ANALYTICAL

BIOCHEMISTRY

69, 339-349 (1975)

The Radioactive lodinated

Labeling Amidination

of Proteins

with

an

Reagent

FREDERICK T. WOOD, MICHAEL M. WV AND JOHN C. GERHART School of Dentistry, University of Pacijic, San Francisco, California 94115, and the Department of Molecular Biology and Virus Laboratory, University of California, Berkeley, California 94720

Received February 3, 1975; accepted June 20, 1975 An iodinated derivative of the imidoester methyl p-hydroxybenzimidate

HCI

(MPHBIM) has been synthesized for the selective labeling of proteins to high specific activity with radioactive iodine. In the first step, MPHBIM is reacted with radioactive iodide in the presence of chloramine T, and the iodinated derivative is precipitated from acidified solution to achieve partial purification. In the second step, the iodinated imidoester is redissolved at slightly alkaline pH and reacted with protein amino groups, to which it couples by amidine linkage. The coupling reaction proceeds in the presence of sulfhydryl reagents used to protect proteins. The main advantage of this two-step labeling procedure is that it avoids direct contact of the protein with potentially deleterious materials such as chloramine T or contaminants of the radioactive iodide.

Several techniques for iodinating proteins have been developed, based on the direct substitution of lz51 onto tyrosyl residues (1,2). These methods involve the use of oxidizing agents which in some cases lead to inactivation of the proteins, presumably by side reactions such as sulfhydry1 oxidation. Recently, Bolton and Hunter (3) and Rudinger and Ruegg (4) reported the synthesis and use of an lz51-labeled acylating agent which is an efficient reagent for introducing radioactive iodine atoms into proteins while eliminating direct contact of the protein with the iodination oxidant. As a further advantage, this reagent allows the introduction of radioactive iodine into proteins and peptides not containing tyrosine, because of its high reactivity toward amines and other nucleophilic groups. We have independently synthesized a comparable reagent, a phenolic imidoester iodinated with lz51, which we report here as having the same advantages as the Bolton and Hunter compound, but which differs from their acylation reagent in the following ways: it is less reactive, and moderately stable in aqueous solutions, even at pH 9.5 and 37°C. It is likely to be more specific in its reaction with proteins in forming protonated amidine linkages with e-amino groups of lysyl residues or a-amino groups of N-terminal residues, as judged from specificity studies with other imidoesters (5-8). Furthermore, this compound is not expected to remove the positive charge from protein amino groups, 339 Copyright @ 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.

340

WOOD

(I) HO

CN

+

ET

CH30H

AL.

+HCl

HO -a-

c\OCH 3 (MPH~IM)

(21

HO

@NH,Cl@ / C \ OCH3

@NH,Cl’ + 2NaI’2S

Oxidiling Agent

C< OCHJ

OH

1. Synthesis and use of an iodinated amidination reagent for the labeling of proteins to high specific activity. Reaction (1): para-hydroxybenzonitrile is converted to methyl phydroxybenzimidate hydrochloride (MPHBIM). Reaction (2): MPHBIM is iodinated to form an iodinated imidoester product (HE), considered to be methyl 35di-iodo-p-hydroxybenzimidate. Reaction (3): IIE adds to amino groups of the protein via amidine linkage, thereby introducing radioactive iodine atoms into the protein. FIG.

as the Bolton-Hunter acylating reagent presumably would, since protonated amidine products in other studies have pK values in the range of 11.5 to 12.5 (15). An outline of the synthesis and use of this imidoester is given in Fig. 1. We have used this imidoester to prepare proteins of high specific radioactivity. A similar iodinated imidoester has been reported (9), but the multistep synthesis of this compound precludes its ready use in radioactive work. Recently, S. Cammisuli and L. Wofsy (personal communication) have independently synthesized and studied the identical imidoesters to those described here, and will report their results elsewhere. MATERIALS

AND METHODS

Materials Para-hydroxy benzonitrile (Eastman Organic Chemical Co.) and reaction grade HCl were obtained commercially and used without further purification. Absolute methanol and diethyl ether (Mallinckrodt Chemical Works) were stored over molecular sieve (Union Carbide) to eliminate water. Other chemicals were obtained commercially as follows: Carrier free lz51 as NaI in 0.05 N NaOH (Schwarz/Mann Chemical Co.), Chloramine T, DMSO (spectrograde) and /3-mercaptoethanol (Math-

