Reassessment of an Innovative Insulin Analogue Excludes Protracted Action yet Highlights the Distinction between External and Internal Diselenide Bridges

Long-acting insulin analogues represent the most prescribed class of therapeutic proteins. An innovative design strategy was recently proposed: diselenide substitution of an external disulfide bridge. This approach exploited the distinctive physicochemical properties of selenocysteine (U). Relative to wild type (WT), Se-insulin[C7U^A, C7U^B] was reported to be protected from proteolysis by insulin-degrading enzyme (IDE), predicting prolonged activity. Because of this strategy's novelty and potential clinical importance, we sought to validate these findings and test their therapeutic utility in an animal model of diabetes mellitus. Surprisingly, the analogue did not exhibit enhanced stability, and its susceptibility to cleavage by either IDE or a canonical serine protease (glutamyl endopeptidase Glu-C) was similar to WT. Moreover, the analogue's pharmacodynamic profile in rats was not prolonged relative to a rapid-acting clinical analogue (insulin lispro). Although [C7U^A, C7U^B] does not confer protracted action, nonetheless its comparison to internal diselenide bridges promises to provide broad biophysical insight.     

Studies on the reaction between reduced riboflavin and selenocystine

Selenocysteine (Sec) is a crucial component of mammalian thioredoxin reductase (TrxR) where it serves as a nucleophile for disulfide bond rupture in thioredoxin (Trx). Generation of the reduced state of Sec in TrxR requires consecutive two electron transfer steps, namely: (i) from NADPH to flavin adenine dinucleotide, (ii) from reduced flavin to the disulfide bond Cys⁵⁹‐S‐S‐Cys⁶⁴, and finally (iii) from Cys⁵⁹ and Cys⁶⁴ to the selenosulfide bond Cys⁴⁹⁷‐S‐Se‐Sec⁴⁹⁸. In this work, we studied the reaction between reduced riboflavin (RibH2) and selenocystine (Sec‐Sec), an oxidized form of Sec. The interaction between RibH2 and Sec‐Sec proceeded relatively slowly in comparison with its reverse reaction, that is, reduction of riboflavin (Rib) by Sec. The rate constant for the reaction between RibH2 and Sec‐Sec was (7.9 ± 0.1) × 10^?2 M^?1 s^?1 (pH 7.0, 25.0°C). The reaction between Rib and Sec proceeded via two steps, namely, a rapid reversible binding of Rib to Sec having a protonated selenol group to form a Sec‐Rib complex, followed by nucleophilic attack of Sec‐Rib by a second Sec molecule harboring a deprotonated selenol group. The equilibrium constant for the overall reduction process of Rib by Sec is (1.2 ± 0.1) × 10⁶ M^?1 (25.0°C). The finding that the interaction of RibH2 with oxidized selenol is reversible with its equilibrium favored toward the reverse reaction provides an additional explanation for the exceptional mechanism of the mammalian Trx/TrxR system involving transient reduction of a disulfide bond.     

Preparation of Selenoinsulin as a Long‐Lasting Insulin Analogue

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^A,C7U^B] 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 _1/2≈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.     

新型桶形芋螺毒素BtIIIB的分离纯化、氨基酸序列测定及二硫键定位研究

采用凝胶色谱、高效液相色谱等方法从栖息于我国南海的桶形芋螺的毒液中纯化出一个新的芋螺毒素BtIIIB．采用氨基酸组成分析、质谱分析和Edman降解测定BtIIIB为15肽, 其氨基酸序列为: CCELPCHGCVPCCWP．结构中含有6个半胱氨酸, 形成3对分子内二硫键．采用部分还原的方法, 分步还原毒素中的二硫键, 用氰基化试剂衍生生成巯基, 然后在碱性条件下将衍生的产物进行裂解, 用MALDI-TOF MS测定裂解后片段的分子量, 确定了其二硫键的配对方式为Cys¹-Cys¹³, Cys²-Cys⁹, Cys⁶-Cys¹²的部分交叉式结构, 是芋螺毒素中比较特别的配对方式．     

Synthese von Humaninsulin. II. Aufbau des cyclischen Fragments A(1–13)

