202
Journal of’the American Cheniiral Society
/
101:l
/ January 3, 1979
3C Nuclear Magnetic Resonance Studies of the Peptide Hormones Oxytocin, Arginine Vasopressin, Isotocin, Mesotocin, Glumitocin, Aspartocin, Related Analogues, and Diastereoisomers. Use of Specifically Deuterated Hormone Derivatives for Assignments and Effects of Structural Changes on 13CN M R Chemical Shifts in Peptides Victor J. Hruby,* K. K. Deb, Arno F. Spatola,lCDonald A. Upson,ld and Diane Yamamofole Contribution from the Department of Chemistry, University of Arizona, Tucson, Arizona 85721. Received April 26, I978
Abstract: The I3C nuclear magnetic resonance ( N M R ) spectra of the naturally occurring peptide hormones oxytocin, arginine vasopressin (AVP), mesotocin, isotocin, aspartocin, and glumitocin were compared. Oxytocin derivatives specifically deuterated in the Half-Cys-I, Tyr-2, Ile-3, Half-Cys-6, Pro-7, or Gly-”2-9 positions were used to make unequivocal assignments of most of the n and @ carbon atoms, and to sort out differences in assignments previously reported. Arginine vasopressin derivativcs, specifically deuterated in the Half-Cys- I , Tyr-2, Phe-3, Half-Cys-6, or Gly-”2-9 positions, also were used for unequivocal assignments. The chemical shifts of invariant residues in these compounds were virtually unchanged from their positions in oxytocin despite the structural changes at positions 8 and/or 4, and differences in biological activities. Analogues with Lamino acid substitutions in the I position [( I-penicillamine]oxytocin).3 position ([Phe3]oxytocin), 4 position ([Leu4]oxytocin). and 2 and 4 positions ( [ Leu2,Leu4]oxytocin, [ Ile2,Leu4]oxytocin,and [ Phe2,Leu4]oxytocin),also generally showed only minor I3C N M R chemical shifts at invariant residues, though there were a few notable exceptions. An interesting observation was that except for the half-cystine residues, the C n I3C chemical shifts of L-amino acid residues were essentially the same whatever their sequence position in the 20-membered disulfide ring moiety of these peptides. However, there were large I3C chemical shift differences (1.4- I .9 ppm) for the C a of equivalent L-amino acids in the same molecule, when one residue was in the 20-membered ring moiety, and the other in the acyclic tripeptide moiety of the hormones. The n chemical shift of residues in the acyclic portion of the molecule were always upfield to those in the ring moiety. When D-half-cystine (positions 1 and 6) or D-tyrosine (position 2) were substituted intooxytocin or AVP, the I3C N M R spectra of the diastereoisomeric peptide hormones often showed significant chemical shift perturbations even for residues quite remote from the substitution position. The similarities and differences of ‘3C chemical shifts a r e briefly discussed in terms of the conformational and biological properties of these peptides
The study of 13C nuclear magnetic resonance ( N M R ) spectra of small (3- 15 residues), biologically active polypeptides has grown steadily recently, and several studies have been directed to correlating chemical shift and relaxation (especially T I )data to conformational and dynamic properties of these molecules in s ~ l u t i o n . When ~ - ~ natural abundance compounds are used, the proper assignment of N M R peaks to particular carbon atoms in specific amino acid residues is of critical importance in interpretation of data. For peptides with more than four residues, assignments are usually made by reference to chemical shifts found in smaller peptides. Summaries of such reference data such as is found in Figure 6 of ref 6 are very useful, and can be used in conjunction with the effects of pH on chemical shifts in terminal residues or in certain acidic and basic side-chain residues, etc., for N M R assignments. However, even in small peptides problems of unambiguous assignments can arise owing to the similarity of chemical shifts on carbons on different amino acid residues, specific effects due to sequence, structure, and conformation, etc. The neurohypophyseal hormones oxytocin, H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NHz, and arginine vasopressin (AVP), H-Cys-Tyr-Phe-Gln-Asn-CysPro-Arg-Gly-N Hz, and a few analogues have been examined in aqueous solution by I3C N M R techniques by several groups,’ - I oand from these studies some tentative conclusions regarding the relationship of these data to biological function have been given.’ In this paper a large number of specifically deuterated derivatives of oxytocin were used to unambiguously establish I3C chemical shifts and to sort out the differences in assignments which have been reported for this h o r m ~ n e . ’ ~ . ~ Similar specifically deuterated arginine vasopressin derivatives 0002-7863/79/1501-0202$01 .OO/O
have also been utilized to establish assignments for this compound. The assignments we obtain generally agree with those of Walter et al.7aThe I3C spectra of a number of other naturally occurring neurohypophyseal hormones” whose I3C N M R spectra have not been reported were then examined. I n general, their I3C chemical shifts are nearly identical with those of oxytocin except for the specific residue changes. A number of oxytocin inhibitors and oxytocin and AVP diastereoisomers also were examined. The inhibitor analogues examined showed a few changes in the I3C N M R chemical shifts (in addition to changes from residue replacements), and i n some cases significant changes were observed. With respect to the oxytocin and AVP diastereoisomers, however, in many cases the chemical shift changes were extensive, both with respect to specifically modified residues as well as other parts of the molecule. These latter changes could not be accounted for by proximal structural changes, but appear to reflect specific conformational factors. Finally a few suggestions regarding the possible relationship of these findings to the biological activities of these compounds are made.
Experimental Section Analytical Methods. Thin layer chromatography (TLC) was done on silica gel G plates using the following solvent systems: (A) l-butanol-acetic acid-water (4:1:5, upper phase only); (B) 1 -butanolacetic acid-pyridine-water ( 1 5:3:10:12); (C) I-pentanol-acetic acid-water (7:7:6); (D) ethyl acetate-pyridine-acetic acid-water (5:5:1:3). Amino acids and peptides were detected by ultraviolet light, iodine vapors, ninhydrin, and fluorescamine. Capillary melting points were determined on a Thomas-Hoover melting point apparatus and are uncorrected. Amino acid analyses were obtained by the method of Spackman, Stein, and MooreI2 on a Beckman 120C amino acid
0 1979 American Chemical Society
Hruby et al.
