Proc. Natl. Acad. Sci. USAVol. 76, No. 7, pp. 3309-3313, July 1979Biophysics

Biofunctional evaluation of a hydrogen bond linking the ring and tail(-turns of oxytocin

(oxytocin analogs/biologically active conformation)

J. Roy, UMA Roy, DIANA C. GAZIS, AND I. L. SCHWARTZDepartment of Physiology and Biophysics, Mount Sinai Medical and Graduate Schools of the City University of New York, New York, New York 10029

Communicated by Vincent P. Dole, March 23, 1979

ABSTRACT Deamino{8-N-methylleucinejoxytocin anddeamino{-a-hydroxyisocaproic acidjoxytocin were synthesizedto study the importance of hydrogen bonding between the car-boxamide carbonyl of asparagine and the peptide N-H of leu-cine in stabilizing the biologically active conformation of oxy-tocin. The analogs were synthesized by coupling deaminotoci-noic acid with Pro-Leu(Me)-Gly-NH2 and Pro-HyIc-Gly-NH2,respectively. (HyIc is a-hydroxyisocaproic acid.) Deamino-8-N-methylleucine]oxytocin was found to possess 48 ± 7 units ofuterotonic activity, 33 ±5 units of avian vasodepressor activity,and 3.15 ± 1.5 units of antidiuretic activity per mg; deamino-[8-a-hydroxyisocaproic acidjoxytocin possessed 134 ± 12 unitsof uterotonic activity, 31 ± 3 units of avian vasodepressor ac-tivity, 9.6 ± 3.0 units of antidiuretic activity, and 0.26 ± 0.02 unitof pressor activity per mg. Neither of the analogs possesses thepeptide N-H at residue 8 required for the formation of a hy-drogen bond with the asparagine carboxamide; however, bothcan assume the conformation needed to evoke the characteristicbiological activities of oxytocin although in lower potency. Itis concluded that such a hydrogen bond does not constitute aconformational constraint that is essential for hormone ac-tion.

Prior to the recent study on [5-aspartic acid]oxytocin (1), allsynthetic analogs of oxytocin, modified at position 5-e.g.,[Ala5]oxytocin (2, 3), [Val5]oxytocin (4), [Ser5]oxytocin (2),[Orn5]oxytocin (5), [Gln5]oxytocin (6), and [Dbu5]oxytocin(7)-have had extremely low or negligible potency with respectto all of the biological activities characteristic of the parenthormone. The asparagine residue has been assigned an im-portant, if not unique, role in the maintenance of the confor-mation of oxytocin in solution (8, 9); namely, it has been pro-posed that a hydrogen bond between the peptide N-H of as-paragine and the C=O of tyrosine stabilizes a 1-turn (com-prising the sequence -Tyr-Ile-Gln-Asn-) in the cyclic part ofoxytocin and the C='O of the asparagine carboxamide may behydrogen bonded to the peptide N-H of leucine, thus stabilizingthe relationship of this 1-turn to a second COOH-terminal1-turn in the molecule composed of the sequence -Cys-Pro-Leu-Gly-NH2. The "cooperative model"-i.e., the "biologicallyactive" conformation of oxytocin (9)-introduced to rationalizethe changes in biological activities accompanying structuralmodification of neurohypophyseal peptides, suggests that anyreplacement of asparagine at position 5 that fails to contributeto the stabilization of the ring (-Tyr-Ile-Gln-Asn-) 1-turn or tothe stabilization of the relationship of the ring and tail 1-turnsto each other would increase the conformational ambiguity ofthe resultant analog with a consequent deterioration of all bi-ological activities. As far as the hydrogen bond in the cyclicmoiety of oxytocin is concerned, there should not be, in allprobability, an absolute requirement for an asparagine residuein position 5-i.e., at least some of the other amino acid residues

ought to be able to replace it. Accordingly, if the conformationalconsiderations that have been adduced do indeed account forthe importance of the asparagine residue for the bioactivity ofoxytocin, the unique character of this residue would dependon the hydrogen bonding of its carboxamide side chain to thepeptide N-H of leucine (the presumption being that this latterhydrogen bond stabilizes the relationship of the two 3-turns inthe oxytocin molecule to provide the requisite structure forbioactivity). Accordingly, inability to form this hydrogen bondhas been offered as the predominant reason to explain thenegligible biological activities of [Dbu5]oxytocin (7).