RADIOACTIVE

IODINATION

OF

PROTEINS

341

eson, Coleman and Bell), bovine plasma albumin (crystallized) (Armor Pharmaceutical Co.), dog serum albumin (Sigma Chemical Co.), sodium borate (Allied Chemical), and sodium pyrophosphate (Mallinckrodt Chemical Works). Deuterium-labeled DMSO and pyridine for the NMR studies were purchased from Bio-Rad. Methods General procedures. The radioactivity of 1251-containing samples was determined with a Nuclear Chicago Mark II gamma counter. Ultraviolet spectra were obtained with a Cary 14 spectrophotometer, and NMR spectra with a Varian T-60 NMR spectrometer. Synthesis of methyl para-hydroxybenzimidate’HC1 (MPHBIM). This synthesis was accomplished by a modification of the method of Hunter and Ludwig (IO). In a SO-ml triple-necked round-bottom flask (21°C). 1.2 g of paru-hydroxy benzonitrile (0.01 mole) was placed to which was added 16 ml of absolute methanol (0.4 mole), 10 ml of diethyl ether, and 5 pellets of molecular sieve to reduce contamination by water. The flask was closed, fitted with a drying tube, and cooled to -20°C in an ice/salt bath, followed by saturation with dried (bubbled through H,SO,) NC1 gas. The solution was then allowed to warm up to 4°C and yielded orange needle-like crystals after 30 min at 4°C. The crystals were quickly filtered at 4”C, washed with cold methanol/ether (1:2) followed by a final cold ether wash, and subsequently stored at 0°C under vacuum desiccation (yield 90%). The product has been kept under these conditions for 6 months without measurable deterioration. The orange crystals eliminated gas upon melting at 17 l-172°C; NMR peaks (D,DMSO) were found at 6 4.25(s), weight 3, assigned to the 0-CH, protons; 6 6.98(d), J = 9 cps, weight 2,6 8.07(d), J = 9 cps, weight 2, assigned to the aromatic protons. [Found: C, 49.5; H, 5.9; N, 7.7: Cl, 18.9%. Calculated for C,H,,NO,Cl (MW = 188 g/mole): C, 51.3; H, 5.3; N, 7.5: Cl, 18.7%.] The radioactive iodination of methyl para-hydroxy (MPHBZM). The iodination of MPHBIM was performed

benzimidate

on a microscale by dissolving 3.7 mg of MPHBIM in 1 ml of 50 mM sodium borate buffer pH 8.5 to obtain a 20 mM stock solution. Brief exposure to 37°C accelerated the dissolving rate. To 1.0 ml of the 20 mM solution of MPHBIM was added 1.0 ml of 40 mM NaI followed by 10 wliters of Na1125 solution (O-2 mCi depending upon the desired specific activity). To this solution was added 1.0 ml of 40 mM chloramine T with rapid mixing. A yellow color appeared and cleared within a few seconds. After 15 min at room temperature (20-22”C), 0.10 ml of 1.0 M P-mercaptoethanol was added to reduce the chloramine T and residual iodine. The pH of the solution was subsequently lowered toward neutrality by adding 20 Fliters of 1.0 M acetic acid whereupon a flocculant white pre-

342

WOOD

ET

AL.

