Enzymic and nonenzymic hydroxylation and chlorination of p-Deuteroanisole

612

COMMUNICATIONS TABLE

II

N-ACETYL-D-GALACTOSAMINYLTRANSFERASE ACTIVITY OF HUMAN MILK Sephadex-treated milk samples from various donors were assayed for N-acetyl-o-galactosaminyltransferase activity as described in the legend of Fig. 1 except that the 2’.fucosyllactose (40 rnp moles) and lacto-iy-fucopentaose I (20 mpmoles) were incubated separately and the incubat,ions were carried out for 18 hours. Toluene, 25 ~1, was added to inhibit bacterial growth. The laCactivity incorporated int,o the oligosaccharides was determined by counting appropriate sections of the chromatograms in a scintillation counter as previously described (1). N-Acetyl-D-galactosamine transferred (cpm) To 2’.Fuco- ro Lacto-.Vsyllactose tucopyltaose

A1 A1 A2 -0 B 0 0 0 AC 0 0

Secretor Secretor Secretor Secretor Secretor Secretor Secretor Secretor Nonsecretor Nonsecretor Nonsecretor

480 230 1250 320 0 0 0 0 540 0 0

340 150 620 250 0 0 0 0 240 0 0

(1Blood typing was performed by Mrs. Mary McGinniss at. the blood bank of the National Institutes of Health. b Secretor status was determined on undiluted saliva by using the standard assay (8). c The A subgroup was not determined. A gene. The correlation of a galactosyltransferase in human milk with blood type B remains to be established. Acknowledgement. We are grateful to Mrs. Barbara Torain for performing the hemagglutination inhibition tests and to the Metropolitan Washington Chapter of the La Leche Society for supplying the samples of milk and blood. REFERENCES 1. &EN, L., GROLLMAN, E., AND GINSBURG, 5'., Proc. Natl. Acad. Sci. U.S. 69, 224 (1968). 2. GROLLMAN, E, KOBATA, A., AND GINSBURG, V., Federation Proc. (In press). 3. MORGAN, W. T. J., Proc. Roy. Sot. (London), Ser. B 161, 308 (1960); WATKINS, W. M., Science 162, 172 (1966).

4. TUPPY, H., AND STAUDENBAUER, W. L., Nature 210,316 (1966). 5. KUHH, R., BAER, H. H., AND GAUHE, A., Ann. Chem. 611, 242 (1958). 6. MONTREUIL, J., Bull. Sot. Chim. Biol. 42, 1399 (1960). 7. HABaD, H., AND HASSID, W. Z., J. Biol. Chem. 241, 2672 (1966). 8. KABAT, E. A., “Blood Group Substances,” p. 56. Academic Press, New York (1956). 9. LUNDBLAD, A., Biochim. Biophys. Acta 148, 151 (1967). AKI RA KOBATA EVELYN F. GROLLMAN T'ICTOR GINSBURG National Institufes of Arthritis and Metabolic Diseases National Institutes of Health Bethesda, A/laryland iOOi.4 Received January 4, 1968

Enzymic ylotion

and

Nonenzymic

and

Chlorination

Hydroxof

p-Deuteroanisole A variety of enzymic hydroxylation reactions with aromatic substrates have been shown to involve a concomitant migration of substituents from the position of oxygen attack to an adjacent position in the aromatic ring (the “NIH Shift”) (1). Cationoid intermediates have been proposed to explain the migration and retention of substituents (2,3). The extent and direction of migration has been correlated with charge stabilization and distribution in such cationoid intermediates (1, 3). While most nonensymic aromatic substitution reactions (Friedel-Crafts alkylation, bromination, nitration, and diazonium coupling) do not exhibit such migrations, a notable exception is the hydroxylation of aromatic compounds with peroxytrifluoroacetic acid (4). Examination of additional aromatic substrates with both enzymic and nonenzymic systems should provide further information as to the nature of the NIH Shift. It was therefore of interest to determine if migration would occur as a result of enzymic chlorinat,ion or whether only hydroxylation reactions allow such migrations. This communication presents the results on both enzymic and nonenzymic chlorination and hydrosylation of 4-deuteroanisole. Materials and methods. p-Deuteroanisole was prepared from p-bromoanisole by reduction in tetrahydrofuran containing D20 and triethylamine with deuterium gas and 57, palladium on charcoal. The product, was purified by fractional