/ I 3C N M R Studies of Peptide Hormones
203
to the disulfide form by oxidation under nitrogen with K3Feanalyzer after hydrolysis in 6 N HCI for 22 h. Optical rotation values (CN)6.15.34The product was purified by partition chromatography were measured at the mercury green line (547 nm) using a Zeiss Old on Sephadex G-2535using the solvent system I -butanol-3.5% aqueous 4 polarimeter. Elemental analyses were performed by Spang Miacetic acid in 1.5% pyridine ( 1 : l ) . The product obtained (Rf0.23) was croanalytical Laboratory or Chemalytics, Inc., and deuterium analyses further purified by gel filtration on Sephadex (3-25 using 0.2 N were performed by Joseph Nemeth, Urbana, 111. aqueous acetic acid as eluent solvent. After lyophilization of the major Materials. In addition to the peptide and amino derivatives whose peak there was obtained 84.2 mg (35%) of [7-[n,B.@,y,y,K,K-'H,Jsyntheses are reported here, the following peptide hormone derivativcs prolineloxytocin [a]:& -22.5" (c 0.5, in HOAc). Amino acid analysis and analogues were prepared and purified as follows: [9-[a,a-*H*]gave the following molar ratios: Asp, I .Ol; Glu, I .OO; Pro, 1.02: Gly glycinaniide]oxytocin according to Glasel et aI.,l3 [ I-he!ni[cu,/j,B0.94; Half-Cys, 1.92; Ile, 1.08; Leu, I.OO;Tyr,0.91; "3, 3.20. T L C ?Hi]cystine]oxytocin by the method of Spatola et aI.:l4 [6-hemi[nin solvent systems A, B, and C gave single uniform spots identical with ?H]cystine]oxytocin. [6-hemi[@,@-2H~]cystine]oxytocin,and [ 6 unlabeled oxytocin. The 300-MHz ' N M R spectrum of the title henii-~-[tr-~H]cystine]oxytocin according to Upson and Hruby;'? [ I-hemi[tu-2H]cystine]oxytocinand [ I-hemi-~-[cu-~H]cystinc]oxy- compound in an aqueous solution was identical with that of oxytocin except for the missing proline-7 residue protons as previously retocin according to Hruby, Upson, and Agarwal;I6 [ l-hemi[cu-2H]ported.36 The milk-ejecting activity37 was found to be identical with cystine, 8-arginine]vasopressin, [ I -hemi-D-[a-2H]cystine, 8-argithat of authentic oxytocin. nine] vasopressin, [ I -hem i [ @,&? H 21 cystine, 8-a rgi ni ne] vasopressi n, [2-DLIa-ZH]TyrosineJoxytocinand Separation of the Diastereomers [2-[ct-*H]tyrosine, 8-arginine]vasopressin, [ 2 - ~ - [ a - * H ] t y r o s i n e , by Partition Chromatography to Give [2-[a-zJ~]Tyrosine]oxytocinand 8-argininelvasopressin. [2-[o,CI,@-'H3]tyrosine, 8-argininelvasoprcshin, [3-[tu-?H]phenylnl;1ninc, 8-arginine]vasoprcssin, and [ O 12-~-[a-~H]TyrosineJoxytocin. Thc solid-phase synthcsis of the precursor nonapeptide rcsin to the title compounds was accomplished [tr,tu-'Hz]glycinamide, 8-argininelvasopressin by the method of Yamamoto et al.;" mesotocin and isotocin by the method of Hruby using procedures similar to those used above. The quantity of Bocglycine substituted resin used was 2.86 g with an amino acid content et a1.;I8 aspartocin by Spatola et al. (unpublished); glumitocin by the method of Gitu;I9 oxypressin by published procedures:'" [desgliiof 0.35 nimol/g resin ( I .OO nimol). S-3.4-DimethyIben7yl protcction was used for cysteine protection, and Boc-Asn and Boc-Gln werc tamine4]oxytocin by the method of Upson;" [4-leucine]oxytocin as previously reported;22 [ 2,4-dileucine]oxytocin by the procedures of coupled as their nitrophenyl esters using N-hydroxybenzotriazole Hruby and Chan:23 and [2-isoleucine, 4-leucine]oxytocin and [2catalysis.I5 The coupling procedures were as before. A three-fold excess of amino acid for DCC couplings and a four-fold exce\s for phenylalanine, 4-leucinc]oxytocin as previously reported.l4 Boc-~t.-[tr-*H]Tyrosineand Boc-[@.@-*H>] tyrosine \vere prcpared nitrophenyl cstcr couplings were uscd. Roc-~~.-[tu-?H] tyrosinet7~ 3 ' ; coupled using DCC (threefold excess of each reagent). There n a s as previously reported." Boc-S-3,4-dimethylbenzylcysteinewas prepared according to the method of Smith.25Other Boc amino acid obtained 3.85 g of Cys(DMB)-Di--[n-*H]Tyr-lle-Gln-Asnderivatives were purchased from Vega-Fox Biochemicals or BiosynCys(DMB)-Pro-L.eu-GIy-0-resin. The peptide was cleaved from the thetica. The polystyrene resin l?6 cross-linked with divinylbcnzene resin using anhydrous ammonia in anhydrous methanol to give 0.879 and chloromethylatcd to an extent of I .07 mmol/g resin was purg (70%) of C y s ( D M B ) - ~ ~ - [ t u - ~ H ] T r y - l l e - G l n - A s n - C y s ( D ~ B ) chased from Lab Systems, Inc., San Mateo, Calif. Solvents were puPro-Leu-Gly-"2, mp 21 8-222 "C. A 280-mg (0.22 mmol) portion rified for partition chromatography as previously described.2h of the nonapeptide was treated with sodium in liquid ammonia to reBoc-[a,B,P,y,y,6,6-zH,lProline. A sample of 0.600 g (4.9 mmol) move the protecting groups and oxidized to the disulfide form with of [tr,b.$,?.*,,6,6-*H7]proline provided by Professor A. T. Blomquist, K3Fe(CN)h to give a crude mixture of [2-~~-[a-~H]tyrosine]oxytocin. Cornell University, was treated with 0.70 g (5.2 mmol) of tert-butyl The diastereomers were separated and purified by partition chroaiidoformate at pH 8.6 in 20 mL ofdioxane-water ( 1 : l ) according matography on Sephadex G-25 using the solvent system I-butanolto the method of SchnabelZ7using a Radiometer Autoburette. After 3.5% aqueous acetic acid in 1 .5% pyridine ( I : I ). The diasiercoisomers the usual workup and recrystallization from ethyl acetate-hexane were nicely separated with [2-[c~-~H]tyrosine]oxytocin eluting at R, there was obtained 0.87 g (83%) of the title compound, mp 133 I35 0.24 and [2-~-[a-*H]tyrosine]oxytocin eluting at R ~ 0 . 