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The synthesis and some pharmacological properties of [fL-DOPA]=oxytocinl B. M. FERRIER AND L. A. BRANDA Department qf'Biochemis~ry and Programme in Reproductive Biology, McMuster University, Hamilton, Ontario H S $98 Received October 20. 1975 Ferrier, B. M. gL Branda, L . A. (1976) The synthesis and some pharmacological properties of 12-L-DOPA)-oxytocin. Can. J . Biochem. 54,507-5 1 1 The first reported synthetic analogue of a naturally occurring peptide with a residue of L-3,6dihydroxyphenylaIanine(L-DOPA) was prepared by coupling N-carbobenzoxy-S-benzylcysteinyl-L-DOPA azide with isoleucylglutaminylasparaginyl-S-benzylcysteinylprolylleucylglycinamide. The protecting groups were removed from the resultant nonapeptide derivative by sodium in liquid ammonia and the peptide analogue was formed by short term oxidation of the dithiol-containing compound. It was isolated by sequential partition chromatography and exclusion chromatography on Sephadex G-25. Et was unstable at neutral or alkaline pH. [2-L-DOPA]-oxytocin was found t o possess a minimum milk-ejection-like activity of 54 2 9 U/mgand uterotonic activity of 26 9 4 Ulmg. These potencies are approximately 12% and 5% of the corresponding potencies of oxytocin. Ferrier, B. M. gt Branda, L. A. (19'76) The synthesis and some pharmacological properties of [2-L-DOPA]-oxytocin. Can. 3. Biochern. 54,507-51 1 Nous avons prepare le premier analogue synthetique r a p p r t e d'un peptide nature1 avec un residu de L-3,4dihydroxyphknylaIanine (L-DOPA) par couplage du N-carbobenzoxy-s-benzylcystkinyl-L-DOPA azide avec l'isoleucy~glutaminylasparaginyl-S-benzylcysteinylprolylleucylglycinamide. Les groupes prstecteurs somt enleves du derive nonapeptidique obtenu B l'aide dca sodium dans I'arnmsniac liquide et le peptide analogue est forme par oxydation a court terme du compose contenant du dithiol. Le peptide est isole par chromatographie de partage dquentielle et chromatographie d'exclmsion sur Sephadex (3-25. El est instable a pH neutre ou alpdin. La [2-L-DOPA]-ocytocine posskde une activite lactsgogue minimum de 54 2 9 Ulmg et une activite uterotonique de 26 + 4 Ulmg. Ces activitk egalent approximativement 12% et 5% de I'activite correspondante de l'acytocine. [Traduit par le journal]
Introduction
carbodiimide (0.23 g) in tetrahydrofuran was added and the mixture stirred in an ice-bath for 30 min and then at room The effect of replacing tyrssine by L-DOPA in temperature for 18 h. The mixture was filtered, the solid on naturally occurring peptides has not been explored, the filter washed with tetrahydrofuran ( 1 ml), rand hexane and relatively few synthetic DOPA-containing pepwas added to the combined filtrate and washings to comtides have been reported. We have prepared the plete the separation of an oil. The oil was dissolved in oxytocin analogue in which tyrosine at position 2 has methanol (5 ml) and hydrazine hydrate (85%, 0.4 ml) was added to the solution which was heated under reflux for 2 h been replaced by L-DOPA (Figs. I and 2) and have determined the effect of this change on two charac- and kept at room temperature for 3 days. The voiume was halved by evaporation and water was added to cause septeristic bio!ogicai activities of oxytocin. aration of a solid. This solid was collected by fiatration and dissolved in a small volume of warm methanol. The soluMaterials and Methods tion was decanted off from a small amount of insoluble oil. Water was added to the solution to precipitate a solid. The N-Curbobenzoxy-S-b,en~yiq.rteinyl-~-3,Cdihydroxyphsn-solid was collected by filtration and crystallized from ylalanine hydrcazide 41) methanol in buff needles 0.13 g, mp 178-182 "C (decamp.) Methyl L-3,ddihydroxyphenylalainate hydrochloride ( 1) [a];" 119.4" (c 1.0 in dimethylformamide). After being (0.25 g) and N-carbobenzoxy-S-benzylcysteine(2) (0.33 g) dried for 24 h over P,O, at room temperature in vasuo, it were suspended in ice-cold tetrahydrofuran (1.5 ml) conwas found t o contain: C, 68.21; H, 5.80; N, BOA%%.(Calc. taining triethylamine (8.I $). A solution d dicyclohexylfor C,,H,,N,O,S: C,60.21; H.5.61; N, 10.40%!. An Asnow test (3) for catechols was applled to this ABBREVIATIONS: L-DOPA, L-3,4dihydroxyphenylalacompound and was strongly positive. %LC on silica in the nine; [2-L-DOPA)-oxytocin,[2-L-3,4dihydroxyphenylala- solvent system l-butanol- acetic acid - pyridine - water, nine]-oxytocin; TLC, thin-layer chromatography. 85:3: 10: 12, showed this material to be separable from the 'This work was supported by grants from the Medical starting amino acid derivatives and the unchamcterized Research Council of Canada. oily dipeptide methyl ester intermediate.
