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PubMed1981

In vitro studies on the metabolic pathway of SQ 14225 (Captopril) and mechanism of mixed disulfide formation.

The metabolic pathway of SQ 14225 (Captopril) and the mechanism of the mixed disulfide formation with endogenous sulfhydryl compounds were studied in in vitro cell free systems. In the rat liver 9000 x g supernatants, SQ 14225-14C was metabolized to one major metabolite, glutathione-SQ 14225 mixed disulfide (GSSQ), and two minor metabolites including SQ 14551, a symmetrical disulfide of SQ 14225. The formation of GSSQ was markedly accelerated by the addition of oxidized glutathione (GSSG), but not affected by the addition of reduced glutathione (GSH), indicating that GSSQ was formed by the thiol-disulfide interchange between SQ 14225 and GSSG. Although the thiol-disulfide interchange was also observed between SQ 14225 and L-cystine, L-homocystine and SQ 14551 as well as GSSG, only the formation of GSSQ was a rapid reaction and markedly decreased by heat treatment of the liver 9000 x g supernatants. These findings demonstrate that the formation of GSSQ is catalyzed by a GSSG specific enzyme which is supposed to be thioltransferase (Glutathione: Disulfide Oxidoreductase). Although GSSQ was stable in the rat liver 9000 x g supernatants, it was rapidly hydrolyzed to cysteine-SQ 14225 mixed disulfide (CySSQ) in the rat kidney 9000 x g supernatants. A specific inhibitor of gamma-glutamyltranspeptidase, anthglutin, inhibited the hydrolysis. GSSQ-14C administered to a beagle dog was excreted into the urine in the form of CySSQ. Thus, it was speculated that CySSQ excreted into the urine as a major metabolite of SQ 14225 was derived from GSSQ formed in the liver followed by hydrolysis in the kidney.

Animals↗
PubMed2005

The genomics of disulfide bonding and protein stabilization in thermophiles.

Thermophilic organisms flourish in varied high-temperature environmental niches that are deadly to other organisms. Recently, genomic evidence has implicated a critical role for disulfide bonds in the structural stabilization of intracellular proteins from certain of these organisms, contrary to the conventional view that structural disulfide bonds are exclusively extracellular. Here both computational and structural data are presented to explore the occurrence of disulfide bonds as a protein-stabilization method across many thermophilic prokaryotes. Based on computational studies, disulfide-bond richness is found to be widespread, with thermophiles containing the highest levels. Interestingly, only a distinct subset of thermophiles exhibit this property. A computational search for proteins matching this target phylogenetic profile singles out a specific protein, known as protein disulfide oxidoreductase, as a potential key player in thermophilic intracellular disulfide-bond formation. Finally, biochemical support in the form of a new crystal structure of a thermophilic protein with three disulfide bonds is presented together with a survey of known structures from the literature. Together, the results provide insight into biochemical specialization and the diversity of methods employed by organisms to stabilize their proteins in exotic environments. The findings also motivate continued efforts to sequence genomes from divergent organisms.

Amino Acid Sequence↗
PubMed2001

Effects of cysteine to serine substitutions in the two inter-chain disulfide bonds of insulin.

Using site-directed mutagenesis we deleted the two inter-chain disulfide bonds of insulin, separately or both, by substitution of the cysteine residues with serine. Deletion of A20-B19 or both of the two inter-chain disulfide bonds resulted in the complete loss of secretion of the mutant single-chain porcine insulin precursor (PIP) from Saccharomyces cerevisiae cells. Removal of the A7-B7 disulfide bond resulted in a large reduction of secretion, but we could obtain the mutant for analysis of its biological and some physico-chemical properties. The A7-B7 disulfide bond deleted insulin mutant retained only 0.1% receptor-binding activity compared with porcine insulin, and its in vivo biological potency measured by mouse convulsion assay was also very low. We also studied some physico-chemical properties of the mutant using circular dichroism, native polyacrylamide gel electrophoresis and reversed-phase HPLC, which revealed some structural changes of the mutant peptides compared to native insulin. The present study shows that the two inter-chain disulfide bonds are important for efficient in vivo folding/secretion of PIP from yeast, especially the A20-B19 disulfide bond, and that the A7-B7 disulfide bond is crucial for maintaining the native conformation and biological activity of insulin.

