Interchain disulfide bridges

Intrachain S—S (disulfide) cross-bridges between cysteine residues in the polypeptide chain are cleaved. (If these disulfides are interchain linkages, then step 2 precedes step 1.)... 

FIGURE 15.3 Proinsulin is an 86-residue precursor to insulin (the sequence shown here is human proinsulin). Proteolytic removal of residues 31 to 65 yields insulin. Residues 1 through 30 (the B chain) remain linked to residues 66 through 87 (the A chain) by a pair of interchain disulfide bridges. 

Disulfide bridges formation ChEs contain 8-10 cysteines six of these form three internal disulfide bridges. The cysteine that is located four amino acids upstream the carboxyl terminus forms a disulfide bridge with a cysteine of an identical subunit, creating an interchain disulfide bridge, which stabilizes the dimeric structure. 

Besides being presently the most efficient method for site-directed interchain disulfide bridging of two different cysteine peptides, this procedure is also recommended for a controlled peptide-protein conjugation by reacting Npys-protected cysteine peptides with properly thiol-functionalized protein carriers. 1761 In this conjugation procedure the amount of peptide grafted to the protein is quantitatively determined by measuring spectro-photometrically at 430 nm the amount of 3-nitropyridine-2(l//)-thione liberated in the reaction. An example of this approach is outlined in Scheme 18. 

A further development of the DMSO/H+ method for oxidation of cysteine peptides led to the cysteine-sulfoxide acid-catalyzed intermolecular disulfide formation with a second S-unprotected or acid-labile protected cysteine component as shown in Scheme 19. 1471 The protonation of the sulfoxide by TfOH in the case of 5(0)-Mob or TFA in the case of 5(0)-Acm derivatives provides electrophilicity to the sulfur atom to allow attack by the second S-unprotected cysteine component (formed by the fast deprotection of the 5-Mob group with TfOH in presence of dimethylsulfide) to generate in a site-directed manner the interchain disulfide bond. Although extensive experience with this method has not been accumulated for interchain disulfide bridging, it has been successfully applied for intrachain site-directed disulfide bond formation in chicken calcitonin-gene-related peptide.1 79 ... 

Alternatively, both peptide chains could be protected at one cysteine residue as a 5-Acm derivative and at the second cysteine residue by an acid-labile [Trt, Mob, Xan, or Bzl(4-Me)], base-labile (Fm), or reduction-labile (5-tBu) group. Both peptide chains may then be separately converted into the free thiol/Acm-protected form for selective activation of one chain as S-SPy or. S -Npys derivatives by reaction with di(2-pyridyl)disulfide or di[5-nitro(2-pyridyl)]disulfide, or as a sulfenohydrazide derivative by reaction with azodicarbocylic acid derivatives for formation of the first interchain disulfide bridge. 

Cleavage of the Trt group of one chain 54 with a weak acid to give 55 and its subsequent thiolysis of the. S -SPy derivative of the second chain 57 directs the formation of the first interchain disulfide bond in 58. The second interchain disulfide bridge is formed between the two Acm-protected cysteine residues of the [bis(Acm), bis(tBu), mono-disulfide]-hetero-dimer 58 by treatment with iodine. Finally, treatment of 59 with chlorosilane/sulfoxide produces the third disulfide bond between the two tBu-protected cysteine residues yielding human insulin (42). 

Scheme 6 Formation of Two Interchain Disulfide Bridges with Different Connectivities Using Two Kinds of Protecting Groups for the Cysteine Residues. Synthetic Schemes for the Two Isomers of the Tryptic/Chymo-tryptic Fragment of (0-Conotoxin-GVIA,62l...

A. General description Eptifibatide is a cyclic heptapeptide containing six amino acids and one mercaptopropionyl residue. An interchain disulfide bridge is formed between the cysteine amide and the mercaptopropionyl moieties. Eptifibatide binds to the platelet receptor glycoprotein (gp) Ilb/IIIa of human platelets and inhibits platelet aggregation. The eptifibatide peptide is produced by solution-phase peptide synthesis, and is purified by preparative reverse-phase liquid chromatography and lyophifized. 

Formation of Disulfide Cross-Links After folding into their native conformations, some proteins form intrachain or interchain disulfide bridges between Cys residues. In eukaryotes, disulfide bonds are common in proteins to be exported from cells. The cross-links formed in this way help to protect the native conformation of the protein molecule from denaturation in the extracellular environment, which can differ greatly from intracellular conditions and is generally oxidizing. 

The disulfide bridges in some proteins are between different peptide chains. Insulin, for instance, has two interchain as well as one intrachain S—S bridges (Figure 25-8). 

Figure 2 Sequences and Stabilities of Interchain Disulfide-Bridged Coiled Coils[151a b c d...

The sequences of synthetic peptides used to form two-stranded a-helical coiled coils with an interchain disulfide bond are shown above. The positions of the cysteine residues are indicated by the solid circles above the sequences. The peptide with a cysteine at position 2 (position a) is designated as C2a. b Line plots (a) A plot of [Gdn HCl]1/2, the transition midpoint of the denaturation profiles in the presence of Gdn HC1 (guanidinium chloride), versus the position of an interhelical disulfide bond at positions 2, 5, 9,12, 16,19,23,26,30, or 33. Open triangles denote the coiled coils with a terminal disulfide bond at either position 2 or position 33. Open squares denote the coiled coils with nonterminal disulfide bonds at position d (5,12,19, or 26). Open circles denote the coiled coils with nonterminal disulfide bonds at position a (9, 16, 23, or 30). Closed symbols denote the coiled coils without a disulfide bridge (reduced cysteine residues). The broken line indicates the [GdnHCl]1/2 of the native coiled coil. 

Monera et a I.135 prepared the three disulfide-bridged two-stranded coiled coils shown in Scheme 7a-c. Two peptides were parallel homostranded coiled coils, differing only by the position of their interchain disulfide bond, which was at the N-terminal (2-2 ) or at the C-terminal (33-33 ). A heterostranded antiparallel coiled coil can be prepared by oxidation of these two reduced peptides to form an interchain disulfide bridge (2-33 ) (Scheme 7c). 

Fig. 5. Schematic representation of the collagen IV molecule, which consists of two al(IV) chains and one a2(IV) chain. The non-triple-helical interruptions of the triple helix are indicated by black bars. The cysteine residues (C) and lysine or hydroxylysine (K) residues putatively involved in intra- or intermolecular bonds are shown. CHO designates a N-giycosidically bound oligosaccharide chain. The subscript numerals indicate the number of residues in a distinct region, summarized for all three a-chains. P designates a main pepsin cleavage site. In interruption 13, the a2(IV) chain forms a 21-residue-long loop, stabilized by an interchain disulfide bridge. NCI, Noncollagenous domain 1 TH, triple-helical domain 7 S, carboxyl-terminal domain.

---