3 9The . former " C (lit. mp of unlabeled Boc-proline, 134-1 36 "C). On T L C in the compound was further purified by gel filtration on Sephadex G-25 solvent systems A, B, and C, a single spot was observed identical with using 0.2 N acetic acid as eluent solvent. There was obtained 45 mg (40%) of [2-[n-2H]tyrosine]oxytocin, [cu];:? -25.3" (c 0.5, 1 N authentic Boc-proline. Proton magnetic resonance indicated deuterium HOAc). Amino acid analysis gave the following molar ratios: Asp, substitution in the labeled positions to be >95oiO. [7-[a,~,~,y,y,6,6-zH~]ProlineJoxytocin. The solid-phase methodZX 0.96; Glu. 1.02; Pro. 1.04; Gly, 1.01; H a l f - C y , 1.88; Ile, 1.00; Leu. was used to synthesize the protected nonapeptide precursor, HI .02; Tyr, 0.93; NH3,3. I O . T L C in solvent systems A , B. and C gave C y ( PMB)-Tyr( Bzl)-lle-Gln-Asn-Cys( P M B)- [a,@,,8,y,y,6,6-*H7]single uniform spots identical with those of authentic oxytocin. Milk-ejecting a ~ t i v i t i e s were ) ~ identical with those of authentic o x j Pro-Leu-Gly-"2. The synthesis was performed on the Vega Model tocin. The D diastereoisomer [2-D-[u-'H] tyrosine]oxytocin was 95 synthesirer, an automated machine similar to that previously described.29The polymer support used was chloromethylated polystyfurther purified by gel filtration on Sephadex (3-25 using 0.2 h acetic rene, 1% cross-linked with divinylbenzene (CI substitution = 1.07 acid as eluent solvent. There was obtained 47 mg (42O/O,41% ovcrall mmol/g resin) which had been substituted with Boc-glycine as preyield of both isomers from nonapeptide), [ t ~ ] : : ~ -83.5" (c 0.51, 1 5 viously reportcdI4 to a glycine level of 0.38 mmol/g resin as measured HOAc). Amino acid analysis gave the following molar ratios: ,4sp, by the modified3' aldimine Boc amino acids were used 0.98; Glu. I .02; Pro, 1.04: Gly, 0.98; Half-Cys. 1 .85: Ile, I .O?:Leu. throughout. The sulfhydryl group of cysteine wasp-methoxylbenzyl I .OO: D-Tyr, 0.91; NH3,3.20. T L C in solvent systems A, B, and C gave protected. and the tyrosine phenol group was benzyl protected. The single uniform spots. procedures for deprotection of Boc protecting groups, neutralization, [2-DL-[B,P-ZH~]Tyrosine]oxytocinand Separation of the Diasteand coupling of amino acid residues to thc growing peptide chain reomers by Partition Chromatography to Gi~e[2-[P,@-~H2]Tyrosine]closely followed those previously used.I4.l5 The exception was the oxytocin and [2-D-[P,~-2Hz]Tyrosine]oxytocin. The solid-phase syncoupling of Bo~-[a,@,P,y.y,K,d-~H7]proline to H-Leu-Gly-0-resin. thetic procedures used to prepare the protected nonapeptide resin to I n this case a single 3-h coupling using a I .25 molar excess of the the title compound were essentially the same as those used to prepare deuterated proline derivative and dicyclohexylcarbodimide (DCC) [2-[a-*H I] tyrosine]oxytocin (vide supra). BOC-DL[@,@-'HZ] tyrosine'" followed by a second coupling using 0.5 mol of each reagent was sufwas added in a single coupling reaction using a twofold excess of the ficient to obtain complete coupling as determined by the ninhydrin amino acid derivative and of DCC. After the synthesis there was obtest.j2 From 3.0 g ( 1 . I mmol) of Boc-glycine-0-resin there was obtained 3.96 g of Cys(DMB)-DL-[@,,8-ZH~]Tyr-lle-Gln-Asntained 4.2 g of H-Cys(PMB)-Tyr(Bz1)-Ile-Gln-Asn-Cys(PMB)- Cys(DMB)-Pro-Leu-Gly-0-resin. The peptide was cleaved from the [a,P,@,y,y,6,6-*H7] Pro-Leu-Gly-0-resin. The peptide was cleaved resin with anhydrous ammonia in anhydrous methanol to give 0.880 from the resin by ammonoloysis in anhydrous methanol saturated with g (70%) of Cys(D.MB)-~L-[~(3-~H2]Tyr-lle-Gln-Asn-Cys(DMB)anhydrous ammonia as previously r e p ~ r t e d . ' ~ There . ' ~ , ~was ~ obtained Pro-Leu-Gly-NHZ. m p 217-221 OC. A 316-mgsample (0.25 mmol) 0.900 g (64%) of Cys(PMB)-Tyr(Bz1)-IIe-Gln-Asn-Cys(PMB)-of the peptide was converted to crude [2-Dl.-[@,P-2H*]tyrosine]oxq[n.B,P,y,y,d,d-2H7]Pro-Leu-Gly-NH~,m p 217-221 "C. A sample tocin as before. The diastereoisomers were separated by partition of 335 mg (0.24 mmol) of the protected nonapeptide in anhydrous chromatography on Sephadex G-25 using the solvent system I-buammonia was treated with N a to remove the protecting groups, and tanol-3.5% aqueous acetic acid in I .5% pyridine ( 1 :1). The D diasteafter removal of the ammonia the remaining peptide was converted reoisomer derivative eluted at R/ 0.41 and the all-L diastereoisomer
Journal of the America,? Chemical Society
204
/ 101:i / January 3, 1979
Oxytocin IpD 6 2 ) (Lptfa E T A L , 1972)
PPhl FROM '3CS2
Figure I . The I3C chemical shift assignments for Ccu. C$, and other aliphatic carbon atonis in oxytocin as determined by Lyerla and Freedman (ref 8 ) . and in oxytocin and arginine vasopressin (AVP) b) Walter et al. (ref 7a). and by this work using 2H-labeled peptides (the asterisk denotes assignments checked in this work by specifically labeled hormone derivatives). Chemical shifts are given in parts per million upfield from I3CS2.and were measured from internal 1.4-dioxane which is 126.1 ppm upfield from ' 3 C S 1( 4 2 ) .
