Int. J. Peptideplotein Res. 9,1977,269-271 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

A N ALTERNATIVE SYNTHESIS O F [ 7-(THIAZOLIDINE-4-CARBOXY LIC ACID)] - 0 X Y T O C I N

MARY BARBER and JOHN H . JONES

The Dyson Perrins Laboratory, University o f Oxford, Oxford, England

Received 1 3 August 1976

An alternative synthesis o f [ 7-(thiazolidine-4-carboxylic acid)]-oxytocin by a 6 + 3 route using a hexapeptide with a preformed disulphide bridge is outlined,

'A recent report (Rosamond & Ferger, 1976) describes the synthesis of an oxytocin ana- 'logue with thiazolidinelcarboxylic acid in qosition 7 which has greatly enhanced oxytocic potency, and we are prompted to outline our own synthesis of this interesting analogue. - We have found the 6 + 3 strategy devised by Muhlemann et af. (1972) for oxytocin (in

+vhich a protected hexapeptide with the di- sulphide bridge preformed is coupled to the C-terminal tripeptide amide) a very successful means of preparing 7-modified oxytocins and have applied it to the 7(thiazolidine4carb- oxylic acid)-analogue as shown in the Scheme. Earlier experience (Fairweather & Jones, 1972) 'had shown that thiazolidine4carboxylic acid ,residues were stable to acidic treatment when in a C-terminal or inchain position, but the #-terminal position is a chemically different proposition, and we therefore took pains to confirm that the acidolysis of (I) with tri- ,f$oroacetic acid gave a product (11) with an intact thiazolidine ring. So far as the optical ktegrity of the thiazolidine4carboxylic acid residue is concerned, we have confidence that 'all is well, as we have shown elsewhere (Barber & Jones, 1976) that derivatives of this imino acid are completely optically stable in the presence of base under conditions which cause rapid racemisation of analogous cysteine 'derivatives. - The approach shown in the Scheme has the important advantage over the published syn- thesis (Rosamond & Ferger, 1976) that the disulphide ring is already formed when the

thiazolidine4-carboxylic acid residue is intro- duced. The final stage was performed under very mild conditions to give chromatographic- ally pure material in good yield: in the pub- lished synthesis (Rosamond & Ferger, 1976) more vigorous and complicated procedures were necessary in the critical last stages, which gave a much lower yield of hormone analogue in which trace impurities were evident even after the extensive chromatographic purifica- tion to which it had been subjected.

EXPERIMENTAL PROCEDURES

Melting points were determined on a Kofler hot-stage apparatus. Optical rotations were determined on a Perkin-Elmer 141 automatic polanmeter using a 1 dm cell. Amino acid analyses were determined on a Jeol JLC-5AH automatic amino acid anlyser. Satisfactory nuclear magnetic resonance spectra in appro- priate solvents were obtained for the amino acid and tripeptide derivatives. Thin-layer chromatography was performed on unbaked Kieselgel G plates eluted with one of the following solvent systems: (A,) n-butanol- acetic acid-water (10: 1 : 3); (A3). n-butanol- acetic acid-water (4: 1 : 1); (H) n-butanol- pyridineacetic acid-water (15: 10: 3: 12).

The preparation of the leucylglycinamide acetate starting material and of protected hexapeptide disulphide derivative (Fairweather & Jones, 1972), which was obtained by a new route using S-trityl protection, will be described in detail later together with related work.

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M. BARBER and J.H. JONES

Cys Tyr Ile Gln Asn Cys Thz Leu Gly

(Ill) Boc

Boc

H

Thz, a residue of thiazolidine4carboxylic acid; Boc, t-butoxycarbonyl; Nsu, succinimido. Other abbreviations are as recommended by the IUPAC-LUB Commission on Biochemical Nomenclature.