Amino Acid Substitution↗
PubMed2004

Replacement of the interchain disulfide bridge-forming amino acids A7 and B7 by glutamate impairs the structure and activity of insulin.

Insulin contains three disulfide bonds, one intrachain bond, A6-A11, and two interchain bonds, A7-B7 and A20-B19. Site-directed mutagenesis results (the two cysteine residues of disulfide A7-B7 were replaced by serine) showed that disulfide A7-B7 is crucial to both the structure and activity of insulin. However, chemical modification results showed that the insulin analogs still retained relatively high biological activity when A7Cys and B7Cys were modified by chemical groups with a negative charge. Did the negative charge of the modification groups restore the loss of activity and/or the disturbance of structure of these insulin analogs caused by deletion of disulfide A7-B7? To answer this question, an insulin analog with both A7Cys and B7Cys replaced by Glu, which has a long side-chain and a negative charge, was prepared by protein engineering, and its structure and activity were analyzed. Both the structure and activity of the present analog are very similar to that of the mutant with disulfide A7-B7 replaced by Ser, but significantly different from that of wild-type insulin. The present results suggest that removal of disulfide A7-B7 will result in serious loss of biological activity and the native conformation of insulin, even if the disulfide is replaced by residues with a negative charge.

Amino Acids↗
PubMed2000

Structures of scrambled disulfide forms of the potato carboxypeptidase inhibitor predicted by molecular dynamics simulations with constraints.

The structures of two species of potato carboxypeptidase inhibitor with nonnative disulfide bonds were determined by molecular dynamics simulations in explicit solvent using disulfide bond constraints that have been shown to work for the native species. Ten structures were determined; five for scrambled A (disulfide bonds between Cys8-Cys27, Cys12-Cys18, and Cys24-Cys34) and five for the scrambled C (disulfide bonds Cys8-Cys24, Cys12-Cys18, and Cys27-Cys34). The two scrambled species were both more solvent exposed than the native structure; the scrambled C species was more solvent exposed and less compact than the scrambled A species. Analysis of the loop regions indicates that certain loops in scrambled C are more nativelike than in scrambled A. These factors, combined with the fact that scrambled C has one native disulfide bond, may contribute to the observed faster conversion to the native structure from scrambled C than from scrambled A. Results from the PROCHECK program using the standard parameter database and a database specially constructed for small, disulfide-rich proteins indicate that the 10 scrambled structures have correct stereochemistry. Further, the results show that a characteristic feature of small, disulfide-rich proteins is that they score poorly using the standard PROCHECK parameter database. Proteins 2000;40:482-493.

Carboxypeptidases↗
PubMed1975

Effects of various protein-modifying agents and the aminonucleoside of puromycin on dithioerythritol-reducible disulfide in glomerular basement membrane.

Comparative study of dithioerythritol-reducible (DTE) disulfide bonds in glomerular basement membranes (GBM) isolated from normal rats and from similar groups of rats treated with the nephrosis-producing aminonucleoside of puromycin emphasize not only the importance of such linkages in the interaction and structural organization of the macromolecular GBM collagen-glycoprotein matrix but also suggest a modality by which GBM semi-permeability might be engendered. Although DTE-reducible disulfide is significantly reduced in GBM of rats as early as the fourth day after administration of a nephrosis-producing dose of the aminonucleoside it has not been possible to demonstrate an unequivocal in vitro or direct effect of the drug on DTE-reducible disulfide in normal GBM. Several-fold increases in DTE-reducible disulfide in GBM subjected to the denaturing action of guanidine-HCl or the proteolytic action of pronase indicates that most of the disulfide lies buried in the GBM. Location of disulfide crosslinks in the innermost regions or core of the GBM might be expected to not only stabilize the membrane but also to protect the GBM from a considerable array of disulfide cleaving (reductases) within the kidney cortex.

Animals↗
PubMed2003

Influence of A7-B7 disulfide bond deletion on the refolding and structure of proinsulin.