Synthesis of human insulin. II. Preparation of the A(1–13) fragment. The present report gives a detailed account of the synthesis of the protected tridecapeptide A(1–13), Boc? Gly? Ile? Val? Glu(OBu^t)? Gln Ser(Bu^t)? Leu? OH ( 20 ), an essential intermediate in the recently published total synthesis of human insulin [1]. The main feature in the synthesis of 20 was the specific formation of a disulfide bond between A6 and A11 in the presence of an additional cysteine residue (A7). The selective ring closure was accomplished with the segment A(6–13), H? Cys(Trt)? Cys(Acm)? Thr(Bu^t)? Ser(Bu^t)? Ile? Cys(Trt)? Ser(Bu^t)? Leu? OH ( 18 ), which was obtained by way of conventional synthesis routes. Treatment of 18 with iodine in trifluoroethanol formed the desired disulfide bridge from the two S-trityl-cysteine residues without affecting the S-acetamidomethyl-protected cysteine A7. A final azide coupling with the N-terminal derivative A(1–5) ( 3 ) provided the tridecapeptide fragment 20 as a crystalline compound.     

Are natural protein networks similar to synthetic elastomeric networks? Structural implications of the characteristic pentapeptide repeat from the elastic protein network of wool

The elasticity of the cross-linked matrix of wool fibers is not well understood. The primary structure of high sulfur proteins of the matrix is known to be dominated by the pentapeptide repeat CysGln²Pro³Thr⁴Cys⁵. The linear proteins containing the repeat are cross-linked by multiple CysCys disulfide bonds to form an elastic network. The existing hypothesis proposes that the disulfide bonds are formed predominantly between Cys¹ and Cys⁵ of the same repeat, which is facilitated by the tight turn induced by the repeat. To elucidate the structural implications of the repeat, we applied solution ¹H NMR to model peptides containing the repeat and also dynamic light scattering and electron microscopy to the polymeric products of their oxidation. The NMR measurements revealed that the repeat in the model peptides does not induce tight turns (water, DMSO). The NOE patterns indicate that to form the Cys¹Cys⁵ intra-repeat disulfide bond, the Gln²-Pro³ peptide bond of the repeat changes its configuration from trans to cis. A substantial energy barrier associated with this transition should decrease the propensity of the repeat for the formation of the Cys¹Cys⁵ bonds. The NMR measurements in DMSO-d₆ revealed that for the repeat with a cycle closed by the Cys¹Cys⁵ bond, a significant population of the conformations adopt a rare type VIa tight turn with cis-Pro³ in position i +2 of the turn. These results suggest that a modified model of the matrix is needed and that the nature of elasticity of the cross-linked polypeptide network from wool fiber might be similar to that of synthetic polymer networks.     

Synthesis of human insulin. III. Preparation of the A(14-21) - B(17-30) fragment (author's transl)]

Synthesis of human insulin. III. Preparation of the A(14-21) - B(17-30) fragment. In the recently published total synthesis of human insulin [1], one of the three principal intermediates is the protected fragment in which sequence 14-21 of the A chain is linked to sequence 17-30 of the B chain by the disulfide bridge between A 20 and B 19. The synthesis of this fragment, and its characterization are described in detail in the present report. This open-chain asymmetrical cystine peptide was prepared by elongating the two chains in the already published intermediate first with fragment A(14–19), Bpoc-Tyr(Bu^t)-Gln-Leu-Glu(OBu^t)-Asn-Tyr(Bu^t)-NH-NH₂ (azide coupling), and secondly with fragment B(21–30), H-Glu(OBu^t)-Arg-Gly-Phe-Phe-Tyr(Bu^t)-Thr(Bu^t)-Pro-Lys(Boc)-Thr(Bu^t)-OBu^t (DCCI/HOBt).     

Synthesis of insulin fragments with disulfide bridges between intact chains A20-B19

