Int. J . PeptideProtein Res. 12, 1978, 164-169 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

SOLID-PHASE SYNTHESIS O F OXYTOCIN, DESAMINOOXYTOCIN A N D 4-THR-OXYTOCIN USING ACTIVE ESTERS IN PRESENCE OF

1 -HYDROXY BENZOTRIAZOLE

SHABBIR AHMED KHAN and K . M . SIVANANDAIAH

Department o f Chemistry, Central College, Bangalore, India

Received 7 February, accepted for publication 25 May 1978

Using appropriate amino acid active esters (3 eq.) in presence of HOBt* ( 1 eq,) and employing DPM protection for the thiol function o f cysteine, a rapid syn- thesis o f oxytocin in the solid phase has been accomplished. The DPMgroup has been removed by sodium-liquid ammonia reduction since boiling TFA is ineffec- tive. Desaminooxytocin and 4-Thr-oxytocin have been synthesized using lesser quantities o f amino acid active esters (1.5 eq.) in presence of HOBt (1 eq.), but the durations o f coupling are longer. The solid-phase synthesis of desamino- oxytocin using appropriate Boc-amino acids in presence o f DCCI in toluene medium has been described. Toluene does not exert any significant accelerating influence on the coupling rate as it does when active esters are employed.

Key words: desaminooxytocin; oxytocin; 4-Thraxytocin; solid-phase peptide synthesis.

Since the first synthesis of oxytocin by du Vigneaud el al. (1954), several syntheses of the hormone have been reported from various laboratories. A number of its analogs have also been synthesized for structure-activity studies. Most of the syntheses of oxytocin proceed via the protected nonapeptide inter- mediate, Z-Cys(Bzl)-Tyr-IleGln-AsnCys(Bzl)- Pro-Leu-Gly-NHz , which on reduction with

* Standard abbreviations for amino acid derivatives and peptides are according to the IUPAC-IUB Com- mission on Biochemical Nomenclature (1975) Biochemistry 14,449-462.

Additional abbreviations used: AcOH, acetic acid; CH, C1, , dichloromethane; DCCI, dicyclohexylcarbo- diimide; DMF, dimethylformamide; DPM, diphenyl- methyl; HOBt, 1-hydroxybenzotriazole; MPA, 3- mercaptopropionic acid; ONp, p-nitrophenyl ester; OPcp, pentachlorophenyl ester; ONSu, N-hydroxy- succinimide ester; OTcp, 2,4,5 -trichlorophenyl ester; TFA, trifluoracetic acid.

164

sodium in liquid ammonia followed by oxi- dation leads to the hormone. However, other protecting groups for the thiol function of cysteine such as carbobenzoxy and benzoyl (Photaki, 1966), trityl (Velluz el al., 1956), ethylmercapto (Inukai el al., 1967), ethyl- carbamoyl (Guttman, 1966), 24N-methyl- benzy1oxycarbamido)ethylcarbamoyl (Jager & Geiger, 1973), and p-methoxybenzyl (Saka- kibara & Shimonishi, 1956) have also found use.

In view of the reported cleavage of the DPM- protecting group from Cys(DPM)containing peptides on boiling with TFA containing phenol (Photaki et al., 1968), we considered it desirable to use this protecting group and synthesize the protected nonapeptide, Boc- Cys(DPM)-Tyr-IleGln-Asn-Cys@PM)-Pro-Leu- Gly-NH2, since treatment of this peptide with boiling TFA-phenol should lead to Cys-Tyr-Ile- Gln-Asn-Cys-Pro-Leu-Gly-NHz (oxytoceine) which could then be oxidized to oxytocin.

SYNTHESIS OF OXYTOCIN AND ANALOGS

The advantage of such a procedure lies in avoiding the less desirable sodium-liquid ammonia reduction which becomes necessary if benzyl group is used for protection of the thiol function.

