Enzymatic Synthesis of Ao,-Halohydrins from Gaseous Alkenes JOHN GEIGERT,* SAUL L

Home , Haloperoxidase

Haloperoxidases: Enzymatic Synthesis of ao,-Halohydrins from Gaseous Alkenes JOHN GEIGERT,* SAUL L. NEIDLEMAN, DEMETRIOS J. DALIETOS, AND SUSANNE K. DEWITT Cetus Corporation, Berkeley, California 94710 Received 6 July 1982/Accepted 24 September 1982

The enzymatic synthesis of a,p-halohydrins from gaseous alkenes is described. The enzymatic reaction required an alkene, a halide ion, dilute hydrogen peroxide, and a haloperoxidase enzyme. A wide range of gaseous alkenes were suitable for this reaction, including those containing isolated, conjugated, and cumulative carbon-carbon double bonds. Chlorohydrins, bromohydrins, and iodohydrins could be formed. The combining of this enzymatic synthesis with a previously described enzymatic synthesis of epoxides from a,p-halohydrins provides an alternate pathway, other than the well-known enzymatic direct epoxidation pathway, from alkene to an epoxide.

Reports of enzyme reactions on gaseous al- was synthesized by the reduction of 2-bromopropionyl kenes have focused primarily on the formation chloride (7). 1-Iodo-2-propanol was synthesized by the of epoxides from these substrates. Hou et al. reaction of 1-bromo-2-propanol and iodide (15). (10) reported that a monooxygenase derived Enzymatic production of halohydrins. The enzymatic reaction mixtures were incubated in 100-ml Pyrex from several methylotrophic bacteria catalyzes flasks equipped with a magnetic stir bar and stirrer. the epoxidation of C2 to C4 n-alkenes, including Each mixture contained 400 ,u of haloperoxidase, 20 butadiene. The monooxygenase derived from mM potassium halide, and 25 ml of 300 mM potassium Mycobacterium sp. strain E20 by DeBont et al. phosphate buffer at various pH values. Because each (5) catalyzes all of the above reactions and haloperoxidase has specific requirements (13), the includes the epoxidation of allene. following conditions were used: CPO, pH 3.0 and Cl-, In the course of our studies on enzymatic Br-, I-; LPO, pH 6.0 and Br-, I-. reactions of industrially important alkenes, we The gaseous alkenes were slowly (10 ml/min) and discovered that a group of enzymes called halo- continuously bubbled through the reaction mixture the formation of during the reaction. Hydrogen peroxide was the last peroxidases catalyze a,,-halo- reagent added, 30 mM final concentration. Because hydrins from the gaseous alkenes. The enzymat- hydrogen peroxide can oxidize certain critical sites on ic reaction occurs in a buffered, aqueous the enzyme molecule (e.g., sulfhydryl groups), thus solution of alkene, halide ion (X-), dilute hydro- damaging its activity, dilute levels of H202 must be gen peroxide (H202), and biocatalyst (Fig. 1). used in the reaction mixture. After initiation, the Microbial sources of haloperoxidase include reaction was allowed to proceed for 15 min. All chloroperoxidase (CPO) from the fungus Cal- reactions were run at room temperature and atmo- dariomyces fumago (14) and bromoperoxidase spheric pressure. from over 50 algae (9). For each alkene tested, a control flask was included. This flask contained everything except haloperoxidase. Under the conditions described, AND experimental MATERIALS METHODS a, -halohydrin product was not detected in the control Biocatalysts. CPO (from C. fumago; 2 mg of protein flasks. per ml) and lactoperoxidase (LPO) (from milk; 5 mg of Reaction mixture analysis. Aliquots of reaction mix- protein per ml) were purchased from Sigma Chemical tures (10 Ill) were injected into a Finnigan 4021 gas Co. (St. Louis, Mo.). LPO is a bromoperoxidase. chromatograph-mass spectrometer equipped with a Gaseous alkenes. Ethylene, propylene, butene-1, bu- coiled, glass column (1.8 m by 4 mm) packed with tene-2 (cis and trans mixture), isobutylene, butadiene, Tenax-GC (80/100 mesh). The carrier gas was helium and allene were purchased from Matheson Gas Prod- set at 25 ml/min. For rapid analysis of the many ucts (Lyndhurst, N.J.). different reaction mixtures, the following temperatures a,P-Halohydrin standards. 2-Chloroethanol, 2-bro- were used: column temperature, programmed from moethanol, 2-iodoethanol, 1-chloro-2-propanol, and 100 to 250°C at a rate of 10°C/min and then held at 1,4-dibromo-2,3-butanediol were purchased from Al- 250°C for 10 min; injector and jet separator tempera- drich Chemical Co. (Milwaukee, Wis.). 1-Bromo-2- tures, set at 260°C. The mass spectrometer was operat- propanol (containing -10%o 2-bromo-1-propanol) and ed in the electron impact mode at 70 eV. The mass 1-bromo-3-buten-2-ol were purchased from Pfaltz and range from m/z 41 to 400 was scanned every 2 s. Bauer, Inc. (Stamford, Conn.). 2-Bromo-1-propanol Confirmation of the identity of the reaction products 366 VOL. 45, 1983 ENZYMATIC SYNTHESIS OF a,-HALOHYDRINS 367