To probe the role of [A7-B7] disulfide bond in the structure and folding of proinsulin, [A7, B7Ser]-proinsulin was prepared. The differences in in vitro refolding, oxidation of free thiol groups, circular dichroism (CD) spectra, antibody and receptor binding assays, and sensitivity to tryptic digestion between the mutant and the wild type proteins were studied. The deletion of [ A7-B7] disulfide bond in proinsulin resulted in a significant decrease of alpha- helix content of the molecule and a great increase in sensitivity to tryptic digestion. The [A7-B7] disulfide bond deleted proinsulin showed a great loss of its receptor binding activity. The in vitro refolding study indicated that the r ate of the oxidation of free thiol groups in the mutant was a little slower at t he later stage as compared to the native molecule, but the deletion of [A7-B7 ] disulfide bond had little effect on the refolding yield. A possible proinsulin folding pathway way was proposed. During the folding, the intra-A chain disulfide bond forms first and very fast. The formation of the [A20-B19] disulfide bond is more crucial than [A7-B7] disulfide bond and forms very likely earlier than the latter.

Amino Acid Substitution↗
PubMed1992

Structure of an antifreeze polypeptide from the sea raven. Disulfide bonds and similarity to lectin-binding proteins.

The antifreeze polypeptide (AFP) from the sea raven, Hemitripterus americanus, is a member of the cystine-rich class of blood antifreeze proteins which enable survival of certain fishes at sub-zero temperatures. Sea raven AFP contains 129 residues with 10 half-cystine residues. We have analyzed these half-cystine residues and established that all 10 of the half-cystine residues appeared to be involved in disulfide bond formation and that disulfide bonds linked Cys7 to Cys18, Cys35 to Cys125, and Cys89 to Cys117. These assignments were established by extensive proteolytic digestions of native AFP using pepsin and thermolysin and purification of the peptides by Sephadex G-15 gel filtration chromatography, anion exchange chromatography, and C18 reverse-phase high performance liquid chromatography. Cystine-containing peptides were detected by a colorimetric assay using nitrothiosulfobenzoate. Disulfide-containing peptides were reduced and alkylated, purified, and analyzed by amino acid analysis. The unreduced disulfide-linked peptides were sequenced directly by automated Edman degradations to confirm the disulfide assignments. Possible arrangements of the two remaining disulfide bonds include linkages Cys69/111 to Cys100/101. The sea raven AFP shares structural similarity with pancreatic stone protein and several lectin-binding proteins, especially with respect to half-cystines, glycines, and bulky aromatic residues. Two of the disulfide linkages we determined for sea raven AFP: Cys7-Cys18 and Cys35-Cys125, are conserved in these proteins. These similarities in covalent structure suggest that the sea raven AFP, pancreatic stone protein, and several lectin-binding proteins comprise a family of proteins which may possess a common fold.

Amino Acid Sequence↗
PubMed1991

Determination of disulfide bond pairs and stability in recombinant tick anticoagulant peptide.

Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of blood coagulation factor Xa (Waxman, L., Smith, D.E., Arcuri, K.E., and Vlasuk, G.P. (1990) Science 248, 593-596). The 60-amino acid sequence of TAP shows limited homology to Kunitz-type inhibitors, including cysteines at positions 5, 15, 33, 39, 55, and 59. For detailed biochemical and pharmacological studies, a recombinant version of TAP (rTAP) has been produced in yeast. To determine the arrangement of the disulfide bonds, rTAP was cleaved with trypsin and chymotrypsin and the purified peptides sequenced using a gas-phase sequenator. The positions of the disulfide bonds were assigned by identifying the cycle(s) at which di-phenylthiohydan-toin-cystine was released. The specific disulfide bridges, Cys-5 to Cys-59, Cys-15 to Cys-39, and Cys-33 to Cys-55, are analogous to those in the prototype Kunitz-type inhibitor, bovine pancreatic trypsin inhibitor (BPTI). While treatment of BPTI with dithiothreitol rapidly and specifically reduced one disulfide bond, the reduction of disulfide bonds in rTAP proceeded at a slower rate and appeared to be nonspecific, reaching a maximum of two disulfides reduced. Reduced rTAP derivatized with either iodoacetic acid or iodoacetamide lost 59% of its inhibitory activity. In contrast, BPTI alkylated with iodoacetic acid inhibited trypsin half as well as the iodoacetamide derivative. Although the arrangement of disulfides in the two inhibitors is the same, their susceptibility to reduction is markedly different.

Amino Acid Sequence↗
PubMed1991

Stabilization by a disulfide bond of the N-terminal domain of a mutant troponin C (TnC48/82).

