carbamylation of amino and tyrosine hydroxyl groups · carbamylation of amino and tyrosine hydroxyl...

THE JOURNAL OF BIOLOGICAL CHEMIWTRY Vol. 242, No. 7, Issue of April 10, pp. 1579..1591, 1967

Printed in U.S.A.

Carbamylation of Amino and Tyrosine Hydroxyl Groups

PREPARATION OF AJS INHIBITOR OF OXYTOCIN WITH NO INTRINSIC ACTIVITY ON THE JSOLATED UTERUS

(Received for publication, March 29, 1966)

DEREK G. SMYTH

From the National Institute for Medical Research, Mill Hill, London, N.W. 7, England

SUMMARY

The reactions of cyanate with model compounds containing amino and tyrosine hydroxyl groups have been studied in detail. The reactions involve nucleophilic addition of the amino or phenoxide group to the molecular form of cyanic acid. The proposed mechanism of the reactions has aided in the selection of optimal experimental conditions for the carbamylation of amino and tyrosine hydroxyl groups in proteins and peptides. Carbamylation represents a useful reaction for the reversible blocking of tyrosine hydroxyl groups, the 0-carbamyl substituent being readily removed by hydrolysis at neutral and alkaline pH values. By application of the cyanate reaction to oxytocin, two new analogues were obtained, IV-carbamyl-0-carbamyloxytocin and N-carbamyl- oxytociu. The sites of reaction and degree of purity were determined by experiments involving degradation of the molecules and by the use of radioactive labeling. On the isolated uterus, IV-carbamyl-0-carbamyloxytocin acts as an inhibitor of oxytocin without itself possessing oxytocic activity; Wcarbamyloxytocin is a feebly active analogue with no inhibitory properties. At pH 7.4 and 37’, the inhibitory analogue undergoes slow hydrolysis to yield the weakly active analogue. The very low activity of IV-carbamyloxytocin provides an explanation for the inactivation of oxytocin by 8 M urea.

During the course of an investigation on the amino acid se- quence of ribonuclease (I), the reaction of cyanate with the amino groups of lysine residues in the S-Peptide was employed to facilitate analysis of the linear structure. The strong interaction that occurs between the ZO-residue S-Peptide and the 194- residue S-Protein of ribonuclease has been discussed as a plausible analogy for the binding of a peptide hormone to its physiological receptor (2). Experience in previous studies on the cyanate end group method (3) and on ribonuclease has stimulated the present investigation in which the cyanate reaction is applied to the peptide hormones. From g-residue peptides containing one NHz- terminal group and one hydroxyl group, carbamyl analogues should be obtainable in the high state of purity necessary for study of the relation of structure to activity at the molecular

level. In order to select the optimum experimental conditions for the preparation of carbamyl derivatives, the reactions of cyanate with model compounds have been studied in detail and are described in this communication.

OH

$3 o=c

9 ? 4”z ? ,CH2-CH-C - NH-CH-C - Ile

s

k I

‘CH,CH-NH- AspNH, - GIuNH,

c=o I Pro - Leu - GlyNH,

?+ 0-q

0

yH2 o=c

qH ? 0

FH2 ? ,CH2-CH-C - NH-CH-C - Ile

5

h I

‘CH,-!H-NH- AspNH, - GIuNH,

c=o

Pro - Leu - GlyNH,

The preparation, characterization, and bioactivity of two new analogues of oxytocin, N-carbamyloxytocin and N-carbamyl-O- carbamyloxytocin, are reported. The finding that N-carbamyl- 0-carbamyloxytocin is an inhibitor of oxytocin without itself stimulating oxytocic activity on the isolated uterus may enable a distinction to be made between structures involved in binding of the hormone to the uterine receptor and structures necessary for activation at the receptor site.

1579

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1580 Carbamylation of Oxytocin Vol. 242, No. 7

50 70 90 110 130 150 170 190 210 230

MA of effluenl (ml.) I -I- t A -I- l5-

FIG. 1. Chromatography of 0-carbamylphenol (0-Cb Phenol). A standard mixture of amino acids (0.2 pmole) was chromatographed at 56’ with 0.7 rmole of 0-carbamylphenol on a column (55 X 0.9 cm) of IR-120 with 0.2 M sodium citrate, pH 3.26 (A) and pH 4.25 (B), as eluent, according to the method of Spackman, Stein, and Moore (8).

EXPERIMENTAL PROCEDURE

Materials

Sodium cyanate and potassium cyanate were obtained from British Drug Houses, Ltd. Potassium cyanate-r4C (7.0 mC per nnnole) was obtained from the Radiochemical Centre, Amersham, England; the compound, isolated from urea-r4C by interaction with potassium n-butoxide (4), was free from cyanide. Amber- lite IRC-50 and Dowex 2-X4 ion exchange resins, analytical grade, were obtained from British Drug Houses Ltd. L-Leucyl- n-leuciner was synthesized according to the method of Vaughan and Osato (5), and L-tyrosyl-n-tyrosiner according to the method of Bergmann et al. (6). Glycylglycylglycine was purchased from Mann Research Laboratories. The purity of the peptides was checked by paper chromatography and by ion exchange chromatography before and after hydrolysis. 0-Carbamylphenol (m.p. 144-145”) was prepared by the method of Loev and Kormendy (7) by the heterogeneous reaction of sodium cyanate with phenol in ether solution in the presence of trifluoracetic acid at 4”, and was recrystallized from water. The elution of 0-carbamylphenol at 50” from a column (55 x 0.9 cm) of Amberlite IR-120 with 0.2 M citrate pH 3.26 as eluent was found to occur at 90 ml, between proline and glycine (Fig. 1).

Synthetic oxytocin was obtained as generous gifts from Dr. K. J&t and Dr. J. Rudinger of the Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Science, Prague (300 units per pmole); from Dr. E. D. Nicolaides of Parke Davis Company, Ann Arbor, Michigan (450 units per rmole); and from Dr. M. Taeschler and Dr. B. Berde of Sandos Ltd., Basle, Switzerland (450 units per pmole). In the first case, the peptide was chromatographed and desalted on a column (150 x 0.9 cm) of Sephadex G-25 with 50% acetic acid as eluent, and was used without further purification. The amino acid composition, determined by acid hydrolysis and quantitative amino acid analy-

1 The peptides n-leucyl-n-leucine and n-tyrosyl-n-tyrosine were synthesized by D. G. S. in the laboratories of Professor J. S. Fruton, to whom I am indebted, in the Biochemistry Department of the Medical School of Yale University, New Haven, Connecti- cut.

sis, was in accord with the theoretical values except that 5% of allo-isoleucine was observed. Solutions of deamino-oxytocin (0.05 pmole per ml) and deamino-deoxy-oxytocin (0.1 pmole per ml) were generously donated by Professor V. du Vigneaud of Cornell University Medical College, New York.

Methods

Reactions at Constant pH-Solutions were maintained automatically at constant pH in an autotitrator, Radiometer TTTl.