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CAW. J. BIBCHEM. VOL. 54. 1976
of sodium nitrite (I8%, 0.1% mi) was added. Stirring was continued at -20 "C for 15 min. Ethyl acetate (7.5 ml) was added. The ~esultant soIution was washed three times with saturated aqueous sodium bicarbonate solt~tiasn (10 ml) and twice with water (10 m1) and then dried over anhydrous sodium sulfate. It was filtered and cooled to -20 "C. N-Carbobenzoxyisoleucy1g1utaminyl~i~paraginy~S - benzylcysteinylpro1y1leucylglyciHsae (II, 250 mg) (4) was dissolved in acetic acid ( 1 ml) and a soHution of hydrogen bromide in acetic acid (33%. B .5 ml). and the solution was stirred for 1 h at room temperature and then poured into anhydrous ether (20 ml). The precipitated heptapeptide hydrobromide was washed twice by decantation of ether. It was twice dissolved in methanol (5 rnl) and re-precipitated by the addition of ether. It was filtered and dissolved in dimethylformarnide ( 5 ml), containing triethylamine (8.7 ml), and this solution was cooled to -20 "C. To it was added the cooled solutic>n of I%rcarbobenzoxy-S-henzylcysteiny1-L-3 ,4dihydroxyphenylalanaine azide previously prepared. The mixture was kept at 2 "C for I6 h and was then stirred at room temperature for 3 h. Ethyl acetate (50 ml) was added and the precipitated solid was filtered, washed exhaustively on the filter with ethyl acetate and air dried. This gave 160 mg of off-white - 49.6" (c 1.0 in solid. rnp 280-215 "6: (decamp.), [a]&" dimethylformamide). The presence sf a catechol group in this material was detected by an Arnow test. [2-L-DOPA]-ox? tocin ClV)
The presumed precursor (111) obtained by coupling N-carbobenzoxy 6 -benzylcysteinyl-L -3,4-dihydrctxyphenyiialanine azide and isoleucylglutaminylasparagiwyI-bybenzylcysteinylpro1y11eucylg1ycinamide 4120 nag) was added to boiling ammonia (120 ml) which had been freshly FIG. 1. Structure of oxytocin and sf [%-L-DOPA]- redistilled from sodium. A sodium stick was held below the oxytocin. Numbers indicate positions of the constituent surface C P the ~ solbltion until a blue coisur pervaded the amino acid residues. entire solution and persisted for 1 min. The ammonia was removed by Hyophilization and the residue dissc~lvedin water (120 ml) to give a solaltion of pH 8. Hydrogen peroxide (0.05 ml, 30%) was added, and the solution allowed to stand at room temperature for I min. The pH was lowered to 5 by the addition of glacial acetic acid and the volume of the solution was lowered to 2 ml by evaporation under reduced pressure at room temperature. The colour of this solution was faintly yellow. This solution was then satuE-Cy s(Bz?)-L-DOPA-HIeGln-Asn-Cy s(Bzl1-P~Q-Leu-Cly- WHz rated with upper phase of the solvent system 1-butanol m pyridine - acetic acid - water, 1000:B5:35:9564. A Sephadex I ) Na, iiq. HHg @-25 colhamn (75 x 1.5 cm) was equilibrated with lower phase of the soHveni system in preparation for partition 21 chromatography (5). The sample was put on the column and eluted in 1.1-ml fractions with upper phase of the (~-L-DBBA)-L%XYT~)CIN solvent system. The eluate was colourless and the yellow D! colour of the applied solution remained on the column. FIG.2. Synthesis of 12-E-DOPA]-oxytocin. Folin-Lowry determination (6) of the peptide content of the eluted fractions showed that there were three regions in Coadpling ~I&T-cc~rboben~~~q-S-he~zy~c'~~.~tei~ty~-~-S,~-dI'which peptide was eluted (Fig. 3). The fractions correhgrdrovphenyla1a~1ine azide and isoleucylglutarnsponding to these regions were pooied, concentrated, after iny laspavcrginyl-S-benzy~cysteiny/pro8ylle~edc$the addition of water, until the organic phase was removed, glycinamide and iyophilized. The products from the two fastest moving M-Carbobenzoxy-S -2senzylcysteinyl-L -3,4-dihydroxy peaks (A and B) were yelowish, somewhat sticky and very phenylalanine hydrazide (I, 150 mg) was dissolved in a small in quantity. The material from peak C was white and solution of hydrogen chloride in 95% aqueous tetrahyfluEy 630 mg). Some of this material (20 mg) was dissolved in glacial acetic acid (0.03 ml) and diluted to 2.5 rnl with drofuran (4.6 N , 1.3 ml). The solution was cooled to water. This solution was put onto the Sephadex 6-25 col-20 "C and stirred vigorously while an aqueous solution
4
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PERRIER AND BRANBA
FRACTION NO.