To obtain the protected nonapeptide resin, Boc-Cy s@PM)-Tyr-Ile-Gln-Asn-Cys(DPM)-Pro- Leu-Gly-OCH2-resin (I), we followed the procedure we had earlier employed for the rapid synthesis of oxytocin on the solid phase (Shabbir Ahmed Khan & Sivanandaiah, 1976~). Thus, starting from Boc-Gly-OCH2 -resin and using appropriate Boc-amino acid active esters in presence of HOBt (Konig & Geiger, 1970), the desired peptide resin was obtained. During this synthesis, the Boc group was removed using 1 N HC1-AcOH except in the case of Boc-Gln-peptide resin when TFA-CH2 C12 (1 : 1) was employed. The same protected nonapep- tide resin could also be obtained starting from Boc-Gly-OCH2 -resin and using Boc-amino acids in presence of DCCI. However, for the incor- poration of Asn, Gln, and Tyr the correspond- ing active esters were employed in presence of HOBt. During this synthesis BF3.Et20- AcOH (Schnabel ef al., 1973) was employed for removal of the Boc group throughout. After completion of the couplings, the peptide was cleaved from the peptide resin by ammon- olysis (Hruby & Barstow, 1972).

For the removal of the DPM group, a portion of the protected peptide was refluxed with TFA containing phenol for l h . Thin-layer and paper chromatography indicated removal of only the Boc group; the DPM group, how- ever, was left unaffected. Therefore, the pep- tide was first treated with TFA to remove the Boc group and the resulting trifluoracetate was then subjected to sodium-liquid ammonia reduction (Siffered & du Vigneaud, 1935) which removed the DPM groups. The oxyto- ceine thus obtained was oxidized using potas- sium ferricyanide (Hope, 1973) and the crude oxytocin was purified on Sephadex (3-15 (Manning et al., 1968). The synthetic product was found to be as active as the natural hor- mone (uterotonic activity-500 I.U./mg).

In one of the above syntheses of oxytocin employing Cys@PM) and in our earlier solid- phase synthesis of oxytocin using Cys(Bzl) (Shabbir Ahmed Khan & Sivanandaiah, 1976a),

Boc-amino acid active esters (3 eq.) were employed in the presence of HOBt (1 eq.). The durations of the reactions varied from 0.5 to 1.5 h except in the case of the hindered Boc-Ile- OPcp when the duration was about 2.5h. This work prompted us to employ the same procedure for the synthesis of the two analogs, desaminooxytocin (Takashima et al., 1968) and 4-Thr-oxytocin (Manning ef al., 1970), which are reported to be about twice as active as the natural hormone. However, for their synthesis we employed only 1.5 eq. of Boc- amino acid active esters and 1 eq. of HOBt for each coupling and it was observed that it is possible to bring about more than 99.4% coupling as determined by the ninhydrin test of Kaiser (Kaiser ef al., 1970), but the duration of the reactions are longer (4 to 6 h).

The S-Bzl-MPA .required for the synthesis of desaminooxytocin was obtained following the procedure (Frankel ef al., 1960) employed for the preparation of S-Bzl-Cys but with some modifications (see Experimental procedures). This is a much simpler procedure than the one reported by Hope et al. (1962) who used sodium in liquid ammonia.

After the completion of the syntheses, the peptide resins, MPA(Bzl)-Tyr-IleGln-Asn- Cys(Bzl)-Pro-Leu-Gly-OCH2 -resin (111) and Z-Cys(Bz1)-Tyr-Ile-Thr-Asn-Cys(Bz1)Pro-Leu- Gly-OCH2 -resin (V) were subjected to ammon- olysis to yield the protected nonapeptide amides MPA(Bzl)-Tyr -1le -Gln -Asn -Cys(Bzl)-Pro-Leu- Gly -NH2 (IV) and Z-Cys(Bzl)-Tyr-Ile-Thr-Asn- Cys(Bzl)-Pro-Leu-Gly-NH2 (VI) respectively. Desaminooxytocin and 4-Thraxytocin were obtained from these nonapeptide amides by reduction with sodium in liquid ammonia followed by oxidation with potassium ferri- cyanide. The resulting crude peptides were purified by gel-filtration on Sephadex (3-15 as described for oxytocin.