HR haloperoxidase RCH=CHR' + X- + H202 + H+ . R H- HR ' + H20

X a .8-hal ohydri n Cl chlorohydrin Br bromohydrin I iodohydrin FIG. 1. General equation for haloperoxidase reaction on alkenes.

TABLE 1. CPO reaction on ethylene and propylene to yield a,4-halohydrinsa Reaction X- Product GC,, (min)b Titer (|ug/mI) on: Ethylene Cl 2-Chloroethanol 4.h 370 Br 2-Bromoethanol 6.4 950 I 2-Iodoethanol 10.7 230 Propylene Cl 1-Chloro-2-propanol (a) 5.5 630 2-Chloro-l-propanol (b) 6.0 60 Br 1-Bromo-2-propanol (a) 8.1 1,910 2-Bromo-1-propanol (b) 8.4 210 I 1-Iodo-2-propanol (a) 10.3 420 2-Iodo-1-propanol (b) 10.5 50 Experimental conditions are described in the text. See Fig. 2. b GC.t, Retention time on gas chromatography.

HO X CH2=CH2 CH2 lH2 X-, H20 ethyl ene (pH 3.01

HCOH OH chl oroperoxi dase CH3CH=CH2 CH3CH--CH2 + CH3CH-CH2 X.9 H09 propylene (pH 3.0) a b

FIG. 2. CPO reaction on ethylene (top) and propylene (bottom) to yield a,-halohydrins. 368 GEIGERT ET AL. APPL. ENVIRON. MICROBIOL.

CH 2C2- 49

M+ I

51 80

.-r I I I I I I.11I . . . . . L82 ...... -. I. I. . . I I, Hlll 1A] I - - I . I , , I 1 I I , , ,1. . . . . I I 0I I M/E 50 60 70 80' 90 100 110 120 130 140 150 160 170

CH Br+ 2 M+. Br+ 93 95 124 126 79 81 1.11I 1. II I .1. I ...... -. . I . -...... , - . . . I . . . - - - . I . , I 11111 I...... -. . . ,I - - . . - I I I I ,1 f . I Ifm.. I I M/E 50 60 70 80 90 100 110 120 130 140 150 160 170

M+ -

172 I+ CH2I + L27 141

M/E 50 60 70 80 90 100 110 120 130 140 150 160 170 FIG. 3. Mass spectra of CPO-produced ethylene chlorohydrin (A), bromohydrin (B), and iodohydrin (C). VOL. 45, 1983 ENZYMATIC SYNTHESIS OF a,P-HALOHYDRINS 369

X + I HO X CH3CH-CH2 * CH31H-CH2

ion a vi isomer a ) CH3CH=CH2 [HOX]

X OH I CH3CH-CH2 CH3CH-CH2

ion b * isomer b 'vi

FIG. 4. Haloperoxidase reaction on propylene.