The synthesis of six insulin fragments is described, in which various sequences of the two chains are linked by the disulfide bridge between A²⁰ and B¹⁹. The fragments in question are: A^20–21–B^19–21, A^20–21–B^18–21, A^20–21–B^17–21, A^19–21–B^19–21, A^16–21–B^18–21 and A^20–21–B^12–21. In order to build up the simpler fragments the disulfide bridge was established by oxidation with iodine of two S-trityl cysteine peptides in which the carboxyl and amino groups were protected by the t-butyl and t-butyloxycarbonyl residue. From the mixture obtained the unsymmetrical cystine peptide was separated in all cases from the two symmetrical ones by counter-current distribution. In the synthesis of the more complex fragments advantageous use was made of smaller unsymmetrical fragments prepared as above but having one amino group protected by the N-trityl residue. After selective elimination of this group it was possible to lengthen the peptide chain at this position. The free peptides were obtained by removal of the protecting groups with strong acids, in particular concentrated hydrochloric acid. While in this deprotecting step the disulfide bond was stable, conditions are discussed under which disproportionation was observed. None of the six synthetic insulin fragments showed activity in stimulating rat adipose tissue to convert ¹⁴C-labelled glucose to CO₂ in vitro.     

The attempted synthesis of open-chain asymmetric cystine peptides by thio-induced fragmentation of sulfonyl thiocarbonates. Insulin fragments with intact disulfide bridges A20-B19

The thiol-induced fragmentations of sulfenyl thiocarbonates (R? S? S? CO? OCH₃) leading to mixed disulfides was applied to the synthesis of open-chain asymmetrical cystine peptides. Various fragments of insulin containing the disulfide bridge between A²⁰ and B¹⁹ were prepared by this method.     

Preparation of Selenoinsulin as a Long-Lasting Insulin Analogue

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^A,C7U^B] 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_1/2≈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.     

Total Chemical Synthesis of an Intra‐A‐Chain Cystathionine Human Insulin Analogue with Enhanced Thermal Stability

Despite recent advances in the treatment of diabetes mellitus, storage of insulin formulations at 4 °C is still necessary to minimize chemical degradation. This is problematic in tropical regions where reliable refrigeration is not ubiquitous. Some degradation byproducts are caused by disulfide shuffling of cystine that leads to covalently bonded oligomers. Consequently we examined the utility of the non‐reducible cystine isostere, cystathionine, within the A‐chain. Reported herein is an efficient method for forming this mimic using simple monomeric building blocks. The intra‐A‐chain cystathionine insulin analogue was obtained in good overall yield, chemically characterized and demonstrated to possess native binding affinity for the insulin receptor isoform B. It was also shown to possess significantly enhanced thermal stability indicating potential application to next‐generation insulin analogues.     

A prediction of the three-dimensional structure of maize NADP+-dependent malate dehydrogenase which explains aspects of light-dependent regulation unique to plant enzymes

Summary A model has been built for the plant NADP-malate dehydrogenase from Zea mays, a key enzyme in photosynthesis, which undergoes light-dependent regulation. The model was based on sequence and presumed structural homology to the known three-dimensional structure of mammalian porcine cytosolic NAD-malate dehydrogenase. A cystine-loop present in an extended C-terminal region of plant NADP-malate dehydrogenases was modelled using molecular mechanics and computer graphical methods, based on the assumption that a disulphide bridge exists in the inactive form of the enzyme between Cys³⁵¹ and Cys³⁶³. The predicted conformation of the intact C-terminal cystine-loop suggests that the extended polypeptide will bind in the active centre and inhibit enzyme activity. Another ionizable cysteine residue in the active site is predicted to control the charge of the catalytic His²¹⁵ and might be responsible for the uniquely tight binding of the positively charged nicotinamide ring of NADP⁺ in this and other C4 and C3 plant NADP-malate dehydrogenases.     

Divergent Synthesis of Bioactive Dithiodiketopiperazine Natural Products Based on a Double C(sp3)−H Activation Strategy

This article provides a detailed report of our efforts to synthesize the dithiodiketopiperazine (DTP) natural products (−)-epicoccin G and (−)-rostratin A using a double C(sp³)−H activation strategy. The strategy's viability was first established on a model system lacking the C8/C8’ alcohols. Then, an efficient stereoselective route including an organocatalytic epoxidation was secured to access a key bis-triflate substrate. This bis-triflate served as the functional handles for the key transformation of the synthesis: a double C(sp³)−H activation. The successful double activation opened access to a common intermediate for both natural products in high overall yield and on a multigram scale. After several unsuccessful attempts, this intermediate was efficiently converted to (−)-epicoccin G and to the more challenging (−)-rostratin A via suitable oxidation/reduction and protecting group sequences, and via a final sulfuration that occurred in good yield and high diastereoselectivity. These efforts culminated in the synthesis of (−)-epicoccin G and (−)-rostratin A in high overall yields (19.6 % over 14 steps and 12.7 % over 17 steps, respectively), with the latter being obtained on a 500 mg scale. Toxicity assessments of these natural products and several analogues (including the newly synthesized epicoccin K) in the leukemia cell line K562 confirmed the importance of the disulfide bridge for activity and identified dianhydrorostratin A as a 20x more potent analogue.     