The regulatory activity of troponin C is reversibly inhibited by a disulfide bridge between cysteine residues introduced by site-directed mutagenesis in positions 48 and 82 (TnC48/82) in the N-terminal domain of rabbit skeletal troponin C (sTnC; Grabarek, Z., Tan, R.-Y., Tao, T., and Gergely, J. (1990) Nature 345, 132-135). In the present work we have investigated the effects of the disulfide on structural properties of TnC48/82 monitored by CD spectroscopy and limited trypsinolysis. The CD spectra of the mutant protein in the oxidized form (oxTnC48/82) with and without Ca2+ are similar to the corresponding ones of the reduced and carboxamidomethylated form (CAMTnC48/82), indicating that the disulfide has essentially no effect on the overall secondary structure. The N-terminal domain of oxTnC48/82 is resistant to thermal unfolding, but that of CAMTnC48/82 is only slightly more stable than the corresponding domain of sTnC. In the presence of Ca2+ oxTnC48/82 is more resistant to trypsinolysis than sTnC whereas the rate of tryptic digestion of CAMTnC48/82 is the same as that of sTnC, indicating that peptide bonds adjacent to lysine residues at position 84 and 88, the sites of tryptic attack, are protected by the disulfide. The disulfide cross-linked N-terminal peptide of TnC48/82 does not bind TnI, unlike its reduced or carboxamidomethylated forms. Our data indicate that the disulfide between Cys48 and Cys82 stabilizes the structure of the N-terminal domain of TnC and blocks its ability to interact with TnI. The effects of the disulfide appear to be restricted to the N-terminal domain of TnC.

Animals↗
PubMed1990

Molecular properties of the histamine H2-receptor. Covalent inhibition by tetraamine disulfides.

A series of tetraamine disulfides related to benextramine (an alpha-adrenoreceptor and H2-receptor antagonist) in which the distance between the inner and the outer nitrogens were changed from five to nine methylenes has been studied. Both effects of the displacement of the disulfide bridge by two methylenes and those of the progressive removal of two of the four nitrogens on the pharmacological profile have been assessed. Peak potency appeared to be associated with eight methylenes between the inner and the outer nitrogens and to four cationic charges as in the most active analogue 4 which was also investigated to assess its receptor specificity towards histamine H1 and muscarinic M2 and M3 receptors. The finding that the carbon analogue 11 (two methylenes for the disulfide bridge) was devoid of activity is consistent with the hypothesis that histamine H2-receptor inhibition is the result of a covalent bond formation by a way of a disulfide-thiol interchange reaction between the disulfide moiety of tetraamine disulfides and a receptor thiol group. However, the possibility that tetraamine disulfides may not act at the H2-receptor but beyond the receptor cannot be excluded.

Animals↗
PubMed1990

The multisubunit structure of synaptophysin. Relationship between disulfide bonding and homo-oligomerization.

Synaptophysin, a major membrane protein of synaptic vesicles, contains four transmembrane regions and two intravesicular loops. Synaptophysin monomers associate into homopolymers that have the potential to form channels in the synaptic vesicle membrane. Here we show that in native synaptophysin, homopolymers are linked by noncovalent forces. The molecule contains unstable intramolecular disulfide bonds that undergo disulfide exchange during solubilization, thereby covalently cross-linking neighboring synaptophysin molecules. The locations of the intramolecular disulfide bonds in synaptophysin were determined, revealing that each of the two intravesicular loops of synaptophysin is circularized by a single disulfide bond. Cross-linking of synaptophysin by disulfide bonds can be triggered in synaptic vesicles and in intact cells by a cycle of reduction and oxidation, suggesting that native synaptophysin is a homomultimer in situ. In addition, chemical cross-linking of native synaptophysin demonstrates that a low molecular weight protein is specifically associated with synaptophysin complexes and is lost upon reduction of the intramolecular disulfide bonds. These data suggest that native synaptophysin forms a noncovalent homomultimeric complex whose structure and interaction with other proteins are dependent on the integrity of its intramolecular disulfide bonds and phospholipid environment.

Adrenal Gland Neoplasms↗
PubMed1990

Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes.

