Regioselective Disulfide Bond Formation Research Articles

Total Synthesis of Human Hepcidin through Regioselective Disulfide‐Bond Formation by using the Safety‐Catch Cysteine Protecting Group 4,4′‐Dimethylsulfinylbenzhydryl

A safety-catch cysteine protecting group, S-4,4'-dimethylsulfinylbenzhydryl (Msbh), was designed and developed to expand the capabilities of synthetic strategies for the regioselective formation of disulfide bonds in cysteine-rich peptides. The directed regioselective synthesis of human hepcidin, which contains four disulfide bonds, was undertaken and led to a high-resolution NMR structure under more physiologically relevant conditions than previously. Conversely, hepcidin synthesized with the formerly assigned vicinal disulfide-bond connectivity displayed significant conformational heterogeneity under similar conditions. The two synthetic forms of human hepcidin induced ferroportin internalization with apparent EC50 values of 2.0 (native fold, 1) and 4.4 nM (non-native fold, 2), with 2 undergoing isomerization to 1 in the presence of ferroportin expressing cells.

Preliminary Structure–Function Relationship Studies on Insulin-Like Peptide 5 (INSL5)

Insulin-like peptide 5 (INSL5) is a two-chain, three-disulfide bonded member of insulin/relaxin superfamily of peptides that includes insulin, insulin-like growth factor I and II (IGFI and IGFII), insulin-like peptide 3, 4, 5 and 6 (INSL3, 4, 5 and 6), relaxin-1 (H1 relaxin), -2 (H2 relaxin) and -3 (H3 relaxin). Although it is expressed in relatively high levels in the gut, its biological function remains unclear. However, recent reports suggest a significant orexigenic action and a role in the regulation of insulin secretion and β-cell homeostasis, which implies that both agonists and antagonists of the peptide may have significant therapeutic applications. Modern solid phase synthesis techniques together with regioselective disulfide bond formation were employed for a preliminary structure–function relationship study of mouse INSL5. Two point mutated analogues, mouse INSL5 A-B(R24A, W25A) and mouse INSL5 A-B(K6A, R14A, Y18A) were chemically prepared, where the residues in the B-chain that may be involved in receptor activation and affinity binding, were respectively mutated. Synthetic mouse INSL5 A-B(R24A, W25A) analogue was inactive on RXFP4, the native receptor for INSL5, suggesting ArgB24 and TrpB25 are probably directly involved in INSL5 receptor activation. Mouse INSL5 A-B(K6A, R14A, Y18A) analogue had both decreased affinity and potency on RXFP4 (pIC50 7.7 ± 0.2, pEC50 7.87 ± 0.18) which indicated that one or more of these residues are critical for the binding to the receptor.

Synthesis of the IGF-II-like hormone vesiculin using regioselective formation of disulfide bonds

Diabetes mellitus, characterised by hyperglycemia and altered β-cell function, is an increasingly common disorder affecting millions of individuals world-wide. While therapeutic regimens exist to manage the condition, diabetic individuals remain prone to complications that are detrimental to both their length and quality of life. An improved understanding of the disease which may then enable development of new treatments is therefore a desirable goal. Vesiculin, a novel IGF-II-like protein was recently isolated from the secretory granules of murine β-cells, and preliminary studies indicate it is capable of signalling via the insulin receptor (IR)/insulin-like growth factor receptor 1(IGF1R) family giving it the potential to elicit both metabolic and mitogenic responses in the beta-cell. In order to facilitate further studies on this new member of the insulin-family of hormones, we undertook a chemical synthesis of the protein using regioselective disulfide bond formation.