In our earlier report (Shabbir Ahmed Khan & Sivanandaiah, 19766) we had observed that toluene accelerates the rate of reaction of active esters in the solid-phase synthesis of oxytocin. We therefore investigated the DCCI- mediated condensation of N-acylamino acids in this solvent. Thus,starting from BocGly-OCH2 - resin the protected nonapeptide resin (III) was obtained using the appropriate Boc-amino

165

S.A. KHAN and K.M. SIVANANDAIAH

acids (3 eq.) and DCCI (3 eq.) at each step. However, for the incorporation of Asn, Gln, and Tyr the respective active esters were employed. At the end of the synthesis, the protected peptide resin was ammonolysed and the released peptide was isolated and purified. During this synthesis, it was observed that the durations of the reactions in toluene medium are the same as those in CH2C12, the solvent normally employed when DCCI is used as coupling agent.

EXPERIMENTAL PROCEDURES

All the amino acids used except glycine are of the L configuration. Melting points (uncor- rected) were determined on a Leitz Wetzlar unit. Thin-layer chromatography was carried out on silica gel G plates using the solvent systems, n-butanol-acetic acid-water (4: 1 :5 ; upper phase); n-butanol-O.l N acetic acid- pyridine (5 :11:3, upper phase); and chlorofom- methanol-acetic acid (40:3:1) and the Rf values are reported as RIA, RfB and RfC. For paper chromatography, Whatman No. 2 paper strips were used in the solvent system n-butanol- acetic acid-water (4: 1 :5; upper phase) and the Rf is expressed as RfD. DPM-cysteine was prepared following the procedure of Zervas (Zervas & Photaki, 1962), and the Boc-amino acids were prepared following the procedure reported by us earlier (Shabbir Ahmed Khan & Sivanandaiah, 1977). The following active esters were employed: Boc-Leu-OPcp, Boc- Pro-ONSu, Boc-Cys(Bzl)-ONp, BocCys(DPM)- ONSu, Boc-Asn-OTcp, BocCln-OTcp, Boc-Thr- OPcp, Boc-Ile-OPcp, MPA(Bzl)-ONp and Z- Cys(Bzl)-OTcp, and these were prepared follow- ing the standard procedures using DCCI. BF3.Et20 was purified as recommended by Fieser & Fieser (1967). The completion of the coupling reactions on the solid phase was determined by Kaiser's test (Kaiser et al., 1970). Boc-Cly-OCH2 -Resin was prepared by the standard procedure (Manning e t al., 1970).

S-Benzylmercaptopropionic acid NEtS (2.8ml, 20mmol) was added followed by benzyl chloride (1.27 ml, 11 mmol) to a well stirred mixture of 3-mercaptopropionic acid (0.87m1, lommol), water (6ml) and

166

ethanol (8ml). After stirring for 1 h, the reaction mixture was diluted with water (50 ml) and extracted with ether (2 x 50ml). The aqueous phase on acidification (pH 4) was stored at 4' for 24h. The deposited crystals were collected, washed with water and dried in vacuo over P 2 0 5 . Yield 1.47g (75%);m.p. 81-82'.

Bo c-Tyr-0 Tcp A solution of Boc-Tyr (1.41 g, 5 mmol) and 2,4,5-trichlorophenol (1 g, 5 mmol) in THF (10ml) was treated with DCCI (1.03g, 5 mmol) at 0' for 4 h. The precipitated dicyclo- hexylurea was removed by filtration and the filtrate was evaporated to dryness. The residue was recrystallized from ethanol. Yield 1.38g (60%); m.p. 173-174.5'; RtC 0 .72 ; [ (~ ]2 - 27.5 ( c 1, DMF).

Anal. Calc. for CzoHzoNOSCl3 (460.8): C, 52.13; H, 4.37; N, 3.04. Found: C, 52.38; H,4.66;N, 3.08.

