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PubMed1998

Mapping of disulfide bridges in antifreeze proteins from overwintering larvae of the beetle Dendroides canadensis.

Antifreeze proteins (AFPs) have been identified in certain high-latitude marine fish, insects and other terrestrial arthropods, and plants. Despite considerable structural variation, the mechanisms of their noncolligative antifreeze activity are probably quite similar. AFPs hydrogen bond onto the surface of potential seed ice crystals at preferred growth sites, thereby preventing growth of the crystals. AFPs from overwintering larvae of the beetle Dendroidescanadensis are among the most active AFPs. These 8.7-kDa proteins consist of seven 12- or 13-mer repeating units. Their most striking feature is the location of cysteines every six residues throughout their length. Consequently, identification of the disulfide linkages of these cysteines is essential to understanding the structure of these AFPs. This study demonstrated that all of the 16 Cys residues in the Dendroides AFPs are disulfide bridged. All of the seven 12- or 13-mer repeats have internal disulfide bridges, and in all but the first repeat the Cys residues at positions 1 and 7 of the repeats are linked. In repeat 1 the Cys at position 1 is linked to the Cys at position 10, rather than the Cys at position 7 as in the other repeats, and the Cys at position 7 of the first repeat is linked to a Cys at position 4 of the second repeat. The disulfide bridges probably function to position the hydrophilic side chains of serine and threonine residues so that they hydrogen bond with ice.

Amino Acid Sequence↗
PubMed1998

Stabilization of ribonuclease HI from Thermus thermophilus HB8 by the spontaneous formation of an intramolecular disulfide bond.

To identify factors that contribute to the thermal stability of ribonuclease HI (RNase HI) from Thermus thermophilus HB8, protein variants with a series of carboxyl-terminal truncations and Cys --> Ala mutations were constructed, and their thermal denaturations were analyzed by CD. The results indicate that Cys41 and Cys149 contribute to the protein stability, probably through the formation of a disulfide bond. Peptide mapping analysis for the mutant protein with only two cysteine residues, at positions 41 and 149, indicated that this disulfide bond is partially formed in a protein purified from Escherichia coli in the absence of a reducing reagent but is fully formed in a thermally denatured protein. These results suggest that the thermal stability of T. thermophilus RNase HI, determined in the absence of a reducing reagent, reflects that of an oxidized form of the protein. Comparison of the thermal stabilities and the enzymatic activities of the wild-type and truncated proteins, determined in the presence and absence of a reducing reagent, indicates that the formation of this disulfide bond increases the thermal stability of the protein by 6-7 degreesC in Tm and approximately 3 kcal/mol in DeltaG without seriously affecting the enzymatic activity. Since T. thermophilus RNase HI is present in a reducing environment in cells, this disulfide bond probably is not formed in vivo but is spontaneously formed in vitro in the absence of a reducing reagent.

Amino Acid Sequence↗
PubMed1998

Determination of disulfide structure in agouti-related protein (AGRP) by stepwise reduction and alkylation.

The agouti-related protein gene (Agrp) plays an important role in body weight regulation. The mature human protein is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The disulfide structure of recombinant human AGRP was determined by chemical methods using partial reduction with tris(2-carboxyethyl)phosphine under acidic conditions, followed by direct alkylation with N-ethylmaleimide or fluorescein-5-maleimide. Partial reduction and alkylation provided several forms of AGRP that were modified in a stepwise fashion. The resulting proteins were characterized by peptide mapping, sequence analysis, and mass spectrometry, showing that AGRP contained a highly reducible disulfide bond, C85-C109, followed by less reactive ones, C90-C97, C74-C88, C67-C82, and C81-C99, respectively. The chemically defined disulfide connectivity of the recombinant human AGRP was homologous to that of omega-agatoxin IVB except for an additional disulfide bond, C85-C109.

Agatoxins↗
PubMed1998

Phorbol ester-induced juxtamembrane cleavage of angiotensin-converting enzyme is not inhibited by a stalk containing intrachain disulfides.

