Synthesis of specifically deuterated S-benzylcysteines and of oxytocin and related diastereomers deuterated in the half-cystine positions

Abstract

S-Benzylcysteine derivatives specifically deuterated at the a carbon only, the p carbon only, and at both the 01 and p carbons have been.synthesized. These labeled compounds have been enzymatically resolved and the enantiomers and reacemates have been converted to the N-tert-hutyloxycarbonyl derivatives. The deuterium labels were not exchanged under the conditions of the syntheses. Condensation of the sodium salt of difthyl a-acetamidomalonate with benzyl chloromethyl sulfide followed by hydrolysis with DCl afforded S-benzyl-DL-[a-*Hl]cysteine. Acetylation followed by treatment with hog renal acylase separated the stereoisomers. A Mannich reaction with [2H2]methylene diacetate, diethyl a-acetamidomalonate, and dimethylamine followed by quaternization of the amino nitrogen with methyl iodide gave diethyl a-acetamido-a-dimethylamino[2H~]methylmalonate methiodide (15). Treatment of 15 with sodium benzylmercaptide gave diethyl a-acetamid~-a-benzylthio[~H~]methylmalonate, which was hydrolyzed with HCl to yield S-ben~yl-DL-[P,/3-~Hz]cysteine or with DCl to afford S-benzyl-DL-[a,P,P-2H3]cysteine. These compounds were resolved as before. The preparation of S-benzyl-DL-[a,P,P-2H3] cysteine required an efficient source of ethanol-d. This deuterated solvent was prepared in quantitative yield in 2 h from tetraethoxysilane, D20, and a catalytic amount of thionyl chloride. The protected deuterated amino acids were used in the preparation of several oxytocin analogues in which the specific deuteration appears in either the 1-hemicystine or the 6-hemicystine residues.

Transfer nuclear Overhauser effect study of the conformation of oxytocin bound to bovine neurophysin I

“…Highly purified oxytocin (>99%) was prepared by direct chemical synthesis as previously reported (Upson & Hruby, 1976). The neurophysin I (NP I) proteins were isolated from freeze-dried bovine pituitaries and purified as previously described (Blumenstein & Hruby, 1976).…”

Conformationally restricted analogs of somatostatin with high mu-opiate receptor specificity.

“…Cysteine was determined as cysteic acid (5). Fast-atom-bombardment mass spectra (FABMS) were obtained on a Varian 311A spectrometer equipped with an Ion Tech Ltd source with xenon as the bombarding gas.Analogs were synthesized by standard solid-phase synthetic techniques (6,7). N`,-t-Butyloxycarbonyl (Boc)-protected amino acids were used throughout the syntheses and were prepared in the usual manner (8,9).…”

“…Analogs were synthesized by standard solid-phase synthetic techniques (6,7). N`,-t-Butyloxycarbonyl (Boc)-protected amino acids were used throughout the syntheses and were prepared in the usual manner (8,9).…”

“…For the preparation of peptides with a COOH-terminal carboxylic acid group, N`a-t-Boc-O-Bzl-L-threonine (Bzl = benzyl) was attached to chloromethylated copoly(styrene-1% divinylbenzene) beads (Lab Systems, 0.71 milliequivalent of Cl per g of resin) by the method of Gisin (10). Carboxamide peptides were prepared with a p-methylbenzhydrylamine resin (threonine substitution, 0.35 mmol/g of resin) as described (7). Preformed symmetrical anhydrides were used in the coupling reactions (11), which were monitored by ninhydrin (12) and/or chloranil (13) protected and removed from the resin with anhydrous liquid HF (10 ml/g of resin) containing 10% anisole.…”

Stereoselective deuterium labeling of proRβ‐protons in the NMR structure determination of a helix‐turn‐helix turn peptide mimic

“…Early on, large quantities of peptide were used to obtain good signal‐to‐noise ratios, although current inverse detected methods require less material for natural abundance long‐range heteronuclear experiments (13). In cases where the Χ 1 angle is involved in a ring, these standard spectroscopic methods should not be used due to the assumption of staggered conformations (14). For example the cystine side chain of oxytocin deviated from the staggered conformation (15).…”