Boc-Cys(DPM)- Tyr-Ile-Gln-Am-Cys(DMP)-Pro- Leu-Gly-OCH2 -resin (I)

(a) By active ester-HOBt procedure. Boc- Gly-OCH2 -resin (2 g, containing 0.92 mmol of Gly) was placed in a manually operated solid- phase synthesis apparatus. The following cycle of deprotection, neutralization and coupling was performed to introduce each new amino acid residue. Unless otherwise stated all the washings were carried out thrice. 1. AcOH wash 2.1N HC1-AcOH (20ml) for 30min; (for removal of Boc- from Boc-Asn- and Boc-Gln- peptide resins, 50% TFA-CH2 C12 was employed for 25 min) 3. AcOH wash 4. DMF wash 5. 10%NEt3-DMF (20ml) for 10min 6. DMF wash 7. The appropriate active ester (3 eq.) and HOBt (1 eq.) wer'e added to a suspension of the resin in DMF (8ml) and the vessel was shaken 8. DMF wash 9. EtOH wash

After the completion of the synthesis, the peptide resin was dried over P 2 0 5 in vacuo and weighed (2.6 9).

SYNTHESIS OF OXYTOCIN AND ANALOGS

(b ) By DCCI procedure. Boc-Gly-OCH2 -resin (2.7 g, containing 0.73 mmol of Gly) was intro- duced into the solid phase synthesis apparatus and the following operations were carried out. 1. AcOH wash 2. (a) BF3.Et20-AcOH (10%) for 45min

fresh reagent 3. AcOH wash 4. CH2 Clz wash 5. 10%NEt3-CH2C12 (30ml) for 10min 6. CH2 C12 wash 7. Addition of the appropriate Boc-amino acid (3 eq.) and DCCI (3 eq.) in CH2C12 (1Oml) and shaking the mixture for 6 h ; washing with CH2C12 (2 x 30ml), repeating the step using 1 eq. of Boc-amino acid and 1 eq. of DCCI, the duration being 6h . For the introduction of Asn, Gln and Tyr, the respective 2,4,5- trichlorophenyl esters (3 en.) in presence of HOBt (1 eq.) in DMF (1Oml) were used as described in procedure (a) 8. CH2 C12 wash 9. EtOH wash

After the completion of the synthesis, the peptide resin was collected on a filter, dried in vacuo over P205 and weighed (3.5 g).

(b) BF3 .Et2 O-AcOH (10%) for 1.5 h with

Bo c-Cys(DPM) -Tyr-Ile-Cln -Am-Cys(D PM)-Pro - LeuCly-NH2 (11) The protected nonapeptide resin (I; 2.6g) was suspended in anhydrous methanol (200 ml) and dry ammonia was bubbled through this suspension at 0" for 2.5h. The mixture was stirred at 06" for 48h, the methanol and ammonia were evaporated and the released peptide was extracted into DMF (2 x 20ml). After evaporation of most of the DMF, 0.1 N HC1 was added and the deposited white solid was isolated and washed with water. The crude peptide was dried in vacuo over P205 and purified once from DMFether. Yield 0.5g (49% based on the amount of Gly originally esterified to the resin); m.p. 190-192"; RfA 0.9, RfC 0.6;[&]

Anal. Calc. for C?4H%N12014S2 (1441.8): C, 61.63; H, 6.71; N, 11.66. Found: C, 61.20; H, 6.50; N, 11.90.

The Boc group from a small quantity of the above peptide was selectively removed using TFA (20min) and the purity of the peptide

- 39.7" (C 0.49, DMF).

trifluoracetate was checked by chromatography; RfA 0.70 (single spot positive to ninhydrin and Pauly reagents), RfD 0.82 (single spot positive to ninhydrin and Pauly reagents).

Attempted removal of DPMgroup from DPM- protected nonapeptide In an attempt to remove both the Boc and the DPM groups, the protected nonapeptide amide (0.05 g) was refluxed with TFA ( 5 ml) contain- ing phenol (0.8ml) for 1 h. The TFA was evaporated in vucuo and the product was examined by t.1.c. and paper chromatography. The mobility of the product was identical with that of S, S'diDPM-nonapeptide amide trifluor- acetate indicating that the DPM groups had not been cleaved from the peptide during the treatment with boiling TFA.