Specialized proteases, referred to as sheddases, secretases, or membrane-protein-solubilizing proteases (MPSPs), solubilize the extracellular domains of diverse membrane proteins by catalyzing a specific cleavage in the juxtamembrane stalk regions of such proteins. A representative MPSP (tumor necrosis factor-alpha convertase) was cloned recently and shown to be a disintegrin metalloprotease that is inhibited by peptide hydroxamates including the compound TAPI. Substrate determinants that specify cleavage by MPSPs remain incompletely characterized, but may include the physicochemical properties of the stalk or unidentified recognition motifs in the stalk or the extracellular domain. We constructed a mutant angiotensin-converting enzyme (ACE) in which the stalk has been replaced with an epidermal growth factor (EGF)-like domain (ACE-JMEGF), to test the hypothesis that MPSP cleavage requires an open, comparatively unfolded or extended stalk. Wild-type ACE is a type I transmembrane (TM) ectoprotein that is efficiently solubilized by a typical MPSP activity. We found that ACE-JMEGF was solubilized inefficiently and accumulated in a cell-associated form on transfected Chinese hamster ovary (CHO) cells; cleavage was stimulated by phorbol ester and inhibited by TAPI, features typical of MPSP activity. Determination of the C-terminus of soluble ACE-JMEGF revealed that, surprisingly, cleavage occurred at a Gly-Phe bond between the fifth and sixth cysteines within the third disulfide loop of the EGF-like domain. Reduction of intact CHO cells with tributylphosphine resulted in the rapid release of ACE-JMEGF (but not wild-type ACE) into the medium, suggesting that a proportion of membrane-bound ACE-JMEGF is cleaved but remains cell-associated via disulfide tethering. The mechanism for the release of ACE-JMEGF in the absence of chemical reduction is unclear. We conclude that the presence of a compact, disulfide-bridged domain does not per se inhibit cleavage by an MPSP activity, but ectodomain release is prevented by disulfide tethering to the TM domain.

Amino Acid Sequence↗
PubMed1999

Cavity-creating mutation at the dimer interface of Plasmodium falciparum triosephosphate isomerase: restoration of stability by disulfide cross-linking of subunits.

Disulfide engineering across subunit interfaces provides a means of inhibiting dissociation during unfolding of multimeric enzymes. Two symmetry-related intersubunit disulfide bridges were introduced across the interface of the dimeric enzyme triosephosphate isomerase from Plasmodium falciparum. This was achieved by mutating a tyrosine residue at position 74 at the subunit interface to a cysteine, thereby enabling it to form a covalent cross-link with a pre-existing cysteine at position 13 of the other subunit. The wild-type enzyme (TIMWT) and the oxidized (Y74Cox) and reduced (Y74Cred) forms of the mutant have similar enzymatic activity, absorption, and fluorescence spectra. All three proteins have similar far-UV CD spectra. The Y74Cred shows a distinct loss of near-UV CD. Thermal precipitation studies demonstrate that TIMWT and Y74Cox have very similar Tm values (Tm approximately 60 degreesC) whereas Y74Cred is surprisingly labile (Tm approximately 38 degreesC). The Y74C mutant results in the creation of a large cavity (approximately 100 A3) at the dimer interface. The crystal structure for the oxidized form of Y74C mutant, crystallized in the presence of low concentrations of dithiothreitol, reveals an asymmetric dimer containing a disulfide bridge at one site and a reduced dithiol cysteine at the other. The crystal structure of the mutant offers insights into the destabilization effects of the interfacial cavities and the role of disulfide tethering in restoring protein stability.

Animals↗
PubMed1998

The longer isoform and Cys-1 disulfide bridge of rat surfactant protein A are not essential for phospholipid and type II cell interactions.