Preparation of canine relaxin by Fmoc-solid phase synthesis and regioselective disulfide bond formation within the A- and B-chains

Abstract Background: The chemical synthesis of multi-disulfide bonded heterodimeric peptides such as insulin has long been of significant scientific and commercial interest as well as a major challenge. The development

A chemical method for investigating disulfide‐coupled peptide and protein folding

Investigations of protein folding have largely involved studies using disulfide-containing proteins, as disulfide-coupled folding of proteins permits the folding intermediates to be trapped and their conformations determined. Over the last decade, a combination of new biotechnical and chemical methodology has resulted in a remarkable acceleration in our understanding of the mechanism of disulfide-coupled protein folding. In particular, expressed protein ligation, a combination of native chemical ligation and an intein-based approach, permits specifically labeled proteins to be easily produced for studies of protein folding using biophysical methods, such as NMR spectroscopy and X-ray crystallography. A method for regio-selective formation of disulfide bonds using chemical procedures has also been established. This strategy is particularly relevant for the study of disulfide-coupled protein folding, and provides us not only with the native conformation, but also the kinetically trapped topological isomer with native disulfide bonds. Here we review recent developments and applications of biotechnical and chemical methods to investigations of disulfide-coupled peptide and protein folding. Chemical additives designed to accelerate correct protein folding and to avoid non-specific aggregation are also discussed.

Chimeric relaxin peptides highlight the role of the A-chain in the function of H2 relaxin

Human gene-2 (H2) relaxin is a member of the insulin–relaxin peptide superfamily. Because of the potential clinical applications of H2 relaxin, there is a need for novel analogs that have improved biological activity and receptor specificity. In this respect, we have chemically assembled chimeric peptides consisting of the B-chain of H2 relaxin in combination with A-chains from other insulin/relaxin family members. The peptides were prepared using solid phase peptide synthesis together with regioselective disulfide bond formation and characterized by RP-HPLC, MALDI-TOF MS and amino acid analysis. Their in vitro activity was assessed in RXFP1 or RXFP2 expressing cells. Replacement of the H2 relaxin A-chain resulted in parallel losses of binding affinity and activity on RXFP1. Not surprisingly H1A-H2B demonstrated the highest activity as the H1 A-chain shares high homology with H2 relaxin whereas INSLA-H2B, which shows low homology, had very poor activity. Importantly A-chain replacements had a dramatic effect on RXFP2 activity similar to previous results demonstrating different modes of activation of A-chain variants on RXFP1 and RXFP2. H3A-H2B is particularly interesting as it displays moderate activity at RXFP1 but poor activity at RXFP2 indicating that it may be a template for specific RXFP1 agonist development. Our study confirms that the activity of H2 relaxin at both RXFP1 and RXFP2 relies on interactions with both the B- and A-chains, and also provide new biochemical insights into the mechanism of relaxin action that the A-chain needs to be in native or near-native form for strong RXFP1 or RXFP2 agonist activity.

Lasiocepsin, a novel cyclic antimicrobial peptide from the venom of eusocial bee Lasioglossum laticeps (Hymenoptera: Halictidae)

In the venom of eusocial bee Lasioglossum laticeps, we identified a novel unique antimicrobial peptide named lasiocepsin consisting of 27 amino acid residues and two disulfide bridges. After identifying its primary structure, we synthesized lasiocepsin by solid-phase peptide synthesis using two different approaches for oxidative folding. The oxidative folding of fully deprotected linear peptide resulted in a mixture of three products differing in the pattern of disulfide bridges. Regioselective disulfide bond formation significantly improved the yield of desired product. The synthetic lasiocepsin possessed antimicrobial activity against both Gram-positive and -negative bacteria, antifungal activity against Candida albicans, and no hemolytic activity against human erythrocytes. We synthesized two lasiocepsin analogs cyclized through one native disulfide bridge in different positions and having the remaining two cysteines substituted by alanines. The analog cyclized through a Cys8-Cys25 disulfide bridge showed reduced antimicrobial activity compared to the native peptide while the second one (Cys17-Cys27) was almost inactive. Linear lasiocepsin having all four cysteine residues substituted by alanines or alkylated was also inactive. That was in contrast to the linear lasiocepsin with all four cysteine residues non-paired, which exhibited remarkable antimicrobial activity. The shortening of lasiocepsin by several amino acid residues either from the N- or C-terminal resulted in significant loss of antimicrobial activity. Study of Bacillus subtilis cells treated by lasiocepsin using transmission electron microscopy showed leakage of bacterial content mainly from the holes localized at the ends of the bacterial cells.