Amino Acid Analysis-Acid hydrolyses were performed in 6 N

hydrochloric acid for 16 hours at 110” in sealed, evacuated tubes (9). Before hydrolysis, air was removed from the hydrolysis mixture by immersing the tube in ethanol-solid carbon dioxide to freeze the sample and evacuating with the aid of an oil diffusion pump to a pressure of 0.5 mm of mercury. The tube was removed from the coolant to allow the sample to thaw slowly under the reduced pressure and then was sealed. After the hydrolysis, hydrochloric acid was removed by rotary evaporation. The residues were dissolved in 0.2 M sodium citrate at pH 2.2 and the solutions were analyzed with the aid of an automatic acid analyzer equipped for measurement of 0.01 pmole of each amino acid (Evans Electroselenium, Ltd., from the original design of Spackman, Stein, and Moore (8)). Alkaline hydrolyses were performed by maintaining solutions in 2.5 N NaOH at 110” for 4 hours in polypropylene tubes. The residues were neutralized with 2.5 N acetic acid and ninhydrin analysis was performed on the resulting solutions at pH 5.

Electrophoretic Analysis-Electrophoresis was performed at room temperature in 0.2 M pyridine acetate, pH 4.8, on What- man No. 1 paper with a voltage gradient of 20 volts per cm. 0-Carbamyl compounds were located by spraying the dried papers with a solution of 0.2yo ninhydrin in 0.1 M acetate buffer at pH 5.3 and heating at 100” for 15 min. This procedure was necessary to ensure release of ammonia by hydrolysis of the 0-carbamyl substituent to give a ninhydrin-positive reaction.

Absorption Spectra-Absorptions over the range of 225 to 325 rnp were measured and recorded automatically on an ultraviolet spectrophotometer (Unicam SPSOO); absorption at single wave lengths was measured on a Unicam SP500 spectrophotometer.


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Issue of April 10, 1967 D. G. #myth 1581

Bioassay-Oxytocic activity was measured on the isolated rat uterus under standard conditions (10) except that Munsick solution (11) was employed. Before use, the solution was thor- oughly saturated with 95% oxygen-5y0 carbon dioxide and pos- sessed a measured pH of 7.5. In the testing of N-carbamyl-O- carbamyloxytocin, it was found critical to ensure that the pH of the solution did not rise above 7.7 in order to prevent significant hydrolysis of the 0-carbamyl substituent from taking place during the assay. The uterus was obtained from rats in which estrus had been induced by prior injection of stilbestrol. Solu- tions of analogues were examined for inhibitory properties by exposure for 1 min to the uterus in a lo-ml tissue bath at 30”; oxytocin was then added without washing of the tissue.

Radioactive Assay-Infinitely thick samples were counted in solution on 2-sq cm circular polythene planchets under a thin window Geiger-Mueller counter. Infinitely thin samples were added to discs of lens tissue fitted into the polythene planchets and were dried in a vacuum before counting. A polythene disc (1 /.L! per g), supplied by the Radiochemical Centre, was used as a I%! standard.

RESULTS

Reaction oj NHe-terrnind Groups uith Cyanate

In order to select the optimum reaction conditions to favor specific carbamylation of the amino group of oxytocin, the rates of reaction of sodium cyanate with an amino acid, two dipeptides, and a tripeptide were determined.

The peptide or amino acid (4 mg) was dissolved in 2 ml of water, 2 ml of 0.4 M sodium cyanate was added, and the reaction mixture was maintained at pH 6.0 and 30” by automatic addition of 2 M acetic acid to the magnetically stirred solution. At intervals, portions (0.2 ml) were added to 2 ml of 0.2 M sodium citrate at pH 2.2 (which destroys cyanate) and the unreacted peptide or amino acid was determined by chromatography at 50” on a column of IR-120 of an automatic amino acid analyzer. Glycyl- glycylglycine emerged from a column (50 x 0.9 cm) at 75 ml with 0.2 M sodium citrate, pH 4.25, as eluent; L-leucyl-L-leucine emerged from a column (5 X 0.9 cm) at 23 ml with 0.2 M sodium citrate, pH 4.25, as eluent; L-tyrosyl-L-tyrosine emerged from a column (15 X 0.9 cm) at 25 ml with 0.4 M sodium citrate, pH 5.28, as eluent; phenylalanine and tyrosine emerged from a column (15 x 0.9 cm) at 18 ml with 0.2 M sodium citrate, pH 4.25, as eluent. In experiments involving the use of 1 M formaldehyde, the samples of the reaction mixture were freed from formaldehyde by concentration in a vacuum before amino acid analysis, and control experiments were performed to ensure that, under the conditions used, formaldehyde does not cause disappearance of phenylalanine or of alanine in the absence of cyanate.

In the presence of 0.2 M sodium cyanate at pH 6.0 and 30”, phenylalanine (pK 9.1) exhibited a half-life of 145 min; leucyl- leucine (pK 8.3), glycylglycylglycine (pK 7.9), and tyrosyltyro- sine (pK 7.7) (12) exhibited half-life values of 19, 13, and 11 min, respectively; oxytocin (pK 6.3) (13) under the same conditions exhibited a half-life of about 4 min (Fig. 2). The rate of reaction and pK value of the NHz-terminal group were thus inversely related. At pH 6 the concentration of the uncharged amino group of an amino acid is very small, that of a peptide is higher, and of oxytocin the highest. The reaction rates are consistent

R-NH, + H-N=C=O + HN=C-OH = H2N-C=O

kHR NHR

MINUTES

FIG. 2. Rates of reaction of 0.2 M cyanate with NHz-terminal compounds. Reactions were performed at pH 6.0 and 30” as described in the text.

with a mechanism of action involving nucleophilic addition of the uncharged amino nitrogen to cyanic acid, the reactive form of sodium cyanate. Indirect evidence favoring this mechanism has also been presented by Stark (14), in a detailed study of the rates of reaction of amines with alkyl isocyanates and cyanate. That the reaction involves molecules and not ions is supported by the finding that the rates of carbamylation of phenylalanine and of alanine are increased when the reaction is performed in

RNHa+ + H+ + RNHs=: RNHCHzOH

the presence of 1 M formaldehyde, which appears to raise the effective concentration of an uncharged form of the NHrtermi- nal group.

In Table I is shown a calculation of the pH dependence of the rate of reaction of sodium cyanate with the NHTterminal group of an ammo acid. The concentration of cyanic acid (pK 3.8) decreases by a factor of 10 for each unit rise in pH whereas the concentration of the uncharged form of the amino group increases correspondingly. The rate of reaction of cyanate with the amino compound is proportional to the product of the concentrations of the reactive species at each pH value. With phenylalanine, for example, the rate would be expected to be constant over the pH range of 5 to 8, at pH 9 to occur at one-half the maximum rate, and at pH 10 at one-tenth the maximum rate (Fig. 3). With oxytocin, the rate of carbamylation of the NH&erminal group (pK 6.3) would be e.xpected to be at a maximum at pH


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1582 Carbamylution of Oxytocin Vol. 242, No. 7

5.3, to take place at one-half this maximum rate at pH 6.3, and at one-tenth the rate at pH 7.3.

