Intramolecular Migrations during Hydroxylation of Aromatic

82 Intramolecular Migrations during

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Hydroxylation of Aromatic Compounds The NIH Shift JOHN DALY, GORDON GUROFF, DONALD JERINA, SIDNEY UDENFRIEND, and BERNHARD WITKOP National Institutes of Health, Bethesda, Md.

When selectively deuterated or tritiated aromatic substrates undergo enzymatic hydroxylation, a fundamental 1,2 shift of deuterium or tritium, from the point of substitution by oxygen, to the adjacent position in the aromatic ring is ob served. Migrations of chloro, bromo, and methyl substituents also occur. The extent and direction of migration observed with tritiated and deuterated aromatic compounds agrees with predictions based on the major canonical forms of cationoid intermediates. Phenolic compounds and aniline show almost negligible migration of tritium while in other compounds such as anisole and the alkylbenzenes the initial positive charge is not delocalized, and significant migrations of deuterium or tritium occur. Migrations are observed dur ing hydroxylation with the electrophilic reagent, peroxytrifluoroacetic acid, but not with other nonenzymatic hydroxyl ating systems. " p v u r i n g studies at N I H , it was discovered that enzymatic hydroxylation of (deuterated or tritiated) substrates leads to a novel and mechanistically important shift of the deuterium or tritium from the point of substitution by oxygen to an adjacent position in the aromatic ring (12). [It has been found recently that arene oxides are likely intermedi ates in the metabolism of aromatic compounds. They rearrange to phenols with concomitant " N I H Shift," and they are enzymatically converted to dihydrodiols and premercapturic acids. In addition, naphthalene oxide has now been demonstrated as an intermediate in the conversion of naph thalene to α-naphthol, iran5-l,2-dihydro-l,2-dihydroxynaphthalene, and 279 In Oxidation of Organic Compounds; Mayo, F.; Advances in Chemistry; American Chemical Society: Washington, DC, 1968.


280

OXIDATION

O F ORGANIC

COMPOUNDS

III

S-(l,2-dihydro-2-hydroxynaphthyl) glutathione. This is the first direct demonstration of an arene oxide as an intermediate in enzymatic hydroxy lation.]. This observation has been extended to include the migration of chlorine, bromine and alkyl groups. W i t h the enzyme, phenylalanine hydroxylase, a variety of substrates have been found to undergo hydroxylation-induced migrations. 4-Deutero- (15), 4-tritio- (14), 4-chloro-, 4-bromo- (13), or 4-methylphenylalanine ( 7 ) are hydroxylated to the corresponding 3-substituted tyrosines, while only small amounts of unsubstituted tyrosine are formed ( Figure 1 ). By contrast, hydroxylation of 4-fluorophenylalanine leads to formation of tyrosine with complete loss of the substituent as fluoride ion (20).

I

II

Φ

I

D M P H

NH + H + NADH + 0 2

R = H, 2

>70

91

—

>85

—

—

54

45

—

—

30

12

—

—

60

4 0

— —

0

—

Enzyme: phenylalanine hydroxylase. Microsomal aryl hydroxylase. Tryptophan-5-hydroxylase. Tyrosine hydroxylase.

aromatic substituents and are compatible with the stabilization or derealization of charge in cationoid intermediates. Compounds which have strong electron donating (ionizable) substituents, such as phenols and aniline exhibit very low degrees of migration and retention. Compounds such as acetanilide, which donate electrons by ionization to a lesser extent, exhibit higher values for migration and retention (17). Compounds, such as tryptophan, phenylalanine, and amphetamine, in which no derealization of charge can take place in a cationoid intermediate, exhibit very high retentions. The charge on the cationoid intermediate formed during 5-hydroxylation of tryptophan is best stabilized in the 4-position. This explains the almost exclusive formation of 4-tritio-5hydroxytryptophan rather than a mixture of the 4- and 6-tritio isomers. Pathways and intermediates in the hydroxylation of various substrates are shown in Figure 6. If the charge is partially delocalized by an unshared pair of electrons from the substituent R, as in 4-tritioacetanil-