Role of the intra-A-chain disulfide bond of insulin-like peptide 3 in binding and activation of its receptor, RXFP2

INSL3 is a member of the insulin-IGF-relaxin superfamily and plays a key role in male fetal development and in adult germ cell maturation. It is the cognate ligand for RXFP2, a leucine-rich repeat containing G-protein coupled receptor. To date, and in contrast to our current knowledge of the key structural features that are required for the binding of INSL3 to RXFP2, comparatively little is known about the key residues that are required to elicit receptor activation and downstream cell signaling. Early evidence suggests that these are contained principally within the A-chain. To further explore this hypothesis, we have undertaken an examination of the functional role of the intra-A-chain disulfide bond. Using solid-phase peptide synthesis together with regioselective disulfide bond formation, two analogs of human INSL3 were prepared in which the intra-chain disulfide bond was replaced, one in which the corresponding Cys residues were substituted with the isosteric Ser and the other in which the Cys were removed altogether. Both of these peptides retained nearly full RXFP2 receptor binding but were devoid of cAMP activity (receptor activation), indicating that the intra-A-chain disulfide bond makes a significant contribution to the ability of INSL3 to act as an RXFP2 agonist. Replacement of the disulfide bond with a metabolically stable dicarba bond yielded two isomers of INSL3 that each exhibited bioactivity similar to native INSL3. This study highlights the critical structural role played by the intra-A-chain disulfide bond of INSL3 in mediating agonist actions through the RXFP2 receptor.

Synthesis of the proteinase inhibitor LEKTI domain 6 by the fragment condensation method and regioselective disulfide bond formation

Proteinase inhibitors are of high pharmaceutical interest and are drug candidates for a variety of indications. Specific kallikrein inhibitors are important for their antitumor activity and their potential application to the treatment of skin diseases. In this study we describe the synthesis of domain 6 of the kallikrein inhibitor Lympho-Epithilial Kazal-Type Inhibitor (LEKTI) by the fragment condensation method and site-directed cystine bridge formation. To obtain the linear LEKTI precursor, the condensation was best performed in solution, coupling the protected fragment 1-22 to 23-68. This method yielded LEKTI domain 6 of high purity and equipotent to the recombinantly produced peptide.

Cysteine and Selenocysteine Deprotection Chemistry in Peptide Synthesis

One of the most challenging areas in the field of peptide science is the chemistry of cysteine and the need for multiple protecting groups that can be removed orthogonally to facilitate regioselective disulfide bond formation. This article reviews the methods and reagents used to remove the various protecting groups of cysteine derivatives with subsequent disulfide bond formation used in peptide synthesis. Also reviewed is the related amino acid selenocysteine, which requires benzyl-type protecting groups during synthesis. As these benzyl groups are difficult to remove using older methods, we highlight the use of electrophilic, aromatic disulfides for the removal of selenocysteine (and cysteine) protecting groups with concomitant disulfide and diselenide bond formation. Keywords: Cysteine, selenocysteine, protecting groups, deprotection, peptides

Use of a Temporary “Solubilizing” Peptide Tag for the Fmoc Solid-Phase Synthesis of Human Insulin Glargine via Use of Regioselective Disulfide Bond Formation