Rat SP-A is a heterooligomer of two closely related isoforms, that requires interchain disulfide linkage for several functions including SP-A-mediated phospholipid vesicle aggregation and modulation of surfactant secretion and uptake by isolated alveolar type II cells. While the Cys6 disulfide bond of rat SP-A is known to be critical for function, the importance of the second interchain disulfide linkage within the N-terminal Isoleucine-3-Lysine-Cysteine-1 (IKC) sequence of the alternatively processed isoform is not clear. To examine the role of the Cys-1-dependent multimerization in SP-A function, we disrupted the Cys-1 disulfide bond by deletion of the IKC sequence (SP-Ahyp, DeltaIKC) or the substitution Cys-1 to Ser (SP-Ahyp,C-1S) in mutant recombinant proteins produced in insect cells. N-terminal sequence analyses revealed that the mutations influenced signal peptidase cleavage specificity, resulting in an increase in the abundance of the longer isoform of SP-Ahyp,C-1S and in N-terminal truncation of a fraction of the SP-Ahyp,DeltaIKC polypeptides at Gly8. On nonreducing SDS-PAGE analysis, both mutant proteins migrated as monomers and dimers but not the higher multimers that are characteristic of the wild-type recombinant protein (SP-Ahyp). Cross-linking analyses demonstrated that the association between trimeric SP-A subunits remained intact despite disruption of the Cys-1 bond. The SP-Ahyp,C-1S eluted in the same volume as SP-Ahyp from the gel sizing column with an apparent mass of 440 kDa, indicative of association of at least 9-10 monomers. The phospholipid binding and aggregation activities of the SP-Ahyp,C-1S were approximately 75% and 55% of the SP-Ahyp, respectively, but the SP-Ahyp,DeltaIKC was functionally comparable to SP-Ahyp. Similarly, both mutant proteins regulated the secretion and uptake of surfactant from isolated type II cells, but the SP-Ahyp,DeltaIKC was somewhat more potent than the SP-Ahyp,C-1S. Competitive binding to the SP-A receptor on type II cells was reduced by both Cys-1 mutations. We conclude that neither Cys-1-dependent multimerization nor the longer SP-A isoform is absolutely required for oligomeric association of trimeric SP-A subunits, SP-A/phospholipid interactions, or the regulation of surfactant secretion or uptake from type II cells by rat SP-A.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1999

Engineering out motion: introduction of a de novo disulfide bond and a salt bridge designed to close a dynamic cleft on the surface of cytochrome b5.

A previous molecular dynamics (MD) simulation of cytochrome b5 (cyt b5) at 25 degrees C displayed localized dynamics on the surface of the protein giving rise to the periodic formation of a cleft that provides access to the heme through a protected hydrophobic channel [Storch and Daggett (1995) Biochemistry 34, 9682]. Here we describe the production and testing of mutants designed to prevent the cleft from opening using a combination of experimental and theoretical techniques. Two mutants have been designed to close the surface cleft: S18D to introduce a salt bridge and S18C:R47C to incorporate a disulfide bond. The putative cleft forms between two separate cores of the protein: one is structural in nature and can be monitored through the fluorescence of Trp 22, and the other binds the heme prosthetic group and can be tracked via heme absorbance. An increase in motion localized to the cleft region was observed for each protein, except for the disulfide-containing variant, in MD simulations at 50 degrees C compared to simulations at 25 degrees C. For the disulfide-containing variant, the cleft remained closed. Both urea and temperature denaturation curves were nearly identical for wild-type and mutant proteins when heme absorbance was monitored. In contrast, fluorescence studies revealed oxidized S18C:R47C to be considerably more stable based on the midpoints of the denaturation transitions, Tm and U1/2. Moreover, the fluorescence changes for each protein were complete at approximately 50 degrees C and a urea concentration of approximately 3.9 M, significantly below the temperature and urea concentration (62 degrees C, 5 M urea) required to observe heme release. In addition, solvent accessibility based on acrylamide quenching of Trp 22 was lower in the S18C:R47C mutant, particularly at 50 degrees C, before heme release [presented in the accompanying paper (58)]. The results suggest that a constraining disulfide bond can be designed to inhibit dynamic cleft formation on the surface of cyt b5. Located near the heme, the native dynamics of the cleft may be functionally important for protein-protein recognition and/or complex stabilization.

Animals↗
PubMed1999

Mapping disulfide connectivity using backbone ester hydrolysis.

The site-specific incorporation of alpha-hydroxy acids into proteins using nonsense suppression can provide a powerful probe of protein structure and function. The resulting backbone ester may be selectively hydrolyzed in the presence of the peptide backbone, providing an "orthogonal" chemistry that can be useful both as an analytical tool and as a structural probe. Here we describe in detail a substantial substituent effect on this hydrolysis reaction. Consistent with mechanistic expectations, the steric bulk of the amino acid immediately N-terminal of the hydroxy acid has a large effect on the hydrolysis rate. On the basis of these results, we also describe a simple protocol for identifying disulfide loops in soluble and membrane proteins, exemplified by the alpha subunit of the muscle nicotinic acetylcholine receptor (nAChR). If a backbone ester is incorporated outside a disulfide loop, hydrolysis alone gives two fragments, but if the ester is incorporated within a disulfide loop, both hydrolysis and reduction are required for cleavage. This test could be useful in characterizing the disulfide topology of complex, membrane proteins.