Whereas there is biological evidence that the reductive cleavage of disulfide bonds is critical for the activation of endocytosed macromolecules such as toxins, immunotoxins, and other drug carriers, virtually nothing is known about the specifics of this cleavage. To study this process, a model compound was synthesized in which a radioiodinated tyramine was linked through a disulfide bond to an undegradable carrier, poly(D-lysine), known to be efficiently endocytosed. Cultured Chinese hamster ovary cells were pulse-labeled with this probe, and the disulfide cleavage was measured as released acid-soluble radioactivity at different times of chase. Pulse-labeled cells were also subjected to subcellular fractionation to identify intracellular structures associated with disulfide cleavage. Cleavage began without lag, amounted to about approximately 7% of the initial cell-bound radioactivity in the first hour and continued for more than 6 h. It was abolished in the presence of N-ethylmaleimide. When sulfhydryl groups present at the cell surface were blocked with cell-impermeant sulfhydryl reagent, the initial phase of disulfide cleavage was inhibited, indicating that cleavage began at the cell surface. A long-lasting intracellular phase of disulfide cleavage began after about approximately 30 min of chase. Subcellular fractionation and kinetic analysis indicated that neither lysosomes nor endosomes were participating in that phase, leaving the Golgi apparatus as the most probable site of endocytic disulfide cleavage.

Animals↗
PubMed1988

Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds.

Ribonuclease T1 has two disulfide bonds linking cysteine residues 2-10 and 6-103. We have prepared a derivative of ribonuclease T1 in which one disulfide bond is broken and the cysteine residues carboxymethylated, (2-10)-RCM-T1, and three derivatives in which both disulfides are broken and the cysteine residues reduced, R-T1, carboxamidomethylated, RCAM-T1, or carboxymethylated, RCM-T1. The RNA hydrolyzing activity of these proteins has been measured, and urea and thermal denaturation studies have been used to determine conformational stability. The activity, melting temperature, and conformational stability of the proteins are: ribonuclease T1 (100%, 59.3 degrees C, 10.2 kcal/mol), (2-10)-RCM-T1 (86%, 53.3 degrees C, 6.8 kcal/mol), R-T1 (53%, 27.2 degrees C, 3.0 kcal/mol), RCAM-T1 (43%, 21.2 degrees C, 1.5 kcal/mol), and RCM-T1 (35%, 16.6 degrees C, 0.9 kcal/mol). Thus, the conformational stability is decreased by 3.4 kcal/mol when one disulfide bond is broken and by 7.2-9.3 kcal/mol when both disulfide bonds are broken. It is quite remarkable that RNase T1 can fold and function with both disulfide bonds broken and the cysteine residues carboxymethylated. The large decrease in the stability is due mainly to an increase in the conformational entropy of the unfolded protein which results when the constraints of the disulfide bonds on the flexibility are removed. We propose a new equation for predicting the effect of a cross-link on the conformational entropy of a protein: delta Sconf = -2.1 - (3/2)R 1n n, where n is the number of residues between the side chains which are cross-linked. This equation gives much better agreement with experimental results than other forms of this equation which have been used previously.

Aspergillus↗
PubMed1989

Determination of three disulfide bonds and one free sulfhydryl in the beta subunit of (Na,K)-ATPase.

The oxidation state of the 7 cyst(e)ine residues of the beta subunit of lamb kidney (Na,K)-ATPase was determined. The fluorescent sulfhydryl-reactive reagent 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole was utilized before and after reduction of disulfide bonds to determine the number and location of disulfide bonds present in the beta subunit. Treatment of tryptic peptides of the beta subunit separated by reverse phase high performance liquid chromatography with a reducing agent and 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole allowed identification of which residues were disulfide-linked. The results indicated that there is only one free sulfhydryl, Cys44, and that Cys125 is disulfide-bonded to Cys148, Cys158 is disulfide-linked to Cys174, and Cys212 is disulfide-bonded to Cys275. All three disulfide bonds were shown to be reduced in the presence of high concentrations of beta-mercaptoethanol and elevated temperature. These conditions also lead to a loss of (Na,K)-ATPase activity.

Amino Acid Sequence↗
PubMed1986

Disulfide-bonded polymerization of plasma fibronectin in the presence of metal ions.

