of a-maltosyl fluoride incubated without enzyme. 

Amylose is a linear polymer of glucose mainly linked with an al,4-glycosidic bond. Maltooligosaccharides have been effectively prepared by polycondensation of a-D-maltosyl fluoride using an a-amylase from Aspergillus oryzae as the catalyst in a mixed solvent of methanol-phosphate buffer (119). 

Kobayashi S, Shimada J, Kashiwa K, Shoda S. Enzymic polymerization of a-D-maltosyl fluoride using a-amylase as catalyst a new approach for synthesis of maltooligosaccharides. Macromolecules 1992 25 3237-3241. 

Active-site structures have been described for these related glycosylases whose reactions with maltosidic substrates and a-maltosyl fluoride proceed with retention of configuration. Their stereochemical behavior with minisubstrates of improper configuration has not been described. 

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