Formation of enantiopure 5-substituted oxazolidinones through enzyme-catalysed kinetic resolution of epoxides

Article abstract of DOI:10.1021/ol800698t

(Chemical Equation Presented) Halohydrin dehalogenase from Agrobacterium radiobacter catalyzed the enantioselective ring opening of terminal epoxides with cyanate as a nucleophile, yielding 5-substituted oxazolidinones in high yields and with high enantiopurity (69-98% ee). This is the first example of the biocatalytic conversion of a range of epoxides to the corresponding oxazolidinones.

Full text of DOI:10.1021/ol800698t

ORGANIC
LETTERS
2008
Vol. 10, No. 12
2417-2420
Formation of Enantiopure 5-Substituted
Oxazolidinones through
Enzyme-Catalysed Kinetic Resolution of
Epoxides
Maja Majeric´ Elenkov,†,‡ Lixia Tang,‡ Auke Meetsma,§ Bernhard Hauer,| and
Dick B. Janssen*,‡
Ru{er BosˇkoVic´ Institute, Bijenicˇka c. 54, 10000 Zagreb, Croatia, and Biochemical
Laboratory, Groningen Biomolecular Sciences and Biotechnology Institute, and Crystal
Structure Center, Zernike Institute for AdVanced Materials, UniVersity of Groningen,
Nijenborgh 4, 9747 AG, Groningen, The Netherlands, and BASF AG, Fine Chemicals
and Biocatalysis Research, Ludwigshafen, Germany
d.b.janssen@rug.nl
Received March 27, 2008
ABSTRACT
Halohydrin dehalogenase from Agrobacterium radiobacter catalyzed the enantioselective ring opening of terminal epoxides with cyanate as
a nucleophile, yielding 5-substituted oxazolidinones in high yields and with high enantiopurity (69-98% ee). This is the first example of the
biocatalytic conversion of a range of epoxides to the corresponding oxazolidinones.
Epoxides are highly valuable compounds in organic synthe-
sis, because the oxirane ring can be chemically transformed
into numerous intermediates.1 To synthesize these important
building blocks in enantiopure form, methods for asymmetric
epoxidation of olefins2 and kinetic resolution of epoxides3
have been developed, mainly based on the use of chiral
catalysts containing transition metals. Biocatalytic methods
are also available,4 including monooxygenase-mediated
epoxidation5 and kinetic resolution of racemic epoxides by
epoxide hydrolases.6,7 Recently, halohydrin dehalogenases
have been explored in the latter conversions.8 These bacterial
enzymes catalyze the reversible conversion of vicinal ha-
loalcohols to epoxides, and their natural role is the metabo-
lism of compounds that possess a vicinal halohydrin group
or of substrates that are degraded via intermediates that carry
such a functionality.
In the epoxide ring opening reactions mediated by the
HheC-type halohydrin dehalogenase from Agrobacterium
radiobacter, various small anionic nucleophiles can be
accepted (Scheme 1). In a recent study on the scope of
† Ru{er Bosˇkovic´ Institute.
‡ Groningen Biomolecular Sciences and Biotechnology Institute.
§ Zernike Institute for Advanced Materials.
| BASF AG, Fine Chemicals and Biocatalysis Research.
(1) Yudin, A. K. Aziridines and Epoxides in Organic Synthesis; Wiley-
VCH: Weinheim, 2006.
(4) de Vries, E. J.; Janssen, D. B. Curr. Opin. Biotechnol. 2003, 14,
414–420
.
(5) Schmid, A.; Hofstetter, K.; Feiten, H.-J.; Hollmann, F.; Witholt, B.
AdV. Synth. Catal. 2001, 343, 732–737
(2) (a) Johnson, R. A. Catalytic Asymmetric Epoxidation of Allylic
Alcohols, Ojima, I. Ed.; Catalytic Asymmetric Synthesis, 2nd ed.; Wiley-
VCH: Weinheim, 2000; pp 231-286. (b) Katsuki, T. Asymmetric Epoxi-
dation of Unfunctionalized Olefins and Related Reactions, Ojima, I. Ed.;
Catalytic Asymmetric Synthesis, 2nd ed.; Wiley-VCH: Weinheim, 2000;
pp 287-326.
.
(6) Archelas, A.; Furstoss, R. Curr. Opin. Chem. Biol. 2001, 5, 112–
119
.
(7) Steinreiber, A.; Faber, K. Curr. Opin. Biotechnol. 2001, 12, 552–
558
.
(8) Janssen, D. B.; Majeric´ Elenkov, M.; Hasnaoui, G.; Hauer, B.; Lutje
Spelberg, J. H. Biochem. Soc. Trans. 2006, 34, 291–295.
(3) Jacobsen, E. N. Acc. Chem. Res. 2000, 33, 421–431.
10.1021/ol800698t CCC: $40.75
Published on Web 05/21/2008
2008 American Chemical Society