613

COMMUNICATIONS distillat,ion (b.p. 152-155”); and its identity was confirmed by mass spectrometry (1.0 atom deut,erium) and proton nuclear magnetic resonance. Chloroperoxidase was generously furnished by Dr. L. P. Hager. Incubations with this preparation were carried out as described (5). The chlorinated anisoles were isolated with unreacted p-deuteroanisole by extraction into ether. The extract was dried over sodium sulfate and concentrated to a small volume prior to gas chromatography-mass spectrometry. Crude preparations of rabbit liver microsomes were obtained by homogenization of liver with 3 volumes of cold 1.15$& KCl, followed by centrifugation at 20,OOOg for 30 minutes to remove cell debris and mitochondria. Incubations were carried out at 37” for 1 hour with the following components: 10 ml of microsome suspension, 3 ml of Tris buffer (0.5 M, pH 8.2), 1 ml EDTA (lo+ M), 10 pmoles of NADP, 5 pmoles of ATP, 25 pmoles of glucose 6-phosphat,e, 10 units of glucose 6-phosphate dehydrogenase, and 50 rmoles of 4-deuteroanisole which w&s added in 0.2 ml of ethanol. The products and unreacted 4-deuteroanisole were extracted into ether. The extract was dried over sodium sulfate and concentrated to a small volume. In some instances the phenolic products were further purified by preparative thin-layer chromatography by using thin-layer plates (silica gel) developed with chloroform, followed by elution of the region to which the phenolic products migrate into ethyl acetate. Nonenzymic chlorination of p-deuteroanisole was performed according to Brown and Hager (5): a mixture of 1 mmole of p-deuteroanisole, 0.5 mmole of sodium hypochlorite (0.5 ml of 4-67’ NaOCl in wat,er), and 100 ml of phosphate buffer (0.1 M, pH 2.8) was agitated in air at 37” for 30 minutes. The products and unreacted starting material were extracted into ether and treated as described above for the enzymic chlorination. Nonenzymic hydroxylation of p-deuteroanisole with peroxytrifluoroacetic acid was carried out in the two-phase system described by Jerina et al. (4). A mixt,ure of 100 pl of p-deuteroanisole, 1 ml of chloroform, 1 ml of trifluoroacetic acid, and 1 ml of 90% hydrogen peroxide was allowed to stand at 5” to -15” for l-l.5 hours. The reactions were terminated by cautious dilution with saturated NaHC03 until all acid present was consumed. After extraction of the products into chloroform, the reaction mixture was purified by preparative thin-layer chromatography on silica gel plates developed with chloroform. The region of the plate to which authentic standards of m- and phydroxyanisole cochromatograph (RF 0.3) was extracted with ethyl acetate and concentrated to a small volume.

ortho

0

L--I , , ,A 6

4

2

8

IO

t (min)

FIG. 1. Gas chromatographic separation of o-, m-, and p-chloroanisoles on a 6-foot X >g-inch OD stainless steel column operated at 115” and packed with 16% carbowax 1000 on 8@100 mesh Gas Chrom P. The total ion monitor of the mass spectrometer is used as a detector.

I 0

4

8

12

t (mid

FIG. 2. Gas chromatographic separation of the o-, m-, and p-hydroxyanisoles on a 6-foot X >4inch OD st.ainless steel column packed with 5% Hi Eff 2B on 60-80 mesh Chromosorb P and operated at 175” with an F & M Hewlett Packard (model 5750) gas chromatograph equipped with a hydrogen flame detector. The deuterium content in the chlorinated and hydroxylated anisoles was determined on an LKB 9ooO gas chromatograph-mass spectrometer at 70 eV. A typical gas chromatographic separation of the isomeric chloroanisoles is shown in Fig. 1 and of the isomeric hydroxyanisoles in Fig. 2.