In order to further understand the overall conformation ofoxytocin and, in particular, to gain more insight into thestructural basis for the stringent requirement for an asparagineresidue in position 5, we have synthesized two analogs ofdeamino-oxytocin-namely, deamino-[8-N-methylleucine]-oxytocin and deamino-[8-a-hydroxyisocaproic acid]oxytocin.In the first compound, the hydrogen of the peptide N-H ofleucine is replaced by a methyl group, and the analog is thusincapable of forming any hydrogen bond involving theC=Oof the asparagine carboxamide. In the second analog, there isalso no possibility for the formation of the hydrogen bond underdiscussion because the residues in positions 7 and 8 are nowlinked through an "ester" rather than an "amide," the prolineresidue in position 7 supplying the carbonyl portion of the ester.Both of the analogs were synthesized by condensation ofdeaminotocinoic acid (10), containing the preformed disulfidebridge of deamino-oxytocin, with the appropriate tail fractionof the respective analogs. The starting point in the synthesis ofthe tail fraction, Pro-Leu(Me)-Gly-NH2 of deamino-[8-N-methylleucineloxytocin was the preparation of N-methylleu-cine or one of its suitable derivatives. Benzyloxycarbonylleucinewas N-methylated with sodium hydride and methyl iodide intetrahydrofuran at room temperature by the method ofMcDermott and Benoiton (11). This method gives virtuallycomplete N-methylation and virtually no racemization. Ben-zyloxycarbonyl-N-methylleucine is a crystalline solid whereasbenzyloxycarbonylleucine is an oil; it was therefore very easyto obtain pure benzyloxycarbonyl-N-methylleucine by crys-tallization. In addition, benzyloxycarbonyl-N-methylleucinewas subjected to hydrogenolysis in aqueous acetic acid over 10%Pd/C catalyst (11) and the purity of N-methylleucine, thusobtained, was checked by chromatography on an amino acidanalyzer; even on overloading, no peak for leucine could bedetected and the N-methylleucine had, if any, less than 1 partof leucine in 1000 parts of N-methylleucine. The synthesis ofthe tripeptide turned out to be more difficult than expected

Abbreviations follow the recommendations of IUPAC-IUB Commis-sion on Biochemical Nomenclature (1972) J. Biol. Chem. 247,977-983;HyIc, a-hydroxyisocaproic acid. In addition, the following abbrevia-tions were used: Boc, tert-butyloxycarbonyl; DCCI, dicyclohexyl-carbodiimide. HyIc and all optically active amino acids have the Lconfiguration.

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because of known problems associated with the use of N-methylamino acids-namely, lack of crystallinity of interme-diates, the lesser reactivity of the methylamino group, and sterichindrance; moreover, particular methods of deprotection andcoupling were required to avoid racemization (12, 13). Boc-Proand Leu(Me)-OBzl were coupled by the mixed anhydrideprocedure (Boc is tert-butyloxycarbonyl); the product wasobtained as a crystalline solid, though in low yield. The productobtained was subjected to hydrogenolysis over Pd/C catalystto yield Boc-Pro-Leu(Me)-OH also as a crystalline solid. Thelatter was then condensed with Gly-NH2 by using dicyclo-hexylcarbodiimide (DCCI)/N-hydroxysuccinimide; thismethod yields tripeptide without any racemization (13). Boc-Pro-Leu(Me)-Gly-NH2 was obtained as a tacky mass andthin-layer chromatography showed the presence of two minorimpurities even after purification by column chromatography.Without any further attempt at purification, the compound wasdeprotected with trifluoroacetic acid and Pro-Leu(Me)-Gly-NH2 was coupled with deaminotocinoic acid by use ofDCCI/1-hydroxybenzotriazole (10). The resulting deamino-[8-N-methylleucine]oxytocin was purified by partition chro-matography (14) followed by gel filtration (15); the analog wasfound to be chromatographically pure and homogeneous onthin-layer chromatography.

