- Building bridges for highly selective, potent and stable oxytocin and vasopressin analogs
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Oxytocin (OT) is an exciting potential therapeutic agent, but it is highly sensitive to modification and suffers extensive degradation at elevated temperature and in vivo. Here we report studies towards OT analogs with favorable selectivity, affinity and potency towards the oxytocin receptor (OTR), in addition to improving stability of the peptide by bridging the disulfide region with substituted dibromo-xylene analogs. We found a sensitive structure-activity relationship in which meta-cyclized analogs (dOTmeta) gave highest affinity (50 nM Ki), selectivity (34-fold), and agonist potency (34 nM EC50, 87-fold selectivity) towards OTR. Surprisingly, ortho-cyclized analogs demonstrated OTR and vasopressin V1a receptor subtype affinity (220 nM and 69 nM, respectively) and pharmacological activity (294 nM and 35 nM, respectively). V1a binding and selectivity for ortho-cyclized peptides could be improved 6-fold by substituting a neutral residue at position 8 with a basic amino acid, providing potent antagonists (14 nM IC50) that displayed no activation of the OTR. Furthermore, xylene-bridged analogs demonstrated increased stability compared to OT at elevated temperature, demonstrating promising therapeutic potential for these analogs which warrants further study.
- Beard, Rhiannon,Stucki, Andy,Schmitt, Muriel,Py, Gabrielle,Grundschober, Christophe,Gee, Antony D.,Tate, Edward W.
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p. 3039 - 3045
(2018/03/29)
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- Discovery of a highly selective and efficient reagent for formation of intramolecular disulfide bonds in peptides
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We have discovered that trans-[Pt(en)2Cl2]2+ (en = ethylenediamine) is a highly selective and efficient reagent for the quantitative formation of intramolecular disulfide bonds in peptides. A series of 14 dithiol peptides which form disulfide-containing rings ranging in size from 14 to 53 atoms were used to characterize the reagent. The dithiol peptides are cleanly and rapidly converted to their disulfide forms by a slight excess of the platinum complex under mild reaction conditions (slightly acidic and neutral media). For all the dithiol peptides studied, including a penicillamine-derived peptide, the oxidation yields range from 97% to 100%. No side reactions were observed, including no oxidation of the methionine side chain. The reaction kinetics for oxidation of reduced pressinoic acid were found to be second order overall: rate = k'[Pt(IV)][dithiol peptide], where k' is a pH-dependent second-order rate constant. Values of 0.60 ± 0.01, 3.5 ± 0.2, and 22 ± 1 M-1 s-1 were determined for k' at pH 3.0, 4.0, and 5.0, respectively (25 °C and 0.45 M ionic strength). A reaction mechanism for oxidation of dithiol peptides by [Pt(en)2Cl2]2+ is proposed. [Pt(en)2Cl2]2+ and its reduction product [Pt(en)2]2+ are essentially substitution inert under the conditions used for disulfide formation, they are nontoxic, and they are readily separated from peptides by HPLC. The characteristics of [Pt(en)2Cl2]2+ and its reaction properties with dithiol peptides suggest that [Pt(en)2Cl2]2+ is a universal reagent for the rapid and quantitative formation of intramolecular disulfide bonds in peptides.
- Shi, Tiesheng,Rabenstein, Dallas L.
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p. 6809 - 6815
(2007/10/03)
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- A rapid and facile method for the preparation of peptide disulfides
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A selective and efficient method for disulfide bond formation in peptides utilizing carbon tetrachloride in dichloromethane in the presence of tetrabutylammonium fluoride (TBAF) is described. The reaction proceeded rapidly and no side reaction was observed with nucleophilic amino acids such as Met, His, Tyr or Trp. This method has been applied to three model peptides using solution and on-the-resin disulfide formation.