Amino Acid Substitution↗
PubMed2000

ATP-dependent dissociation of non-disulfide-linked aggregates of coagulation factor VIII is a rate-limiting step for secretion.

Deficiency in coagulation factor VIII leads to the bleeding disorder hemophilia A. Previous studies demonstrated that factor VIII secretion is limited due to an ATP-requiring step early in the secretory pathway. In this report, we identified that this ATP-dependent rate-limiting step involves the dissociation of non-disulfide-linked aggregates within the endoplasmic reticulum (ER). In contrast to the numerous examples of interchain disulfide-linked aggregates, factor VIII is the first protein characterized to form non-disulfide-linked high molecular weight aggregates within the ER. Approximately a third of newly synthesized factor VIII was detected in high molecular weight aggregates. These aggregates disappeared over time as functional factor VIII appeared in the medium. The aggregated complexes did not require proteasomal degradation for clearance. Aggregate formation was enhanced by ATP depletion, and upon restoration of metabolic energy, these aggregates were dissociated and secreted. With the coexpression of von Willebrand factor (vWF), a small portion of vWF coaggregated with factor VIII. However, vWF dissociated from the aggregates more rapidly than factor VIII, supporting that these aggregates are dynamic. An increase in the factor VIII expression level elicited a corresponding increase in the fraction of factor VIII that was aggregated. In addition, a 110 amino acid sequence containing a hydrophobic beta-sheet within factor VIII was identified that may predispose factor VIII to aggregation. These data show that formation and ATP-dependent dissolution of nondisulfide-linked factor VIII aggregates is a dynamic, rate-limiting step during the folding process in the early secretory pathway. In summary, we have identified an unprecedented requirement for protein transport out of the ER that involves an ATP-dependent dissociation of non-disulfide-linked aggregates within the ER.

Acetylcysteine↗
PubMed2000

Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme.

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.

Circular Dichroism↗
PubMedDate not supplied

Disulfide cross-linked hyaluronan hydrogels.

A new disulfide cross-linking strategy was developed to prepare hyaluronic acid (HA) hydrogel from thiol-modified HA. First, dithiobis(propanoic dihydrazide) (DTP) and dithiobis(butyric dihydrazide) (DTB) were synthesized and then coupled to HA with carbodiimide chemistry. Next, disulfide bonds of the initially formed gel were reduced using dithiothreitol (DTT) to give, after exhaustive dialysis, the corresponding thiol-modified macromolecular derivatives HA-DTPH and HA-DTBH. The degree of substitution of HA-DTPH and HA-DTBH could be controlled from 20% to 70% of available glucuronate carboxylic acid groups. The pK(a) values of the HA-thiol derivatives were determined spectrophotometrically to be pK(a) = 8.87 (HA-DTPH) and pK(a) = 9.01 (HA-DTBH). The thiol groups could be oxidized in air to reform disulfide linkages, which resulted in HA-DTPH and HA-DTBH hydrogel films. Further oxidation of these hydrogels with dilute H(2)O(2) created additional cross-links and afforded poorly swellable films. The disulfide cross-linking was reversible, and films could be again reduced to sols with DTT. Release of blue dextran from cross-linked films was used as a model for drug release. The rapid gelation of the HA-DTPH solution under physiological conditions was also achieved, which demonstrated the capacity for in situ cell encapsulation. Thus, L-929 murine fibroblasts were encapsulated in HA-DTPH hydrogel; these cells remained viable and proliferated during 3 days of culture in vitro.

Animals↗
PubMedDate not supplied

Stable, long-term bacterial production of soluble, dimeric, disulfide-bonded protein pharmaceuticals without antibiotic selection.