cipitate formed. Under these conditions unreacted MPHBIM, iodide, and chloramine T remained soluble. The precipitate of the iodinated imidoester was collected by centrifugation at 10,000 rpm for 5 min, dissolved in 2 ml of 50 mM sodium borate buffer, pH 8.5, at 37°C for a few minutes, and was stored at 0°C or frozen at -20°C. Frozen samples have been kept for 7 days without detectible decomposition, as judged from ultraviolet spectral characteristics. The product is referred to as “iodinated imidoester” (IIE). This material was compared to synthetically prepared methyl 3,5-di-iodo-p-hydroxybenzimidate (hydrochloride salt) and proved to be indistinguishable in absorption spectra at pH 2, 6, 8 and 12, as described in Results. The yield of IIE after acid precipitation was 60-80% on the basis of absorbance at 339 nm at pH 6.0. The incorporation of Nalz5 I into IIE agreed within 5% of the amount expected based on the yield of the di-iodo derivative of MPHBIM estimated from ultraviolet absorbance. Synthesis of methyl 3,5-di-iodo-p-hydroxybenzimidate. The precursor material, 3,5-di-iodo-p-hydroxybenzonitrile, was synthesized, according to the method of Sumimoto (16), as follows: Chlorine gas (120 ml) was bubbled into a suspension of iodine ( 1.3 g) in acetic acid (2.0 ml) at 22°C followed by 30 min of stirring at 30°C and warming to 50°C for 20 min with subsequent cooling to 22°C for 40 min. To this iodinating solution was added 0.6 g of p-hydroxybenzonitrile and 20 ml of H,O with vigorous stirring at 50°C for 17 min and then at 80°C for 20 min with subsequent rapid cooling in ice. The precipitated excess I, was dissolved by adding 0.8 ml of 10% Na$O,. The final yellow precipitate was collected by filtration, washed with H,O and dried, with a 75% yield. The tan crystals melted at 204-206°C and exhibited an NMR spectrum in D, pyridine of 6 8.0(s) assigned to the two aromatic protons. The synthesis of methyl 3,5-di-iodo-p-hydroxybenzimidate hydrochloride was accomplished from this precursor using the same procedure as in the synthesis of MPHBIM, as described previously. The yield was approximately 60%, of light orange crystals melting at 250-252°C with gas elimination, and exhibiting NMR peaks (D,DMSO) at S 8.37(s), weight 2, assigned to the aromatic protons, 6 4.20(s), weight 3, assigned to the 0-CH, protons. [Found: C, 22.0; H, 1.8; N, 3.2; I, 58.1; Cl, 8.1%. Calculated for C,H,NO,I,Cl (MW = 439 g/mole): C, 21.9; H, 1.8; N, 3.2; I, 57.9; Cl, 8.0%.] Spectral properties are described in the Results. RESULTS Ultraviolet spectra. Spectra of synthetically prepared methyl phydroxybenzimidate*HCl (MPHBIM) and methyl 3,5-di-iodo-p-hydroxybenzimidateeHC1 were obtained within the pH range of 2-12 as shown in Figs. 2 and 3.

RADIOACTIVE

220

240

IODINATION

260

260

OF

300 A (nm)

343

PROTEINS

320

340

360

FIG. 2. Ultraviolet absorption spectra of methyl para-hydroxybenzimidate.HCl (MPHBIM). A 10 mM stock solution (5.5 mg in 2.95 ml sodium borate, pH 8.5) was prepared with brief warming at 37°C. Spectra were obtained at 21°C within 10 min after dilution, 1:300, in the following solutions: pH 12, 10 mM NaOH; pH 8.6, 40 mM sodium borate: pH 7.55, 40 mM sodium phosphate; pH 2.0, 10 mM HCI. The molar absorptivity, E, is given with the units 10e3 M-I cm-r.

0 220

I 240

I 260

I 280

I 300 X

'.... ... . .._... : I ", -_ _,,,_.I.. . .. . 320 340 360

hml

3. Ultraviolet absorption spectra of methyl 3,5-di-iodo-p-hydroxybenzimidate*HCl. This compound was synthesized as described in Materials and Methods and was dissolved as a 2.0 mM solution in 40 mM sodium borate buffer, pH 8.5. with brief warming to 37°C. Spectra were obtained at 21°C within 10 min after dilution, 1:50, in the following solutions: pH 12, 10 mM NaOH; pH 8.1, 40 mM sodium phosphate; pH 5.98, 40 mM sodium phosphate; pH 2.0, 10 mM HCI. The molar absorptivity, E, is given with the units 1O-3 M-* cm-‘. FIG.

344

WOOD

ET

AL.