For deamino-[8-a-hydroxyisocaproic acid]oxytocin, the tailfraction Pro-HyIc-Gly-NH2 was synthesized through the stepsdetailed below (HyIc is a-hydroxyisocaproic acid). For thecompound, Boc-Pro-HyIc-OBzl, the ester bond was formed byreaction with benzenesulfonyl chloride in pyridine by theprocedure of Shemyakin et al. (16). The product was isolatedand purified by column chromatography on silica gel; the re-sulting oil was subjected to hydrogenolysis over Pd/C catalyst.The free acid thus obtained was condensed with Gly-NH2 bythe mixed anhydride procedure. The crystalline Boc-Pro-HyIc-Gly-NH2 was deprotected with trifluoroacetic acid andthe product was coupled with deaminotocinoic acid by use ofDCCI/1-hydroxybenzotriazole. Deamino-[8-a-hydroxyiso-caproic acidloxytocin, thus obtained, was purified in the usualmanner by partition chromatography followed by gel filtration;the analog was found to be chromatographically pure and ho-mogeneous on thin-layer chromatography.

EXPERIMENTALAll melting points (uncorrected) were determined with aThomas-Hoover capillary melting point apparatus. Opticalrotations were measured with a Carl Zeiss precision polarimeter(0.001°). Elemental analyses were performed by GalbraithLaboratories (Knoxville, TN). The analogs were hydrolyzed,in the usual way, with 6 M HCI for 22 hr and the hydrolysatewas analyzed (17) on a Beckman 121C amino acid analyzer.With the hydrolysate of deamino-[8-N-methylleucine]oxytocin,the analyzer was operated with the eluting buffer at half-nor-mal flow rate for determination of N-methylleucine (18).Thin-layer chromatography was performed on Quantum silicagel G plates with the following solvent systems, and the productswere detected with the chlorine/tolidine reagent (19): (A)benzene/MeOH, 19:1; (B) CHCl3/MeOH, 6:1; (C) benzene/MeOH/HOAc, 17:3:1; (D) CHC1s/MeOH, 9:1; (E) 1-BuOH/H20/HOAc, 100:35:15; and (F) upper phase of 1-BuOH/EtOH/H20 containing 3.5% HOAc and 1.5% pyridine,7:2:9.

Uterotonic activity was measured on isolated horns fromvirgin rats in natural estrus by the method of Holton (20), asmodified by Munsick (21), with Mg2+-free van Dyke-Hastingssolution. Avian vasodepressor assays were performed on con-scious chickens by the method of Coon (22), as modified by

Munsick et al. (23). The antidiuretic activity was determinedon anesthetized male rats by the method of Jeffers et al. (24),as modified by Sawyer (25). Pressor assays were performed onanesthetized male rats according to the method in the U.S.Pharmacopeia (26). The four-point assay design (27) was usedwith the U.S. Pharmacopeia posterior pituitary referencestandard.

Boc-Pro-Leu(Me)OBzl. A solution of Boc-Pro (2.96 g, 13.7mmol) and N-methylmorpholine (1.51 ml, 13.7 mmol) inEtOAc (20 ml) was cooled to -15oC and was treated, withstirring, with isobutylchloroformate (1.78 ml, 13.7 mmol); vo-luminous solid separated. After 5 min, the reaction mixture wastreated with Leu(Me)-OBzl in dimethylformamide (20 ml) [thefree base was liberated by treatment of Leu(Me)-OBzl-TosOH(13) (5.6 g, 13.7 mmol) with Et3N (1.91 ml, 13.7 mmol) in di-methylformamide]. The stirring was continued at -15'C foran additional 15 min and then at room temperature overnight.The reaction mixture was concentrated under reduced pressureand the residue was partitioned between EtOAc and water. TheEtOAc layer was washed three times with 5% NaHCO3 solution(30 ml), once with water (25 ml), three times with 1 M HCl (30ml), and then three times with water (30 ml). The organic layerwas dried (Na2SO4) and was concentrated to a thin oil thatdeposited needle-shaped crystals from n-hexane/petroleumether (1:5, 60 ml). The dried material weighed 0.8 g (13.5%):melting point = 85-87°C; homogeneous by thin-layer chro-matography (solvent A); [a]24 = -95.3 (1 g in 100 ml ofMeOH). Analysis: calculated for C24H36N205: C, 66.64; H, 8.39;N, 6.48. Found: C, 66.57; H, 8.46; N, 6.37.