Ox y tocin The protected nonapeptide amide (0.078 g, 0.05 mmol) was treated with TFA (1.2 ml) for 30min and then the TFA was removed in vucuo. Treatment of the residue with ether deposited the trifluoracetate as a solid. The DPM groups in this peptide were removed by reduction with sodium in liquid ammonia as described earlier and the resulting thiol was oxidized (Hope, 1973). The product was purified on a column (1.2 x 1lOcm) of Sephadex G-15 (Manning et al., 1968) to yield 22 mg (40%) of oxytocin as a white solid; RIA 0.62, RfB 0.68. It was found to have a n , uterotonic activity of 500 I.U./mg.

MPA (Bz1)-Tyr-Ile-Gln-Asn-Cys(Bz1)-Pro-Leu- Cly-NH2 (IV)

(a) Using active esters and HOBt. Starting from BocGly-OCH2 -resin (1 .Og, containing 0.25 mmol of Gly), the peptide resin (III) was built employing the appropriate active esters (1.5 eq.) in presence of HOBt (1 eq.) in DMF following essentially the procedure described earlier for the synthesis of oxytocin. After completion of the synthesis, the peptide resin (1 .O g) was subjected to ammonolysis and the released protected nonapeptide amide (IV) was isolated by extraction into DMF. The DMF extract was evaporated to a small volume and 0.1N HCl was added when a white solid

167

S.A. KHAN and K.M. SIVANANDAIAH

separated. The crude peptide was collected by filtration and reprecipitated from DMF- water. Yield 25mg (11%); m.p. 239-241'; [a] &' - 39.6" ( C 1, DMF); RfA 0.86.

(b ) Using DCCI in toluene. The protected nonapeptide resin (111) was obtained starting from Boc-Gly-OCHz-resin (1.4 g, containing 0.36mmol of Gly) following the procedure described for the synthesis of oxytocin using active esters with the following change in the coupling step. The appropriate Boc-amino acid (3 eq.) was added to a suspension of the resin in toluene (6ml) and the vessel was shaken for 15min. DCCI (3 eq.) was then introduced and shaking was continued. For the incorporation of Asn, Gln, And Tyr, the corresponding 2,4,5-trichlorophenyl ester was dissolved in a small quantity of DMF and added to a Suspension of the resin in toluene.

After the completion of the synthesis, the resin was collected and dried (1.45 g). The nonapeptide resin was ammonolysed, the protected peptide (IV) was extracted into DMF and isolated and purified as described earlier. Yield 105mg (21%); m.p. 237-239";[a]g - 41.8" (C 1, DMF); RfA 0.90.

Desaminooxytocin The S-Bzl groups from the protected nonapep- tide (IV; 0.059 g, 0.05 mmol) were removed by reduction with sodium in liquid ammonia and the resulting thiol was oxidized with potassium ferricyanide as described for oxytocin. The crude peptide was purified on Sephadex (3-15 to furnish 22mg (44%) of a white solid having an uterotonic activity of 837 I.U./mg; RfA 0.43;[a] 2: - 96" (c 0.5,lM AcOH).

Z -Cys (Bzl) - Tyr-Ile-Thr-Asn -Cys (Bzl) -Pro-Leu -

Starting from Boc-Gly-OCH2 -resin (1.5 g, con- taining 0.47 mmol of Gly) the protected nona- peptide resin, Z-Cys(Bzl)Tyr-ne-Thr-Asn- Cys(Bzl)Pro-LeuGly-OCH2 -resin 0, was ob- tained using the appropriate active esters (1.5 eq.) in presence of HOBt (1 eq.) following the experimental procedure described for oxytocin. The Boc group was removed using 1N HC1-AcOH at each stage. After the com- pletion of the synthesis, the protected nonapep-

168

Gly-NHz (VI )

tide resin (1.7 g) was subjected to ammonolysis and the released peptide (VI) was isolated by extraction into DMF. The DMF was evaporated in vucuo and the residue was treated with ether. The resulting solid was warmed with 95% ethanol, cooled to room temperature, filtered and washed successively with 95% ethanol and ethyl acetate. Yield 215 mg (42%); m.p. 227-

Anal. Calc. for C64H15Nl1014SZ (1296.6): C, 60.09; H, 6.61 ; N, 11 9 . Found: C, 59.70; H, 7.00; N. 11.58.