These predictions correspond closely with the values obtained experimentally. The measured half-life of phenylalanine at pH 9.0 and 30” in 0.4 M sodium cyanate was 124 min; at pH 8.0, 69 min; and at pH 6.0, 75 min. The measured rates of carbamylation of tyrosine in 0.4 M cyanate at pH 8 and pH 6 showed half-life values of 78 min and 70 min, respectively. The similar rates of reaction of cyanate with phenylalanine and with tyrosine indicates that carbamylation at the hydroxyl group of tyrosine,

TABLE I Predicted pH dependence in reaction of 0.4 M sodium

cyanate with 0.002 M phenylalanine

The concentrations of cyanic acid and of the uncharged form of NH,-terminal group of phenylalanine were calculated assuming pK values of 4.0 and 9.0, respectively. The relative reaction rates were calculated as the product of the values given in Col- umns 2 and 3. No correction was made for the decomposition of cyanate at acid pH.

PH

4

5 6 7

8 9

10

T

Concentration of cyanic acid

.M

2 x 10-r 4 x lo-2 4 x lo-3 4 x lo-4 4 x lo-6 4 x 1cV 4 x lo-7

-

-

Concentration of uncharged NH%- terminal group

M

2 x lo-8 2 x lo-7 2 x lo-8 2 x lo-6 2 x 10-d

1 x lo-3 2 x 10-a

4 x lo-8 8 x 10-Q 8 X 1OP 8 x lo-9 8 x lo-9 4 x lo-9 8 x lo-‘0

if it occurs, must be much slower than carbamylation at the NHzterminal group.

To obtain information on the reaction of the NHz-terminal group of oxytocin with cyanate, the rates of disappearance of biological activity of the hormone in the presence of cyanate were studied with the use of the following procedure. Syntocinon (1 ml, Sandoz Ltd., 10 oxytocic units; 0.02 Nmole of oxytocin) was diluted with 2 ml of water, and 3 ml of 0.08 M sodium cyanate was added. The solution was maintained at pH 6.0 and 30’ for 2 hours by automatic addition of 3 M sodium dihydrogen phosphate. At intervals of 30 min, portions (0.4 ml) were removed in duplicate and were diluted with 5 ml of 0.02 M sodium phosphate at pH 8.0. Residual oxytocic activity in these solutions was determined by bioassay on the isolated rat uterus. The same procedure was employed in determining the rates of inactivation of oxytocin, deamino-oxytocin, and deamino-deoxy- oxytocin. To confirm that the reaction of cyanate with oxytocin was stopped by diluting and raising the pH to 8.0, a portion (0.4 ml) of a solution of 0.2 M sodium cyanate was diluted with 5 ml of 0.2 M sodium phosphate, pH 8.0, and 0.5 ml of Syntocinon solution (5 units) was added; bioassay of the solution before and after storage for 4 hours at 30” confirmed that no loss of activity occurred.

It was observed that the relative rates of inactivation of oxytocin over the pH range of 5 to 8 (Table II) correspond approxi- mately with the predicted pH dependence for the reaction of cyanate with the NHS-terminal group (Fig. 3), and the results are in accord with the assumption that loss in activity is asso- ciated principally with carbamylation at this group. In addition, deamino-oxytocin (which contains a tyrosine hydroxyl group but no NH2 terminus) showed no detectable loss of activity

FIG. 3. Predicted pH dependence in the reaction of cyanate with the NHI-terminal groups of phenylalanine and oxytocin. The series of values calculated for the reaction of cyanate with oxytocin are not to be compared quantitatively with the values calculated for the reaction with phenylalanine. No correction has been made for the decomposition of cyanate at acid pH.


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Issue of April 10, 1967 D. G. Smyth 1583

when exposed to 0.04 M cyanate for 2 hours at pH 6. That these conditions resulted in almost complete inactivation of oxytocin is further evidence that inactivation of the hormone by cyanate is caused principally by a reaction at the NHz-terminal group. It must be noted, however, that a loss of activity of up to 5% would not be detected by bioassay and therefore this procedure can provide only an approximate indication of the specificity of the reaction.

Reaction oj NH&O-terminal Groups with Cyan&

The reaction of cyanate with urea to form biuret was studied as a model system to show the possibility of linear carbamylation of an N-carbamyl peptide to form an N-carbamocarbamyl peptide. The rate of this reaction would be expected to increase by a factor of 10 for each unit fall in pH.

NHzCONHR + HNCO + NHzCONHCONHR

Urea (50 mg) was dissolved in 5 ml of water, and 5 ml of 2 M

sodium cyanate were added. A portion (0.2 ml) of the reaction mixture was added to 1 ml of 1 N hydrochloric acid and the urea content was determined on the amino acid analyzer with a column (150 x 0.9 cm) of IR-120 operated at 50” with 0.2 M sodium citrate, pH 3.25, as eluent. A second portion (0.2 ml) of the reaction mixture was added to a column (5 x 0.4 cm) of Dowex 2-X4 (acetate form, washed with water) and elution was performed with 10 ml of water. The total eluate was concentrated to dryness in a vacuum, the residue dissolved in 2 ml of 0.2 M

citrate at pH 2.2, and the solution was analyzed for urea as above. The reaction mixture containing urea and 1 M cyanate was held at pH 6.0 and 30” by automatic addition of 5 M acetic acid. After 1 hour, a portion (0.2 ml) was removed, added to 1 ml of 1 N

hydrochloric acid, and the residual urea was determined; a second portion (0.2 ml) was removed, separated from excess cyanate on Dowex 2, and residual urea was determined. In a control experiment, 1 M cyanate was subjected to the same procedure in the absence of added urea. The amounts of urea formed were subtracted from the respective amounts found in the above experiment.

It was found that urea may be recovered quantitatively from a reaction mixture containing urea and 1 M cyanate provided cyanate is removed by ion exchange at neutral pH. When the cyanate was destroyed by acidification, a 10% decrease in the recovery of urea was noted. On the other hand, no carbamylation of urea could be detected when urea was maintained at pH 6 in 1 M sodium cyanate at 30” for 1 hour. At lower pH values, with the concentration of cyanic acid increasing lo-fold per pH unit, the conversion of urea to biuret (or of N-carbamyl peptide to N-carbamocarbamyl peptide) can be substantial.

In preparing carbamyl derivatives, it is important to avoid acidification in the presence of cyanate. The reagent can be removed on an anion exchange column at neutral pH or, in the case of a protein, by dialyzing against water or a neutral buffer at low temperature. In general, the concentration of cyanic acid should be maintained at a minimum and carbamylation should preferably be carried out at as high a pH value as is consistent with a convenient rate of reaction.

Reaction of Phenolic Hydroxyl Groups with Cyanate

To investigate the optimum conditions for formation of O-carbamyl derivatives of tyrosine residues in proteins and peptides, studies were performed on the carbamylation of phenol in aque-

TABLE II Rates of inactivation of oxytocin by cyanate

The rates were measured as described in the text.