was made by gas chromatography retention time and positional isomers formed (-9:1, isomer a/ mass spectral comparison with authentic standards isomer b) correlated with the stability of the whenever available. Not all reaction conditions were postulated halonium ion intermediates (Fig. 4). optimized nor was there complete conversion of sub- Ion a would be more stable than ion b due to strate. the electron-donating .ability of the methyl group. This ratio of positional isomers that was RESULTS AND DISCUSSION formed enzymatically matches the ratio that one A range of substrates are-known to be haloge- would obtain by chemical addition of hypohal- nated by haloperoxidases, including ,-keto ac- ous acid to propylene (16). Although the gas ids, cyclic 1-diketones, and phenols (14). How- chromatograph separation did not provide com- ever, the ability of haloperoxidases to yield a,3- plete resolution of the positional isomers, this halohydrins from carbon-carbon double bonds is separation coupled with the diagnostic fragmen- not so well known. Previous studies with al- tation of halohydrins in the mass spectrometer kenes have included a few steroids and a vinyl did permit unambiguous assignment of each phosphate (11, 14). In this report we describe the positional isomer (Fig. 5 illustrates this for pro- formation of a,p-halohydrins from ethylene, pylene bromohydrin). propylene, butene-1, butene-2 (cis and trans), Butadiene, being a conjugated alkene, could isobutylene, butadiene, and allene. Since the have yielded both 1,2- and 1,4-adduct products, formation ofa,3-halohydrins from alkenes in the due to the possible resonance of the intermedi- presence of hypohalous acid (HOX) is a well- ate allylic halonium ion (Fig. 6). However, only known preparative and industrial procedure, the the 1,2-adducts were detected (Table 2, Fig. 7). products formed in the enzymatic reactions Such exclusive formation of the 1,2-adduct has were interpreted in terms of hypohalous acid also been previously observed in the chemical addition across the carbon-carbon double bond. addition of HOBr to butadiene (4). Ethylene yielded a single a,,B-halohydrin The 1,2-adduct products could have further product with each halide ion used (Table 1, Fig. reacted to yield dihalohydrins (Fig. 8). Howev- 2). Halogen on the product was confirmed by the er, under the conditions tested, only minor isotopic abundances for chlorine (35Cl-37CI, 3:1) amounts of these dihalohydrins were produced. and bromine (79Br-81Br, 1:1), whereas iodine Allene, being a cumulative alkene, could have was noted by the presence of the I+ mass ion yielded two halohydrin positional isomers (Fig. (m/z 127) in the mass spectra (Fig. 3). Detection 9). However, none of the positional isomer that of molecular ions in the mass spectra confirmed would have tautomerized to the a-haloketone that halohydrin formation and not halogenation was detected in the enzymatic reaction (Table 2, had indeed occurred. Fig. 7). Exclusive addition of the halogen on the Propylene, being an asymmetric alkene, yield- central carbon and of the hydroxyl group on the ed two halohydrin positional isomers with each terminal carbon has also been previously ob- halide ion used (Table 1, Fig. 2). The ratio of served in the chemical addition of HOCI to a OH Br A CH 3CH-CH2

r OH B B CH 3CH -CH 2

220 240 260 2 0 3do SCAN 7:3 8:1 9:0 9:4 10:2 TIME (MIN) b A F2OX B 45 Ili CH 3CH

Br+ CH3 H 109 CH-CH2

123 1 125

CHI Br + 2 93 95 Br+ 79 81 M-H+

137 139 11 .Il.l1 .,I ..1 . 11U , I I I I I I II I . . ,I . I ,I, I I I. , I M/E 50 60 70 80 90 100 110 120 130 140 M/E 50 60 70 80 90 1A0 110 120 130 140 FIG. 5. Reconstructed ion chromatogram (a) and distinctive mass spectrum for each positional isomer of propylene bromohydrin (b). A, 1-Bromo-2-propanol; B, 2-bromo-1-propanol.