derivative at HfO.23. After gel filtration on Sephadex (3-25 there was obtained 47 mg (37.6%) of [2-[@,fi-2H2]tyrosine]oxytocin. [a]::, -23.8' ( e 0.5 1 . 1 N HOAc). Amino acid analysis gave the following molar ratios: Asp, I .OO: Glu, 1.03; Pro, I .02; Gly, I .OO: Half-Cys, 1.92; IIc. 0.98: Leu. 0.97: T \ r . 1.00. "3, 3.10. T L C in solvent systems A, B. and C gave single uniform spots identical with those of authentic oxytocin. Milk-ejecting activities were identical with those of oxytocin. Gel filtration purification on Sephadex (3-25 of [2-D-[@,fi-2Hl]tyrosineloxytocin was also performed to give 53 mg (42.4%, 40% overall yield of both diastereoisomers from the nonapeptide), [CY]:& -79' (c 0.54, I N HOAc). Amino acid analysis gave the following molar ratios: Asp. 0.98; Clu. 0.96; Pro, 1.08: Gly, 0.97: Half-Cys, 1.93: Ile. I .OO: Lxu. 1.05: Tyr. I .02; S H 3 , 2.90. Thin layer chromatography in solvent systems A, B, and C gave single uniform spots. I3C NMR Spectral Determinations. The I3C N M R spectra were measured on peptide samples of 20-80 mg/mL in DzO at 22.63 M H z using a Bruker W H - 9 0 FT spectrometer interfaced with a BNC-12 computer, Diablo disk system, and Nicolet 293 controller system. The protons were decoupled with continuous proton noise decoupling at power levels sufficient to completely remove '3C-'H scalar coupling. The decoupler power and modulation frequency were maintained constant in recording individual spectra. The FID was stored in 8 K memory locations, and generally 5 X lo4 scans or more were taken for each spectrum. The FID generally was Fourier transformed to a 6000-Hz sweep width. All measurements wcre made at a probe temperature of 26 f 2 "C. Sample tubes I O mm in diameter were used and the pH was adjusted using CDlCOzD and NaOD. The pH values were determined before and after the "C N M R experiment with a calibrated Radiometer pH meter, and the values obtained (0.05 pH units) are given as pD ( p D = pH 0.4).38
+
Results Previously two groups have made assignments of the I3C N M R resonances of o x y t o ~ i in n ~aqueous ~ ~ ~ solution and assignments have also been made for arginine vasopressin in this In the former case there are a number of differences. The correct assignments are critical to interpretation of chemical shift and relaxation data in terms of conformational and dynamic properties of the peptide. Therefore, for the purpose of establishing spectral assignments, specifically deuterium-labeled derivatives of oxytocin and of arginine vasopressin were prepared and used to make assignments. W e have previously shown4a that deuterated peptides can be used to make unequivocal I3C assignments. As a further aid the correlation table of Boveyh was used, and the I3CN M R spectra of other naturally occurring neurohypophyseal hormone derivatives, mesotocin ( [Ile8]oxytocin),37isotocin ( [Ser4, IIe8]oxytocin),40 glumitocin ( [Ser4, GIn8]oxytocin),41aspartocin ( [Asn4]oxytocin),and also oxypressin ( [Phe3]oxytocin),20were examined. The latter compounds have close sequence homol-
ogy to oxytocin (and vasopressin). The results of these studies are shown in Figure 1, and the chemical shift data are tabulated in Table I. For further confirmation of the arginine vasopressin assignments, the previous I3C N M R studies of Walter et aI.':I of the naturally occurring neurohypophyseal peptides 8-arginine vasotocin ( [ Phe3, Arg8]vasopressin) and 8-lysine vasopressin ( [Lys8]vasopressin)] were valuable. As shown in Figure 1, all the asterisked carbon atoms were unambiguously determined by use of the deuterated analogues. Since we had oxytocin derivatives with deuterium substitutions a t the Half-Cys-1 N and 0.Tyr, a , ,8, 3' and 5'. Ile a , HalfCys-6 a and 0,Pro CY, @, ?, and 6, Leu a , and Gly a carbon atoms it was possible to settle all of the previous disagreements in a s ~ i g n m e n t(Figure ~ ~ , ~ I , Table I). Previous differences in the assignments of the C a resonances of Half-Cys-6 and Gln-4 had been n 0 t e d ~ ~(see 3 ~ Figure 1). Also some ambiguity remained for the Tyr-2 C a resonance which was reported as upfield to Gln-4 Cct in Table I of Walter et but indicated as downfield in Figure 2 of the same paper.7a Use of [2-[ct-*H]tyrosine]oxytocinclearly showed the Tyr-2 CLY downfield to the glutamine resonance in agreement with Figure 2 in Walter et al.7aA I3CN M R spectrum of [6hemi[a-*H]cystine]oxytocinshowed the Half-Cys-6 C a to be a t 141.4 ppm,42 in agreement with Walter et al. I n addition assignments previously made7a.8 for the C a of Half-Cys- 1, Ile-3, Pro-7. and Gly-9 were corroborated using specific deuterated derivatives. In the C 6 region of oxytocin previous assignment differences were noted for the Half-Cys-1 and Leu-8 resonance^.'^.^ Using [ 1 -hemi[a.~,@-2H~]cystine]oxytocin, the assignments of Walter et al,7awere confirmed. In addition the previous assignments for the Tyr-2, Half-Cys-6, and Pro-7 6-carbon resonances were corroborated using deuterated analogues. In residues with Cy or further removed carbon atoms, the only difficulties in assignment are attributed to the y carbons of the Pro-7, Ile-3, and Leu-8 residues all of which have resonances near 168 ppm. These resonances appear as a very closely spaced unysmmetrical doublet or triplet depending upon the PH.'",~ By use of [7-[ct,P,P,y,y,6,6-2H2]proline]oxytocin it could be shown that the most downfield absorption was due to the Pro-7 ?-carbon resonance. In oxypressin (Table I), which possesses Leu-8 and Pro-7 residues, but has a Phe residue in place of Ile in position 3, both of the y carbons in Leu-8 and Pro-7 appear at 168.0 ppm, which is slightly downfield of the Cy of L e u 4 and Ile-3 in oxytocin. On the other hand, in mesotocin, isotocin, and glumitocin (Table I ) , which possess no L e u 4 residue, but which all contain an Ile-3 residue, the resonance for the Cy carbons in this region always
Hruby et al.