The spectrum of MPHBIM at pH 8.5 showed no detectable change after 5 hr although considerable alteration had occurred after 7 days at 21°C. The spectra for MPHBIM showed different isosbestic points in the 5-7 pH range (258,304 nm) compared with the 8-10 range (260,3 11 nm). This behavior suggests the occurrence of at least three acid-base forms of the compound in the 2-12 pH range, as would be expected since MPHBIM has two ionizable groups, the phenolic group and the imido group. At pH 7.7, the MPHBIM absorption peak at 326 nm reached a maximum molar absorptivity of 5000 ~-l cm-l. This absorption decreased to one-half value as the pH was raised to 9.0 or lowered to 6.4. In this neutral pH range, the major species exhibiting long wavelength absorption at 326 nm is presumably either the doubly charged (zwitterion) intermediate or the uncharged intermediate, depending upon the relative pK’s of the phenolic and the imido groups. The molar absorptivity of the intermediate species may exceed 5000 M-’ cm-l at 326 nm, since this species will never comprise the entire molecular population if the two pK values are close. The spectra of methyl 3,5-di-iodo-p-hydroxybenzimidate resemble those of MPHBIM and also indicate the presence of at least three species in the 2.0-12 pH range. In the neutral pH range, an absorption maximum for this iodinated derivative occurs at 339 nm and reaches a molar absorptivity of 23,000 at pH 5.5. The absorption maximum at 339 nm decreases to one-half of the maximal value as the pH is raised to 7.4 or lowered to 3.2. Thus, the pK’s of the ionizing groups are shifted approximately two pH units to the acid side by the introduction of two iodine atoms onto the phenolic group of MPHBIM. The di-iodo compound possesses the same two possibilities for ionization intermediates as does MPHBIM. The presence of an isoelectric intermediate (either uncharged or zwitterionic) in the neutral pH range might be expected to decrease the solubility of the compound in polar solvents at neutral pH values and explain why it can be precipitated under such conditions and yet dissolved in 5% trichloroacetic acid or at high pH. Preliminary observations indicate that MPHBIM and the di-iodo derivative undergo large spectral changes upon reaction with NH, or Eaminocaproic acid. These spectral changes have not been pursued in the present study. The effect of pH, temperature, and concentration on the protein amidination. Radioactive iodine was introduced into MPHBIM by the chloramine T reaction, as described in Materials and Methods. Spectral analysis of this iodinated imidoester (IIE) at pH 2, 6, 8, and 12 showed this material to be indistinguishible from methyl 3,5-di-iodo-p-hydroxybenzimidate synthesized from 3,5-di-iodo-p-hydroxybenzonitrile. The effect of the pH and the temperature on rate of reaction of bovine

RADIOACTIVE

IODINATION

OF

PROTEINS

345

FIG. 4. Progress curves for amidination of bovine plasma albumin (BPA) with radioactive HE. The reaction mixture (volume 1.0 ml), contained 20 mg of albumin, 4 mM HE containing 5.6 X 106 cpm lz51, and 50 mM buffer (sodium borate for pH 9.5 and sodium pyrophosphate for pH 7.5. The reaction was initiated by the addition of IIE and allowed to proceed at 37°C. Samples of 0. I-ml volume were removed at intervals and mixed with 1.O ml of 5% trichloroacetic acid at 0°C. After 30 min (or longer), the precipitates were collected by filtration on Whatman GF/C filters. Radioactivity was determined by y-solid crystal spectrometry. Recovery of 5.6 X lo5 cpm per sample was considered to be 100% yield. The control reaction contained IIE, 50 mM sodium borate, pH 9.5, 37°C. without

BPA.

plasma albumin with radioactive IIE are illustrated in Figs. 4 and 5. Figure 4 is a progress curve of the derivatization of albumin with IIE at pH values of 7.5 and 9.5 and a temperature of 37°C. When the molar ratio of IIE to albumin was 14: 1, and albumin was present at a concentration of 20 mg/ml, the maximum rate of incorporation of radioactivity was l-2% per hr, with a maximum incorporation of 30% of the P5 input. These data illustrate the rather slow reaction rate of IIE. In tests of specificity of the reaction, it was found that 50 mM P-mercaptoethanol had no detectable effect on the reaction rate, indicating the usefulness of this reaction for proteins which must be kept in the presence of sulfhydry1 agents. Figure 5 indicates that the reaction velocity increased by a factor of 3 as the temperature increased by 10°C in the range of 0-40°C. Also, as the pH increased 1.4 units, the reaction velocity doubled in the pH 6.5-9.5 range. At this time it is not possible to evaluate the contribution to the pH-rate dependence made by ionization of IIE, of the protein amino groups, and of the IIE-amino group transition state (1 l), although it is apparent from the spectral studies that the ionization of IIE is essentially complete by pH 8.4 and should not enter as a factor above this PH.