Incubation of human plasma fibronectin in the presence of low concentrations of FeCl3 or CuSO4 led to the formation of disulfide-bonded multimers as revealed by analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing or reducing conditions. The polymers induced by FeCl3 did not enter the spacer gel, and those induced by CuSO4 migrated to the top of the running gel, indicating that the former polymers were larger than the latter, which in gel filtration experiments appeared to be larger than Mr 670,000. The polymerization occurred between pH 7 and 9 and more rapidly at 22 or 37 degrees C than at 4 degrees C and was inhibited by metal-chelating reagents. NaCl, heparin, spermine, urea, or guanidine hydrochloride did not appreciably affect the reaction, whereas dithioerythritol enhanced the CuSO4-induced polymerization of fibronectin. When incubated in the presence of FeCl3, the Mr 30,000 NH2-terminal, Mr 40,000 gelatin-binding, and the Mr 120,000-140,000 COOH-terminal fragments of fibronectin formed disulfide-bonded polymers, whereas only the Mr 140,000 fragment was polymerized in the presence of CuSO4. Disulfide-bonded polymers were also formed in the presence of FeCl3 but not CuSO4 when the free sulfhydryl groups of fibronectin were blocked by N-ethylmaleimide. The results suggest that in the presence of CuSO4, disulfide-bonded polymerization of fibronectin may involve predominantly the free sulfhydryl groups, whereas in the presence of FeCl3, also the intramolecular disulfides may exchange to form disulfides between separate fibronectin molecules. Thus, under different conditions, different parts of fibronectin may be susceptible to disulfide-bonded polymerization.

Chelating Agents↗
PubMed1994

Disulfide bond formation between dimeric immunoglobulin A and the polymeric immunoglobulin receptor during hepatic transcytosis.

The polymeric immunoglobulin receptor on rat hepatocytes binds dimeric IgA on the sinusoidal surface and mediates its transport to the canaliculus, where the complex of dimeric IgA and secretory component, the cleaved extracellular domain of polymeric immunoglobulin receptor, is secreted into bile. This process is unique in that disulfide bonds are formed between dimeric IgA and polymeric immunoglobulin receptor during transcytosis, permanently preventing their dissociation. Here we present three lines of evidence that disulfide bonding between dimeric IgA and polymeric immunoglobulin receptor occurs predominantly in a late transcytotic compartment and that hepatic transcytosis can proceed in the absence of disulfide bond formation. First, throughout the course of transcytosis the percentage of intracellular dimeric IgA disulfide bonded to polymeric immunoglobulin receptor is less than half that in bile, suggesting that disulfide bond formation is a late event in transcytosis. Second, dimeric IgA that recycles from early endocytotic compartments into the circulation is mostly noncovalently bound to secretory component. Finally, the rate of transcytosis of dimeric IgA and its appearance in bile are not affected when disulfide bond formation with polymeric immunoglobulin receptor is inhibited by blocking of free thiol groups on dimeric IgA with iodoacetamide. These results are consistent with other findings in the literature and indicate that the main physiological role of disulfide bond formation between dimeric IgA and polymeric immunoglobulin receptor is not to facilitate transcytosis but, rather, to stabilize the dimeric IgA-secretory component complex after its release into external secretions such as bile and intestinal secretions.

Animals↗
PubMedDate not supplied

Regioselective formation of the three disulfide bonds of a 35-residue insect peptide.

PMP-D2, a 35-residue peptide containing three disulfide bonds, was synthesized on solid-phase using 9-fluorenylmethoxy-carbonyl (Fmoc) as alpha-NH2 protection and simultaneous air oxidation of the six cysteines for formation of its disulfide bonds. The overall yield was 13%. As very little research has been done on the regioselective formation of three disulfide bonds, we decided to investigate different strategies using either trityl (Trt), acetamidomethyl (Acm) and methoxybenzyl (Mob), or methoxytrityl (Mmt), trityl and acetamidomethyl, as cysteine-protecting groups and Fmoc as alpha-NH2 protection. In the first strategy, the first disulfide bond was formed by air oxidation and the second was formed by iodine oxidation of the Cys(Acm). Then, the Cys (Mob) was deprotected using TFMSA/TFA treatment for formation of the third disulfide bond. This last step was poorly reproducible on a large scale. The overall yield was 2.5%. In the second strategy, the first disulfide was formed on the resin after removal of the methoxytrityl group, and the two remaining disulfide bonds were formed classically in solution. The overall yield was 2%. From the overall yields using these strategies, it appears clear that simultaneous oxidation of the six cysteines is particularly appropriate for the synthesis of PMP-D2.

Acetamides↗