Deuterated Oxytocins: The Synthesis and Biological Properties of a Crystalline Analog of Deamino-Oxytocin Deuterated in the 5-Asparagine Position

Abstract

Abstract.-This paper describes the synthesis and biological activity of [5-L-asparagine-a4,34,3-d3]-deamino-oxytocin. The model peptide S-benzyl-3-mercaptopropionyl-L-asparaginyl-a4,3,-d3-glycinamide was also prepared to observe the effect of the conditions of peptide synthesis on the deuterium content. It was found that normal synthetic procedures could be used without a significant loss in the deuterium content.The [5-L-asparagine-a,,S,-d3 1-deamino-oxytocin had, within experimental error, the same avian vasodepressor and oxytocic activities as deamino-oxytocin itself.In a continuation of a program to evaluate the effect on the biological activity of deamino-oxytocin (Fig. 1) (1) Synthesis of N-carbobenzoxy-L-asparagine-a,,f-d3 (II) and p-nitrophenyl N-carbobenzoxy-L-asparaginate-a,fid-d3 (III): The procedure reported for the protio analog was followed.4 L-Asparagine-a,#,#-d3,5 2.0 gm (0.015 mole), 3.0 gm of potassium bicarbonate and 2.68 gm of benzyl chloroformate in 40 ml of water gave 3.2 gm (89%) of the acid II: mp 160(161' (cor). The acid II was dissolved in 32 ml of ethanol, filtered through glass, cooled immediately,4 and dried to give 2.48 gm (69%) of the acid II:3 mp 165-166°(cor);[a]D23-6.2' (c 1, 1 N NaHCO3). Mass spectrum: d2, 12.3%; d3, 87.7% (96% D).A modification of the procedure reported for the protio analog was followed.4 A solution of 2.30 gm (0.008 mole) of the acid II, 1.45 gm of p-nitrophenol, and 1.95 gm of dicyclohexylcarbodiimide (DCC) in 18 ml of dimethylformamide (DMF), dried and freshly distilled,6 was stirred for 1 hr at 00 and at room temperature for 15 min. Glacial acetic acid (0.3 ml) was added, the solution was stirred for 15 min, cooled to -20°, and filtered. The product III was precipitated by the addition of 70 ml of water, and was isolated by filtering the cooled mixture. After being dried in vacuo for 24 hr, there was 263

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

“…[l-Hemi-DL-[a,d,/3-2Hs]cystine]oxytocm (5) and Separation of the Diastereomers. The same process described for the preparation of 4 was repeated, substituting 18 for 16.…”

“…If the label is introduced at nonexchangeable positions, it provides a durable marker which does not significantly alter the properties of the molecule. [3][4][5][6][7][8][9][10][11][12][13] The usefulness of such labels has been demonstrated for proteins in studies of structure and folding.14-19 Unambiguous proton8-11'20-22 and carbon-1312'18 '23 nuclear magnetic resonance assignments for many peptides have been made using specifically deuterated derivatives. The development of deuteron magnetic resonance spectroscopy has opened the way for a direct study of the microdynamical behavior of specific segments of the neurohypophyseal peptide hormones and their binding to the neurophysins, their biological carrier proteins.24 '25 These studies have begun to provide insights into the conformational aspects of these biologically active compounds, giving more direct evidence for structure-activity relationships than has been available from amino acid substitutions, which are more likely to perturb the structure of the molecule.…”

“…The specifically deuterated cysteine derivatives were also resolved into their enantiomeric pairs without loss of the label, and one was used for the synthesis of the specifically labeled oxytocin [6-hemi[lS,/3-2H2]cystine]oxytocin (2). The race-1-1 H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 1234 5 6789 1 mic compounds were used to incorporate specifically deuterated cysteine into the half-cystine positions of the oxytocin derivatives [6-hemi-DL-[a-2Hi]cystine]oxytocin (3), [l- hemi-DL-[d,d-2H2]cystine]oxytocin (4), and [1-hemi-DL-[ ,ß,ß-2 3] cystine]oxytocin (5) and the diastereomeric pairs were separated by partition chromatography on Sephadex G-25.31•32 '45 The synthesis of each of the partially deuterated S-benzylcysteine derivatives was accomplished using different approaches. The S-benzyl-DL-[a-2Hi] cysteine (6) was made by displacement of chloride from benzyl chloromethyl sulfide by sodium diethyl acetamidomalonate followed by hydrolysis of"the adduct with 11 N DC1 in D20 (Scheme I).…”