370 VOL. 45, 1983 ENZYMATIC SYNTHESIS OF a,4-HALOHYDRINS 371

x X OH I1+ I1 I CH2=CH-CH-CH2 D CH2=CH-CH-CH2 (1,2-adduct)

H? X [X CH2=CH-CH=CH2 CH2=CH-CH-CH2 CH2=CH-CH-CH2 (1,2-adduct) +I_ OH X I CH2-CH=CH-CH2 CH2-CH=CH-CH2 (1 ,4-adduct) FIG. 6. Haloperoxidase reaction on 1,3-butadiene.

TABLE 2. Haloperoxidase reaction on butadiene and allene to yield aL,4-bromohydrinsa Haloperoxidase Prod GC, Titer reaction uct (min)b (ILg/nil) LPO on butadiene 1-Bromo-3-buten-2-ol (a) 10.5 1,395 2-Bromo-3-buten-1-ol (b) 11.2 160 1,4-Dibromo-2,3-butanediol (c) 18.3 23 CPO on allene 2-Bromo-2-propen-1-ol (a) 8.1 420 a Experimental conditions are given in the text. See Fig. 7. b GCr,, Retention time on gas chromatography.

HO Br Br OH lactoperoxidase I I I I CH2=CH-CH=CH2 CH2=CH-CH-CH2 + CH2=CH-CH-CH2 Br-, H202 a b butadi ene (pH 6.01 evi

Br HO OH Br I I I I CH2-CH-CH-CH2

_r OH chl oroperoxi dase I CH2=C=CH2 co . CH2=C-CH2 Br-,, H6,02 allene (pH3)3 a FIG. 7. LPO (top) and CPO (bottom) reactions on butadiene (top) and allene (bottom) to yield a,p- bromohydrins. 372 GEIGERT ET AL. APPL. ENVIRON. MICROBIOL. OH X [HOX] I A,> CH2=CH-J-H'-H2 CH2-CA- H- H2 FIG. 8. Haloperoxidase conversion of 1,3-butadiene to monohalohydrins and dihalohydrins.

X OH CH2=4-CH2 CH2=C-CH2 [CHOX) CH2=C=CH2'

OH O X +CC C 11 41 CH2=C-UH2 H2=C-CH2 CH3C- H2

FIG. 9. Haloperoxidase reaction on allene.

allenic hydrocarbons (2). Under the conditions sponsible for this reaction was named halohy- listed in Materials and Methods, formation of drin epoxidase. the dihalohydrin product was not observed (Fig. The combining of halohydrin epoxidase with 10). The remaining gaseous alkenes also were haloperoxidase provides an alternate pathway, shown to react with haloperoxidase to yield the other than the well-known direct epoxidation expected halohydrin products (Fig. 11). pathway, from an alkene to an epoxide (Fig. 12). Therefore, although the mechanism of cataly- Biological systems, especially those existing in sis by the haloperoxidase enzymes is still a an environment containing available halide salts matter of discussion (8, 12), the products formed (e.g., marine organisms), may use such an enzy- in these reactions are consistent with free hypo- matic strategy in various biosynthesis pathways. halous acid- being generated by the enzyme. The numerous haloperoxidases, a,4-halohy- Regiospecific and stereospecific properties of drins, and epoxides found in such systems sup- the formed a,,B-halohydrins were not investigat- port this thought (6, 9). ed in the study. However, haloperoxidases are reported not to produce optically active halohydrin products (11). Castro and Bartnicki (1, 3) observed an enzy- X OH f OH matic route to epoxides that involved I a,P-halo- CH2=C-CH2 CH2-C-H2 hydrins. They isolated an organism, a Flavobac- I I terium sp., that converted the chlorohydrin of WI HO X allyl chloride and the bromohydrins of allyl alcohol, allyl bromide, propylene, and 2-butene FIG. 10. Haloperoxidase does not convert allene to to their respective epoxides. The enzyme re- its dihalohydrin. VOL. 45, 1983 ENZYMATIC SYNTHESIS OF a,A-HALOHYDRINS 373 HO X

[HOXJ H- CH4 CH3CH2CH=CH2 CH3CH2CH-CH butene-1

HO X EHOX]I CH3CH=CHCH3 CH3CH-CHCH3 butene-2 (cis and trans mixtiure)