- Maruyama, Toshihiro,Ikeo, Takayoshi,Ueki, Masaaki
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p. 5031 - 5034
(2007/10/03)
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- Trans-Dichlorotetracyanoplatinate(IV) as a Reagent for the Rapid and Quantitative Formation of Intramolecular Disulfide Bonds in Peptides
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Oxidation of cysteine thiol groups by trans-dichlorotetracyanoplatinate(IV) to form intramolecular peptide disulfide bonds has been studied for a series of dithiol peptides ranging from 4 to 15 amino acid residues in length. The dithiol peptides are rapidly and quantitatively transformed to their intramolecular disulfide forms by a slight excess of [Pt(CN)4Cl2]2-, as shown by HPLC. Quantitative analyses by HPLC and by spectrophotometric titration confirm a [Pt(IV)]:[dithiol peptide] stoichiometry of 1:1. Under the low pH conditions used, oxidation to form a 38-membered ring in the case of reduced somatostatin is as rapid as that to form much smaller rings, suggesting that ring closure is not the rate-determining step. The oxidation rates increase as the pH is increased. Time-resolved spectra show two isosbestic points, indicating that no peptide-platinum intermediates accumulate to a significant amount. A reaction mechanism similar to that for reduction of [Pt(CN)4Cl2]2- by monothiols is proposed. [Pt(CN)4Cl2]2- is a mild oxidant and essentially substitution inert; its reduction product, [Pt(CN)4Cl2]2-, is stable, has no redox chemistry with peptides, and does not form complexes with peptides. Moreover, [Pt(CN)4Cl2]2- and [Pt(CN)4Cl2]2- are nontoxic and readily separable from peptides by HPLC, and the cost of the Pt(IV) complex is negligible compared with that of peptides. The only unwanted side reaction observed with [Pt(CN)4Cl2]2- is oxidation of the sulfur of methionine to the sulfoxide form. These characteristics and the results of this study suggest that [Pt(CN)4Cl2]2- is an excellent reagent for the formation of intramolecular peptide disulfide bonds.
- Shi, Tiesheng,Rabenstein, Dallas L.
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p. 4590 - 4595
(2007/10/03)
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- Thiol/disulfide exchange reactions of captopril and penicillamine with arginine vasopressin and oxytocin
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The kinetics and equilibria of the reaction of the thiol-containing drugs captopril (D-3-mercapto-2-methylpropanoyl-L-proline, CpSH) and penicillamine (β, β-dimethylcysteine, PSH) with the disulfide bonds of the neurohypophyseal peptide hormones arginine vasopressin (AVP) and oxytocin (OT) have been characterized. CpSH reacts with AVP and OT by thiol/disulfide interchange to form two peptide-CpSH mixed disulfides, which in turn react with another molecule of CpSH to form the reduced peptide and CpSSCp. Forward and reverse rate constants and the equilibrium constant are reported for both steps in the reaction of CpSH with AVP and OT at pH 7.00. The rate constant for the first step (k1) is much larger than that for the second step (k2). Also, once formed, the peptide-CpSH mixed disulfides rapidly undergo intramolecular thiol/disulfide interchange with reformation of the cyclic peptide and CpSH. PSH reacts with AVP and OT by the same two-step reaction sequence; however, the rate of the second step is very slow due to steric hindrance from the methyl groups of PSH and the PSH moiety of the peptide-PSH mixed disulfides. Using rate constants determined in this study and PSH levels in the plasma of patients on PSH therapy, it is predicted that in vivo reduction of the disulfide bonds of AVP and OT by PSH and CpSH has little effect on the plasma half-lives of AVP or OT.
- Rabenstein, Dallas L.,Yeo, Pauline L.
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p. 109 - 118
(2007/10/02)
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- A MODIFIED BENZHYDRYLAMINE - A USEFUL HANDLE REAGENT FOR Fmoc BASED SOLID PHASE SYNTHESIS OF PEPTIDE AMIDES
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Usefulness of a dimethoxybenzhydrylamine derivative, 3-(3-(Fmoc-amino-4-methoxyphenylmethyl)-4-methoxyphenyl)propionic acid, for Fmoc-based solid phase synthesis of peptide amides was demonstrated by preparation of three biologically active peptide amides, i.e. tetragastrin, neuromedin B and vasopressin. 1M trimethylsilyl bromide-thioanisole (molar ration 1 : 1) in trifluoroacetic acid was recommended as a deprotecting reagent for releasing the peptide amides from the resin.
- Funakoshi, Susumu,Murayama, Eigoro,Guo, Lili,Fujii, Nobutaka,Yajima, Haruaki
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p. 2791 - 2800
(2007/10/02)
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