614

COMMUNICATIONS TABLE

I

COMPARISON OF MIGRATION AND RETENTION OF DEUTERIUM DURING HYDROXYLATION WITH MICROSOMES .~ND WITH PEROXYTRIFLUOROACETIC ACID

R = ‘H,

‘H, Cl. etc.

X = OCH,, NHCOCH,,

Substrate Cl, etc.

e

CH3C

‘N _ + H@

dependent

H ‘0

Peroxytrifluoroacetic acid

migra-

Results. The enzymic chlorination of p-deuteroanisole with chloroperoxidase led to approximately a 1:l mixt,ure of p-chloroanisole and o-chloroanisole. The p-chloroanisole contained no deuterium, but the o-chloroanisole, as expected, retained 1.0 atom of deuterium. The nonenzymic chlorination with hypochlorous acid gave a mixture of ~(23 parts)- and o-chloroanisole (10 parts), again with essentially 0 and 1 atom of deuterium. 2,4-Dichloroanisole (2 parts) was also observed as a reaction product. Microsomal oxidation of p-deuteroanisole gave two major products, viz., p-deuterophenol, resulting from oxidative demethylation, containing 0.95 deuterium atom, and p-hydroxyanisole with 0.60 atom of deuterium. The production of p-hydroxyanisole and phenol approximated 5 pmoles each

0 + H@

H\g,H

Microsomes at

pll 8

FIG. 3. Steps for hydroxylation-induced tions during aromatic hydroxylat,ion.

PH

Retention in para hydroxylated product (“/)

H ‘N

I

I

a HO

D

+H

0

FIG. 4. Possible stabilization of cationoid intermediates by a route other than migration.

p-Deuteroanisole p-Deuterochlorobenzene p-Deuteroacetanilide a Ref. 2. * Ref. 6. c J. Daly, unpublished ported as 15% in Ref. 7. d Ref. 4.

60 54a

8 70h

300

7d

results;

previously

re-

from 50 rmoles of anisole. In addition, small amounts of o-hydroxyanisole containing 0.88 atom of deuterium were detected both by thinlayer chromatography and gas chromatography. The formation of m-hydroxyanisole could not be detected by gas chromatography. Furthermore, mass spectra taken at several points throughout the p-hydroxyanisole peak showed the deuterium content to be constant. Nonenzymic hydroxylation of p-deuteroanisole with peroxytrifluoroacetic acid afforded both oand p-hydroxyanisole in addition to other unidentified products. The p-hydroxyanisole contained 0.08 deuterium atom. This retention was independent of whether the hydroxylation was conducted for 1 hour at 5”, 1 hour at -5”, or 1.5 hours at -15”. The deuterium-labeled phenol and o- and p-hydroxyanisole obtained from a microsomal hydroxylation of p-deuteroanisole were subjected to the peracid oxidizing system at - 15” for 1 hour. The reaction was worked up in the usual manner and the remaining st,arting materials were isolated by thin-layer chromatography. Examination by combined gas chromat.ography-mass spectrometry showed each recovered compound to have retained all of its initial deuterium, and proved that there was no exchange during the reaction or the isolation. The possibility of exchange during gas chromatography was not explored. Discussion. The discovery of the hydroxylationinduced migrations of substituents during enzymic hydroxylation of aromatic rings has permitted new insights into the mechanism of enzymic aryl hydroxylation (1). The deuterium and tritium