FIG.4. E X C ~ U S chromatography ~OH on Sephadex G-25 in FRACTBON NO. 0.2 N acetic acid of [%-L-DOPA]-oxytocinfrom peak C , FIG. 3. Partition chromatography of crude [%-I,- Fig. 3: fraction volume, 3.0 ml. Fractions were pooled as indicated by the broken lines. DOPA]-oxytocin on Sephadex G-25 in the solvent system I-butan01 - pyridine - acetic acid - water 1000: 15: as 1: aspartic acid 1 .Q, gllatarnic acid 1 .a, proline 0.9, hemi35:958; fraction volume, 1 . 1 ml;eIution pattern shown by cystine 1.8, leucine 1.0, isoleucine 1.0 and ammonia 2.8. Folin-Lowry determination of pepaide content in eluate. A standard sf L-DOPA was treated under the hydrolysis Fractions were pooled as indicated by the broken lines. conditions and was shown by comparison with an untreated standard to have been partially destroyed. The presence urnn from which the organic phase had keen discharged of 1 moi of 1.-DOPA. in the peptide was confirmed by a with a mixture of pyridine and 0.2 N acetic acid 4 1 :4) and quantitative Arnow test, ucjing a mixture of t-DOPA which was then equilibrated with 0.2 N acetic acid. This and equimslar amounts of the other constituent amino came solvent was used to elute 3.8-ml fractions. Measureacids as the standard. ment s f the absorbance of these fractions at 280 wm showed The milk-ejection-like activity c~f[2-~-D&PPA1-oxytc1cin a region of peptide material eluted as a symmetrical peak was determined by measurement of the contraction of an with a shoulder on its leading edge (Fig. 4). The elution isolated strip of mammary gland from a lactating mouse (4). volume sf the peak was 105 ml. Oxytscin is eluted at 112 ml The uterotonic activity was measured on an isolated under the same conditions. The fractions corresponding to uterine horn from a virgin Wistar rat (81, using MLIHSIC~ the major peak (Fig. 4) were concentrated under reduced solution (9). pressure and lyophiliaed to give 12 mg white, fluffy amorphous material, which gave a positive Arnow test for the presence of a catechol group: [a]%" 322.' (c 0.5 in 1N Results m d Discussion acetic acid). After being dried in vnclts for 24 h over phos[2-L-DOPA]-oxytocin(IV), a hormone analogue phorous pentoxide at room temperature it was found to contain: C, 50.25; H, 4.48; N, 16.80%. (Calc. for with a residue of L-DOPA in place of the residue of G43H66N12013SZ: C , 50.47: I%, 6.50; N 16.43%.) A scan of tyrosine in oxytocin, was synthesized (Fig. 2) by the BSV absorbance of this material in 0.2 N acetic acid short-term oxidation of the dithiol-containing preshowed two maxima: A ,,,,,, (e 2.83 x 103),Amak2,, ( E 4.68 cursor. The precursor was produced by treatment x B03). Approximately 4% s f the absorption of by sodium in liquid ammonia of N-carbsbenzoxy-S12-e-DOPA]-oxytocin at 282 nm can be ascribed to its benzylcysteinyl - L- 3,4 - dihydroxyphenyla1s~ny1isocystine content. The spectrum ofr.-DOPA iaa 0.2 N acetic ( E 2.56 x 109),A , ,,,,, (e leucylglutaminylasparaginy1-S- b e m z y 1 c y s ~ acid showed two maxima: A 2-64x lo3). Neither [2-e-DOPA)-oxytocin nor L-DOPA lylleucylglycinamidg (111) which had been syntheshowed any absorption at wave lengths greater than 300 nrn sized by coupling N-carbobenzowy-S-benZylcy(Fig. 5). TEC of [2-L-DOPA]-oxytocimon silica showed it steinyl-L-3,4dihydroxygRenylr?1anineazide with to be homogeneous in the solvent systems I-butanol - isoIeucyIglutaminylasparagimyI- S - benzylcysteinylacetic acid - water (4:I:5) (Rf 0.89), and 1-butanol - ben- proBylleucylglycinamide. The hormone analogue zene - pyridine - acetic acid - water (30:10: 1:2:37) (Rf was isolated by sequential partition chromatography 0.17). For amino acid analysis, a sample was hydrolyzed at and exclusion chromatography on Sephadex G-25. The difficulty in preparing peptides containing 110 "C for 22 h in 6 N HC1. The amino acids and ammonia were found in the following ratios, with glycine being taken DOPA lies in the ready oxidation s f DOPA to the
,,,,,,
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CAN. J. BIQCHEM. VOL. 54, 1976
WAVELENGTH (nmB FIG. 5 . Ultraviolet absorption spectrum of [2-LDOPAl-oxytocin (-) and 0 % ' ~ - D O P(-----), A I x M in 0.2 N acetic acid.
quinone. Felix cb a / . (lo), who prepared several L-DOPA-containing dipeptides and tripeptides, found it desirable to protect the catecho1 hydroxyl groups to prevent oxidation. Bn the presently described synthetic scheme, there necessarily is a ring-clcdsing oxidation step after the cleavage of the protecting groups and in light of this we decided that the protection might not be a significant advantage. The synthesis was designed so that the residue of L-DOPA was exposed to as few synthetic steps as possible, that the pH whenever possible was kept below 7 and that the oxidation step was carried out with the shortest convenient exposure to the oxidizing agent. A yellow ccplour did develop during the oxidative ring closure step in the synthesis, indicating that some oxidation had occurred. This colour was retained on the Sephadex cc~lurnnduring partition chromatography and the final product was white. The UV absorption spectrum which showed no absorption at wavelengths greater than 300 nm (Fig. 51, indicated that the material did not contain any oxidized products. The molar extinction coefficient for [ZL-DOPA]-oxytocinat 282 nm was shown to be 2.8%x lo3, of which approximately 4% can be ascribed to the cystine residue. The extinction coefficient corrected for cystine is thus similar
to that of L-DOPA at its corresponding maximum absorption (2.55 x 1Q3 at 280 nm). Oxidation of L-DOPA gives materials which absorb more intensely throughout the UV spectrum (1 I), The potencies of the two biological activities assayed for [2-L-DOPA]-oxytocinwere found to be 54 5 9 U/mg for milk-ejection-like activity and 26 9 4 U/mg for uterotonic activity. Oxytocin when assayed in this laboratory for milk-ejection-like activity against U.S.P. Posterior Pituitary Reference Standard (12) was found to have a potency of 459 ? 23 U/mg. Its uterotonic potency is 546 L- 18 U/mg (13). The replacement of the tyrosine residue in oxytocin by a residue of L-DOPA has, therefore, resulted in a decrease in potency of these biological activities and a change in the ratio of their potencies. The biological assay of 12-L-DOPA]-oxytoci was complicated by the fact that prior dilution with assay medium resulted in rapid Boss of activity. The solution prepared as the stock solution for assay sonsisted of [2-L-DOPA]-oxytocinin 0.2 N acetic acid. For assay, this stc~cksolution must be diluted with Tyrode solution or Munsick solution, both of which have been pretreated with a mixture of 0, and CO, (95:s). The high oxygen concentration in these solutions and the elevation of the pH of the peptide containing solution from acidic to somewhat above 7 presumably combined to cause rapid oxidation of the DOPA residue. The assays were, therefore, done by diluting the stock solution immediately before injecting it into the assay bath. Although the results from different biological assays were reproducible within the experimental error of the assay, it is possible that even with this minimum exposure to oxidizing conditions, there is some Boss of biological activity and we therefore consider that the figures which we quote for [ZL-DOPA)-oxytocinare minimum potencies. ~ e v e r ahormone l analogues have heon prepared in which the tyrosine residue of oxytocin has been replaced by another amino acid residue and have given some information which may lead to an understanding of the structural requirements at position 2 for the expression of biological activity. Low biological activity of several analogues of oxytocin with aliphatic amino acids in place of tyrosiwe had suggested that an aromatic nucleus was necessary at position 2 for the expression of a high degree of activity. However, the observation that 12-isoleucine]-oxytocin possesses moderate biological activity put that assumption in question (18). [2-Phenylalaninel-oxytocin,with phenylalanine in place of tyrosine had reduced but still very considerable potency in those activities characteristic of oxytocin 14-17). This suggested that the phenolic hydroxyl group in tyrosine was facilitating rather than necessary for activity. The synthesis of [2-I.-DOPA]-oxytocin allows for examination of the
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FEWRIER AND BRANBA
TABLE 1. The effect of some changes in ~ubetitutionof the tyrosine ring on the uterotonic activity of oxytacin -
Compound
Amino acid at position 2
[2-PheragrHalabwine]-oxflmin Oxfloein [2-L-DOPA]-oxytmin [2-(3-Poddyrmi~1e)]-oxytwin
PhenyBalanine 4-HydroxyphenylaIanine 3,L$DihydrcsxyphenyIalanine 3-Podo-4-hydroxyphenyIaIanine
-
--
[2(3-Methyltyrosine)]-oxytwin
-.