Boc-Pro-Leu(Me). Boc-Pro-Leu(Me)-OBzl (0.78 g, 1.8 mmol)was subjected to hydrogenolysis over 10% Pd/C (100 mg) in90% EtOH (100 ml) for 5 hr; the catalyst was separated by fil-tration and the filtrate was concentrated under reduced pres-sure; a solid was obtained. This was crystallized from EtOAc/petroleum ether (1:1, 40 ml): weight = 0.45 g (72.6%); meltingpoint = 168.5-169.5°C; homogeneous by thin-layer chroma-tography (solvents B and C); [a]24 = -51.3° (1 g in 100 ml ofdimethylformamide). Analysis: calculated for C17H30N205: C,59.63; H, 8.83; N, 8.18. Found: C, 59.73; H, 8.90; N, 8.17.

Deamino-8-N-methylleucineloxytocin. Boc-Pro-Leu(Me)(376 mg, 1.1 mmol), Gly-NH2 [obtained by hydrogenolysis ofZ-Gly-NH2 (Z is benzyloxycarbonyl) over 10% Pd/C catalystin 95% EtOH] (100 mg, 1.32 mmol), and N-hydroxysuccini-mide (253 mg, 2.2 mmol) were dissolved in dimethylformamide(25 ml). The solution was cooled to -22°C and was treated withDCCI (317 mg, 1.54 mmol) dissolved in a minimum of di-methylformamide; the reaction mixture was stirred at -220Cfor 1 hr and then at room temperature for 22 hr. The mixturewas concentrated under reduced pressure and the residue wasextracted with EtOAc; the extract was washed twice with water(25 ml), three times with 5% NaHCO3 solution (25 ml), threetimes with 1 M HCl (25 ml), and finally three times with water(25 ml). This was dried (Na2SO4) and concentrated under re-duced pressure; a gummy product was obtained. The productwas chromatographed on silica gel and was eluted with 1%MeOH in CHCI3. The solvents were removed and a tacky masswas obtained: weight = 0.30 g; thin-layer chromatography(solvents C and D) showed two minor impurities. This prepa-ration of Boc-Pro-Leu(Me)-Gly-NH2 gave satisfactory resultsin the synthesis of deamino-[8-N-methylleucine]oxytocin asdetailed below.

Boc-Pro-Leu(Me)-Gly-NH2 (120 mg, 0.3 mmol) was treatedwith trifluoroacetic acid (2 ml) in the cold and the solution wasleft at room temperature for 30 mmin Trifluoroacetic acid wasevaporated under reduced pressure and the residue was dilutedtwice with ether and then twice more with benzene; each time

