RING-OPENING AND CLOSING RATES FOR THIOSUGARS

Reaction of 4, 4ʹ-dipyridyl disulfide (4PDS) with thiols requires the RS- form, and shows a Brønsted βnncvalue of 0, 34. The second-order rate constants for the process are 6.6 X 104, 4.6 X 106, and 6.9 X 107 M-1 s-1 for reaction of mercaptoethanol (pK = 9.5) with the neutral, mono-, and diprotonated forms of 4PDS. With 2, 2ʹ-dipyridyl disulfide (2PDS) the corresponding rate constants are 5.6 X 104, 1.5 X 108, and 1.7 X 1010 M-1 s-1. The increased reactivity of the protonated forms of 4PDS and 2PDS permits the dipyridyl disulfides to be used as reagents for thiols over the pH range -1 to 9. Ring opening and closing rates and the proportion of the sugar with a free SH group have been measured for several thiosugars by reaction with 4PDS, 2PDS, and 5, 5ʹ-dithiobis(2-nitrobenzoic acid) (DTNB). Ring opening and closing are base catalyzed from pH 6 to 10, and at lower pH values a pH-independent rate is seen. For most thiosugars, base-catalyzed ring opening shows kB= 40-150 M-1 s-1, but the value for 6-thio-D-fructose is 17 200 M-1 s-1. The pH-independent rate for 6-thio-D-fructose is also 180 times that for 5-thio-D-glucose. Mutarotation of 5-thio-α-D-glucopyranose ([α]25D +215°) to the equilibrium mixture (80% a and 20% β; [α]25D+188°) is base catalyzed at high pH, acid catalyzed below pH 1, and pH independent between pH 1 and 3?with the base-catalyzed and pH-independent rates being 520 times faster than and roughly equal to, respectively, the corresponding ring-opening rates. Base-catalyzed mutarotation is postulated to proceed through intermediates in which the sulfur has a negative charge and forms an induced dipolar bond with the carbonyl carbon of the aldehyde. Rotation of the aldehyde group and ring closure produce mutarotation, while ring opening, as measured by reaction with disulfide reagent, requires movement of the sulfur away from the aldehyde so it can react. 6-Thio-β-D-fructopyranose is converted to the β-furanose form at a rate sufficient for it to be a substrate for fructokinase (which is specific for the β-furanose) with a Vmaxthe same as that of D-fructose and a of 6-10 mM, although at high enzyme levels ring opening becomes rate limiting. The disulfide of 6-thio-D-fructose and 4-mercaptopyridine (wnich should exist 82% as the β-furanose) has a Vmax16% that of D-fructose and a Kmof M MM, the lowest known for this enzyme; the calculated values for the β-furanose forms of this disulfide and D-fructose are identical. By assuming the Kmof the β-furanose of 6-thio-D-fructose also to be the same as that of D-fructose, and the relative amounts of the anomeric forms with a free SH group to be the same as with D-fructose, 6-thio-D-fructose is calculated to contain 0.6%β-furanose, 0.11% α-furanose, and 0.02% acyclic form. At pH 7 the ring-opening rate for theβ-furanose is 0.19 s-1, while the ring-closing rates to β-furanose and β-pyranose are 6 and 9 s-1. Equilibration between the furanoses of 6-thio-D-fructose, and presumably also of D-fructose, is thus rapid (estimated half-time 1.2-3.5 s at 25 °C). © 1979, American Chemical Society. All rights reserved.