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.

The complex-type N-linked octasaccharide oxazoline having LacNAc as the nonreducing end sugar was efficiently synthesized using the benzyl-protected LacNAc, mannose, and β-mannosyl GlcNAc units as key building blocks. To achieve a highly β-selective glycosylation with the LacNAc unit, the N-trichloroacetyl group was used for the protection of the amino group in the LacNAc unit. After complete assembly of these units and deprotection, the obtained free sugar was successfully derivatized into the corresponding sugar oxazoline. On the other hand, the N-acetylglucosaminylated saposin C, a hydrophobic lipid-binding protein, was chemically synthesized by the native chemical ligation reaction. On the basis of the previous results related to the synthesis of the nonglycosylated saposin C, the O-acyl isopeptide structure was introduced to the N-terminal peptide thioester carrying GlcNAc to improve its solubility toward aqueous organic solvents. The ligation reaction efficiently proceeded with the simultaneous O- to N-acyl shift at the O-acyl isopeptide moiety. After the removal of the cysteine-protecting group and folding, saposin C carrying GlcNAc was successfully obtained. The synthetic sugar oxazoline was then transferred to this glycoprotein using the mutant of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) (glycosynthase), and the saposin C carrying the complex-type nonasaccharide was successfully obtained.

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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...

The chemical synthesis of human interleukin-2 (IL-2) , having a core 1 sugar, by a ligation method is reported. Although IL-2 is a globular glycoprotein, its C-terminal region, in particular (99-133), is extremely insoluble when synthesized by solid-phase method. To overcome this problem, the side-chain carboxylic acid of the Glu residues was protected by a picolyl ester, thus reversing its polarity from negative to positive. This reverse polarity protection significantly increased the isoelectric point of the peptide segment and made it positive under acidic conditions and facilitated the purification. An efficient method to prepare the prolyl peptide thioester required for the synthesis of the (28-65) segment was also developed. These efforts resulted in the total synthesis of the glycosylated IL-2 having full biological activity.