Numerous biopharmaceuticals and other recombinant biotechnology products are made in prokaryotic hosts. However, bacterial production of native, biologically active eukaryotic proteins is rarely possible for disulfide-bonded and/or multisubunit proteins. We previously described the production of soluble, native disulfide-bonded dimeric proteins in the Escherichia coli cytoplasm (Miele et al., 1990; Mantile et al., 1993). Native, biologically active proteins with up to six disulfide bonds have been produced with our expression system (Garces et al., 1997). However, plasmid instability during induction limited its usefulness. We now report the stable, high-level expression of soluble, disulfide-bonded human uteroglobin without antibiotic selection. We designed a new vector containing a multifunctional stabilization region that confers complete plasmid stability and increased protein yields without copy number increases. Recombinant expression remains fully inducible after long-term continuous culture in nonselective liquid medium (at least 260 generations). This system may significantly expand the applications of bacterial expression to recombinant production of soluble, bioactive proteins for biochemical studies and biopharmaceutical/industrial purposes. As a result of the very broad activity spectrum of the stabilization region we selected, its use could be extended to bacterial hosts other than enterobacteria.

Anti-Bacterial Agents↗
PubMed2005

Disulfide bond cleavage induced by a platinum(II) methionine complex.

The cleavage of a disulfide bond and the redox equilibrium of thiol/disulfide are strongly related to the levels of glutathione (GSH)/oxidized glutathione (GSSG) or mixed disulfides in vivo. In this work, the cleavage of a disulfide bond in GSSG induced by a platinum(II) complex [Pt(Met)Cl2] (where Met = methionine) was studied and the cleavage fragments or their platinated adducts were identified by means of electrospray mass spectrometry, high-performance liquid chromatography, and ultraviolet techniques. The second-order rate constant for the reaction between [Pt(Met)Cl2] and GSSG was determined to be 0.4 M(-1) s(-1) at 310 K and pH 7.4, which is 100- and 12-fold faster than those of cisplatin and its monoaqua species, respectively. Different complexes were formed in the reaction of [Pt(Met)Cl2] with GSSG, mainly mono- and dinuclear platinum complexes with the cleavage fragments of GSSG. This study demonstrated that [Pt(Met)Cl2] can promote the cleavage of disulfide bonds. The mechanistic insight obtained from this study may provide a deeper understanding on the potential involvement of platinum complexes in the intracellular GSH/GSSG systems.

Glutathione↗
PubMed2002

Design and construction of an open multistranded beta-sheet polypeptide stabilized by a disulfide bridge.

The design and characterization of an open eight-stranded beta-sheet in a synthetic, 2-fold symmetric 70-residue peptide is described. The design strategy involves the generation of a 35-residue four-stranded beta-sheet peptide in which successive hairpins are nucleated by appropriately positioned (D)Pro-Xxx sequences. Oxidative dimerization using a single Cys residue positioned at the center of the C-terminal strand results in a disulfide-bridged eight-stranded structure. Nuclear Overhauser effects firmly establish an eight-stranded beta-sheet in methanol. In water, the outer strands are frayed, but a well-defined four-stranded beta-sheet stabilized by a disulfide bridge and a hydrophobic cluster is determined from NMR data. Comparison of the precursor peptide with the disulfide-bridged dimer reveals considerable enhancement of beta-sheet content in the latter, suggesting that the disulfide cross-link is an effective strategy for the stabilization of beta-sheets.

Amino Acid Motifs↗
PubMed2003

Steric effects governing disulfide bond interchange during thermal aggregation in solutions of beta-lactoglobulin B and alpha-lactalbumin.

Intermolecular disulfide bond formation in pure beta-lactoglobulin (beta-Lg) B and in its 1:1 mixture with alpha-lactalbumin (alpha-La), heated at 85 degrees C for 10 min in solutions of low and high (100 mM NaCl) ionic strength and pH 6.0, was studied by reverse-phase HPLC and MALDI-TOF mass spectrometry. Disulfide bonding between beta-Lg monomers was more extensive than reported in the literature for a temperature of 68.5 degrees C, including formation of trimers connected by two of the three adjacent cysteines, C106/C119/C121. The participation of the different thiol groups in disulfide bonds appeared to depend on their location in the native structure, with surface-located cysteines more involved than internally located ones. This also applied to alpha-La-beta-Lg interactions, where the predominant participants were the surface-located alphaC111, alphaC120, alphaC61, and alphaC6. The least active participant was alphaC28, suggesting that it becomes sterically inaccessible during unfolding of the protein. High ionic strength apparently promoted disulfide bonding. The order of cysteine participation at the high ionic strength was similar to that at low ionic strength, with fewer native-location bonds observed and a lower activity of some groups, such as beta-C106/C119/C121 and alphaC61.