346

WOOD I

I

37-c

,I

3.2

ET AL. I

I

I

‘c

21% 3.3 T-‘113

3.4

3.5

3.6

3.7

?I(-‘,

FIG. 5. The pH and temperature dependence of the rate of amidination of BPA with radioactive IIE. The reaction was performed as described in the legend of Fig. 4, with the following conditions: (O-O), pH 7.5, 100 mM sodium pyrophosphate; (A-A), pH 8.4, 100 mM sodium borate; (O--O), pH 9.5, 100 mM sodium borate. Progress curves throughout 24 hr were obtained for each condition of temperature and pH, and data were extrapolated back to zero time to evaluate initial velocity, V,, shown on the ordinate. The units of Vi are percentage of input counts incorporated per hour into protein (TCAinsoluble material).

The dependence of reaction rate on the concentration of the two reactants was explored briefly under conditions in which each reactant was singly held constant and in large excess while the other was varied (pseudo first-order conditions). As shown in Fig. 6, the reaction velocity was linearly dependent on the variable reactant under these conditions, suggesting that the reaction is second order with respect to IIE and protein, together. There was no indication of the steep dependence on protein concentration mentioned by Bolton and Hunter (3) for their acylating agent. Analysis of the amidinated protein. Dog serum albumin was reacted with IIE (G 100 &il~moIe) for 1 day at 21°C and then dialyzed extensively against 0.15 M NaCl containing 5 mM sodium phosphate, pH 7.4. The specific activity of this preparation was approximately 10 $Zi per mg of protein. This material was analyzed by SDS gel electrophoresis (12) and formed a sharp major band of radioactivity at the serum albumin position (67 X lo3 daltons) and a minor band at the dimerized albumin position. Together the radioactivity at these positions comprised 80% of the input radioactivity. This result suggests that the radioactive

RADIOACTIVE

0

IODINATION

OF PROTEINS

347

0.05

0.10 0. I 5 0.20 0.25 IBPAIIIIEI, hM)2 FIG. 6. The dependence of amidination rate on reactant concentrations. The reaction volume of 0.15 ml contained 50 mM sodium borate, pH 8.5, and concentrations of BPA and IIE as follows: Condition 1 (indicated by circles), BPA varied from 0 to 0.098 mM (6.6 mglml) while IIE remained constant at 1.07 mM and 10’ cpm: or condition 2 (indicated by squares), IIE varied from 0 to 2.14 mM and 2 X 10’ cpm, while BPA remained constant at 0.098 mM. The concentrations of IIE were estimated spectrophotometrically at pH 6, taking the authentic di-iodo compound to have a molar absorptivity of 23 X lo3 M-I cm-i at 339 nm. The amidination reaction was initiated by the addition of IIE, was allowed to proceed for 1 hr at 37°C and was stopped by the addition of 1 ml of 5% trichloroacetic acid and chilled to 0°C. After 16 hr, the precipitates were collected and counted as described in the legend of Fig. 4. The rate of formation of the amidine linkage was taken as the radioactivity incorporated into acid-insoluble materials in the first hour of reaction.

imidoester had become attached to the protein by covalent linkage and not by noncovalent adsorption. Stability ofZZE. Since the reaction rate under pseudo first-order conditions provided a means to measure IIE concentration on a relative scale, a test was made of the stability of IIE at pH 8.5 and 21°C. Samples from a stock solution of IIE (approx. 1.1 mM) were removed at intervals and reacted for 1 hr with a standard amount of protein (10 mglml). The results indicated that after 24 hr of incubation the stock solution still contained greater than 80% of the initial concentration of IIE. In addition, spectral studies with nonradioactive IIE have shown that solutions of IIE at pH 8.5 (50 mM sodium borate) undergo no detectable spectral change after a week of storage at -20°C. Thus, it should be possible to

348

WOOD

ET

AL.

prepare and store the radioactive reagent for prolonged periods. Hunter and Ludwig reported the related compound, methyl benzimidate, to have a half-life for hydrolysis of greater than 50 hr at pH 8.5 and 39°C (10). DISCUSSION