Solid-phase peptide synthesis has been refined to a stage where efficient preparation of long and complex peptides is now achievable. However, the postsynthesis handling of poorly soluble peptides often remains a significant hindrance to their purification and further use. Several synthetic schemes have been developed for the preparation of such peptides containing modifications to aid their solubility. However, these require the use of complex chemistry or yield non-native sequences. We describe a simple approach based on the use of penta-lysine "tags" that are linked to the C-terminus of the peptide of interest via a base-labile linker. After ready purification of the now freely solubilized peptide, the "tag" is removed by simple, brief base treatment giving the native sequence in much higher overall yield. The applicability of the method was demonstrated by the novel preparation of insulin glargine via solid-phase synthesis of each of the two chains--including the notoriously poorly soluble A-chain--followed by their combination in solution via regioselective disulfide bond formation. At the conclusion of the chain combination, the solubilizing peptide tag was removed from the A-chain to provide synthetic human glargine in nearly 10% overall yield. This approach should facilitate the development of new insulin analogues as well as be widely applicable to the improved purification and acquisition of otherwise poorly soluble synthetic peptides.

Synthesis, Conformation, and Activity of Human Insulin‐Like Peptide 5 (INSL5)

Insulin-like peptide 5 (INSL5) was first identified through searches of the expressed sequence tags (EST) databases. Primary sequence analysis showed it to be a prepropeptide that was predicted to be processed in vivo to yield a two-chain sequence (A and B) that contained the insulin-like disulfide cross-links. The high affinity interaction between INSL5 and the receptor RXFP4 (GPCR142) coupled with their apparent coevolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for RXFP4. Given that the primary function of the INSL5–RXFP4 pair remains unknown, an effective means of producing sufficient quantities of this peptide and its analogues is needed to systematically investigate its structural and biological properties. A combination of solid-phase peptide synthesis methods together with regioselective disulfide bond formation were used to obtain INSL5. Both chains were unusually resistant to standard synthesis protocols and required highly optimized conditions for their acquisition. In particular, the use of a strong tertiary amidine, DBU, as Nα-deprotection base was required for the successful assembly of the B chain; this highlights the need to consider incomplete deprotection rather than acylation as a cause of failed synthesis. Following sequential disulfide bond formation and chain combination, the resulting synthetic INSL5, which was obtained in good overall yield, was shown to possess a similar secondary structure to human relaxin-3 (H3 relaxin). The peptide was able to inhibit cAMP activity in SK-N-MC cells that expressed the human RXFP4 receptor with a similar activity to H3 relaxin. In contrast, it had no activity on the human RXFP3 receptor. Synthetic INSL5 demonstrates equivalent activity to the recombinant-derived peptide, and will be an important tool for the determination of its biological function.

Disulfide bond rearrangement during regioselective oxidation in PhS(O)Ph/CH3SiCl3 mixture for the synthesis of α‐conotoxin GI

Rearrangement of disulfide bonds during the synthesis of alpha-conotoxin GI using PhS(O)Ph/CH(3)SiCl(3) oxidation procedure was observed. We have demonstrated that the protecting scheme (order of acetamidomethyl (Acm) and (t)Bu protecting groups) of the Cys residues as well as the reaction time influenced the ratio of the native and the mispaired compounds, while the temperature of the reaction mixture had no significant effect. However, in all cases the nonnative derivative was produced in high amount. The structure of the isomers was identified by the combination of enzymatic digestion and mass spectrometry measurements. We conclude that the air oxidation followed by the application of Tl(tfa)(3) for the regioselective formation of disulfide bonds leads up to the appropriate compound in the case of the synthesis of alpha-conotoxin GI, while the oxidation procedure using PhS(O)Ph/CH(3)SiCl(3) system resulted in the nonnative disulfide isomer.