Scheme 1
.
Halohydrin Dehalogenase Catalysed Ring Opening
Reactions
halohydrin dehalogenase catalyzed ring opening of epoxides,
we have discovered that cyanate (OCN-) is accepted as a
nucleophile.9 Due to cyclization, the products that are formed
undergo conversion to 2-oxazolidinones, which are interest-
ing building blocks for the preparation of pharmaceutical
intermediates10 and have wide application as chiral auxiliaries
and ligands.11
Figure 1. Epoxides tested as substrates in cyanate-mediated ring
opening reaction catalysed by HheC.
Enzymatic conversions were first performed on analytical
scale. Reactions were monitored by extracting products from
the aqueous phase, followed by GC analysis. We found that
only five substrates (1a-1e) were converted at a reasonable
rate. Only very low or no activity was observed toward
substrates 1f-1l (less than 0.2 µmol·min-1·mg-1). For
example, no reaction of NaOCN with 1i or 1k took place,
even though formation of the cyanoalcohols 3-cyclohexyl-
3-hydroxypropionitrile and 3-hydroxy-4-methylpentanenitrile
in a CN--mediated ring opening reaction with the same
Although chemical ring opening reactions of epoxides with
different nucleophiles have been studied extensively, little
work has been done on the reaction with inorganic cyanates
and the transformation of the products to 2-oxazolidino-
nes.12,13 Dyen and Swern14 reported that reaction of potas-
sium cyanate with epoxides in DMF/H2O produced 2-ox-
azolidinones in moderate yield. This method is applicable
to terminal alkyl epoxides only. The main obstacle for the
direct conversion of epoxides to oxazolidinones is the weak
nucleophilicity of the cyanate ion.15 In 2005, Bartoli et al.
reported a method for the synthesis of enantiopure 5-sub-
stituted oxazolidinones from racemic epoxides by CoIIIsalen-
catalyzed kinetic resolution, using urethane as the nucleo-
philic reagent.16 Despite the established relevance of
oxazolidinones in synthetic chemistry, their preparation by
other means has been limited to a few methods.17,11 Of these,
an indirect procedure involving preparation of amino alcohols
and subsequent cyclization emerged as a versatile route, in
part because of the availability of substrates in both racemic
and enantiomerically pure form.11
substrates occurred at a good rate (1.1 and 0.8 µmol·min-1
·
mg-1, respectively).19 Previously, the biocatalytic potential
of HheC has been explored by using spectrophotometric
assays based on reaction of p-nitrostyrene oxide with
different nucleophiles.20 It was observed that the enzyme
does not catalyze a reaction between this substrate and
OCN-, whereas Br-, Cl-, N3 , NO2 , and CN- were
accepted as nucleophiles, affording the corresponding ring
opening products. These results indicate that HheC has a
more restricted substrate range when OCN- is the nucleo-
phile than in azide- and cyanide-mediated ring opening
reactions.
-
-
Due to the ambident nature of the OCN-, ring opening of
epoxides can proceed Via nitrogen (a) or oxygen (b) attack,
leading to two isomeric products, ꢀ-hydroxy isocyanate and
ꢀ-hydroxycyanate, respectively (Scheme 2). Organic cyanates
Here, we report that cyanate ion can act as a nucleophile
in the ring opening of epoxides catalyzed by halohydrin
dehalogenase to form highly enantioenriched 5-substituted
2-oxazolidinones.
The A. radiobacter halohydrin dehalogenase (HheC) was
explored as a biocatalyst in the reaction of epoxides with
OCN-. A range of epoxides that are known from previous
studies18 to be accepted by HheC when CN- is the
nucleophile was tested in these experiments (Figure 1).
Scheme 2
(9) Hasnaoui-Dijoux, G.; Majeric´ Elenkov, M.; Lutje Spelberg, J.; Hauer,
B.; Janssen, D. B. ChemBioChem 2008, 7, 1048–1051.
(10) Diekema, D. J.; Jones, R. N. Drugs 2000, 59, 7–16.
(11) Ager, D. J.; Prakash, I.; Schaad, D. R. Chem. ReV. 1996, 96, 835–
875.
(12) Thomsen, A. L. Ber. 1878, 11, 2136–2139
.
(13) Schierle-Arndt, K.; Kolter, D.; Danilemeier, K.; Steckhan, E. Eur.
J. Org. Chem. 2001, 2425–2433
are unstable compounds and undergo isomerization to
isocyanate at room temperature.21 The reaction is faster in
polar than in nonpolar solvents. On the other hand, ꢀ-hydroxy
isocyanates cannot be isolated, because they spontaneously
cyclize to oxazolidinones.
To identify products formed, semipreparative scale reactions
(0.50 g, 100-250 mM) were performed (Table 1). Racemic
.
(14) Dyen, M. E.; Swern, D. J. Org. Chem. 1968, 33, 379–384.
(15) Jencks, W. P.; Carriuolo, J. J. Am. Chem. Soc. 1960, 82, 1778–
1786.
(16) Bartoli, G.; Bosco, M.; Carlone, A.; Locatelli, M.; Melchiorre, P.;
Sambri, L. Org. Lett. 2005, 7, 1983–1985.
(17) Dyen, M. E.; Swern, D. Chem. ReV. 1967, 67, 197–246
.
(18) Majeric´ Elenkov, M.; Hauer, B.; Janssen, D. B. AdV. Synth. Catal.
2006, 348, 579–585.
2418
Org. Lett., Vol. 10, No. 12, 2008