Compound PH [NaCNOl Half-life for inactivation

Oxytocin

Deamino-oxytocin 6

Deamino-deoxy- oxytocin

6.5

.- M

0.04 0.02 0.2 0.02 0.08 0.1 0.2

0.04

1.0

min

17 30 4

100 27

180 75

No loss of activity in 2 hrs

No loss of activity in 4 hrs

2is 2io 360

MWhngth milliins

FIG. 4. Absorptions of phenol (0.85 pmole per ml) and of O- carbamylphenol (0-Cb Phenol) (4.2 pmole per ml) in water. Ab- sorptions were measured on a Unicam SP800 spectrophotometer.

ous solution. Synthetic 0-carbamylphenol, prepared by reaction of phenol with cyanate in an organic solvent, was found to possess only & the molar absorbance of phenol at 275 rnM (Fig. 4), and this feature was used to advantage in analyzing reaction mixtures containing phenol and 0-carbamylphenol. Further- more, 0-carbamylphenol was found to be ninhydrin-positive (due to release of ammonia by hydrolysis under the conditions of the ninhydrin reaction) whereas phenol is ninhydrin negative, and this allowed the concentration of 0-carbamylphenol to be determined by amino acid analysis (Fig. 1). Thus the rate of disappearance of phenol and the rate of formation of O-carbamylphenol were accurately measured.

Phenol (20 mg) was dissolved in 5 ml of water, and 5 ml of 2 M

sodium cyanate were added. The solution at 30” was maintained at pH 8, 7, 6.5, or 6 by automatic addition of 5 M acetic acid. At intervals, portions (0.5 ml) were removed, added to 4.5 ml of water, and acidified immediately to about pH 2 by addition of


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Carbamylation of Oxytocin Vol. 242, No. 7

Hours

FIQ. 5. Rates of formation of 0-carbamylphenol (0-G phenol) from phenol. Reactions were carried out at 30’ with 2 X 10-Z M phenol and 1 M sodium cyanate.

concentrated hydrochloric acid. (Cyanic acid, released from the cyanate at pH 2 is present at 10-I M concentration before rapid destruction; in the reaction mixture containing 1 M cyanate at pH 6, the cyanic acid concentration is lo-* M; and at pH 8, lo-’ M.) Residual phenol in each solution was determined after 5-fold dilution with Hz0 by measurement of absorption at 275 rnp, and 0-carbamylphenol in each diluted sample of the reaction mixture was determined by chromatography of portions (0.5 ml) at 50” on a column (15 X 0.9 cm) of the automatic amino acid analyzer with 0.4 M citrate, pH 5.28, as eluent. O-Carbam- ylphenol emerged at 24 ml of effluent, coincident with the elution position of the synthetic compound prepared by reaction in organic solvent.

When phenol was allowed to react with 1 M cyanate at 30”, the half-life of formation of 0-carbamylphenol was found to be in the region of 2 hours (Fig. 5). The initial rate was observed to be nearly the same at pH 6 as at pH 7, which is consistent with a mechanism involving cyanic acid and the negatively charged phenoxide ion. Thus the rate of formation of 0-carbamyl deriva-

\ \

tives may be expected to be independent of pH over the range of 5 to 9.

Hydrolysis of 0-Carbamylphenol

Base Hydrolysis-O-Carbamylphenol (7 mg) was dissolved by warming in 10 ml of 0.01 M acetic acid. The pH of the well stirred solution was cautiously adjusted to 7.4 by addition of 3 M

disodium hydrogen phosphate from a microsyringe, and the solution was maintained automatically at this pH and 37”. At intervals of 0, g, 1, 2, and 4 hours, portions (0.5 ml) were removed, added to 5 ml of HzO, and treated with concentrated hydrochloric acid (0.05 ml). Portions (1 ml) of the samples were analyzed at pH 5.28 and 50” on a column (15 x 0.9 cm) of the amino acid analyzer; other portions were used, after suitable dilution with Ha, for measurement of absorption at 275 mn (cf. Fig. 4).

Acid Hydrolysis-0-Carbamylphenol (13.7 mg) was dissolved in 10 ml of 0.1 M acetic acid. A portion (1 ml) was treated with 1 ml of concentrated hydrochloric acid, the mixture was frozen, the hydrolysis tube was evacuated, and the tube sealed without thawing of the sample. The solution was maintained at 110’ for 16 hours, then was frozen to condense acid and phenol from the upper walls of the tube. After the tube was opened, the con- tents were allowed to thaw. The solution was diluted lo-fold with Hz0 without removal of the hydrochloric acid, and phenol was determined by measurement of absorption at 275 mp. This procedure was necessary because of the volatility of phenol under the conditions normally used for removal of 6 N hydrochloric acid from hydrolysis mixtures. The yield of phenol released from 0-carbamylphenol under these conditions was quantitative. In a control experiment, phenol was submitted to the same conditions; the phenol was recovered without decomposition. In addition, 0-carbamylphenol was found to be completely stable when stored for 5 days at pH 2.0 and 30”.

The rapid hydrolysis of 0-carbamylphenol in neutral and alkaline solution (Fig. 6) enforces the use of a rather low pH value for the preparation of 0-carbamylphenol from phenol, but even at pH 6.5 in the presence of 1 M cyanate, 0-carbamylphenol undergoes slight hydrolysis (Fig. 7). In more acid solution, the

OOH e (-Jo.coNH2

instability of cyanate (half-life of 1 M cyanate at pH 6 and 30” is 65 min) prevents extended use of the reagent in high concentration at pH values below 5.5, and precipitation of cyanuric acid formed from the cyanate can interfere with the reaction. After 2 hours at pH 8, however, an 18% yield of 0-carbamylphenol can be obtained with negligible decomposition of cyanate. The conditions employed, therefore, for obtaining maximum yield of 0-carbamylphenol from phenol were 1 M sodium cyanate for 2 hours at pH 8.0, 3 hours at pH 7.0, 2 hours at pH 6.5, and then 2 hours at pH 6.0. Under these conditions, a 73% yield of 0-carbamylphenol was achieved (Table III), the remaining 27 % being accounted for as residual phenol. After 6 hours with this reaction procedure, a fine crystalline precipitate separated on cooling and sufficient material was obtained for identification by melting point and mixed melting point as 0-carbamylphenol. The reaction can be taken further by removal of the by-products

FIG. 6. Hydrolysis of 0-carbamylphenol (0-G phenol) at pH 7.4 and 37”. The rate of hydrolysis was measured as described in the text.


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derived from cyanate at acid pH and resubmitting the mixture TABLE III

to additional reaction with cyanate at pH 6.0. This over-all Rates of formation of 0-carbamylphenol from phenol, and

procedure involving stepwise alteration of pH is suitable for the of carbamylation at hydroxyl group of tyrosine, in

carbamylation of amino and tyrosine hydroxyl groups in linear presence of 1 M NaCNO at 90”

peptides where the groups are freely available, and will result in The rates of the reactions were measured as described in the

Phenol, initial E276

quantitative reaction at amino groups and up to 96% reaction text.

at tyrosine hydroxyl groups. PH HOWS

Reaction of Cyan&e with 0-Carbamyl Derivatives

The stability of 0-carbamylphenol in the presence of cyanate % was examined as a model system to test the possibility that ex- 8.0 0 100 posure of an 0-carbamyl peptide to cyanate might result in the 1 84 formation of an 0-carbamocarbamyl peptide. This reaction 2 80

would be favored at low pH. 7.0 3 61

0 O.CONHz + HNCO + 0 4 52

0.CONH.CONHz 5 46

0-Carbamylphenol (10.1 mg) was dissolved by warming in 2.5 ml of 0.01 M acetic acid, and 2.5 ml of 2 M sodium cyanate previously adjusted to pH 6 were added. The solution at 30” was maintained at pH 6.5, 7.0, or 8.0 by automatic addition of 5 M acetic acid. Samples (0.5 ml) were removed at intervals of 0, 1, 2, and 4 hours; each was added to 5 ml of water and was acidified to pH 2 with concentrated hydrochloric acid. Phenol

6.5 6 39 7 36

6.0 8 32 9 30

I

TABLE IV

-

%

0

12 18

% 100 90 87

41 60 48 57 51 52

62 66

70 73

45 43

35 37

Tyrosine- ydmxyl grow,

initial En0

was estimated after 5-fold dilution by measurement of absorption Hydrolysis of 0-carbamylphenol in presence of 1 AI

at 275 rnp. Residual 0-carbamylphenol was determined by cyanate at SO”

amino acid analysis; 0-carbamocarbamylphenol would not be The experimental details are described in the text. detected on the amino acid analyzer because the urea released from it (by hydrolysis in the analyzer coil) is only very weakly reactive to ninhydrin.