t - Butoxycarbonyl -L -thiazolidine-4-carboxylic acid L-Thiazolidine-4-carboxylic acid (1.67 g, 10 mmol) was dissolved in aqueous dioxan (SO%, 20ml), and the pH was increased to 9.5 by addition of sodium hydroxide (4 N). t-But- oxycarbonyl azide (1.7 g, 11 mmol) was added in portions over a period of 3 h, the pH being maintained at 9.5 by addition of sodium hydroxide (4N). After 2 4 h the mixture was washed with ether (2 x 20ml) and the pH of the aqueous layer was slowly reduced to 3.0 by careful addition of hydrochloric acid (5 N) with stirring and cooling to 0°C. The white suspension which formed was extracted into ethyl acetate (3 x 25 ml) without delay. The combined organic layers were dried and evaporated. Trituration with light petroleum gave a white powder which was recrystallised from ethyl acetate-light petroleum to give acylimino acid (1.15g, 50%) of m.p. 126- 130°, [ a ] g -99.0' (c 1 in MeOH). [Lit. (Felix et al., 1973) m.p. 127-129", [ a ] g - 118' (cO.9 in MeOH).] (Found: C, 46.5; H, 6.5;N, 5.7;S, 13.5. Calc. for CsHl5N04S: C,46.3;H,6.4;N,6.O;S, 13.7%.)

t - Butoxycarbonyl- L - thiazolidine -4 - carboxylic acid succinimido ester t - Butoxycarbonyl- L - thiazolidine4-carboxylic acid (0.466 g, 2 mmol) was dissolved in ethyl acetate (5 ml) with N-hydroxysuccinimide (0.250g, 2mmol) and the solution was cooled to 0' C. Dicyclohexylcarbodiimide (0.458 g, 2.2mmol) was added slowly and after stirring the reaction mixture overnight at room tem-

perature, the dicyclohexylurea (0.46 g) was removed by filtration. The filtrate was washed4 quickly with sodium hydrogen carbonate (lo%, 5 ml), water (5 ml), dried and evaporated to give an oil. Precipitation from ethyl ace ta te by addition of light petroleum gave the active ester (0.45g, 66%) as a white powder of m.p. 130-131', [a12 - 102.7' (c 1 in CHC13).

HI8NZO6S requires: C, 47.3; H, 5.5; N, 8.S; (Found: C, 47.4; H, 5.8; N, 8.3; S , 9.7. C13-

s, 9.7%.)

t -Butoxycarbonyl- L - thiazolidine-4-carbonyl- L -1eucylglycinamide (I ) L-Leucylglycinamide acetate (0.49 g, 2 mmol) and triethylamine (0.41 g, 4 mmol) were dis- solved in dimethylformamide (5 ml), and t-butoxycarbonyl-L-thiazolidine4-carboxylic acid succinimido ester (0.66 g, 2 mmol) was added with stirring st room temperaturb After 16h , the solvent was evaporated and the resulting oil was distributed between water (10 ml) and ethyl acetate (10 ml). The aqueou4 layer was washed with ethyl acetate (2 x lOml) and the combined organic layers were dried and evaporated to give a colourless oil which gave a white powder on trituration' with ether. Reprecipitation from chloroform- light petroleum gave the required acyltri- pepride amide (0.46g, 56%) as a fine white powder of m.p. 151-153', [a],"-90.3' (c0.8 in CHC13). (Found: C, 50.5; H, 7.3, N, 13.6. Cl7H%NOSS requires: C, 50.7. H, 7.5; N, 13.9%.)

The use of the corresponding pentachloro- phenyl ester [m.p. 127-129', [a]," -57.0'

270

AN ALTERNATIVE SYNTHESIS OF [ 7-(THIAZOLIDINE4CARBOXYLIC ACID)] 6XYTOCIN

(cO.8 in CHC13)] for the preparation of this protected tripeptide gave a product which could not easily be separated from penta- chlorophenol.