Uterotonic activity (isolated rat uterus) -
-
-
-
38" 546 f lgB 26 4 Nonec (inhibitor of oxytocin) 3-MethyI-4-hydrsxyphenyla1anine Nonee (inhibitor of oxfloein)
°[Ref. IS), &(Refe13, C(Ref.19).
biological effects of introducing a second hydrcsxyl group into phenylalanine. It appears that the rernoaral of the hydsoxyl group t o give E2-phenylalanine]-oxytocira and the introduction of a second hydroxyl group to give [2-L-DOPA]-oxytocin have similar effects on the activities caf oxytocin (Table I). Marbach and Rudinger (19) have reported the synthesis s f [2-(3-iodotyrosine)mxytscin and [2(3-methyltyrassine)]-oxytocinn These analogues inhibit the uterotsnic activity of oxytocin and d c ~not possess any uterotonic activity themselves. Comparison of the biological activities of the three disubstituted [2-phenylalaninel-oxytscin analogues (Table 1) suggests that a11 three peptides bind to the oxytocin receptors on the rat uterus. The differences in activity must, therefore, occur subsequent to binding. Marbach and Rudinger conclude that the similarity in the result s f introducing an iodo car a methyl sbabstituent (~rrho to the hydroxyl group must be due t o steric effects and not tia alterations in the pK, of the hydroxyl group. The two different substituents would alter the pK, in opposite directions. A hydroxyl group is isosteric with a methyl g o u p and it must be concluded that the 3-hydroxyl substitraent in [2-L-DOPA]-oxytocin has an effect which is not simply steris. It is possible that the 3-hydroxyl group reacts with some other site in such a way that the molecule is oriented to be in an oxytocin-like presentation.
We are grateful to Mrs. C. Cardy and Mr. D. Bargavel for performing the biological assays.
1. 09Neill, 9. J., Veitch, F. P. & Wagner-Jauregg, T. (1956) J . Org. Chem. 21,363-364 2. Haringtom, G. R. & Mead, T. H. (1936) Bic~chem.J. SO, 1598-161 1 3. Arnow. &. E. (1937) 3. B i d . Chem. 118,531-537 4. Bodanszky, M. & du Vigneaod. V . ( 1 9 9 ) J . Am. Chem. Soc. 81,5688-5691 5. Yamaskiro, D. (1964) Nature (London) 201,76-77 6. Lowry, 0. W., Rosebrough, N. J., h r r , A. L. & Randall, R.J. (1951) J. Biol. Cirern. 193,265-275 7. Fielitz, G., Rosa, R., Mattei, A., Melander, S., Garofalo, E., Gioia de Goch, M N . & Coch, J . A. (1970)Proc. SOL..Exp. Blol. Med. 133, 1155-1 157 8. Holton, P. A. (19448) Br. S. Pharmacof. 3,328-336 9 . Munsick. R. A. (1966) Endoc.rino&ogy66,451457 10. Felix, A. M . , Winter, D. P., Wang, S.-S., Kuleska. I. B . , Pod. W. R . , Hang, D. L. & Skeppard, W. (1974)J. Med. Chern. 17,422426 1 1 . Yasunobu. #. T., Peterson, E. W. & Mason, H. S. Q 1959)J. Biol. C h e a . 234,3241-3295 12. The Phaurnacopeia of'the United States: of America (1970) 18th revision, p. 469, Mack Publishing Go., Easton, Pa. 13. Ghan, W. Y., O'Gonnell, M. & Pomeroy, S. R. (1963) Endocrinology 72,279-282 14. JoSt, K., Wudinger, 3. & Sorrn, F. (1963) Cofiect. Czech. C h e a . Cornmun. 28, 1706-17 14 15. Bodanszky, M. & du Vigneaeed, V. (1959) J. A m . Chem. Soc. 81,6072-6075 16. Jaquenoud, P. A. & Boissonnas, R . A. (1959) Helv. Chirn. act^ 42,788-793 17. Konzete, H. & Berde, B. (1959)Br. J . Pharmacol. 14, 133-136 18. Bsamda, k. A. & du Vigneaud, V. (1967) Moi. Bharrnucol. 3,248-253 19. Marback, P. & Rudinger, J. (1974) Expeaicntia 30.696
The synthesis and some pharmacological properties of [fL-DOPA]=oxytocinl B. M. FERRIER AND L. A. BRANDA Department qf'Biochemis~ry and Programme in Reproductive Biology, McMuster University, Hamilton, Ontario H S $98 Received October 20. 1975 Ferrier, B. M. gL Branda, L . A. (1976) The synthesis and some pharmacological properties of 12-L-DOPA)-oxytocin. Can. J . Biochem. 