In Oxidation of Organic Compounds; Mayo, F.; Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

284

OXIDATION

O F

ORGANIC

COMPOUNDS

III

ide, the label is partially lost via path A . This path will be favored at higher p H , and the degree of tritium retention during hydroxylation of 4-tritioacetanilide decreases with increasing p H . As expected, the loss of deuterium by this path was higher than that of tritium. The degree of retention in the final product w i l l depend not only on the relative impor tance of paths A and B, but also on the ratio of paths C and D (k /k ) in the rearomatization of the cyclohexadienone intermediate. Doublelabeling experiments with 3,5-dideutero-4-tritioacetanilide indicate that the latter reaction involves a large isotope effect in analogy to the rather large (kn/k >—' 6-7) isotope effects found for the enolization of simple ketones. This was expected from the data on phenylalanine hydroxylation, where direct loss via path A is very small (less than 6% ), so that the observed 94% retention is the result of a large isotope effect in the enolization of the intermediate cyclohexadienone.


H

T

T

It is known that the metabolism of chlorobenzene in vivo leads to a variety of products including 4-chlorophenol, 4-chlorocatechol, 4-chlorophenylmercapturic acid, and 4-chloro-l,2-fran.9-dihydrodihydroxybenzene (26). When the metabolism of 4-deuterochlorobenzene was studied in this laboratory, 3-chlorophenol and an O-methylated chlorocatechol were isolated i n addition to the products reported above (19). The products isolated, the proposed intermediates, and their deuterium contents are summarized in Figure 7. A n intermediate, X , is postulated for this scheme

τ Figure 6.

Η

Postulated intermediates formed during enzymatic hy droxylation and their relation to the NIH Shift


82.

DALY

E T

AL.

Intramolecular Migrations

285


ci

OH 0%D

OH l%D

OH 23% D

Figure 7. Metabolism in vivo of 4-deuterochlorobenzene and the acid-catalyzed dehydration of 4-chloro-l ,2-dïhydrodihydroxybenzene-l -H 2

of enzymatic oxidation. This X could be a cationoid structure or an epoxide, either of which could react with nucleophiles, such as "activated glutathione" (2), or with water to form dihydrobenzene derivatives. X could also undergo rearomatization reactions as shown earlier (Figure 6). The 4-chlorophenol retained 54% of the deuterium while the 4-chlorocatechol and the O-methyl-4-chlorocatechol contained only insignificant amounts of deuterium. This indicates that the catechol derivatives arise by dehydrogenation of the diol rather than by hydroxylation of the 4-chlorophenol; if the catechols had arisen from 4-chlorophenol, they would have contained at least one-half of its deuterium content (27% ). The possibility that they arise from 3-chlorophenol is under investigation. The 3-chlorophenol, contained 84% of the deuterium, but its metabolic origin is not certain. The mercapturic acid contained very little 2% ) deuterium, and its formation is being studied further. As expected, the isolated 4-chloro-l,2-fran.s-dihydrodihydroxybenzene contained one deu-



286

OXIDATION

O FO R G A N I C

COMPOUNDS

III

terium atom. O n treatment with I N H C l at 100 °C. this material de hydrates to form 4-chlorophenol with a concomitant migration and retention of 2 3 % of the deuterium. This nonenzymatic migration and retention of deuterium demonstrates that cationoid intermediates of the type generated i n this dehydration are compatible with the migrations observed during enzymatic hydroxylations. Similar studies are planned with 4-substituted-l-deutero-l,2-benzene epoxides. The migration of alkyl substituents during the hydroxylation of 4-hydroxyphenylpyruvic acid (25) and 4-fluorophenylpyruvic acid (27) ( Figure 8 ) provides another example of the N I H Shift and was formerly thought to be a unique phenomenon. It is i n a sense atypical since a decarboxylation accompanies the hydroxylation and side chain migration. Nonenzymatic examples of intramolecular migrations are known, such as the migration of tritium during oxidative polymerization of -OH