/ "C N M R Studies of Peptide Horniones
205
Table 1. Chemical Shifts and Assignments of C,, Cg, C.,, Ca, and Aromatic Carbon Resonances of Amino Acid Residues of Mesotocin, Isotocin, Glumitocin, Aspartocin, Oxypressin, Oxytocin, and Arginine Vasopressin in D2O"
carbons
PD
oxytocin 4.9
I 50.6 140.07 153.40 168.34 170.55 171.98
[ Ile8]oxytocin (mesotocin) 4.0
[Ser4,1 le8]oxytocin (isotocin) 4.2
[Ser3,GIn8]oxytocin (glumitocin) 4.0
150.67
150.66
150.53
[Asn4]oxytocin (aspartocin) 3.0
[Phe3]oxytocin (oxypressin) 3.0
[Args]-, vasopressin 5.2
150.49 140.12 153.43 168.23 170.57 172.13
150.53 140.04 153.13 168.02 170.48 171.91
I 50.56
139.06 164.73 168.28 152.13 36.82 133.96 156.55 167.95 177.68 182.45
133.96 156.84 168.08 178.02 182.5 I
132.07 163.47h 168.1 5 h 144.75
132.21 163.34 167.95 144.59
132.2 I 163.46 168.02 144.76
139.42 166.19 161.58 131.82 163.41 167.96 144.75
141.37h 154.44h 142.34 156.7 I
141.37 154.26 142.35 156.55
141.50 154.50 142.41 156.84
141.43 154.50 142.34 156.90
137.54 166.9 I 161.65
137.50 166.65 161.58
I 32.72h 156.7 I 168.15 177.83 182.06
132.53 156.55 167.95 177.68 182.06
135.39 132.2 I 132.2 1 156.84 168.08 178.02 182.21
135.40 132.27 132.27 156.90 167.96 177.96 182.12
132.21 163.67 168.23 144.91
132.01 163.40 168.02 144.62
131.98 163.30 167.64 144.56
141.62 154.43 142.46 157.08 142.46 157.08
141.52 153.96 142.21 156.32
141.40 153.94 142.38 156.32
137.27 166.91 161.58
137.63 166.54 161.65
I 36.92h 156.49 56.97 63.95 63.95 65.77 137.41 1 56.32b 65.55 62.64 77.21 38.8 I 1 52.29h
132.21 157.08 168.23 178.00 182.19
I 37.2g6 156.7 1 65. I O 62.89 77.26 39.20
137.28 156.55 65.20 62.70 77.48 38.80
137.21 156.84 65.32 62.67 71.26 38.58
137.27 156.32 65.72 62.67 77.40 39.03
137.60 156.30 65.60 62.70 77.40 39.10
137.27 156.32 57.15 64.22 64.00 65.98 137.83 156.00 65.55 62.45 77.48 39.03
140.46 1 53.20b
140.40 152.97
140.33 152.76
140.39 153.04
140.90 153.13
140.04 153.13
140. I O b
a Chemical shifts are reported in parts per million upfield from I3CS2 measured from internal I ,4-dioxane ( I 26. I ppm). On this scale, Me4Si (external) is 193.7 ppm upfield from I3CS2.Conversion to the Me& scale then is dc = 193.7 ppm - 6csZ (this table). The chemical shifts are accurate to 0.05 ppm. Assignment check by use of specifically deuterated derivative (see text).
appears at 167.9-168.1 ppm. In all cases this is downfield to the resonances for lle-3 or Leu-8 in oxytocin. On this basis we tentatively assign the more downfield resonance to the Ile-3 y carbon and the more upfield resonance to the Leu-8 y carbon. Owing to the complexity of the off-resonance ' H decoupled spectrum of oxytocin in this region, it was not possible to use this method to distinguish between these carbons. All
of the aromatic carbon assignments correspond to those of Walter et a]. and are not repeated here. The structure of 8-arginine vasopressin (AVP) differs from oxytocin in two respects: a Phe residue replaces Ile at position 3 and an Arg residue replaces Leu at position 8. Previously Walter et al. have assigned the I3C N M R spectra of AVP based on its structural similarity to oxytocin and the close
206
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similarities of their 13C N M R spectra. In the C a region, the however, should be noted. The lle-3 C a moved downfield 0.5 1 Arg-8 C a can be readily assigned a t 139.1 ppm by reference ppm in isotocin and aspartocin and 0.45 ppm in glumitocin to Figure 6 in ref 6. The only difficult assignments in this region relative to its chemical shift in oxytocin, though none of the arise for the Phe-3, Gln-4, and Tyr-2 C a resonances which other lle-3 aliphatic carbon resonances were similarly affected, occur as a closely spaced triplet near 137 ppm. By utilizing the nor were the adjacent Asn-5 C a and Cg chemical shifts. Inpartially deuterated hormone derivatives, [2-[cu-*H]tyroterestingly the Tyr-2 CP chemical shift was shifted downfield sine]-8-arginine]vasopressin and [3-[a-2H]phenylalanine]about 0.41 ppm in glumitocin and aspartocin but no similar 8-arginine]vasopressin, we were able to show unambiguously effect was seen in isotocin. It is perhaps not surprising that a that the downfield resonance at 136.9 ppm was due to the change of the Leu-8 residue in oxytocin to the Ile-8 in mesoPhe-3 Ca and the middle resonance at 137.4 ppm was due to tocin and isotocin should have only a minor effect in the the Tyr-2 C a . By elimination then, the upfield peak was due chemical shifts of the adjacent Gly-9 and Pro-7 residues. to the Gln-2 C n (Table I ) . All of the other Ca resonances However, even replacement with Gln-8 in glumitocin produced correspond closely to those found in oxytocin, though we did only minor changes with the largest being a 0.25-ppm downcorroborate the Half-Cys-l resonance by use of [ I-hemi[afield shift for the Pro-7 Ca. In oxypressin a few more signifi2H]cystine]-8-arginine]vasopressin and the GIy-9 resonance cant chemical shifts were seen relative to oxytocin. For exby use of [9-[a,a-2H]glycinamide]-8-arginine]vasopressin. ample, the Tyr-2 C p was shifted 0.7 pprn downfield and the I n the Cp region two areas of ambiguity presented themselves. Tyr-2 Cn 0.55 ppm upfield in oxypressin relative to its position The first, which involved a closely spaced doublet at about 152 i n oxytocin, perhaps as a result of the proximity of the Phe-3 ppm was resolved by use of [ 1-hemi[P,P-2H2]-cystine]-8-(which replaces Ile-3) and its associated aromatic ring current argininelva~opressin~~ and showed the upfieid absorption at in the latter compound. On the other hand, the Gln-4 C a and pD 5 to be due to that of the Half-Cys- 1 Cp. A more difficult C p carbons are hardly affected with the largest shift being a problem was posed by a closely spaced unsymmetrical