54,507-5 1 1 The first reported synthetic analogue of a naturally occurring peptide with a residue of L-3,6dihydroxyphenylaIanine(L-DOPA) was prepared by coupling N-carbobenzoxy-S-benzylcysteinyl-L-DOPA azide with isoleucylglutaminylasparaginyl-S-benzylcysteinylprolylleucylglycinamide. The protecting groups were removed from the resultant nonapeptide derivative by sodium in liquid ammonia and the peptide analogue was formed by short term oxidation of the dithiol-containing compound. It was isolated by sequential partition chromatography and exclusion chromatography on Sephadex G-25. Et was unstable at neutral or alkaline pH. [2-L-DOPA]-oxytocin was found t o possess a minimum milk-ejection-like activity of 54 2 9 U/mgand uterotonic activity of 26 9 4 Ulmg. These potencies are approximately 12% and 5% of the corresponding potencies of oxytocin. Ferrier, B. M. gt Branda, L. A. (19'76) The synthesis and some pharmacological properties of [2-L-DOPA]-oxytocin. Can. 3. Biochern. 54,507-51 1 Nous avons prepare le premier analogue synthetique r a p p r t e d'un peptide nature1 avec un residu de L-3,4dihydroxyphknylaIanine (L-DOPA) par couplage du N-carbobenzoxy-s-benzylcystkinyl-L-DOPA azide avec l'isoleucy~glutaminylasparaginyl-S-benzylcysteinylprolylleucylglycinamide. Les groupes prstecteurs somt enleves du derive nonapeptidique obtenu B l'aide dca sodium dans I'arnmsniac liquide et le peptide analogue est forme par oxydation a court terme du compose contenant du dithiol. Le peptide est isole par chromatographie de partage dquentielle et chromatographie d'exclmsion sur Sephadex (3-25. El est instable a pH neutre ou alpdin. La [2-L-DOPA]-ocytocine posskde une activite lactsgogue minimum de 54 2 9 Ulmg et une activite uterotonique de 26 + 4 Ulmg. Ces activitk egalent approximativement 12% et 5% de I'activite correspondante de l'acytocine. [Traduit par le journal]
Introduction
carbodiimide (0.23 g) in tetrahydrofuran was added and the mixture stirred in an ice-bath for 30 min and then at room The effect of replacing tyrssine by L-DOPA in temperature for 18 h. The mixture was filtered, the solid on naturally occurring peptides has not been explored, the filter washed with tetrahydrofuran ( 1 ml), rand hexane and relatively few synthetic DOPA-containing pepwas added to the combined filtrate and washings to comtides have been reported. We have prepared the plete the separation of an oil. The oil was dissolved in oxytocin analogue in which tyrosine at position 2 has methanol (5 ml) and hydrazine hydrate (85%, 0.4 ml) was added to the solution which was heated under reflux for 2 h been replaced by L-DOPA (Figs. I and 2) and have determined the effect of this change on two charac- and kept at room temperature for 3 days. The voiume was halved by evaporation and water was added to cause septeristic bio!ogicai activities of oxytocin. aration of a solid. This solid was collected by fiatration and dissolved in a small volume of warm methanol. The soluMaterials and Methods tion was decanted off from a small amount of insoluble oil. Water was added to the solution to precipitate a solid. The N-Curbobenzoxy-S-b,en~yiq.rteinyl-~-3,Cdihydroxyphsn-solid was collected by filtration and crystallized from ylalanine hydrcazide 41) methanol in buff needles 0.13 g, mp 178-182 "C (decamp.) Methyl L-3,ddihydroxyphenylalainate hydrochloride ( 1) [a];" 119.4" (c 1.0 in dimethylformamide). After being (0.25 g) and N-carbobenzoxy-S-benzylcysteine(2) (0.33 g) dried for 24 h over P,O, at room temperature in vasuo, it were suspended in ice-cold tetrahydrofuran (1.5 ml) conwas found t o contain: C, 68.21; H, 5.80; N, BOA%%.(Calc. taining triethylamine (8.I $). A solution d dicyclohexylfor C,,H,,N,O,S: C,60.21; H.5.61; N, 10.40%!. An Asnow test (3) for catechols was applled to this ABBREVIATIONS: L-DOPA, L-3,4dihydroxyphenylalacompound and was strongly positive. %LC on silica in the nine; [2-L-DOPA)-oxytocin,[2-L-3,4dihydroxyphenylala- solvent system l-butanol- acetic acid - pyridine - water, nine]-oxytocin; TLC, thin-layer chromatography. 85:3: 10: 12, showed this material to be separable from the 'This work was supported by grants from the Medical starting amino acid derivatives and the unchamcterized Research Council of Canada. oily dipeptide methyl ester intermediate.