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the solvent was evaporated under reduced pressure, and the,residue was finally dried over P205 under reduced pressure.The deprotected tripeptide salt was dissolved in dimethyl-formamide (2 ml) and the solution, at 00C, was neutralized withN-methylmorpholine (0.035 ml, 0.3 mmol). After addition ofdeaminotocinoic acid (145 mg, 0.2 mmol) and 1-hydroxyben-zotriazole (54 mg, 0.4 mmol), the solution was treated with solidDCCI (52 mg, 0.25 mmol). The reaction mixture was stirredat 00C for 2 hr and then at room temperature for 24 hr. Thesolid that separated was removed by filtration and was washedwith dimethylformamide; the combined filtrate was concen-trated under reduced pressure to a thick oil. The oil was dis-solved in a mixture of lower phase (1 ml) and upper phase (2ml) of the solvent system I-BuOH/C6H6/3.5% HOAc con-taining 1.5% pyridine (1:1:2); the solution was subjected topartition chromatography on a column (3.1 X 69.5 cm) ofSephadex G-25 (fine). The column was eluted with the upperphase of the solvent system at 30 ml/hr and the peptide materialwas detected by the Folin-Lowry method (28); a major peakemerged at RF 0.42, mostly separated from several minor peaks.The fractions corresponding to this peak were pooled and di-luted with twice the volume of water; the solution was con-centrated under reduced pressure and then lyophilized. Thematerial, 105 mg, was purified by gel filtration in 0.2 M HOAcon a column (2.8 X 113.5 cm) of Sephadex G-25 (superfine) ata flow rate of 25 ml/hr. The compound emerged as a sharpsymmetrical peak with maximum at 78.2% of the column bedvolume: weight = 83 mg (41.3% based on deaminotocinoicacid); homogeneous by thin-layer chromatography (solventsF and F); [a]f = -95.3 (0.5 g in 100 ml of 1 M HOAc).Analysis: calculated for C44H67NI1O12S2: C, 52.52; H, 6.71; N,15.31. Found: C, 52.18; H, 6.83; N, 15.04. Amino acid analysisgave the following molar ratios: Asp, 1.03; Glu, 1.00; Pro, 0.99;Gly, 1.00; Ile, 0.99; Tyr, 0.96; and NH3, 3.06. A sample hy-drolyzed after performic acid oxidation by the method ofMoore (29) gave the ratios: cysteic acid, 1.17; Asp, 1.00;Leu(Me), 0.93.

Boc-Pro-HyIc-OBzl. Benzenesulfonyl chloride (3.1 g, 17.6mmol) was added with stirring, over a period of 10 min, to asolution of Boc-Pro (3.4 g, 15.8 mmol) in pyridine (17 ml) andtetrahydrofuran (17 ml) cooled to -10°C. After 15 min,HyIc-OBzl (30) (3.5 g, 15.8 mmol), in a solution of pyridine (6ml), was added to the solution above; the reaction mixture wasstirred at 0°C for 1 hr and then at room temperature overnight.The reaction mixture was poured into ice water (300 ml) andthe oil that separated was extracted with EtOAc; the extract waswashed three times with 2% HCl solution (25 ml), three timeswith 5% NaHCO3 solution (25 ml), and three times with water(25 ml). This was dried (Na2SO4) and, on evaporation of thesolvent, a thick liquid was obtained. The product was purifiedby column chromatography on silica gel and was eluted with1% MeOH in CHC13. On evaporation of the solvents, a thickliquid was obtained: weight = 4.7 g (70.8%); homogeneous bythin-layer chromatography (solvent A); [a]24 = -80.2° (2.0 gin 100 ml of MeOH). Analysis: calculated for C23H3N06: C,65.85; H, 7.93; N, 3.34. Found: C, 65.90; H, 8.03; N, 3.47.

Boc-Pro-HyIc-Gly-NH2. Boc-Pro-HyIc-OBzl (3.5 g, 8.3mmol) was hydrogenolyzed in MeOH (100 ml) in the presenceof 10% Pd/C (300 mg); after 3 hr the catalyst was separated byfiltration and the solvent was removed under reduced pressure.The residual oil was dissolved in CHC13 (20 ml) together withN-methylmorpholine (0.84 g, 8.3 mmol); the solution wascooled to -100C and then was treated with ethyl chloroformate(0.90 g, 8.3 mmol). After 15 min a solution of glycinamide hy-drobromide (1.3 g, 8.3 mmol) in dimethylformamide (20 ml),containing N-methylmorpholine (0.84 g, 8.3 mmol) and cooled