doublet 0.27-ppm downfield shift for the Cn of Gln-4. However, the at about 156.5 ppm (Figure I ) which contained the Tyr-2, Half-Cys-6 and Asn-5 P carbons are shifted downfield 0.5 and Phe-3, and Asn-5 CP resonances. Use of the specifically deu0.4 ppm, respectively, relative to their chemical shifts in oxytered analogue [2-[cu,P,P-2H3]tyrosine]-8-arginine]vaso- tocin. It is significant to note that in mesotocin and isotocin the Ile-8 and Ile-3 residues have identical PCH, -yCH2, and ? C H , pressin'' showed the Tyr-2 CP to be located at the downfield chemical shifts and only slightly different 6CH3 I3Cchemical more intense portion of the doublet. Examination of the oxypressin proton decoupled I3C N M R spectrum did not resolve shifts (Table I). However, the C n chemical shifts of Ile-8 and the remaining assignment unambiguously, since in this case Ile-3 are very different, differing by 1.4 ppm in mesotocin and the upfield side of the doublet is more intense. We have ten1.8 ppm in isotocin. In this regard it is interesting to note that the Gln-8 C a in glumitocin has a 1.9-ppm chemical shift diftatively placed Phe-3 CP in agreement with Walter et al.7a since in arginine vasotocin the Asn-5 and Tyr-2 Cp chemical ference from the Gln-4 C a in oxytocin and mesotocin; in all shifts are identical, but feel that further confirmatory work is of these cases the residue 8 C a is upfield relative to the residue needed. All other AVP assignments of y and further removed 3 (or 4) C a . carbon atoms including aromatic carbon atoms were readily We next examined a number of analogues of oxytocin with made by reference to tables of Bovey6 and to the spectrum of single or double substitutions in the 2 and 4 positions which we had previously shown to be inhibitors of the neurohypooxytocin, and are in agreement with those of Walter et al.7a physeal hormones.43 These included [ L e ~ ~ ] o x y t o c i n , ~ * - ~ ~ Having settled the assignments for oxytocin and AVP we next turned our attention to the naturally occurring and closely [ Leu2,Leu4]o x y t o ~ i n , ~[ Ile*,Leu4] ~ , ~ ~ o x y t ~ c i n , ~and ~,~~ [ P h e * , L e ~ ~ ] o x y t o c iI n .addition ~ ~ ~ ~ ~ we examined [ 1 -penistructurally related analogues of oxytocin: mesotocin, which cillamine]oxytocin ( [ P e n l ] o x y t ~ c i n )The . ~ assignments and differs from oxytocin only in the replacement of the Leu-8 chemical shift parameters for these compounds are shown in residue by an Ile; isotocin, which in addition to the position 8 Table 11. In most cases the assignments followed directly from replacement in mesotocin also has a Ser-4 residue in place of these previously made for oxytocin. For [Leu2,Leu4]]-and a Gln-4 residue; glumitocin, which also has a Ser-4 in place of [ Ile2,Leu4]oxytocin the chemical shifts for all carbons of the Gln-4 residue, and a GIn-8 residue in place of the Leuresidue; and aspartocin, which differs only by an Asn-4 residue constant residues were essentially the same ( f 0 . 4 ppm) as in place of the Gln-4 residue found in oxytocin. The I3C N M R those found in oxytocin. The lle-2 and Ile-3 residues I3C chemical shifts were identical except for a small (0.25 ppm) spectra of the aliphatic region of three of these compounds are difference for the Cp. The Leu-4 C n was considerably downshown in Figure 2. In addition we examined oxypressin field (1.6 ppm) of the Leu-8 C a in [Ile2,Leu4]oxytocinand this ( [ Phe3]oxytocin).which possesses the ring structure of vasowas also found to varying degrees in all the other L e u 4 subpressin and the tripeptide side chain of oxytocin, and differs stituted analogues (Table 11). The Leu-2 Cn in [Leu2.Leu4]from oxytocin only at position 3 where Phe replaces the Ile oxytocin was somewhat less downfield (0.32 ppm) relative to residue. The assignments and chemical shifts for the C a , alithe Leu-8 C a . All other leucine carbon atoms showed either phatic, and aromatic carbons of all of these compounds are identical or only slightly shifted chemical shifts. In [Leu4]given in Table I . oxytocin a few small chemical shift perturbations (0.6-0.8 The 13C N M R assignments for these compounds were ppm) relative to the same carbon atoms i n oxytocin were obgenerally very readily made owing to the remarkable constancy of the chemical shifts of the carbons i n the invariant residues served at Tyr-2 C a , Asn-5 Cp, Leu-8 6CH3, and Ile-3 yCH3. They were all relative upfield shifts. In [Phe2,Leu4]oxytocin, (Table I ) . Chemical shift changes greater than f 0 . 3 0 ppm most chemical shifts were similar to those in oxytocin and the were very rare (Table I ) , and when changes did occur the shift other analogues, but significant changes relative to oxytocin could be readily observed and the assignment made by inwere observed, expecially at residue positions 5 , 6 , and 7. The spection. For example, the Ile-3 aCH3 was found at 182.1 ppm Pro-7 CP and Asn-5 C n were 1.4 and 1.8 ppm downfield, rein both oxytocin and mesotocin, and thus the Ile-8 aCH3 in the spectively, of their position in oxytocin, and the Half-Cys-6 C a latter compound could be assigned to the slightly upfield resonance at 182.5 ppm. In isotocin the Ile-3 moved 0.15 ppm and C p were about 0.8 ppm downfield. I n [Penl]oxytocin virtually all the chemical shifts are the same as found in oxyupfield relative to its chemical shift in mesotocin (Table I), and tocin for the equivalent residues. The half penicillamine 1 Cn the Ile-8 nCH3 also moved slightly upfield (0.06 ppm; see Table I ) . Even residues adjacent to those where substitutions and C p resonances were readily assigned by their pH depenhad been made showed little variation. A few exceptions, dence8" and by reference to the spectrum of [Pen',Leu2]oxy-
Hruby et al.