Mapping disulfide bonds in insulin with the route 66 method: Selective cleavage of S-C bonds using alkali and alkaline earth metal enolate complexes

Simple and fast identification of disulfide linkages in insulin is demonstrated with a peptic digest using the Route 66 method. This is accomplished by collisional activation of singly and doubly charged cationic Na⁺ and Ca²⁺ complexes generated using electrospray ionization mass spectrometry (ESI-MS). Collisional activation of doubly charged metal complexes of peptides with intermolecular disulfide linkages yields two sets of singly charged paired products separated by 66 mass units resulting from selective S-C bond cleavages. Highly selective elimination of 66 mass units, which corresponds to the molecular weight of hydrogen disulfide (H₂S₂), is observed from singly charged metal complexes of peptides with disulfide linkages. The mechanism proposed for these processes is initiated by formation of a metal-stabilized enolate at Cys, followed by cleavage of the S-C bond. Further activation of the products yields sequence information that facilitates locating the position of the disulfide linkages in the peptic digest fragments. For example, the doubly charged Ca²⁺ complex of the peptic digest product GIVEQCCASVCSL/FVNQHLCGSHL yields paired products separated by 66 mass units resulting from selective S-C bond cleavages at an intermolecular disulfide linkage under low-energy collision-induced dissociation. Further activation of the product comprising the A chain reveals the presence of a second disulfide bridge, an intramolecular linkage. Experimental and theoretical studies of the disulfide linked model peptides provide mechanistic details for the selective cleavage of the S-C bond.     

Semi-synthesis of disulfide-linked branched tri-ubiquitin mimics

Ubiquitin (Ub) chain isopeptide bond mimics are useful molecules for biochemical and biophysical studies. Herein, we report the semi-synthesis of the disulfide-linked K11/K48-branched tri-Ub (Ub₃^11/48(S–S)), the first example of an isopeptide mimic for the branched Ub chains, which have recently emerged as an interesting category of Ub modifications. Our strategy comprised the E1-dependent synthesis of the Ub conjugate of aminoethanethiol, followed by disulfide formation with Ub(K11C, K48C). The structure of the synthetic isopeptide bond mimics was verified by the crystal structure of Ub₃^11/48(S–S). Deubiquitination and pulldown assays indicated that the synthetic Ub₃^11/48(S–S) could be hydrolyzed by linkage-specific deubiquitinases (K11-specific Cezanne and K48-specific OTUB1), and recognized by proteasomal ubiquitin receptor S5a.     

Total synthesis of human insulin under directed formation of the disulfide bonds

A preliminary account is given of a total synthesis of human insulin involving directed formation of the three disulfide bonds at different stages of the fragment-condensation approach. The synthesis was facilitated by the application of two new methods for the selective removal of protecting groups. In the first, two S-Trt-protected cysteine residues are converted to the disulfide without affecting S-Acm-protected cysteine residues. The second new method consists in a very mild, pH-controlled, acidolysis of N(α)-Trt, leaving intact N(α)-Bpoc and other acid-labile protecting groups. The last step of the synthesis was the formation of the disulfide bridge between the Acm-protected cysteine residues A7 and B7 by iodine. Extensive counter-current distribution yielded the synthetic hormone in pure form. It was compared and found to be identical with natural human insulin. Identification was achieved by means of thinlayer chromatography and electrophoretic procedures, as well as by comparing the pattern of break-down by enzymes (finger-printing). The natural and synthetic hormones were crystallized under identical conditions. The synthetic human insulin was found to possess full biological activity in an in vitro system.     