to -10°C, was added to the reaction mixture above. Stirringwas continued in the cold bath for 10 min and then at roomtemperature overnight. The solvent was evaporated under re-duced pressure and the residue was partitioned between EtOAcand water. The organic layer was washed three times with 1 MHCI (25 ml), three times with 5% NaHCO3 solution (25 ml), andthree times with water (25 ml). The organic layer was dried(Na2SO4) and the solvent was evaporated under reducedpressure. The oil thus obtained crystallized from EtOAc/n-hexane (1:2, 30 ml): weight = 2.0 g (62.5%); melting point =102-104'C; homogeneous by thin-layer chromatography(solvent D); [a]24 =-78.7° (1.0 g in 100 ml of MeOH). Analysis:calculated for C18H31N306: C, 56.09; H, 8.11; N, 10.90. Found:C, 56.04; H, 8.02; N, 10.72.

Deamino-[8-a-hydroxyisocaproic acid]oxytocin. Boc-Pro-HyIc-Gly-NH2 (35 mg, 0.09 mmol) was treated with tri-fluoroacetic acid (0.5 ml) in the cold and the solution was leftat room temperature for 30 min. Trifluoroacetic acid wasevaporated under reduced pressure and the residue was dilutedwith benzene. The solvent was evaporated under reducedpressure; the process was repeated twice more and the residuewas then dried under reduced pressure over P205. The de-protected tripeptide salt was dissolved in dimethylformamide(0.5 ml) and the cold solution was neutralized with N-methyl-morpholine (0.01 ml, 0.09 mmol). After addition of deamino-tocinoic acid (43.5 mg, 0.06 mmol) and 1-hydroxybenzotriazole(16.2 mg, 0.12 mmol), the solution was cooled to0C and wastreated with DCCI (14.8 mg, 0.072 mmol) in dimethylform-amide (0.2 ml). The reaction mixture was stirred at that tem-perature for 2 hr and then at room temperature for 24 hr. Thesolid was separated by filtration and was washed with di-methylformamide; the combined filtrate was concentratedunder reduced pressure to an oil. This was dissolved in a mixtureof lower phase (0.5 ml) and upper phase (1.5 ml) of the solventsystem 1-BuOH/CrH6/3.5% HOAc containing 1.5% pyridine(1:1:2); the solution was subjected to partition chromatographyon a column (3.1 X 68cm) of Sephadex G-25 (fine). The columnwas eluted with the upper phase of the solvent system at a flowrate of 31 ml/hr; the product emerged as a major peak at RF0.52. The product, 58 mg, obtained after lyophilization as be-fore, was further purified by gel filtration in 0.2 M HOAc ona column (2.8 X 115.0 cm) of Sephadex G-25 (superfine) at aflow rate of 23 ml/hr. The compound was eluted as a sharpsymmetrical peak with maximum at 81.9% of the column bedvolume: weight = 36mg (60% based on deaminotocinoic acid);homogeneous by thin-layer chromatography (solvents E andF); [a]24 = -75.20 (0.5 g in 100 ml of 1 M HOAc). Analysis:calculated for C4sH64N1oO13S2: C, 52.00; H, 6.50; N, 14.10.Found: C, 51.81; H, 6.56; N, 13.96. Amino acid analysis gavethe following molar ratios: Asp, 1.00; Glu, 1.01; Pro, 0.88; Gly,1.00; 1/2 Cys, 0.45; mixed disulfide of cysteine and 3-mercap-topropionic acid, 0.35; lie, 0.99; Tyr, 0.89; and NH3, 2.70.

RESULTS AND DISCUSSIONThe analogs were tested for some of the biological activitiescharacteristic of oxytocin, and the results are summarized inTable 1. Hruby et al. (33) observed that the ring moiety ofoxytocin or deamino-oxytocin possesses low uterotonic activitybut no detectable avian vasodepressor activity. The restorationof the characteristic biological activities of oxytocin anddeamino-oxytocin on addition of the tripeptide tail, Pro-Leu-Gly-NH2, to the ring structure might be due to the addition ofresidues at positions 7 and 8 which, together with residues atpositions 3 and 4, provide binding sites for interactions with the

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Table 1. Comparison of biological activities of deamino-oxytocin and its analogs with amino acidsubstitutions in position 8*Uterotonic Vasodepressor Antidiuretic Pressor