/ I3CN M R Studies of Peptide Hormones
207
I
I
1
130
I40
150 PPM
I
I
I
1
160
170
IS0
167
UPFIELD FROM vCS2
Figure 2. ' ? C Nuclear magnetic resonance spectrum of the Ca. Cp, and other aliphatic carbon atoms of the peptide hormones glumitocin. mesotocin, and isotocin. Chemical shifts are in parts per million upfield from I3CS1,and were measured from internal 1.4-dioxane which is 126.1 ppm upfield from l3CS*(42).
t o ~ i nand , ~ ~in both cases these resonances are considerably downfield relative to the comparable C a and CP carbons in Half-Cys-1 in oxytocin (8.8 and 1 1 . 1 ppm, respectively). Significant chemical shift differences relative to oxytocin were noted only for the Half-Cys-6 C a and the Asn-5 Cp. The resonance at 155.8 ppm was previously assigned8h to the HalfCys-6 CP and the downfield resonance a t 153.0 ppm to the Asn-5 CP, primarily based on the N T I for the former resonance which was similar to that of the other aliphatic carbon atoms (mostly Ccu) which make up the 20-membered disulfide-containing ring in [Pen'loxytocin. This places the Half-
Cys-6 CP 1.4 ppm upfield from its position in oxyotcin and the Asn-5 CP at 3.7 ppm downfield from its position in oxytocin. If the assignments were switched the Half-Cys-5 CP would be 1.4 ppm downfield from its position in oxytocin and the Asn-5 CP 0.9 ppm downfield from its position in oxytocin. I n either case they undoubtedly reflect the conformational differences between oxytocin and [ P e n l l o x y t ~ c i n . ~ As previously reported in our studies on the synthesis of specifically deuterated o ~ y t o c i n ' ~and - ' ~arginine vasopressin' derivatives, we often prepared and separated diastereomeric analogues of the hormones. We report here the l3C N M R of
208
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Table 11. Assignments of I3C Resonances of a , b,?, and 6 and Aromatic Carbon Resonances of Oxytocin, [Penl]Oxytocin, [Leu2s4]Oxytocin, [Leu4]0xytocin, [Ile2,Leu4]Oxytocin,and [Phe2,Leu4]0xytocinin DzO' carbons
DD Gly-9 aCH2 Leu-8 aCH
PCH2 ?CH 6CH3 6CH3 Pro-7 aCH PCH2 rCHr 6CHz HalfC ~ S - aCH 6 BCH2 Asn-5 aCH KH2 GIn-4 nCH BCH2 rCH2 Leu-4 aCH BCH2 YCH dCH3 dCH3 lle-3 aCH KH rCHz rCH3 dCH3 Tyr-2 nCH BCH2 CI c2.6 c3.5
c4
oxytocin 4.9
[Pen']oxytocin 4.3
[ oxytocin 4.4
[ Leu4]oxytocin 5.2
[lle2,Leu4]oxytocin 5.1
[Phe2,Leu4]oxytocin 5.3
150.60 140.07 153.40 168.34 170.55 171.98 132.07 163.47 168.15 144.75
150.50 140.15 153.22 167.61 171.0 172.0 131.65 163.35 167.6 144.7
150.53 139.94 153.26 168.26 170.85 171.98 131.94 163.34 168.00 144.82
150.78 140.14 153.54 168.51 171.14 172.42 132.03 163.95 168.10 145.02
150.60 140.01 153.33 168.40 170.94 172.10 132.08 162.93 168.12 144.8 1
150.41 140.53 153.65 168.21 170.49 171.92 132.33 162.1 168.21 144.46
141.37 154.44 142.34 156.7 1 137.54 166.9 1 161.65
141.03 155.8 142.50 153.0 137.44 166.72 161.60
140.98 154.17 142.21 156.29
141.33 153.70 142.38 156.96
140.92 154.50 142.22 156.45
140.53 153.65 141.53 156.7 1
138.44 153.26 168.00 170.85 171.98 133.44 156.97 168.28 177.63 182.12
139.10 153.54 168.10 171.14 172.40 132.24 157.35 168.51 178.49 182.21 138.05 156.96 65.55 62.45 77.48 39.2
138.44 153.02 168.12 170.94 172.10 133.3 1 157.23 168.40 177.63 182.25
138.30 153.27 168.21 170.49 171.92 132.60 156.7 1 168.21 177.83 182.06
132.72 156.7 1 168.15 177.83 182.06 137.28 156.7 1 65.10 62.89 77.26 39.20
133.1 157.4 168.0 178.24 181.66 137.05 157.0 65.99 62.89 77.48 39.20
Leu-2 aCH
139.62 152.7 1 168.28 170.55 171.58
PCH2 yCH bCH3 dCH3 Ile-2 tvCH BC H rCH2 rCH3 bCH, Phe-2 nCH BCH2 CI
133.3 1 157.23 168.40 177.63 182.25 137.93 157.72 57. I5 64.22 64.22 65.55
c2.6 c3,5 c4
HalfCys-1 CUCH OCH2
139.94 153.26
140.46 153.40
140.14 153.54
140.40 153.33
140.53 153.27
HalfPen-I aCH
PC Y(CH3)2
131.65 142.13 167.01
Chemical shifts are reported in parts per million upfield from '3CSz measured from internal 1,4-dioxane (1 26.1 ppm). On this scale, Me4Si (external) is 193.7 ppm upfield from ITS2.Conversion to the MedSi scale then is 6~ = 193.7 ppm - 6cs2Scale (this table). The chemical shifts are accurate to 0.05 ppm. five of t h e s e c o m p o u n d s ( T a b l e I I I). T h e n o n d e u t e r a t e d oxytocin derivatives were known previously and t h e i r biological activities a r e reported in T a b l e 1V along with those of t h e other c o m p o u n d s whose I3C N M R s p e c t r a are examined here. The vasopressin diastereoisomers are newer compounds and t h e i r biological activities are still under investigation. The a s s i g n m e n t s of all of t h e '3C resonances of t h e five di-
a s t e r e o m e r i c derivatives of oxytocin a n d t w o diastereomeric derivatives of vasopressin (Table I 11) were m a d e by comparison with o u r previous results with oxytocin, vasopressin, a n d their analogues. In addition we utilized t h e results from o u r previous work with 13C-labeled derivatives of oxytocin and vasopressin44-47t o establish s o m e of t h e assignments. T h e r e w e r e a number of significant c h a n g e s in chemical shift, b u t except ior
Hruby et al.
1 I3CN M R Studies of Peptide Hormones
209
Table 111. Chemical Shifts and Assignments of Carbon Resonances of Amino Acid Residues of Oxytocin, [ 1-Hemi-~-[n-*H]Cys]oxytocin, [ I-Hemi-~-[P~-~H~]Cys]oxytocin, [2-~-[a-~H]Tyr]Oxytocin, [2-D-[P/3-*Hz]Tyr]Oxytocin,[6-Hemi-~-[n-~H]Cys]oxytocin. AVP, [ 1 Hemi-D-[a-*H]Cys]AVP,and [ 2-D-[ a-*H]Tyr]AVP in D 2 0 a
carbons
oxytocin
PD
4.9 150.60 140.07 153.20 168.15 170.55 171.98
[h-hemi-D2-D12-D[ I-hemi-D- [ I -hemi-D- [ n-*H]Cys]- [a-*H]Try]- [@@-‘HI] [a-*HICys]- [@@-*H2] oxytocin Cysloxytocin oxytocin Tyrloxytocin oxytocin 6.2 150.60 140.00 152.74 168.34 170.55 171.98
6.0 ..