Chromatography, High Pressure Liquid↗
PubMed2004

Effects of designed sulfhydryl groups and disulfide bonds into soybean proglycinin on its structural stability and heat-induced gelation.

The gel-forming ability of glycinin is one of soybean's most important functional properties. The proglycinin A1aB1b homotrimer was engineered to introduce sulfhydryl groups and disulfide bonds, and their effects on the structural stability and the heat-induced gelation were evaluated. On the basis of the crystal structure, five mutants were designed and prepared: R161C and F163C forming an interprotomer disulfide bond with the inherent free cysteine residue of Cys377, N116C/P248C forming a new intraprotomer disulfide bond, and N116C and P248C introducing a new sulfhydryl group. Mutants of R161C, F163C, and N116C/P248C formed a new disulfide bond as expected. N116C/P248C was significantly more stable than the wild type against chemical and thermal denaturation and more resistant to alpha-chymotrypsin digestion, whereas F163C showed significantly increased thermal stability. All mutants exhibited greater hardness of heat-induced gels than wild type, and in particular, N116C/P248C gave the hardest gel. This result indicates that it is possible to increase hardness of glycinin gel by introduction of cysteine residues using protein engineering.

Disulfides↗
PubMed1997

Synthesis and pharmacology of novel analogues of oxytocin and deaminooxytocin: directed methods for the construction of disulfide and trisulfide bridges in peptides.

Using as models the neurohypophyseal nonapeptide hormone oxytocin and its analogue deaminooxytocin, several directed routes to formation of sulfur-sulfur bridges have been developed and evaluated. The linear sequences (through common octapeptide-resin intermediates) were assembled smoothly on tris(alkoxy)benzylamide (PAL) poly(ethylene glycol)-polystyrene (PEG-PS) graft supports, using stepwise Fmoc solid-phase chemistry. Side-chain protection of beta-mercaptopropionic acid (Mpa) and/or cysteine (Cys) was provided by S-2,4,6-trimethoxybenzyl (Tmob), S-acetamidomethyl (Acm), and/or a series of sulfenyl thiocarbonate and carbamoylsulfenyl protecting/activating groups: S-(methoxycarbonyl)sulfenyl (Scm), S-(methoxycarbonyl)disulfanyl (Sscm), S-(N-methyl-N-phenylcarbamoyl)sulfenyl (Snm), and S-(N-methyl-N-phenylcarbamoyl)disulfanyl (Ssnm). Thiolytic displacement of S-Snm (preferred) or S-Scm provided intramolecular cyclized peptide disulfides, and homologation of the chemistry with S-Ssnm (again preferred) and S-Sscm provided the corresponding trisulfides along with smaller amounts of disulfides and tetrasulfides. These chemistries could be implemented both in solution and in solid-phase modes. Various parameters were studied systematically and optimized, and the novel trisulfides of oxytocin and deaminooxytocin were synthesized and purified to homogeneity. The trisulfide compounds were evaluated in three assays: uterotonic in vitro, uterotonic in vivo, and pressor tests, and they showed substantial potencies, ranging from 5% to 40% of the parent (disulfide) activities, as well as protracted actions. The affinities of the peptide trisulfides to uterine membrane receptors were only 3.3-3.6-fold lower than those of the parent disulfides. Possible explanations of the biological results are discussed.

Animals↗
PubMed2006

Inexpensive, one-pot synthesis of unsymmetrical disulfides using 1-chlorobenzotriazole.

A new synthesis of unsymmetrical disulfides is described. The reaction of a thiol R1SH with 1-chlorobenzotriazole (BtCl) at -78 degrees C in DCM affords a high-yielding conversion to R1SBt without appreciable formation of the symmetrical disulfide R1SSR1. R1SBt is then reacted with R2SH to form the unsymmetrical disulfide in a one-pot sequence with green character that avoids the use of toxic and harsh oxidizing agents. The methodology has been developed for synthesis of various types of disulfides.

Azoles↗