The hydrolysis reactions of three (2-deoxy-/?-D-glucopyranosyl)pyridinium salts exhibit first-order rate constants that are independent of pH in the range of 4.4-10.1 pH units. Derived second-order rate constants for the hydrolysis reactions of (2-deoxy-/J-D-glucopyranosyl)-4'-bromoisoquinolinium bromide (5b) conducted in the presence of nucleophilic monoanions (µ = 2.0) including AcO-, Cl-, Br-, and N3-exhibit a Swain-Scott parameter (s) of 0.03 ± 0.05, indicating that these reactions show no sensitivity to the nature of the anion. However, a substantial quantity of the (2-deoxyglucopyranosyl)pyridinium salt hydrolysis product is formed as a result of a post-ratelimiting reaction involving a nucleophilic anion. Analysis of the product ratios indicates that the first-formed intermediate in the hydrolytic reaction is a solvent-separated ion painmolecule encounter complex. The data allow a calculated estimate of greater than 2.5 x 10-12 s for the lifetime of the glucopyranosyloxocarbenium ion in aqueous solution.
A series of human insulin maleimido derivatives with short and long linkers was synthesized by exploiting the variations in the pK(a) values and environment of the three amino groups present in the protein. The syntheses were accomplished in organic solvent because of maleimide's instability in basic aqueous media. The derivatives thus obtained were conjugated to the free thiol on Cys34 of human serum albumin (HSA) and purified. A structure-activity relationship based on in vitro receptor binding and activation results for this series of insulin-HSA conjugates showed that the best compounds were attached at the B1 position of insulin with either short or long linkers. Two conjugates were administered subcutaneously to streptozotocin-induced diabetic rats and found to possess blood glucose normalizing activity up to 8 h post-administration. The return to diabetic plasma glucose levels was not observed within the time frame of the experiment (48 h). In comparison, the insulin-treated group's normalization activity lasted 2 h and returned to a diabetic level at 8 h. The onset of the conjugate activities were delayed by 1 h when compared to the activity of human insulin. The study results led to the identification of CJC-1575 as a potent and long lasting human insulin analogue.
Kinetic isotope effects (KIEs) on the yeast R-glucosidase-catalyzed hydrolysis of two R-D-glucopyranosyl pyridinium salts were measured at 25 °C and pH ) 6.8. The measured KIEs on k cat for the 2 H-2, 13 C-1, and 15 N-1′ labeled substrates R-D-glucopyranosyl pyridinium bromide (1) and R-D-glucopyranosyl isoquinolinium bromide (2) were, respectively, 1.115 ( 0.006 and 1.106 ( 0.009, 1.028 ( 0.006 and 1.027 ( 0.005, and 1.019 ( 0.007 and 0.985 ( 0.005. KIEs for the spontaneous hydrolysis of R-D-glucopyranosyl 4′-bromoisoquinolinium bromide (3) were measured at 80 °C and pH ) 6.8. The measured KIEs on k hyd for the 2 H-1, 2 H-2, 13 C-1, and 15 N-1′ labeled substrate R-D-glucopyranosyl 4′-bromoisoquinolinium bromide (3) were, respectively, 1.189 ( 0.009, 1.094 ( 0.010, 1.005 ( 0.002, and 1.015 ( 0.004. The KIEs obtained in this study of yeast R-glucosidase-catalyzed hydrolysis reactions are consistent with a transition state which involves a small degree of nucleophilic attack by an enzyme active site carboxylate group at the anomeric carbon center, whereas the corresponding transition state for the uncatalyzed reaction in water does not involve any nucleophilic assistance. In addition, the equilibrium isotope effects for deprotonation of the conjugate acid (K a 14 /K a 15 ) of isoquinoline was measured to be 1.0216 ( 0.0005.
Peptide inhibitors corresponding to sequences in the six helix bundle structure of the fusogenic portion (gp41) of the HIV envelope glycoprotein have been successfully implemented in preventing HIV entry. These peptides bind to regions in HIV gp41 transiently exposed during the fusion reaction. In an effort to improve upon these entry inhibitors, we have successfully designed and tested peptide analogs composed of chemical spacers and reactive moieties positioned strategically to facilitate covalent attachment. Using a temperature-arrested state prime wash in vitro assay we show evidence for the trapping of a pre-six helix bundle fusion intermediate by a covalent reaction with the specific anti-HIV-1 peptide. This is the first demonstration of the trapping of an intermediate conformation of a viral envelope glycoprotein during the fusion process that occurs in live cells. The permanent specific attachment of the covalent inhibitor is projected to improve the pharmacokinetics of administration in vivo and thereby improve the long-term sustainability of peptide entry inhibitor therapy and help to expand its applicability beyond salvage therapy.
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