Redox activity and multiple copper(I) coordination of 2His–2Cys oligopeptide

Copper binding motifs with their molecular mechanisms of selective copper(I) recognition are essential molecules for acquiring copper ions, trafficking copper to specific locations and controlling the potentially damaging redox activities of copper in biochemical processes. The redox activity and multiple Cu(I) binding of an analog methanobactin peptide‐2 (amb₂) with the sequence acetyl–His₁–Cys₂–Tyr₃–Pro₄–His₅–Cys₆ was investigated using ion mobility–mass spectrometry (IM‐MS) and UV–Vis spectrophotometry analyses. The Cu(II) titration of amb₂ showed oxidation of amb₂ via the formation of intra‐ and intermolecular Cys–Cys disulfide bridges and the multiple Cu(I) coordination by unoxidized amb₂ or the partially oxidized dimer and trimer of amb₂. The principal product of these reactions was [amb₂ + 3Cu(I)]⁺ which probably coordinates the three Cu(I) ions via two bridging thiolate groups of Cys₂ and Cys₆ and the δN₆ of the imidazole groups of His_6, as determined by geometry optimized structures at the B3LYP/LanL2DZ level of theory. The products observed by IM‐MS showed direct correlation to spectral changes associated with disulfide bond formation in the UV–Vis spectrophotometric study. The results show that IM‐MS analysis is a powerful technique for unambiguously determining the major ion species produced during the redox and metal binding chemistry of oligopeptides. Copyright © 2015 John Wiley & Sons, Ltd.     

A Facile Method for Producing Selenocysteine‐Containing Proteins

Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo‐tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNA^Ser. Ser‐allo‐tRNA was converted into Sec‐allo‐tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec‐allo‐tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDH_H with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo‐tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo‐tRNA^UTu system offers a new selenoprotein engineering platform.     

Enhancing the Thermostability of a Cold-Active Lipase from Penicillium cyclopium by In Silico Design of a Disulfide Bridge

Cysteine mutants of a cold-active lipase (PcLipI) from Penicillium cyclopium were designed by the software Disulfide by Design Ver. 1.20 in an effort to improve enzyme thermostability by addition of a disulfide bridge. Those mutants predicted by molecular dynamics simulation to have better thermostability than the wild type were first expressed in Escherichia coli BL21(DE3) and then, for further investigation, in Pichia pastoris GS115. By replacing Val²⁴⁸ and Thr²⁵¹ with cysteines to create a disulfide bridge, the recombinant lipases reE-PcLip^V248C-T251C (expressed in E. coli) and reP-PcLip^V248C-T251C (expressed in P. pastoris) were obtained. Both had enhanced thermostability with half-lives at 35 °C about 4.5- and 12.8-fold longer than that of the parent PcLipI expressed in E. coli and P. pastoris, respectively. The temperature optima of reE-PcLip^V248C-T251C and reP-PcLip^V248C-T251C were 35 and 30 °C, which were each 5 °C higher than those of the parent PcLipI expressed in E. coli and P. pastoris. The K _ms of reE-PcLip^V248C-T251C and reP-PcLip^V248C-T251C toward tributyrin were 53.2 and 39.5 mM, while their V _maxs were 1,460 and 3,800 U/mg, respectively. PcLip^V248C-T251C had better thermostability and catalytic efficiency than the other mutants and the parent PcLipI.     

The S−S Bridge: A Mixed Experimental-Computational Estimation of the Equilibrium Structure of Diphenyl Disulfide

The disulfide bridge (−S−S−) is an important structural motif in organic and protein chemistry, but only a few accurate equilibrium structures are documented. We report the results of supersonic-jet microwave spectroscopy experiments on the rotational spectra of diphenyl disulfide, C₆H₅−S−S−C₆H₅ (including all ¹³C and ³⁴S monosubstituted isotopologues), and the determination of the equilibrium structure by the mixed estimation (ME) method. A single conformation of C₂ symmetry was observed in the gas phase. This disulfide is a challenging target since its structure is determined by 34 independent parameters. Additionally, ab initio calculations revealed the presence of three low-frequency vibrations (<50 cm⁻¹) associated to phenyl torsions which would prevent the calculation of an accurate force field. For this reason, instead of the semiexperimental method, we used the mass-dependent (r_m) method to fit the structural parameters concurrently to moments of inertia and predicate parameters, affected with appropriate uncertainties. The predicates were obtained by high-level quantum-chemical computations. A careful analysis of the results of different fits and a comparison with the ab initio optimizations confirms the validity of the used methods, providing detailed structural information on the title compound and the disulfide bridge.