COOH

(F)

HO

(F)

HO'

,ΧΟΟΗ + C 0

2

Figure 8. Migration of the side chain during hy droxylation of phenylpyruvates with (hydroxy) phenylpyruvic acid oxidase 4-tritio-2,6-xylenol (Figure 9) for which a cationoid intermediate has also been proposed (6). Oxidation of 4-alkylphenols with Caro's reagent or lead tetraacetate (30, 31) leads to migration of the alkyl substituent and formation of 2-alkylhydroquinones (Figure 10). Caro's acid is a source of O H as is peroxytrifluoroacetic acid. W i t h the latter reagent, methyl migrations occur during oxidation of 1,2,3,4-tetramethylbenzene (Figure +

10) ( 5 ) .

The enzymatic migration of chloro and bromo groups, but not of fluoro substituents, by a cationoid mechanism is supported by observations on aluminum chloride-catalyzed isomerizations of halo arenes (22), where a l l halogens except fluorine migrate by a mechanism involving cationoid intermediates. F o r the enzymes, however, fluorine appears to be removed reductively, so that it is lost as fluoride ion rather than as positive fluorine. The reagent, peroxytrifluoroacetic acid, also causes migrations of tritium and deuterium during 4-hydroxylation of 4-tritio ( deutero ) acetanilide and 4-deuterochlorobenzene (Figure 11). The retentions with the acetanilides are lower (8 and 10% vs. 30 and 45% ) than those obtained on enzymatic hydroxylation while the retention obtained with the chlorobenzene is higher (70% vs. 54% ) than the value obtained on enzymatic hydroxylation. Other hydroxylating systems as those of


82.

DALY

E T

A L .


Figure 9.

287

Intramolecular Migrations

Migration of tritium during oxidative polymerization of 4-tritio-2,6-xylenol

Figure 10.

Migration of alkyl substituents during nonenzymatic hydroxylations

2

H

Figure 11. Migration of tritium and deuterium during hydroxylation of acetanilides and chlorobenzene with peroxytrifluoroacetic acid Fenton (4), Udenfriend ( 3 ) , Hamilton (16), or Viscontini (1) which have been considered as possible models for enzymatic hydroxylation do not lead to significant migration and retention of tritium during the formation of 4-hydroxyacetanilide ( Table II ). Thus, only model hydroxylating systems proceeding via cationic intermediates produce the migration of substituents characteristic of enzymatic hydroxylation. It appears


288

OXIDATION

Table II.

O F ORGANIC

COMPOUNDS

III

Retention of Tritium in 4-Hydroxyacetanilide Formed from 4-Tritioacetanilide with a Variety of Nonenzymatic Hydroxylating Systems ( 18 ) a

Hydroxylating System F CC0 H H 0 , Fe , EDTA H 0 , catalytic amounts of Fe 0 , Fe , ascorbic acid, EDTA 0 , Fe , DMPH , EDTA 3

2


2

2

2

2

2+

2+

2+

4

9.6 1.9 1.0 1.2 1.9

b

8

2

Retention of Tritium, %

H+

and catechol

" The 2- and 3-hydroxyacetanilides produced retained at least 80% of the original ac tivity of 4-tritioacetanilide ( DMPH = dimethyltetrahydropteridine ). Retention of deuterium with this reaction was 7.5%. 4

6

most likely that the migrations observed on enzymatic hydroxylation are a function of both the structure of the substrate and the nature of the oxygenating species. The discovery of the N I H Shift has provided new insights into the mechanisms of aromatic hydroxylation and a new criterion for studying model hydroxylating systems. It is also important i n studies on drug metabolism and has led to the development of new enzyme assays for at least two important hydroxylases, phenylalanine hydroxylase (10) and tryptophan hydroxylase (23).