COMMUNICATIONS retentions in a variety of substrates have been correlated with the influence of the various subst,ituents on charge dist,ribution in the proposed cationoid intermediates (2, 3). The mechanism proposed for the migration (Fig. 3) requires a hydroxyl group for the stabilization of the rearranged product as the cyclohexadienone. On this basis one would predict that other enzymic or chemical substitutions of aromatic rings, such as chlorination (5), which could not lead to cyclohexadienone type stabilization, would lead to loss rather than migration of substituents. Confirmation of t,his expectation was found in the essentially complete loss of deuterium from p-deuteroanisole on either enzymic or nonenzymic chlorinations in the para position. Hydroxylation of the same substrate with microsomal preparations produced p-hydroxyanisole with 60% of the initial deuterium via a migration and retent,ion pathway. This retention is higher in relationship to migrat,ion with other substrates than would be predicted on the basis of delocalization of chargein cationoid intermediates byelectron-donating substituents (3). As shownin Table I, which presents data from previous studies as well as from the present experiments, p-deuterochlorobenzene with an electron-attracting substituent, and p-deuteroanisole with an electrondonating substituent, exhibit similar retentions on microsomal p-hydroxylation. p-Deuteroacetanilide exhibit,s a much lower retention which in the case of p-tritioacetanilide was shown to be strongly pH-dependent (7). These observations and the fact that phenols and anilines show very low retention of tritium (3) on hydroxylation suggest another cyclohexadienone-type intermediate which inhibits rather than promotes migration (Fig. 4). Presumably, stabilization of a cationoid hydroxylation intermediate via a 2,5-cyclohexadienone structure removes the driving force for the NIH Shift. A variety of other deuterated aromatic substrat,es are being investigated to provide evidence for or against this interpretation of hydroxylation-induced migration and retention of deuterium. These studies are being paralleled by an investigation of retentions shown by various model hydroxylating systems. None of these nonenzymic model systems have led to migration of tritium or deuterium as a result of hydroxylation, except for the electrophilic reagent, peroxytrifluoroacetic acid (4). However, as shown in Table I, nonenzymic migration and retention with this reagent

615

does not correspond quantitatively with those of microsomal hydroxylation of the same substrates. The peroxytrifluoroacetic acid system affords higher retentions with p-deuterochlorobenzene and much lower retentions with p-deut.eroacetanilide and p-deuteroanisole than the retentions observed with the microsomal system. Large pH differences and binding in the enzyme-substrate complex may partially explain the variation in retention between the model and the enzyme systems. A variety of other hydroxylating systems are currently under investigation. AcknozuZedgr,lents. The authors gratefully acknowledge the advice and encouragement of Drs. Bernhard Witkop and Sidney Udenfriend. They would also like to acknowledge the generous cooperation of Rlr. Ray Pittman in obtaining the mass spectral measurements. REFERENCES 1. GUROFF,

G., DALY, J., JERINA, D., RENSON, J., B., AND UDENFRIEND, S., Science 157, 1524 (1967). JERINA, D., DALY, J., AND WITKOP, B., J. Am. Chem. Sot. 89, ES38 (1967). DALY, J., GUROFF, G., UDENFRIEHD, S., AND WITKOP, B., Arch. Biochem. Biophys. 122, 218 (1967). JERINA, D., DALY, J., LANDIS, W., WITEOP, B., AND UDENFRIEND, S., J. Am. Chem. Sot. 89, 3317 (1967). BRO\VPI’, F. S., AND HAQER, L. P., J. Am. Chem. Sot. 89, 719 (1967). DALY, J., GUROFF, G., JERINA, D., UDENB., “Proceedings of FRIEND, S., AND WITKOP, the International Oxidation Symposium, August 1967, San Francisco.” Sdvan. Chem. Ser. (In press). UDENFRIEND, S., ZALTZMAN-NIRENBERG, P., DALY, J., GUROFF, G., CHIDSEY, C., AND WITKOP, B., Arch. Biochem. Biophys. 120, 413 (1967). WITKOP,

2.

3.

4.

5. 6.

7.

DONALD JERINA GORDON GUROFF JOHN DALY

Laboratory of Chemistry, National Institute of Arthritis and Metabolic Diseases Laboratory of Clinical Biochemistry, National Heart Institute National Institutes of Health Bethesda, MarzJland Received January 2, 1968