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CAW. J. BIBCHEM. VOL. 54. 1976
of sodium nitrite (I8%, 0.1% mi) was added. Stirring was continued at -20 "C for 15 min. Ethyl acetate (7.5 ml) was added. The ~esultant soIution was washed three times with saturated aqueous sodium bicarbonate solt~tiasn (10 ml) and twice with water (10 m1) and then dried over anhydrous sodium sulfate. It was filtered and cooled to -20 "C. N-Carbobenzoxyisoleucy1g1utaminyl~i~paraginy~S - benzylcysteinylpro1y1leucylglyciHsae (II, 250 mg) (4) was dissolved in acetic acid ( 1 ml) and a soHution of hydrogen bromide in acetic acid (33%. B .5 ml). and the solution was stirred for 1 h at room temperature and then poured into anhydrous ether (20 ml). The precipitated heptapeptide hydrobromide was washed twice by decantation of ether. It was twice dissolved in methanol (5 rnl) and re-precipitated by the addition of ether. It was filtered and dissolved in dimethylformarnide ( 5 ml), containing triethylamine (8.7 ml), and this solution was cooled to -20 "C. To it was added the cooled solutic>n of I%rcarbobenzoxy-S-henzylcysteiny1-L-3 ,4dihydroxyphenylalanaine azide previously prepared. The mixture was kept at 2 "C for I6 h and was then stirred at room temperature for 3 h. Ethyl acetate (50 ml) was added and the precipitated solid was filtered, washed exhaustively on the filter with ethyl acetate and air dried. This gave 160 mg of off-white - 49.6" (c 1.0 in solid. rnp 280-215 "6: (decamp.), [a]&" dimethylformamide). The presence sf a catechol group in this material was detected by an Arnow test. [2-L-DOPA]-ox? tocin ClV)
The presumed precursor (111) obtained by coupling N-carbobenzoxy 6 -benzylcysteinyl-L -3,4-dihydrctxyphenyiialanine azide and isoleucylglutaminylasparagiwyI-bybenzylcysteinylpro1y11eucylg1ycinamide 4120 nag) was added to boiling ammonia (120 ml) which had been freshly FIG. 1. Structure of oxytocin and sf [%-L-DOPA]- redistilled from sodium. A sodium stick was held below the oxytocin. Numbers indicate positions of the constituent surface C P the ~ solbltion until a blue coisur pervaded the amino acid residues. entire solution and persisted for 1 min. The ammonia was removed by Hyophilization and the residue dissc~lvedin water (120 ml) to give a solaltion of pH 8. Hydrogen peroxide (0.05 ml, 30%) was added, and the solution allowed to stand at room temperature for I min. The pH was lowered to 5 by the addition of glacial acetic acid and the volume of the solution was lowered to 2 ml by evaporation under reduced pressure at room temperature. The colour of this solution was faintly yellow. This solution was then satuE-Cy s(Bz?)-L-DOPA-HIeGln-Asn-Cy s(Bzl1-P~Q-Leu-Cly- WHz rated with upper phase of the solvent system 1-butanol m pyridine - acetic acid - water, 1000:B5:35:9564. A Sephadex I ) Na, iiq. HHg @-25 colhamn (75 x 1.5 cm) was equilibrated with lower phase of the soHveni system in preparation for partition 21 chromatography (5). The sample was put on the column and eluted in 1.1-ml fractions with upper phase of the (~-L-DBBA)-L%XYT~)CIN solvent system. The eluate was colourless and the yellow D! colour of the applied solution remained on the column. FIG.2. Synthesis of 12-E-DOPA]-oxytocin. Folin-Lowry determination (6) of the peptide content of the eluted fractions showed that there were three regions in Coadpling ~I&T-cc~rboben~~~q-S-he~zy~c'~~.~tei~ty~-~-S,~-dI'which peptide was eluted (Fig. 3). The fractions correhgrdrovphenyla1a~1ine azide and isoleucylglutarnsponding to these regions were pooied, concentrated, after iny laspavcrginyl-S-benzy~cysteiny/pro8ylle~edc$the addition of water, until the organic phase was removed, glycinamide and iyophilized. The products from the two fastest moving M-Carbobenzoxy-S -2senzylcysteinyl-L -3,4-dihydroxy peaks (A and B) were yelowish, somewhat sticky and very phenylalanine hydrazide (I, 150 mg) was dissolved in a small in quantity. The material from peak C was white and solution of hydrogen chloride in 95% aqueous tetrahyfluEy 630 mg). Some of this material (20 mg) was dissolved in glacial acetic acid (0.03 ml) and diluted to 2.5 rnl with drofuran (4.6 N , 1.3 ml). The solution was cooled to water. This solution was put onto the Sephadex 6-25 col-20 "C and stirred vigorously while an aqueous solution
4
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PERRIER AND BRANBA
FRACTION NO.