The present paper describes a method of introducing radioactive iodine into proteins by way of the iodinated imidoester, methyl 3,5-diiodo-para-hydroxybenzimidate. The parent imidoester, methyl paruhydroxybenzimidate, is synthesized from p-hydroxybenzonitrile and methanol. The MPHBIM is then reacted with Nalz51 in the presence of an oxidant, chloramine T, to form the iodinated derivative which can be isolated by acid precipitation and stored frozen at -20°C for at least a week without hydrolytic decomposition. This radioactive di-iodo compound can then be reacted with the protein in a 7.5-9.5 pH range and between 21 and 37°C with a yield of 20-30% of the radioactivity incorporated into the protein in 24 hr. Preparations exceeding lo4 cpm per pg protein have been obtained by this procedure; specific activities tenfold higher may, in practice, be the upper limit obtainable with these procedures, unless the acid precipitation step is omitted and conditions are found for more rapid amidination, both modifications allowing work with smaller amounts of iodinated imidoester. The main advantage from the use of this imidoester, as with the Bolton and Hunter iodinated acylating reagent (3), is that the protein need never be exposed to oxidizing agents, such as chloramine T used to accomplish iodination, nor to the lz51-labeled NaI commercial preparations which are thought to contain deleterious impurities (13). Furthermore, these reagents may be used to introduce radioactive iodine into protein lacking tyrosine. On the basis of spectral changes, the iodinated imidoester was found to be a moderately acidic compound, undergoing ionization of its phenolic hydroxyl and imido groups below pH 8. Iodination is known to lower the pH of phenolic ionization by more than 3 units, from 10.1 in the case of tyrosine to 6.5 in the case of di-iodo tyrosine (14) and the same effect is apparent in the comparison of MPHBIM and its di-iodo derivative, as reported in the Results. As a consequence, the amidine derivative formed by the reaction of the di-iodo reagent with protein probably bears a negative charge on its phenol group at neutral pH values and above. This charge alteration would also occur with the Bolton and Hunter reagent, as well as with di-iodo tyrosyl residues produced in proteins by direct iodination techniques (1,2). It is characteristic of imidoesters that the amidine group formed by their reaction with amino groups preserves the positive charge of the amine, now borne by the amidine nitrogens with pK values in the 11.5-12.5 range (15). There-

RADIOACTIVE

IODINATION

OF PROTEINS

349

fore, the positive charge of e-amino groups of lysyl residues or of (Yamino groups of N-terminal residues may be preserved during the reaction with IIE, whereas these charges would probably be lost upon reaction with the Bolton and Hunter acylating reagent. ACKNOWLEDGMENTS The authors thank Dr. Stephen Kent for his help in interpreting the NMR spectra and M. Bothwell for suggesting acid-precipitation as a method of purification of the radioactive imidoester. This research was supported by U.S. Public Health Service Research Grant GM I9363 from the National Institute of General Medical Sciences.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1 I. 12. 13.

Greenwood, F. C., Hunter, W. M., and Clover, .I. S. (1963) Biochem. J. 89, 114-123. Marchalonis, J. J. (1969) Biochem. J. 113, 299-305. Bolton, A. E., and Hunter, W. M. (I 973) Biochem. J. 133, 529-538. Rudinger, J., and Ruegg, U. (1973) B&hem. J. 133, 538-539. Haynes, R., and Feeney, R. E. (1968) Biochemistry 7, 2879-2885. Perham, R. N., and Richards, F. M. (1968)J. Mo!. Biol. 33, 795-807. Reynolds, J. H. (1968) Biochemistry 7, 3131-3135. Wofsy, L., and Singer, S. J. (1963) Biochemistry 2, 104-I 16. Riley, M., and Perham, R. N. (1973) Biochem. J. 131, 625-635. Hunter, M. J., and Ludwig, M. L. (1962)J. Amer. Chem. Sot. 84, 3491-3504. Hand, E. S., and Jencks, W. P. (1962) J. Amer. Chem. Sot. 84, 3805-35 14. Weber, K., and Osborn, M. (1969) J. Bid. Chem. 244,4406-4412. Hunter, N. M. (1971) in Radioimmunoassay Methods: European Workshop (Kirkham, K. E.. and Hunter, N. M., eds.), pp. 3-23, Churchill Livingstone,Edinburgh and London. 14. Roche, J., and Michel, R. (1951) Advan. Protein Chem. 6, 253-297. 15. Albert, A., Goldacre, R., and Phillips, J. (1948) J. Chem. Sm. Lo&on, 2240-2249. 16. Sumimoto, Shinzaburo (Shionogi and Co., Ltd.) Ger. Offen. 1,96 1,289 (Cl. CO7bcd, A 61 k), 09 July 1970.