At pH 7.0, 6.5, and 6.0, no evidence of reaction of O-carbamylphenol with cyanate was found. The disappearance of O-carbamylphenol at the higher pH values (Pig. 7) was accounted for by appearance of the corresponding amount of phenol formed by hydrolysis (Table IV). Similarly in the preparation of O-carbamylphenol from phenol and cyanate, the disappearance of phenol was accounted for quantitatively by the appearance of

PH Constituents of reaction

mixture

6.5

7.0

8.0

--

0-Carbamylphenol Phenol

0-Carbamylphenol loo 85 70 62 Phenol 17 25 34

0-Carbamylphenol 100 32 16 16 Phenol 63 79 78

0-carbamylphenol (Table III). I Jnc ler the conditions to be employed in the preparation of N-carbamyl-0-carbamyloxytocin, therefore, allophanate formation was not expected to occur to a significant extent. It was noted, however, that when a solution of 0-carbamylphenol containing 1 M cyanate at pH 6 was acidified to pH 2, some disappearance of 0-carbamylphenol (approxi- mateIy 20%) occurred, in contrast with the quantitative recovery of 0-carbamylphenol that was obtained when cyanate was removed without acidification. This result emphasizes that at the end of a carbamylation reaction, cyanate should not be destroyed by addition of strong acid. The excess reagent should be re-

Found

Ohr / lhr j2hrs 4 hrs

% % loo 95

4

%

93 5

% 87 12

pH 8-O moved by ion exchange or by dialysis against weak acid buffer at low temperature.

I I 0 1 2 3 4 Reaction of Cyanate with Tyrosine

Hours n-Tvrosine (3 mg) was dissolved in 5 ml of HzO, and 5 ml of

FIG. 7. Rates of hydrolysis of 0-carbamylphenol (0-Cb phenol) 2 M sbdium cyana& was added. The solution was held for 2

in the presence of 1 M cyanate. Reactions were performed at hours at pH 8.0 and 30” by automatic addition of 5 M acetic acid. 30’ with 2 X 10-* M 0-carbamylphenol and 1 M sodium cyanate. The pH was lowered to 7.0 for 3 hours, 6.5 for 2 hours, and then


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to 6.0 for 2 hours. At intervals of 1 hour, samples (0.5 ml) were removed, diluted with 5 ml of HtO, and concentrated hydrochloric acid was added to pH 2. Residual tyrosine hydroxyl- group was estimated by measurement of absorbance at 280 rnh. Duplicate samples (0.5 ml) were removed from the reaction mixture and the solutions were acidified and analyzed at 50” on the amino acid analyzer with the use of a column (50 x 0.9 cm) operated at 50” with 0.4 M citrate, pH 5.28, as eluent.

when tyrosine was allowed to react with 1 M sodium cyanate at pH 8 for 1 hour and the solution acidified, amino acid analysis revealed the presence of two new ninhydrin-positive substances in addition to unreacted tyrosine (Fig. 8). Neither product was formed when phenylalanine was exposed to the same conditions, and in a control experiment omitting the tyrosine only the small intermediate peak (probably cyanuric acid or a carbamyl derivative of cyanuric acid) was seen. When carbamylation was carried out at pH 7, Peak I was found to increase to a maximum at 4 hours and Peak II rose to a maximum at 1 hour and then de- clined. On a preparative scale, the two derivatives of tyrosine were isolated from a reaction mixture at pH 7 by chromatography on a 150-cm column. Crystallization occurred on cooling of fractions containing Peak II. Evidence was obtained that

FIG. 8. Chromatography of N-carbamyltyrosine (Peak I) and 0-carbamyltyrosine (Peak II) derived from tyrosine and cyanate. Tyrosine (1.6 X 10-a M) was allowed to react with 1 M sodium cyanate at pH 8 and 30” for 1 hour; chromatography was performed at 50” on a column (56 X 0.9 cm) of IR-120 with 0.4 M sodium citrate, pH 5.28, as eluent.

Peak I contained N-carbamyl-0-carbamyltyrosine and that Peak II contained 0-carbamyltyrosine.

Both substances were found to be stable at pH 2 and 30” for 24 hours. When the material in Peak II was maintained at pH 8.0, on the other hand, the compound was found to disappear rapidly and free tyrosine was formed (Fig. 9). The rate of disappearance of 0-carbamyltyrosine was noted to equal the rate of appearance of tyrosine. Similarly, Peak I underwent rapid hydrolysis in neutral or alkaline solution but no ninhydrin-positive product was observed (N-carbamyltyrosine is ninhydrin- negative; N-carbamyl-0-carbamyltyrosine is ninhydrin-positive because ammonia is released by hydrolysis of the 0-carbamyl group at 100” during the ninhydrin reaction). The 0-carbamyl derivatives are esters of carbamic acid and it is no surprise to find that they are comparatively stable at acid pH and that they undergo hydrolysis easily in mild alkali. Further support for the structures proposed for the materials in Peak I and Peak II was obtained from the results of electrophoresis at pH 4.8 and of chromatography at pH 3.2, 4.2, and 5.3.

As a direct test for whether the NHrterminal group of each tyrosine derivative had been carbamylated, N-carbamyl-O-carbamyltyrosine (Peak I) and 0-carbamyltyrosine (Peak II) were subjected to the acidic conditions suitable for cyclization to tyrosine hydantoin. Only an N-carbamyl derivative is capable of forming a hydantoin.

c ONHl CO-NH

N~c~cooH

I I N? p”

OH’ Nq tooH ,

CH,

iH H+ (:

’ P HZ

H i4 OH

2

The resulting solutions were added to a column of Dowex 50 (H+ form), which was eluted with water. The eluates were concentrated to dryness and submitted to alkaline hydrolysis (cf. Reference 3). The eluate containing tyrosine hydantoin would thereby give rise to free tyrosine. Peak I yielded tyrosine, confirming that it contained an N-carbamyl compound; Peak II did not give tyrosine, as would be expected since the free NH&erminal group of 0-carbamyltyrosine caused the compound to be retained on the Dowex 50 column.