Peptide (rat) (fowl) (rat) (rat)Deamino-oxytocint 803 + 36 975 A 24 t19 1.44 i 0.06Deamino-[8-N-methylleucine]-

oxytocin 48 7 33 + 5 3.15 1.5Deamino-[8-a-hydroxyisocaproic

acid]oxytocin 134 12 31 + 3 9.60 3.0 0.26 + 0.02* Values are expressed in U. S. Pharmacopeia (USP) units/mg ± SEM.t Values taken from ref. 31.Apparently, deamino-[8-N-methylleucine]oxytocin has a slight depressor effect which is maskedin some injections by the pressor effect of the vehicle. The depressor effect did not appear inphenoxybenzamine-treated rats, presumably because such rats have a lower basal blood pressure.Ten micrograms of the analog showed no inhibitory effect on the pressor response to vasotocin. Ithas been shown (32) that [4-leucinejoxytocin also produced a weak and transient depressor response,but [4-leucinejoxytocin had no inhibitory activity on the vasopressor response to vasopressin.

receptors. However, the effect of the interaction of the completenatural acyclic tripeptide with the ring moiety has also to beconsidered (9, 34-37). For the two analogs reported here, bio-logical activities characteristic of oxytocin have been restored(avian vasodepressor activity) and enhanced (uterotonic ac-tivity) on attachment of the tail fractions to the ring moiety ofdeamino-oxytocin; thus, either the N-methylleucine-containingor the hydroxyisocaproic acid-containing tail fraction canpartially substitute for the natural acyclic tripeptide in adducingthe biological activities of oxytocin. After we had completedthe synthesis of these two analogs and were carrying out thebioassay, the synthesis and some pharmacological propertiesof [8-N-methylleucine]oxytocin were published by Hlavaceket al. (38), who reported uterotonic activity of 45 units/mg,galactogogic activity of 46 units/mg, and antidiuretic activityof 0.27 unit/mg; avian vasodepressor activity was not re-ported.

In view of the present work and that of Hlavacek et al. (38),the "cooperative model" (9, 35) for the biologically activeconformation of oxytocin can no longer include an absoluterequirement for hydrogen bonding between the carboxamidecarbonyl of asparagine and the peptide N-H of leucine becausethe oxytocin analogs described herein can assume the confor-mation needed to evoke the characteristic biological activitiesof oxytocin-albeit with lower potency-even though thishydrogen bond cannot form.The preferred conformation of oxytocin in aqueous medium

is not the same as the preferred conformation in dimethylsulfoxide (39-41). Therefore, any comprehensive hypothesisconcerning the biofunctional conformation of oxytocin and itscongeners must await the development of insight into thequestion of whether and to what degree the biologically sig-nificant interaction of the hormone with its receptors requiresan aqueous or a nonaqueous environment.

Uterotonic activity of deamino-[8-a-hydroxyisocaproicacid joxytocin is more than 4 times greater than that of the avianvasodepressor activity of deamino-[HyIc]oxytocin, whereas fordeamino-[8-N-methylleucine]oxytocin, the ratio of uterotonicto avian vasodepressor activity is approximately 1.5. Stericcrowding due to the methyl group may or may not be respon-sible for lowering the relative uterotonic potency of the lattercompound. NMR studies may shed some light on conforma-tion-bioactivity relationships of these analogs relative to thoseof oxytocin and deamino-oxytocin. In any case, compared toother analogs of oxytocin and deamino-oxytocin, the ratio ofthe uterotonic activity to avian vasodepressor activity is highfor both of the analogs here reported and particularly so in thecase of the hydroxyisocaproic acid analog.

We thank Ms. Lea Hillel-Deborah and Mr. David Rubin for carryingout the bioassays and Dr. David Live of the Rockefeller University forsome of the amino acid analyses reported in this study. This work wassupported by Grant AM-10080 of the National Institute of Arthritis,Metabolism, and Digestive Diseases and by the Life Sciences Foun-dation, Inc.

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