Synthetic insulin analogues with a long lifetime are current drug targets for the therapy of diabetic patients. The replacement of the interchain disulfide with a diselenide bridge, which is more resistant to reduction and internal bond rotation, can enhance the lifetime of insulin in the presence of the insulin-degrading enzyme (IDE) without impairing the hormonal function. The [C7U ,C7U ] variant of bovine pancreatic insulin (BPIns) was successfully prepared by using two selenocysteine peptides (i.e., the C7U analogues of A- and B-chains, respectively). In a buffer solution at pH 10 they spontaneously assembled under thermodynamic control to the correct insulin fold. The selenoinsulin (Se-Ins) exhibited a bioactivity comparable to that of BPIns. Interestingly, degradation of Se-Ins with IDE was significantly decelerated (τ ≈8 h vs. ≈1 h for BPIns). The lifetime enhancement could be due to both the intrinsic stability of the diselenide bond and local conformational changes induced by the substitution.
Electrochemical microreactors, which have electrodes integrated into the flow path, can afford rapid and efficient electrochemical reactions without redox reagents due to the intrinsic properties of short diffusion distances. Taking advantage of electrochemical microreactors, Kolbe electrolysis of di-and trifluoroacetic acid in the presence of various electron-deficient alkenes was performed under constant current at continuous flow at room temperature. As a result, di-and trifluoromethylated compounds were effectively produced in either equal or higher yields than identical reactions under batch conditions previously reported by Uneyamas group. The strategy of using electrochemical microreactor technology is useful for an effective fluoromethylation of alkenes based on Kolbe electrolysis in significantly shortened reaction times.
The protein disulfide isomerase (PDI) family, found in the endoplasmic reticulum (ER) of the eukaryotic cell, catalyzes the formation and cleavage of disulfide bonds and thereby helps in protein folding. A decrease in PDI activity under ER stress conditions leads to protein misfolding, which is responsible for the progression of various human diseases, such as Alzheimer's, Parkinson's, diabetes mellitus, and atherosclerosis. Here we report that water-soluble cyclic diselenides mimic the multifunctional activity of the PDI family by facilitating oxidative folding, disulfide formation/reduction, and repair of the scrambled disulfide bonds in misfolded proteins.
Synthetic insulin analogues with al ong lifetime are current drug targets for the therapyo fd iabetic patients.T he replacement of the interchain disulfide with adiselenide bridge, which is more resistant to reduction and internal bond rotation, can enhance the lifetime of insulin in the presence of the insulin-degrading enzyme (IDE) without impairing the hormonal function. The [C7U A ,C7U B ]v ariant of bovine pancreatic insulin (BPIns) was successfully prepared by using two selenocysteine peptides (i.e., the C7U analogues of A-and Bchains,r espectively). In ab uffer solution at pH 10 they spontaneously assembled under thermodynamic control to the correct insulin fold. The selenoinsulin (Se-Ins) exhibited ab ioactivity comparable to that of BPIns.I nterestingly, degradation of Se-Ins with IDE was significantly decelerated (t 1/2 % 8hvs. % 1hfor BPIns). The lifetime enhancement could be due to both the intrinsic stability of the diselenide bond and local conformational changes induced by the substitution.Insulin, as mall globular protein (5.8 kDa), comprises two peptide chains,t he A-chain (Ins-A, 21 amino acid residues) and B-chain (Ins-B,3 0a mino-acid residues). Then ative structure in am onomeric active state is stabilized by two interchain disulfide bridges,Cys A7 -Cys B7 and Cys A20 -Cys B19 ,in addition to one intrachain disulfide linkage,C ys A6 -Cys A11 . [1] Considerable efforts have been directed toward development of various insulin analogues [2] which imitate either bolus secretion of insulin for expeditiously reducing postprandial blood glucose levels [3] or basal secretion of insulin to control the glucose level for an entire day. [4] Thel atter long-acting analogues have been designed so that insulin forms infusible precipitates or soluble oligomers (hexamer or dihexamer) under physiological conditions and slowly releases active insulin monomers.In contrast, the insulin-degrading enzyme (IDE) is ap ossible alternative target for diabetes therapy.I DE, which is involved in clearance of insulin and amyloid b (Ab), [5] is found in the liver and kidneys.Recent research has revealed that synthetic IDE inhibitors increase circulation of insulin by preventing its degradation in the liver,t hus resulting in improvement of the postprandial glucose tolerance. [6] However,other research suggests that IDE inhibitors could induce accumulation of Ab in the brain, [7] and would lead to Ab-mediated cognitive impairment. Hence,the design of long-lasting insulin analogues resistant against IDE would be desirable. [8] In this study,wehave attempted anew approach to alonglasting insulin analogue by exploiting the unique chemical properties of adiselenide bond. Namely,i ntroduction of two juxtaposed selenium atoms to the insulin analogue could lead to ah igher kinetic and thermodynamic stability than that of the wild-type without affecting the bioactivity.T his new strategy is based primarily on the higher rotational barrier of aSe À Se bond (ca. 4kcal mol À1 )than that of an S À Sbond (ca. 3kcal mol À1 ), [9] and se...
To elucidate the reaction mechanism of the disulfide (SS) bond formation reaction of a polypeptide molecule with a water-soluble selenoxide reagent, trans-3,4-dihydroxyselenolane oxide (DHS(ox)), short-term oxidation experiments were carried out for the reduced state (R) of a recombinant hirudin CX-397 variant at pH 7.0 and 25 °C. In the reaction, R was oxidized sequentially to one-SS, two-SS, and three-SS intermediate ensembles within 1 min. The kinetic analysis revealed that the three second-order rate constants for the SS formation are proportional to the number of thiol groups existing in the reactant SS intermediates, indicating the stochastic nature of the SS formation. Ab initio calculation at the HF/6-31++G(d,p) level in water by using the polarizable continuum model suggested that the SS formation reaction is highly exothermic and proceeds via a reactive thioselenurane intermediate with a distorted linear O-Se-S linkage. The results clearly demonstrated that the rate-determining step of the SS formation reaction is the first bimolecular process between a thiol substrate and DHS(ox) rather than the subsequent process to release a SS product.
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