Reaction of cyanate with tyrosine thus results in the competi- tive formation of two different, monosubstituted derivatives. Both N-carbamyltyrosine and 0-carbamyltyrosine undergo further reaction to yield N-carbamyl-0-carbamyltyrosine. The 0-carbamyl substituents undergo hydrolysis at neutral and alka-

N-carbamyltyrosine

/ \ Tyrosine

\

N-carbamyl-0-carbamyltyrosine

/ 0-carbamyltyrosine

line pH values. N-Carbamyl-0-carbamyltyrosine formed from tyrosine and 1 M cyanate at pH 7 was obtained in 45% yield, being present in equilibrium with 55% of N-carbamyltyrosine.


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Issue of April 10, 1967 D. G. Xmyth 1587

FIG. 9. Hydrolysis of 0-carbamyltyrosine at pH 8.0 and 30”. Solutions were analyzed by chromatography at 50” on a column (50 X 0.9 cm) of IR-120 with 0.4 M sodium citrate, pH 5.28, as eluent.

When the reaction was performed with stepwise alteration of pH, a 65% yield of N-carbamyl-0-carbamyltyrosine was obtained (Table III). The reaction of cyanate with the NHz-terminal group of tyrosine (pK 9.2) is about 5 times as fast as the reaction at the hydroxyl group. The hydroxyl group of tyrosine residues in peptides possess pK values a little lower than the hydroxyl group of free tyrosine and would be expected to undergo carbamylation a little more rapidly.

The reaction of cyanate with tyrosine hydroxyl groups has not been reported previously. Chen, Grossberg, and Pressman (15) reported that tyrosine exposed to 1 M potassium cyanate at pH 8 for 17 hours did not undergo change in absorbance at 280 mn. On the basis of the present experiments, the reaction conditions used by Chen, el al. would result in equilibrium mixture containing about 15% N-carbamyl-0-carbamyltyrosine and 85?& N-carbamyltyrosine (see Table III). Failure to detect the 0-carbamylation reaction may have been caused by dilution of samples of the reaction mixture before measurement of absorption. In the diluted solutions at pH 8, hydrolysis of the O-carbamyl substituent would greatly exceed recarbamylation and the initial absorbance would be restored.

Preparation of N-Carbamyloxytocin

Oxytocin (5 mg) was dissolved in 6 ml of 0.04 M sodium cyanate, and the solution was maintained for 2 hours at pH 6.0 and 30” by automatic addition of 3 M sodium diiydrogen phosphate. On the basis of the measured rates of carbamylation of the model compounds (Fig. 2) and of the rates of inactivation of the hormone by cyanate (Table II), carbamylation of the NHzterminal group of oxytocin was expected to go nearly to completion and to occur about 200 times as fast as carbamylation at the tyrosine hydroxyl group. To remove residual cyanate, the solution was added to a column (5 x 0.4 cm) of Dowex 2-X4 in the acetate form and 10 ml of Hz0 eluate were collected. The resulting solution, which contained N-carbamyloxytocin together with

traces of N-carbamyl-0-carbamyloxytocin and oxytocin, was adjusted to pH 8.5 with 1 M sodium carbonate and was maintained at 30” for 2 hours. This procedure ensured that any dicarbamyl analogue was converted to N-carbamyloxytocin by

Oxytocin --t N-carbamyloxytocin ti N-carbamyl-0-carbamyloxytocin

mild hydrolysis. Hydrochloric acid (1 M) was then added to pH 3, and the solution (which contained N-carbamyloxytocin and traces of unreacted oxytocin) was added to a column (5 x

0.4 cm) of IRC-50 in the sodium form. N-Carbamyloxytocin was eluted at room temperature in a total volume of 20 ml of 0.05 M acetic acid. This step ensures removal of any unreacted oxytocin. Final purification and desalting of N-carbamyloxytocin were performed by gel filtration. The solution was concentrated in a vacuum down to about 2 ml, and was added to a column (150 x 0.9 cm) of Sephadex G-25 with 50% acetic acid as eluent; the peptide was located by alkaline hydrolysis and ninhydrin analysis of portions of alternate fractions. The acetic acid was removed by evaporation in a vacuum, the product was dissolved in 5 ml of Hz0 and stored at -15”. The concentration of the peptide was determined by acid hydrolysis and quantitative amino acid analysis; an over-all yield of 44% was obtained. Bioassay of this preparation of N-carbamyloxytocin on the isolated rat uterus (10, 11) showed 0.1% of the specific activity of oxytocin; no inhibitory properties were present.

In control experiments, the ability of the IRC-50 column to retain oxytocin was determined under the above conditions; when 1 mg of oxytocin was applied at acid pH, no detectable amino acids (less than 0.01 pmole) were found on acid hydrolysis and amino acid analysis of the total eluate from the column. To confirm that the preparation of N-carbamyloxytocin was free from unreacted oxytocin, a portion of the solution was passed through an additional column of IRC-50 under the standard conditions; no change in specific biological activity resulted. To


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1588 Carbamylation of Oxytocin Vol. 242, No. 7

confirm that the preparation of N-carbamyloxytocin was free from small amounts of N-carbamyl-0-carbamyloxytocin, a portion of the solution was incubated at pH 8.5 and 30” for 1 hour; bioassay again revealed no change in oxytocic activity.

Identiification of N-Carbamyloxytocin

N-Carbamyloxytocin (2.0 mg) was oxidized according to the method of Hirs (16). The product was maintained in 6 N hydrochloric acid at 100” for 1 hour to form the hydantoin and after removal of the hydrochloric acid in a vacuum, the residue was dissolved in water. The solution was applied to a column (10 x 0.9 cm) of Dowex 50-X2 prepared in the H+ form and washed with water. The first 30 ml of Hz0 eluate were concentrated to about 2 ml, and cyst&c acid hydantoin was isolated on a column (10 x 0.9 cm) of Dowex l-X8 operated at 25” with 0.2 N hydrochloric acid as eluent. The position of the eluted hydantoin, located by alkaline hydrolysis of portions of each fraction (2 hours at 110”) and by ninhydrin analysis (17), was identical with that of cysteic acid hydantoin prepared from carbamyl cysteic acid (3). Alkaline hydrolysis of the combined fractions which formed the peak from the Dowex 1 column was performed for 22 hours in 0.2 N sodium hydroxide at 110’. The released cysteic acid, determined by automatic amino acid analysis, was obtained in 52% yield.

Reaction of Oxytocin with Potassium Cyanate-14C

Oxytocin (2.0 mg) was dissolved in 3 ml of a solution containing 9.7 mg of potassium cyanateJ4C (activity specified as 0.47 PC per Bmole; as determined by radioassay, 0.46 PC per pmole), and the pH of the mixture was adjusted to 6.0 with 3 M sodium

dihydrogen phosphate. After incubation at pH 6 and 30” for 2 hours, the reaction mixture was freed from cyanate by washing through the column of Dowex 2, and freed from N-carbamyl-O- carbamyloxytocin by maintaining at pH 8.5 and 30” for 2 hours. The mixture was freed from residual oxytocin by addition to the column of IRC-50, and 20 ml of H20 eluate were collected. N- Carbamyloxytocin-14C was separated from low molecular weight by-products of cyanate by gel filtration on Sephadex G-25 with 50% acetic acid as eluent. (When 0.2 M acetic acid was used as eluent, no separation of radioactive peptide from small molecular weight material was obtained.) The elution position of the radioactive peptide, 38 to 68 ml, was located by radioactivity assay of infinitely thin samples from alternate fractions. The fractions containing the first radioactive peak were combined, concentrated to about 3 ml, and the solution made up to 5 ml with 50% acetic acid. A l-ml portion of this solution was submitted to acid hydrolysis and quantitative amino acid analysis. Three portions (0.2 ml) were added to 2-sq cm planchets and were dried in a vacuum; 0.2 ml of Hz0 was added to each for counting as infinitely thick samples. Appropriate dilutions of the initial potassium cyanate were assayed in the same way immediately before use in the carbamylation reaction. The specific radioactivity of the isolated N-carbamyloxytocin was found to be 0.47 PC per Imole.