The Chemistry and Biology of Human Relaxin‐3

A novel member of the human relaxin subclass of the insulin superfamily was recently discovered during a genomics database search and named relaxin-3. Like human relaxin-1 and relaxin-2, relaxin-3 is predicted to consist of a two-chain structure and three disulfide bonds in a disposition identical to that of insulin. To undertake detailed biophysical and biological characterization of the peptide, its chemical synthesis was undertaken. In contrast to human relaxin-1 and relaxin-2, however, relaxin-3 could not be successfully prepared by simple combination of the individual chains, thus necessitating recourse to the use of a regioselective disulfide bond formation strategy. Solid phase synthesis of the separate, selectively S-protected A and B chains followed by their purification and the subsequent stepwise formation of each of the three disulfides led to the successful acquisition of human relaxin-3. Comprehensive chemical characterization confirmed both the correct chain orientation and the integrity of the synthetic product. Relaxin-3 was found to bind to and activate native relaxin receptors in vitro and stimulate water drinking through central relaxin receptors in vivo. Recent studies have demonstrated that relaxin-3 will bind to and activate human LGR7, but not LGR8, in vitro. Secondary structural analysis showed it to adopt a less ordered confirmation than either relaxin-1 or relaxin-2, reflecting the presence in the former of a greater percentage of nonhelical forming amino acids. NMR spectroscopy and simulated annealing calculations were used to determine the three-dimensional structure of relaxin-3 and to identify key structural differences between the human relaxins.

Unambiguous synthesis of stromal cell-derived factor-1 by regioselective disulfide bond formation using a DMSO–aqueous HCl system

Total synthesis of human and murine stromal cell-derived factor-1s (SDF-1s) (residues 1–67) containing two disulfide bonds and an oxidation-sensitive Trp residue was achieved unequivocally by stepwise disulfide-forming reactions: A silver trifluoromethanesulfonate (AgOTf)–(DMSO)–aqueous HCl system was used in combination with air oxidation to form regioselectively each disulfide bond.

Peptides corresponding to the second epidermal growth factor-like domain of human blood coagulation factor VII: synthesis, folding and biological activity.

Factor VIIa (FVIIa) is the enzymatically active constituent of the FVIIa/tissue factor (TF) complex, the initiator of the extrinsic pathway of blood coagulation. The zymogen FVII and FVIIa are composed of discrete domains, two of which are homologous to the epidermal growth factor (EGF). This investigation examined the significance of the FVII EGF-2 domain in the processes leading to activation of factor X (FX). Peptides 47 residues in length and corresponding to the amino acid sequence of the EGF-2 domain of human FVII were prepared by solid-phase synthesis methods. Peptide variants with all six Cys residues replaced by L-2-aminobutyryl residues (1), or containing one (2a-c), two (3a,b) or three (4) disulfide bonds, were obtained by application of various S-protecting groups and oxidation methods. Peptide 4, containing the cystine bridge arrangement corresponding to that found in the native protein, was prepared by a two-step regioselective disulfide bond formation method. An evaluation of the anti-coagulant properties of peptides 1-4 revealed that all peptides, with the exception of the two-cystine isomer containing non-native disulfide pairings (3b), were potent inhibitors of TF/FVIIa-mediated activation of FX. The fully constrained peptide 4 was found to be twice as active as its completely non-constrained counterpart 1, the two peptides showing IC50 values of 1.6 +/- 0.5 microM (1) and 0.8 +/- 0.2 microM (4) with respect to TF/FVIIa-dependent FX activation. The results of this study demonstrate the functional importance of the EGF-2 domain of FVII in the induction of coagulation by the extrinsic pathway.

Synthesis of protegrin-related peptides and their antibacterial and anti-human immunodeficiency virus activity.

All disulfide analogs (types I, II and III) of protegrin (PG)-1, an 18-residue antimicrobial peptide having two intramolecular disulfide bonds, were synthesized using regioselective disulfide bond formation. Random air-oxidation of the fully reduced PG-1 formed the type III PG-1. In addition, a type III analog containing an amidated carboxy-terminal residue was also prepared. Each analog showed significant and different antibacterial and anti-human immunodeficiency virus (HIV) activity. Deletion of two disulfide bridges caused a significant decrease in activity.