FIG.4. E X C ~ U S chromatography ~OH on Sephadex G-25 in FRACTBON NO. 0.2 N acetic acid of [%-L-DOPA]-oxytocinfrom peak C , FIG. 3. Partition chromatography of crude [%-I,- Fig. 3: fraction volume, 3.0 ml. Fractions were pooled as indicated by the broken lines. DOPA]-oxytocin on Sephadex G-25 in the solvent system I-butan01 - pyridine - acetic acid - water 1000: 15: as 1: aspartic acid 1 .Q, gllatarnic acid 1 .a, proline 0.9, hemi35:958; fraction volume, 1 . 1 ml;eIution pattern shown by cystine 1.8, leucine 1.0, isoleucine 1.0 and ammonia 2.8. Folin-Lowry determination of pepaide content in eluate. A standard sf L-DOPA was treated under the hydrolysis Fractions were pooled as indicated by the broken lines. conditions and was shown by comparison with an untreated standard to have been partially destroyed. The presence urnn from which the organic phase had keen discharged of 1 moi of 1.-DOPA. in the peptide was confirmed by a with a mixture of pyridine and 0.2 N acetic acid 4 1 :4) and quantitative Arnow test, ucjing a mixture of t-DOPA which was then equilibrated with 0.2 N acetic acid. This and equimslar amounts of the other constituent amino came solvent was used to elute 3.8-ml fractions. Measureacids as the standard. ment s f the absorbance of these fractions at 280 wm showed The milk-ejection-like activity c~f[2-~-D&PPA1-oxytc1cin a region of peptide material eluted as a symmetrical peak was determined by measurement of the contraction of an with a shoulder on its leading edge (Fig. 4). The elution isolated strip of mammary gland from a lactating mouse (4). volume sf the peak was 105 ml. Oxytscin is eluted at 112 ml The uterotonic activity was measured on an isolated under the same conditions. The fractions corresponding to uterine horn from a virgin Wistar rat (81, using MLIHSIC~ the major peak (Fig. 4) were concentrated under reduced solution (9). pressure and lyophiliaed to give 12 mg white, fluffy amorphous material, which gave a positive Arnow test for the presence of a catechol group: [a]%" 322.' (c 0.5 in 1N Results m d Discussion acetic acid). After being dried in vnclts for 24 h over phos[2-L-DOPA]-oxytocin(IV), a hormone analogue phorous pentoxide at room temperature it was found to contain: C, 50.25; H, 4.48; N, 16.80%. (Calc. for with a residue of L-DOPA in place of the residue of G43H66N12013SZ: C , 50.47: I%, 6.50; N 16.43%.) A scan of tyrosine in oxytocin, was synthesized (Fig. 2) by the BSV absorbance of this material in 0.2 N acetic acid short-term oxidation of the dithiol-containing preshowed two maxima: A ,,,,,, (e 2.83 x 103),Amak2,, ( E 4.68 cursor. The precursor was produced by treatment x B03). Approximately 4% s f the absorption of by sodium in liquid ammonia of N-carbsbenzoxy-S12-e-DOPA]-oxytocin at 282 nm can be ascribed to its benzylcysteinyl - L- 3,4 - dihydroxyphenyla1s~ny1isocystine content. The spectrum ofr.-DOPA iaa 0.2 N acetic ( E 2.56 x 109),A , ,,,,, (e leucylglutaminylasparaginy1-S- b e m z y 1 c y s ~ acid showed two maxima: A 2-64x lo3). Neither [2-e-DOPA)-oxytocin nor L-DOPA lylleucylglycinamidg (111) which had been syntheshowed any absorption at wave lengths greater than 300 nrn sized by coupling N-carbobenzowy-S-benZylcy(Fig. 5). TEC of [2-L-DOPA]-oxytocimon silica showed it steinyl-L-3,4dihydroxygRenylr?1anineazide with to be homogeneous in the solvent systems I-butanol - isoIeucyIglutaminylasparagimyI- S - benzylcysteinylacetic acid - water (4:I:5) (Rf 0.89), and 1-butanol - ben- proBylleucylglycinamide. The hormone analogue zene - pyridine - acetic acid - water (30:10: 1:2:37) (Rf was isolated by sequential partition chromatography 0.17). For amino acid analysis, a sample was hydrolyzed at and exclusion chromatography on Sephadex G-25. The difficulty in preparing peptides containing 110 "C for 22 h in 6 N HC1. The amino acids and ammonia were found in the following ratios, with glycine being taken DOPA lies in the ready oxidation s f DOPA to the
,,,,,,
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CAN. J. BIQCHEM. VOL. 54, 1976
WAVELENGTH (nmB FIG. 5 . Ultraviolet absorption spectrum of [2-LDOPAl-oxytocin (-) and 0 % ' ~ - D O P(-----), A I x M in 0.2 N acetic acid.
quinone. Felix cb a / . (lo), who prepared several L-DOPA-containing dipeptides and tripeptides, found it desirable to protect the catecho1 hydroxyl groups to prevent oxidation. Bn the presently described synthetic scheme, there necessarily is a ring-clcdsing oxidation step after the cleavage of the protecting groups and in light of this we decided that the protection might not be a significant advantage. The synthesis was designed so that the residue of L-DOPA was exposed to as few synthetic steps as possible, that the pH whenever possible was kept below 7 and that the oxidation step was carried out with the shortest convenient exposure to the oxidizing agent. A yellow ccplour did develop during the oxidative ring closure step in the synthesis, indicating that some oxidation had occurred. This colour was retained on the Sephadex cc~lurnnduring partition chromatography and the final product was white. The UV absorption spectrum which showed no absorption at wavelengths greater than 300 nm (Fig. 51, indicated that the material did not contain any oxidized products. The molar extinction coefficient for [ZL-DOPA]-oxytocinat 282 nm was shown to be 2.8%x lo3, of which approximately 4% can be ascribed to the cystine residue. The extinction coefficient corrected for cystine is thus similar
to that of L-DOPA at its corresponding maximum absorption (2.55 x 1Q3 at 280 nm). Oxidation of L-DOPA gives materials which absorb more intensely throughout the UV spectrum (1 I), The potencies of the two biological activities assayed for [2-L-DOPA]-oxytocinwere found to be 54 5 9 U/mg for milk-ejection-like activity and 26 9 4 U/mg for uterotonic activity. Oxytocin when assayed in this laboratory for milk-ejection-like activity against U.S.P. Posterior Pituitary Reference Standard (12) was found to have a potency of 459 ? 23 U/mg. Its uterotonic potency is 546 L- 18 U/mg (13). The replacement of the tyrosine residue in oxytocin by a residue of L-DOPA has, therefore, resulted in a decrease in potency of these biological activities and a change in the ratio of their potencies. The biological assay of 12-L-DOPA]-oxytoci was complicated by the fact that prior dilution with assay medium resulted in rapid Boss of activity. The solution prepared as the stock solution for assay sonsisted of [2-L-DOPA]-oxytocinin 0.2 N acetic acid. For assay, this stc~cksolution must be diluted with Tyrode solution or Munsick solution, both of which have been pretreated with a mixture of 0, and CO, (95:s). The high oxygen concentration in these solutions and the elevation of the pH of the peptide containing solution from acidic to somewhat above 7 presumably combined to cause rapid oxidation of the DOPA residue. The assays were, therefore, done by diluting the stock solution immediately before injecting it into the assay bath. Although the results from different biological assays were reproducible within the experimental error of the assay, it is possible that even with this minimum exposure to oxidizing conditions, there is some Boss of biological activity and we therefore consider that the figures which we quote for [ZL-DOPA)-oxytocinare minimum potencies. ~ e v e r ahormone l analogues have heon prepared in which the tyrosine residue of oxytocin has been replaced by another amino acid residue and have given some information which may lead to an understanding of the structural requirements at position 2 for the expression of biological activity. Low biological activity of several analogues of oxytocin with aliphatic amino acids in place of tyrosiwe had suggested that an aromatic nucleus was necessary at position 2 for the expression of a high degree of activity. However, the observation that 12-isoleucine]-oxytocin possesses moderate biological activity put that assumption in question (18). [2-Phenylalaninel-oxytocin,with phenylalanine in place of tyrosine had reduced but still very considerable potency in those activities characteristic of oxytocin 14-17). This suggested that the phenolic hydroxyl group in tyrosine was facilitating rather than necessary for activity. The synthesis of [2-I.-DOPA]-oxytocin allows for examination of the
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FEWRIER AND BRANBA
TABLE 1. The effect of some changes in ~ubetitutionof the tyrosine ring on the uterotonic activity of oxytacin -
Compound
Amino acid at position 2
[2-PheragrHalabwine]-oxflmin Oxfloein [2-L-DOPA]-oxytmin [2-(3-Poddyrmi~1e)]-oxytwin
PhenyBalanine 4-HydroxyphenylaIanine 3,L$DihydrcsxyphenyIalanine 3-Podo-4-hydroxyphenyIaIanine
-
--
[2(3-Methyltyrosine)]-oxytwin
-.