Literature Cited (1) Bobst, Α., Viscontini, M., Helv. Chim. Acta 49, 884 (1966). (2) Booth, J., Boyland, E., Sims, P., Biochem.J.79, 516 (1961). (3) Brodie, B., Axelrod, J., Shore, P., Udenfriend, S.,J.Biol. Chem. 208, 741 (1954). (4) Breslow, R., Lukens, L. N.,J.Biol. Chem. 235, 292 (1960). (5) Buehler,C.,Hart, H.,J.Am. Chem. Soc. 85, 3177 (1963). (6) Butte, W. Α., Jr., Price, C.C.,J.Am. Chem. Soc. 84, 3567 (1962). (7) Daly, J., Guroff, G., Arch. Biochem. Biophys. 125, 136 (1968). (8) Daly, J., Guroff, G., Udenfriend, S., Witkop, B., Arch. Biochem. Biophys. 122, 218 (1967). (9) Daly, J. W., Guroff, G., Udenfriend, S., Witkop, B., Biochem. Pharmacol. 17, 31 (1968). (10) Guroff, G., Abramowitz, Α., Anal. Biochem. 19, 548 (1967). (11) Guroff, G., Daly, J., Arch. Biochem. Biophys. 122, 212 (1967). (12) Guroff, G., Daly, J. W., Jerina, D., Renson, J., Witkop, B., Udenfriend, S., Science 157, 1524 (1967). (13) Guroff, G., Kondo, K., Daly, J., Biochem. Biophys. Res. Commun. 25, 622 (1966). (14) Guroff, G., Levitt, M., Daly, J., Udenfriend, S., Biochem. Biophys. Res. Commun. 25, 253 (1966). (15) Guroff, G., Reifsnyder, C. Α., Daly, J., Biochem. Biophys. Res. Commun. 24, 720 (1966). (16) Hamilton, G., Hanifin, J., Friedman, J., J. Am. Chem. Soc. 88, 5266 (1966). (17) Jerina, D., Guroff, G, Daly, J., Arch. Biochem. Biophys. 124, 612 (1968).



82. DALY ET AL. Intramolecular Migrations 289

(18) Jerina, D., Daly, J., Landis, W, Witkop, B., Udenfriend, S.,J.Am. Chem. Soc. 89, 3347 (1967). (19) Jerina, D., Daly, J., Witkop, B.,J.Am. Chem. Soc. 89, 5488 (1967). (20) Kaufman, S., Biochim. Biophys. Acta 51, 619 (1961). (21) Levitt, M., unpublished data. (22) Olah, G. Α., Tolgyesi, W. S., Dear, R. Ε. Α.,J.Org. Chem. 27, 3441 (1962). (23) Renson, J., unpublished data. (24) Renson, J., Daly, J., Weissbach, H., Witkop, B., Udenfriend, S., Biochem. Biophys. Res. Commun. 25, 504 (1966). (25) Schepartz, B., Gurin,S.,J.Biol. Chem. 180, 663 (1949). (26) Smith, J. N., Spencer, B., Williams, R. T., Biochem. J. 47, 284 (1950). (27) Taniguchi, K., Kappe, T., Armstrong, M. D.,J.Biol. Chem. 239, 3389 (1964). (28) Udenfriend, S., Zaltzman-Nirenberg, P., Daly, J., Guroff, G., Chidsey, C., Witkop, B., Arch. Biochem. Biophys. 120, 413 (1967). (29) Ullrich, V., Wolf, J., Amadori, E., Staudinger, H., Z. Physiol. Chem. 349, 85 (1968). (30) Witkop, B., Goodwin, S., Experientia 8, 377 (1952). (31) Witkop, B., Goodwin, S.,J.Am. Chem. Soc. 79, 179 (1957). RECEIVED September 10, 1967.


Intramolecular Migrations during Hydroxylation of Aromatic

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