L - Thiazolidine - 4 - carbonyl- L - leucylglycina- mide hemihydrate (II) t - Butoxycarbonyl - L - thiazolidine-4-carbonyl- L - leucylglycinamide (30 mg) was dissolved in 90% trifluoroacetic acid (1 ml) and the solution was set aside for 15min at room temperature. The solvents were evaporated to give an oil which gave a white powder on trituration with ether. Reprecipitation from methanol-ether gave the trifluoroacetate salt as a slightly hydro- scopic powder. This was dissolved in methanol (1 ml), and passed down a column of Amberlyst A-21 (OH- form). The fractions showing absorption at 280 nm were pooled and evapora- ted. Trituration with ether gave tripeptide amide hemihydrate (22 mg, 92%) of m.p. 150-153", [ a ] g -57.4' (c0.5 in H20). (Found: C, 46.6; H, 7.7; N, 17.8. ClzH22 'N403S.fHz0 requires: C, 46.3; H, 7.4; N, .18.0%.)

t - Butoxycarbonyl - [7-(L - thiazolidine - 4 - carboxylic acid)] -0xytocin The protected hexapeptide disulphide deriva- tive (HI), (20 mg, 24 prnol), L-thiazolidine- 4-carbonyl-L-leucylglycinamide (1 0.9 mg, 35 pmol) was 1 -hydroxybenzotriazole (4.8 mg, 36 pmol) were dissolved in dimethylformamide (0.4ml) and cooled to 0°C. A solution of dicyclohexylcarbodiimide in dimethylforma- mide (0.1 M, 0.3m1, 30pmol) was added and the reaction mixture was left at room tem- perature for 16h . The product was isolated exactly as in the published procedure (Muhle- mann et al., 1972) for the preparation of t- but oxycarbonyl-oxytocin. Lyophilisation gave t - butoxycarbonyl - [ 7- (L - thiazolidine4carb- oxylic acid)J -0xytocin as a fluffy white powder (10.2mg, 42%) of RF (A2) 0.50 and RF '(A3) 0.51. Amino acid analysis: Asp 1.00; Clu 0.98; Cly 1.02; Ile 1.01; Leu 0.98; Tyr '0.88. (Found: C, 47.8; H, 6.7; N, 14.1. C4,- .HnN12014S3.3HzO requires: C, 47.9; H, 6.6; N, 14.2%.)

7 - ( L -Thiazolidine-4-carboxylic acid)-oxytocin t - Butoxycarbonyl - [7 - (L - thiazolidine -4 -car- boxylic acid)] -0xytocin (7 mg, 6pm0l) was treated with 90% trifluoroacetic acid (1 ml) for 15 min at room temperature. The solution was diluted with water to 40ml, evaporated to a volume of about 5 ml, and finally lyophilised. The residue was chromotographed on a column (1.5 x 100cm) of Sephadex G-15 in 0.2M acetic acid at a flow rate of 12mlh- ' . The eluant, which was monitored by U.V. absorption, was collected in fractions of 2.2ml per tube. Fractions 47-53 inclusive were pooled and lyophilised to give [ 7-(L-thiazolidine-4-car- boxylic acid)/-oxytocin (4.2 mg, 70%) as a fluffy white powder of RF (A3) 0.20 and RF (H) 0.56. Amino acid analysis: Asp 1.00; Glu 1.01; Gly 1.04; Pro 0.98; Ile 0.99; Leu 1.00; Tyr 0.93. (Found: C, 50.2; H, 6.3; N, 15.2. C42 HMN12 012 S3. AcOH requires: C, 48.7; H, 6.3; N, 15.5%.)

ACKNOWLEDGEMENTS

This synthesis was undertaken at the suggestion of the late Professor J . Rudinger. M.B. thanks the SRC for a maintenance grant.

REFERENCES

Rosamond, J.D. & Ferger, M.F. (1976) J. Med. Chem.

Muhlemann. M., Titov, M.I. Schwyzer, R. & Rudinger, J. (1972) Helv. Chim. Acta. 55,2854-2860

Fainveather, R . & Jones, J.H. (1972) J. Chem. Soc. Perkin Trans. I , 2475-1481

Barber, M. & Jones, J.H. Proceedingsof the European Peptide Symposium at Wepion, Belgium, April 1976, (A. Loffet, ed.), p. 109, Editions de I'Universitk de Bruxelles, 1976

Felix, A.M., Jimenez, M.H., Vergona, R. & Cohen, M.R. (1973) Int. J. Pept. Prot. Res. 5,201-206

19,873-876

Address: Dr. J.H. Jones The Dyson Perrins Laboratory South Parks Road Oxford OX1 3QY United Kingdom

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