[ HOX) HO) .H (CH3)2C=CH2 (CH3) RH2

i sobutyl ene FIG. 11. Haloperoxidase reaction on butene-1, butene-2, and isobutylene.

a OH X Hal operoxi dase R H + RCH=CHR' + H202 + X- + H+ - * RR H-CHR '+ H200

OH X 0 Halohydrin RCH-CHR I RCH-CHR' + X- + H Epoxi dase

b ~~~~~~~Monooxygenase , RCH=CHR' + O + NADH + H+ WI RCH-CHR' + NAD+ + H20 FIG. 12. Two enzymatic pathways to an epoxide from an alkene: (a) haloperoxidase coupled with halohydrin epoxidase and (b) direct epoxidation with a monooxygenase. 374 GEIGERT ET AL. APPL. ENVIRON. MICROBIOL.

ACKNOWLEDGMENT Am. Chem. Soc. 73:5063-5067. 8. Harrison, J. E., and J. Schultz. 1976. Studies on the We thank M. Moreland for the synthesis of several a,1- chlorinating activity of myeloperoxidase. J. Biol. Chem. halohydrin standards. 251:1371-1374. 9. Hewson, W. D., and L. P. Hager. 1980. Bromoperoxi- dases and halogented lipids in marine algae. J. Phycol. LITERATURE CITED 16:340-345. 1. Bartnicki, E. W., and C. E. Castro. 1969. Biodehalogena- 10. Hou, C. T., R. Patel, A. I. Laskin, and N. Barnabe. 1979. tion. The pathway for transhalogenation and the stereo- Microbial oxidation of gaseous hydrocarbons: epoxida- chemistry of epoxide formation from halohydrins. Bio- tion of C2 to C4 n-alkenes by methylotrophic bacteria. chemistry 8:4677-4681. Appi. Environ. Microbiol. 38:127-134. 2. Bianchini, J. P., and M. Cocordano. 1970. Experimental 11. Kollonitsch, J., S. Marburg, and L. M. Perkins. 1969. and theoretical study of the addition of hypochlorous acid Enzymatic formation of chiral structures in racemic form. to allenes. Tetrahedron 26:3401-3411. J. Am. Chem. Soc. 92:4489-4490. 3. Castro, C. E., and E. W. Bartnicki. 1968. Biodehalogena- 12. Libby, R. D., J. A. Thomas, L. W. Kaiser, and L. P. tion. Epoxidation of halohydrins, epoxide opening, and Hager. 1982. Chloroperoxidase halogenation reactions. transhalogenation by a Flavobacterium sp. Biochemistry Chemical versus enzymatic halogenating intermediates. J. 7:3213-3218. Biol. Chem. 257:5030-5037. 4. Dalton, D. R., and R. M. Davis. 1972. Bromohydrin for- 13. Morrison, M., and G. R. Schonbaum. 1976. Peroxidase- mation in dimethyl sulfoxide. The reaction on conjugated catalyzed halogenation. Annu. Rev. Biochem. 45:861- dienes. Tetrahedron Lett., p. 1057-1060. 888. 5. DeBont, J. A. M., M. M. Atwood, S. B. Primrose, and W. 14. Neldleman, S. 1975. Microbial halogenation. Crit. Rev. Harder. 1979. Epoxidation of short chain alkenes in Microbiol. 5:333-358. Mycobacterium E20: the involvement of a specific mono- 15. Stewart, C. A., and C. A. VanderWerf. 1954. Reaction of oxygenase. FEMS Microbiol. Lett. 6:183-188. propylene oxide with hydrogen halides. J. Am. Chem. 6. Fenical, W. 1982. Natural products chemistry in the Soc. 76:1259-1264. marine environment. Science 215:923-928. 16. Weissermel, K., and H. J. Arpe. 1978. Secondary prod- 7. Fickett, W., H. K. Garner, and H. J. Lucas. 1951. The ucts of propene, p. 235-272. In Industrial organic chemis- configuration of optically active 1,2-dichloropropane. J. try. Verlag Chemie, New York.