6.1
5.9
5.2
6.2
6.I
150.60 140.07 152.84 168.01 170.55 172.12
150.52 139.90 153.16 168.18 170.45 171.82
150.41 139.89 153.15 167.64 170.12 171.62
150.47 140.13 153.59 168.21 170.48 172.17
150.56
150.66
150.6 I
139.06 164.73 168.28 152.13 36.82 131.98 163.30 167.64 144.56
139.16 164.83 168.28 152.16 37.04 132.14 163.27 168.28 144.68
139.1 1 164.73 168.24 152.10 37.08 132.02 163.26 167.22 144.73
14 I .40 153.94 142.38 156.32 137.63 166.54 161.65
141.44 154.37 142.28 155.80 138.12 167.17 161.39
141.98 153.95 141.98 156.90 138.35 165.77 161.61
136.75 157.30 56.53 64.22 64.22 66.2 I 137.28 157.30 65,55 62.45 77.70 39.03
137.63 156.90 57. I5 64.66 64.22 65.98
137.28 156.58 65.55 62.68 77.48 39.03
136.92 156.49 56.97 63.95 63.95 65.77 137.41 156.32 65.55 62.64 77.21 38.8 I
155.70 65.55 62.89 77.70 39.03
140.52 153.59
140.10 152.29
152.16
139.12 152.10
132.07 163.34 168.08 144.75
I 3 I .94 163.43 167.80 144.92
132.04 163.24 168.18 144.7 1
132.1 I 163.14 167.64 144.88
13 1.04 163.27 168.21 144.42
141.37 154.44 142.34 156.7 1 137.54 166.9 I 161.65 132.72 156.7 1 168.34 177.83 182.06
141.25 I 54.04 142.02 156.45 138.25 166.6 I 161.39 132.72 157.36 168.08 177.37 182.3 I
141.17 154.26 141.02 156.45 138.18 166.65 161.38 132.79 157.16 168.01 177.65 182.74
141.61 154.53 141.79 156.47 139.90 165.80 161.55 132.70 I 57.52 167.88 177.92 182.6 I
141.79 153.72 141.79 156.59 139.58 165.61 161.46 132.95 157.48 167.64 177.63 182.43
140.53 153.59 142.21 156.58 137.9 I 166.91 161.52 133.18 156.58 168.21 177.76 182.19
136.92 156.66 65.38 62.96 77.48 39.25
140.46 153.40
152.74
AVP
6.0
132.07 163.47 168.15 144.75
137.28 156.7 I 65.10 62.89 77.26 39.20
[ I -hemi-D-
[n-?H]Cys]-2-D-[u->H]AVP TjrIAVP
136.82 156.60 65.10 62.45 77.13 38.58 140.07
137.15 157.00 65.67 62.67 77.48 39.24 139.90 151.84
65.77 62.45 77.48 38.94 139.89
Chemical shifts are reported in parts per million upfield from I3CS2 measured from internal 1.4-dioxane at 126.1 ppm from the I3CS2 resonance. On this scale, Me4Si (external) is 193.7 ppm upfield from I3CS2.Conversion to the MejSi scale then is bc = 193.7 ppm - 6cs2*tale (this table). The shifts are accurate to 0.05 ppm, Obtained from I3C Y M R spectrum of [6-hemi-D-[d.~-’H~]C~s]ox~.tocin at pD 6.0. Otherwise spectra were virtually identical
a I-ppm downfield chemical shift for the Pro-7 Coc in [6hemi-D-cystine]oxytocin, none of these were on the tripeptide side chain residues, which were essentially unchanged throughout. Changes, of course, were seen at the D residues. In the D-Half-Cys-1 derivatives both the (Y and /3 I3C resonances are downfield from their positions in oxytocin, but both are quite pH dependent,45 47 and since they move upfield with an increase in pH, the effects seen in Table 111 are not as great as seen at identical pH conditions. The other major chemical shift differences seen were at the residues adjacent to the D residues. For example, in [2-D-tyrosine]oxytocin the HalfCys- 1 C u and C/3 were shifted downfield about 0.6 and I .6 ppm, respectively, relative to their chemical shift positions in oxytocin. Interestingly the Ile-3 carbon resonances were hardly affected except for the Cp. I n [2-D-tyrosine, 8-arginineIvasopressin, however, the Half-Cys- 1 C u was shifted downfield about 1 ppm, but the C p was hardly changed relative to its
positions i n AVP. On the other hand, the Phe-3 C(Ymoved upfield about 1 ppm and the C$ about 0.5 ppm upfield relative to their positions in AVP. Interestingly the largest chemical shift observed was for the Gin-4 C a in [2-D-tyrosine]oxqtocin, which was over 2 ppm upfield from its position in oxytocin. Even the CB of Gln-4 was about 0.8 ppm dorrtgield from its position in oxytocin. I n general, however, except for chemical shift changes due to stereochemical changes at adjacent residue, there were only minor shifts. It should be emphasized that for these compounds, however, some of the C a and Cp’ ‘1ssignments must be viewed as tentative. Discussion Although assignment of the I3C N M R spectra of peptides of the structural complexity of oxytocin, arginine vasopressin and its derivatives and analogues to specific carbon atoms of individual residues is often possible by reference to correlation
210
Journal of the American Chemical Society
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/ January 3, 1979
Table IV. Biological Activities of Oxytocin, Arginine Vasopressin, Mesotocin, Isotocin, Glurnitocin, Aspartocin, and Analogues
compd
oxytocin
AVD
biological activities, units/mg pressor
oxytocin arginine vasopressin inesotocin
546 f 1 8 a 12 f 0.2'
507 f 23b 100 f 15'
3.1 f O . l a 487 f 1 5 c
2.7 f 0.2a 501 f 53'
291 f 21e
502 f 37'
6.3 f 0.8'
1.1 f 0 . 1 '
145 f 12f
310 f 15J
0.06 f 0.0 I f
0.18 f 0.03f
isotocin g I u m i toci n
antidiuretic
milk ejecting 410 f 16' 116 f 19d 30 - 120' 330 f 21 471 f 7 0 d 219 f 15f
-8f
531
XI f 6 d 298 f 128g
aspartocin 107 f 29g oxypressin 20h [ 1 -hemi-D-cystine]oxy- -1.9 f 0.1'
201 f 12g 30h -0.02'
0. I 3 f 0.03g 3h