Uterotonic activity (isolated rat uterus) -
-
-
-
38" 546 f lgB 26 4 Nonec (inhibitor of oxytocin) 3-MethyI-4-hydrsxyphenyla1anine Nonee (inhibitor of oxfloein)
°[Ref. IS), &(Refe13, C(Ref.19).
biological effects of introducing a second hydrcsxyl group into phenylalanine. It appears that the rernoaral of the hydsoxyl group t o give E2-phenylalanine]-oxytocira and the introduction of a second hydroxyl group to give [2-L-DOPA]-oxytocin have similar effects on the activities caf oxytocin (Table I). Marbach and Rudinger (19) have reported the synthesis s f [2-(3-iodotyrosine)mxytscin and [2(3-methyltyrassine)]-oxytocinn These analogues inhibit the uterotsnic activity of oxytocin and d c ~not possess any uterotonic activity themselves. Comparison of the biological activities of the three disubstituted [2-phenylalaninel-oxytscin analogues (Table 1) suggests that a11 three peptides bind to the oxytocin receptors on the rat uterus. The differences in activity must, therefore, occur subsequent to binding. Marbach and Rudinger conclude that the similarity in the result s f introducing an iodo car a methyl sbabstituent (~rrho to the hydroxyl group must be due t o steric effects and not tia alterations in the pK, of the hydroxyl group. The two different substituents would alter the pK, in opposite directions. A hydroxyl group is isosteric with a methyl g o u p and it must be concluded that the 3-hydroxyl substitraent in [2-L-DOPA]-oxytocin has an effect which is not simply steris. It is possible that the 3-hydroxyl group reacts with some other site in such a way that the molecule is oriented to be in an oxytocin-like presentation.
We are grateful to Mrs. C. Cardy and Mr. D. Bargavel for performing the biological assays.
1. 09Neill, 9. J., Veitch, F. P. & Wagner-Jauregg, T. (1956) J . Org. Chem. 21,363-364 2. Haringtom, G. R. & Mead, T. H. (1936) Bic~chem.J. SO, 1598-161 1 3. Arnow. &. E. (1937) 3. B i d . Chem. 118,531-537 4. Bodanszky, M. & du Vigneaod. V . ( 1 9 9 ) J . Am. Chem. Soc. 81,5688-5691 5. Yamaskiro, D. (1964) Nature (London) 201,76-77 6. Lowry, 0. W., Rosebrough, N. J., h r r , A. L. & Randall, R.J. (1951) J. Biol. Cirern. 193,265-275 7. Fielitz, G., Rosa, R., Mattei, A., Melander, S., Garofalo, E., Gioia de Goch, M N . & Coch, J . A. (1970)Proc. SOL..Exp. Blol. Med. 133, 1155-1 157 8. Holton, P. A. (19448) Br. S. Pharmacof. 3,328-336 9 . Munsick. R. A. (1966) Endoc.rino&ogy66,451457 10. Felix, A. M . , Winter, D. P., Wang, S.-S., Kuleska. I. B . , Pod. W. R . , Hang, D. L. & Skeppard, W. (1974)J. Med. Chern. 17,422426 1 1 . Yasunobu. #. T., Peterson, E. W. & Mason, H. S. Q 1959)J. Biol. C h e a . 234,3241-3295 12. The Phaurnacopeia of'the United States: of America (1970) 18th revision, p. 469, Mack Publishing Go., Easton, Pa. 13. Ghan, W. Y., O'Gonnell, M. & Pomeroy, S. R. (1963) Endocrinology 72,279-282 14. JoSt, K., Wudinger, 3. & Sorrn, F. (1963) Cofiect. Czech. C h e a . Cornmun. 28, 1706-17 14 15. Bodanszky, M. & du Vigneaeed, V. (1959) J. A m . Chem. Soc. 81,6072-6075 16. Jaquenoud, P. A. & Boissonnas, R . A. (1959) Helv. Chirn. act^ 42,788-793 17. Konzete, H. & Berde, B. (1959)Br. J . Pharmacol. 14, 133-136 18. Bsamda, k. A. & du Vigneaud, V. (1967) Moi. Bharrnucol. 3,248-253 19. Marback, P. & Rudinger, J. (1974) Expeaicntia 30.696
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