Isolation of Cysleic AcidJ4C Hydantoin from N-Carbamyloxytocin-W

N-Carbamyloxytocini4C (0.4 pmole) was oxidized with per- formic acid according to the method of Moore (18). Cycliza- tion was performed in 6 N hydrochloric acid, and cysteic acid-W hydantoin was isolated from the reaction mixture as described

above. All the radioactivity applied to the Dowex 50 column appeared in the first 30 ml of Hi0 eluate. Infinitely thin samples from portions of alternate fractions from the Dowex 1 column were assayed; the position of the principal radioactive peak coin- cided with the elution position of cysteic acid hydantoin. The solution containing the combined fractions of this peak (30 ml to 44 ml) was found to contain 59% of the radioactivity of the N-carbamyloxytocin used, a yield similar to the yield of cysteic acid hydantoin obtained from nonradioactive N-carbamyloxytocin.

Treatment of N-Carbamyloxytocin with Potassium Cyanale-W

A solution of 2 mg of N-carbamyloxytocin in 2 ml of water was treated with 1 ml of a solution containing 9.7 mg of potassium cyanateJ4C (0.51 PC! per pmole; final cyanate concentration, 0.04 M). The reaction was allowed to proceed for 2 hours at pH 6 and 30”. Separation of the peptide from radioactive and other contaminants was effected by passage through the columns of Dowex 2 and IRC-50, exposure to pH 8.5 for 2 hours, and by gel filtration on Sephadex G-25. The peptide was located by alkaline hydrolysis and ninhydrin analysis, and radioactivity assay was performed on infinitely thin samples of the combined fractions containing peptide. No significant radioactivity could be detected.

In summary, the lack of retention of N-carbamyloxytocin by a cation exchange resin is evidence that the compound prepared possesses no free NH&erminal groups, and its oxidation to cysteic acid hydantoin in moderate yield proves that the NH2- terminal group was carbamylated. Further evidence for the site and specificity of the reaction was obtained with the use of potassium cyanate-i4C which demonstrated an equimolar reaction of cyanate with oxytocin, and degradation of N-carbamyloxytocin- 14C showed the radioactivity to be derived from a carbamylated half-cystine residue in the NHz-terminal position. Treatment of N-carbamyloxytocin with 0.04 M potassium cyanate-14C under the same conditions used in its preparation resulted in no detectable binding of radioactivity. In addition, deamino-oxytocin (which lacks a NH&erminal group and possesses a high biological activity) was not inactivated to a detectable degree by 0.04 M

cyanate. The results of these experiments constitute strong evidence confirming the structure and purity of N-carbamyloxytocin prepared by the procedure described in this communication.

Preparation of N-Carbamyl-0-Carbamyloxytocin

In view of the ease of hydrolysis of the 0-carbamyl substituent by base, the conventional methods of peptide synthesis from the constituent amino acids cannot be employed in the preparation of N-carbamyl-0-carbamyloxytocin. In addition, the use of alkaline pH values in the presence of sulfhydryl reagents, such as cyanate (19), is undesirable because of the possibility of hydrolysis at the disulfide bond of oxytocin.

Oxytocin (2.0 mg) in 1.5 ml of Hz0 was mixed with 1.5 ml of 2 M sodium cyanate. The pH of the solution was adjusted to and maintained for 3 hours at 7.0 and 30” by automatic addition of 5 M acetic acid. The pH of the solution was lowered to 6.5 for 2 hours, and then for an additional a-hour period the pH of the solution was maintained at 6.0. The reaction mixture was freed from residual cyanate by washing through a column (5 X 0.4 cm) of Dowex 2-X4 in the acetate form. The Hz0 eluate (10 ml) was immediately acidified to pH 2, concentrated in a


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FIG. 10. Bioassay of N-carbamyl-0-carbamyloxytocin on the isolated rat uterus. When the uterus was exposed at 30” for 1 min to N-carbamyl-0-carbamyloxytocin at a concentration of 2 X 10-6 pmole per ml and then to oxytocin at a concentration of

vacuum to about 2 ml, and 2 ml of glacial acetic acid were added. The resulting solution was transferred quantitatively to a column (150 x 0.9 cm) of Sephadex G-25, and 50% acetic acid was used as eluent. The reaction product, located by alkaline hydrolysis and ninhydrin analysis, emerged in a symmetrical peak at 70 ml of effluent and was free from salts. The acetic acid was removed by concentration in a vacuum, and a 95% over-all yield of peptide mixture was obtained. Although 0-carbamylphenol could be separated from phenol on a column of Sephadex G-25, at- tempts to separate N-carbamyl-0-carbamyloxytocin from N-carbamyloxytocin on this column were not successful.

To assess the extent of carbamylation at the tyrosine hydroxyl group in this preparation of N-carbamyl-0-carbamyloxytocin, the procedure was repeated with the use of radioactive cyanate and the extent of incorporation of isotope was measured. N-Car- bamyloxytocin was dissolved in 4 ml of 1 M potassium cyanate to which was added 2.0 mg of potassium cyanaW4C (resulting net specific activity, 0.050 PC per pmole). The pH of the reaction mixture was adjusted to 7.0 and the solution was treated in the manner described above for the preparation of N-carbamyl- 0-carbamyloxytocin. The specific activity of the isolated peptide mixture was 0.033 PC per pmole, which corresponds with 66% reaction at the tyrosine hydroxyl group of N-carbamyloxytocin. The reaction conditions employed in this preparation of N-carbamyl-0-carbamyloxytocin result in complete carbamyld- tion of the NHn-terminal group and about 70% carbamylation of the hydroxyl group. In addition, treatment of the active

8 X 10-T pmole per ml (4 milliunits/lO ml; molar ratio of inhibitor to oxytocin 25:1), the response of the uterus to 4 milliunits was inhibited to that normally given to 2 milliunits of oxytocin (A). At a molar ratio of 60:1, total inhibition was observed (B).

analogue deamino-deoxy-oxytocin (0.01 pmole per ml) with cyanate under the same conditions as were used in preparing N-carbamyl-O-carbamyloxytocin resulted in complete retention of the bioactivity, which confirms that the carbamylation of oxyto- tin involved only NH&erminal and hydroxyl groups.

On the isolated rat uterus, N-carbamyl-0-carbamyloxytocin prepared as described exhibited no oxytocic activity up to the highest concentration tested (10m3 pmole per ml), which is 1000 times the concentration of oxytocin used to elicit a normal response. When N-carbamyl-0-carbamyloxytocin was tested for inhibitory properties at an analogue to hormone ratio of 25 : 1, the response of the uterus to oxytocin (4 milliunits in 10 ml) was 50% inhibited; at a ratio of 60: 1, total inhibition occurred (Fig. 10). The ability of the uterus to recover its response to oxytocin was fully restored on incubation for 8 min at pH 7.4, with washing of the tissue; when higher concentrations of inhibitor were used, recovery of the uterus was delayed. Responses of the uterus to bradykinin and angiotensin were unaffected by 16 times the concentration of N-carbamyl-0-carbamyloxytocin necessary for total inhibition of oxytocin.