231";[c~]g -32" (C 1, DMF); RIA 0.81.

4 - Th r-oxy t o cin The protected nonapeptide amide (VI; 0.065 g, 0.05 mmol) was reduced with sodium in liquid ammonia and the resulting thiol was oxidized with potassium ferricyanide. The crude product was purified on Sephadex G-15 as described for oxytocin to yield 22mg (42%) of 4-Thr- oxytocin with an uterotonic activity of 899 I.U./mg; RfB 0.7 1.

REFERENCES

du Vigneaud, V., Ressler, C., Swan, J.M., Roberts, C.W. & Katsoyannis, P.G. (1954) J. Am. Chem.

Fieser, L.F. & Fieser, M. (1967) Reagents for Organic Synthesis, vol. 1 , p. 70, John Wiley & Sons, New York

Frankel, M., Gertner, D., Jacobson, H. & Zilkha, A. (1960) J. Chem. Soc. (q, 1390-1393

Guttmann, S. (1966) Helv. Chim. Acta 49,83-96 Hope, D.B. (1973) Experienfia 29,389 Hope, D.B., Murti, V.V.S. & du Vigneaud, V. (1962)

J. Biol. Chem. 237,1563-1566 Hruby, V.J. & Barstow, L.E. (1972) inMacromolecular

Synthesis, (Bailey, W.J., ed.), vol. 4 , pp. 91-94, John Wiley & Sons, Inc., New York

Inukai, N., Wakano, K. & Murakami, M. (1967) Bull. Chem. SOC. Japan 4 0 . 2 9 13 -29 18

Jiger, G. & Geiger, R. (1973) in Peptides 1972, (Hansen, H. & Jakubke, H.D., eds.), pp. 90-92, North-Holland Publishing Company, Amsterdam

Kaiser, E., Colescott, R.L., Bossinger, C.D. & Cook, P.I. (1970) Anal. Biochem. 34,595-598

Konig, W. & Geiger, R. (1970) Chem. Eer. 103,788- 798

Manning, M., Wuu, T.C. & Baxter, J.W.M. (1968) J. Chromafog. 38,396-398

Manning, M., Coy, E. & Sawyer, W.H. (1970) Bio- chemistry 9,3925-3930

SOC. 76,3115-3121

SYNTHESIS OF OXYTOCIN AND ANALOGS

Photaki, I. (1966) J. Am. Chem. SOC. 88, 2292- 2299

Photaki, I., Bardakos, V., Lake, A.W. & Lowe, G. (1968) J. Chem. SOC. (CJ 1860-1864

Sakakibara, S . & Shimonishi, Y. (1965) BUN. Chem. SOC. Japan 34,1412-1413

Schnable, E., Klostermeyer, H. & Berndt, H. (1973) in Peptides 1971, (Nesvadba, H., ed.), pp. 69-76, North-Holland Publishing Company, Amsterdam

Shabbir Ahmed Khan & Sivanandaiah, K.M. (19760) Tetrahedron Letters 199-200

Shabbir Ahmed Khan & Sivanandaiah, K.M. (19766) Synthesis 6 14 -6 1 5

Shabbir Ahmed Khan & Sivanandaiah, K.M. (1977) Indian J. Chem. 15.80

Siffered, R.H. & du Vigneaud, V. (1935) J. Biol. Chem. 108,753-158

Takashima, H., du Vigneaud, V. & Merrifield, R.B. (1968) J. Am. Chem. SOC. 90,1323-1325

Velluz, L., Amiard, G., Bartos, J., Coffmet, B. & Heymes, R. (1956) Bull. SOC. Chim. France 1464- 1467

Zervas, L. & Photaki, I. (1962) J. Am. Chem. Soc. 84, 3887-3897

Address: Dr. K.M. Sivamndaiah Professor of Chemistry Central College Bangalore 560001 India

169