Since the activity of N-carbamyloxytocin is extremely weak, 0.1 y0 of the activity of oxytocin, the measured biological properties of this preparation of N-carbamyl-0-carbamyloxytocin on the isolated uterus are those of the dicarbamyl analogue; the inhibitory action of the N-carbamyl-0-carbamyloxytocin totally conceals the weak activity of the N-carbamyloxytocin present in the mixture. When N-carbamyl-0-carbamyloxytocin is main-


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tained for 2 hours at pH 8.5 and 30”, however, the inhibitory properties disappear and the product exhibits the weak stimu- lant activity of N-carbamyloxytocin.

DISCUSSION

In preparing a chemical derivative of a biologically active compound, it is essential that the required product be obtained free even from traces of contaminating biologically active material, lest a potent property of the contaminant conceal or color a weak property of the principal constituent. In an earlier experiment, a reaction mixture that contained 99% N-carbamyloxytocin and 1% N-carbamyl-0-carbamyloxytocin, as shown by measurements with radioactivity, was found to possess no oxytocic activity on the isolated uterus but exerted a weak inhibitory action against oxytocin, and these properties were erroneously attributed to the monocarbamyl analogue (20). Removal of the small amount of N-carbamyl-0-carbamyloxytocin from the mixture is found to eliminate the inhibitory properties and uncover the weak stimu- lant activity of N-carbamyloxytocin. In the preparation of N-carbamyl-0-carbamyloxytocin, on the other hand, although the level of contamination by N-carbamyloxytocin is rather high, the chemical nature of the contaminant and its very weak activity are known with precision; the net biological property exhibited on the isolated uterus i.e. of inhibition without oxytocic activity, is that of N-carbamyl-0-carbamyloxytocin.

In order to anticipate and control the reaction of cyanate at a site other than the NH&erminal group of oxytocin, the study with model compounds has proved invaluable. The rates of formation and the ease of hydrolysis of 0-carbamylphenol and 0-carbamyltyrosine, learned from these experiments, opens the way to general application of this new reversible reaction to tyrosine residues in proteins and peptides. Carbamylation of amino and tyrosine hydroxyl groups can now be usefully applied in studies on the “active sites” of enzymes and other proteins.

Where an 0-carbamyl derivative possesses a potent biological property not exhibited by the parent hydroxyl compound, as in the present experiments, exposure to pH 7.4 and 37” (physiological conditions of pH and temperature) sets a predictable limit to the persistence of this property. Thus the inhibitor, N-carbamyl-O-carbamyloxytocin, gradually loses its inhibitory powers at pH 7.4 and 37” and releases N-carbamyloxytocin, a weakly active analogue with no inhibitory properties. To avoid un- wanted hydrolysis, therefore, care must be taken in the handling of N-carbamyl-0-carbamyloxytocin during experimental work. In general, the hydrolysis of 0-carbamyltyrosine derivatives at pH 7.4 might be exploited to advantage. If an 0-carbamyl compound were applied as a drug and the parent hydroxyl compound were inactive, then the action of the drug could be expected to have a limited duration. Alternatively, where an 0-carbamyl derivative is comparatively inert and the parent hydroxyl compound highly active, exposure to physiological conditions of temperature and pH may result in a slow generation of biological activity. For example, incubation of a crude preparation of deamino-O-carbamyloxytocin at pH 7.4 and 37” is found to be accompanied by the protracted release of biological activity,2 as measured on the isolated uterus. The relatively inactive compound deamino-O-carbamylo@ocin has the potential to act as a hormonogen in viva by releasing the high activity of deamino- oxytocin over a period of time.

z D. G. Smyth, unpublished experiments.

The very low activity of N-carbamyloxytocin as well as the ease with which oxytocin undergoes inactivation in the presence of 0.04 M cyanate (21) could explain the irreversible inactivation that takes place when oxytocin is exposed to 8 M urea (22). Since a concentration of 0.02 M cyanate is present at equilibrium in 8 M urea (23, 24), the inactivation by urea could be attributed to

NHZ-C-NH2 s NHh+ + CNO’

b

a chemical reaction between oxytocin and cyanate rather than to a physical effect involving change in molecular conformation. It was, in fact, cautioned in the original report on the inactivation of oxytocin by urea that chemical change should be ruled out before concluding that inactivation is caused by change in spatial structure.

That N-carbamyloxytocin possesses very weak oxytocic activity with no inhibitory properties on the isolated uterus is in accordance with the similar properties exhibited by other derivatives of oxytocin in which the NH&erminal group is modified (25-27). Deamino-oxytocin, on the other hand, is highly active (28). The amino group of oxytocin appears not to perform an essential role per se in the over-all activity of the hormone, either in binding or in stimulation of activity. This group, however, must occupy a region in the oxytocin molecule that is disturbed by the introduction of even a small sub&ituent. The very weak activity of N-carbamyloxytocin, compared with oxytocin, could be caused by steric hindrance introduced directly between the analogue and that area of the receptor involved in binding, or between the analogue and that area of the receptor involved in slimulation. In either case, the carbamyl substituent might have an adverse effect on a particular conformation of the hormone that is required for a specific association with the receptor. By measurement of diffusion rates during dialysis, however, Craig found little difference in the preferred conformations of oxytocin and N-carbamyloxytocin in the absence of a tissue receptor (29).

That N-carbamyl-0-carbamyloxytocin acts as a powerful inhibitor of oxytocin without stimulating contraction of the isolated uterus, exerting 50% inhibition of the activity of oxytocin at an analogue to hormone ratio of about 25 : 1, indicates that this compound retains an ability to bind to the receptor and must possess a topography suitable for binding. Further studies on this interesting analogue will delineate the range of conditions over which the inhibitory properties are effective.

AckncMuZedgments-I wish to thank Dr. K. Jijst and Dr. J. Rudinger of the Institute of Organic Chemistry and Biochemis- try, Czechoslovak Academy of Science, Prague, Dr. E. D. Nico- laides of Parke Davis Ltd., Ann Arbor, Michigan, and Dr. M. Taeschler and Dr. B. Berde of Sandoz Ltd., Basle, Switzerland, for generous gifts of oxytocin; and Dr. V. du Vigneaud for gifts of deamino-oxytocin and deamino-deoxy-oxytocin. Miss M. Ellis has given valuable technical assistance.

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Derek G. SmythTHE ISOLATED UTERUS

AN INHIBITOR OF OXYTOCIN WITH NO INTRINSIC ACTIVITY ON Carbamylation of Amino and Tyrosine Hydroxyl Groups: PREPARATION OF

1967, 242:1579-1591.J. Biol. Chem.

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http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/242/7/1579.full.html#ref-list-1

http://www.jbc.org/

carbamylation of amino and tyrosine hydroxyl groups · carbamylation of amino and tyrosine hydroxyl...

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