detectable product and reactions occurred with no evidence
of formation of the other regioisomer. The exclusive forma-
tion of 5-substituted isomers indicates that attack at the less
substituted position of the oxirane ring was highly predomi-
nant. This is consistent with our previous observations on
the regioselectivity of HheC.9,18,19
Table 1. Conversion of Epoxides 1a-1e with Cyanate
Catalysed by HheCa
Ring opening of racemic epoxides 1a-1e resulted in a
kinetic resolution. Conversion of 1b-1d proceeds rapidly
to 50%, after which it drastically slows down, indicating high
enantioselectivity of the biotransformations. Very high ee’s
of 5-methyl substituted 2-oxazolidinones 2b-2d were ob-
tained (93-98% ee, entries 2, 3, and 4). HheC was less
enantioselective toward monosubstituted epoxides. Ring
opening of 1a and 1e resulted in moderate enantioselectivity
(69-80% ee, entries 1 and 5). As previously observed,19
HheC exhibited very high enantioselectivity toward 2,2-
disubstituted epoxides, with E values (kcat,R/Km,R/(kcat,S/Km,S
)
from 85 to over 200. This property makes the enzyme a
valuable tool for the preparation of enantiomerically pure
tertiary alcohols and, in this case, oxazolidinones with
quaternary stereocenters. Conversion of racemic epoxides
was achieved within 1-5 h, pointing to a good enzyme
activity in such a non-natural reaction.
HheC can catalyze a racemization of the enantiomers of
epichlorohydrin (1e) when Cl- is present in the incubation
mixture.22 Upon ring opening by halide, epichlorohydrin is
converted to prochiral 1,3-dichloro-2-propanol. Racemization
is caused by the reversibility of this ring opening reaction,
of which the equilibrium is toward the epoxide. Treatment
of highly enantioenriched (R)-1e with HheC led to nearly
racemic epoxide (20% ee) within 1 h (Figure 2). Traces of
a For reaction conditions and procedures see Supporting Information.
b Isolated yield. c Determined by chiral GC analysis. d Apparent inversion
of configuration in a series of halogen-containing epoxides compared to
alkyl epoxides is due to different substituent priority according to CIP rule,
not to a different stereochemical preference of the enzyme. e Conversion
was 49-50%. f Substrate was completely consumed; 100% conversion.
epoxides 1a-1e were converted employing catalytic amounts
of purified enzyme (2-5% w/w) in Tris-SO4 buffer (0.5 M,
pH 7.5). Reactions were carried out at room temperature and
stopped when substrate conversion approached 50% (entries
1-4) or 100% (entries 5 and 6). After usual workup, enantio-
merically enriched products 2a-2d were isolated in high yields,
44-47%, whereas some loss of material was observed with 2e
(entries 5 and 6). In control reactions, in which the enzyme
was omitted, formation of products was not observed. The
structure of products 2a-2e was confirmed by NMR, MS, and
IR analysis. All products show an infrared band at around 1740
cm-1, characteristic for the carbonyl in 2-oxazolidinones. To
further prove the structure of the products, racemic 2a-2d were
synthesized (2e was commercially available) and NMR spectra
were found to match the products isolated from the enzymatic
reactions.
Figure 2. Racemization of (R)-1e catalysed by HheC. The reaction
Formation of 2-oxazolidinones (2a-2e) was a clean and
highly regioselective reaction, with no observable formation
of byproduct. None of the two possible intermediates
(Scheme 2) could be detected by GC. In each case, the
corresponding 5-substituted 2-oxazolidinone was the only
was carried out with 25 mg (100 mM) of (R)-1e and 0.75 mg of
HheC in 2.5 mL of Tris-SO4 in the presence (sOs) and absence
(s•s) of 25 mM NaCl.
Cl- present in sample were enough to trigger this reaction.
However, the rate of racemization became faster by adding
a small amount of Cl- (Figure 2).
(19) Majeric´ Elenkov, M.; Tang, L.; Hauer, B.; Janssen, D. B. AdV.
Synth. Catal. 2007, 349, 2279–2285.
(20) Lutje Spelberg, J. H.; Tang, L.; van Gelder, M.; Kellogg, R. M.;
Janssen, D. B. Tetrahedron: Asymmetry 2002, 13, 1083–1089.
(21) Patai, S. Chemistry of Cyanates and Their Thio DeriVatiVes; John
Wiley and Sons: New York, 1977; Part 1, pp 415-517.
(22) Lutje Spelberg, J. H.; Tang, L.; Kellogg, R. M.; Janssen, D. B.
Tetrahedron: Asymmetry 2004, 15, 1095–1102.
Org. Lett., Vol. 10, No. 12, 2008
2419

A ring opening reaction of 1e with OCN- resulted in a
dynamic kinetic resolution due to the fact that enantioselec-
tive ring opening and substrate racemization occurred
simultaneously. The reaction of rac-1e with OCN- without
additional optimization (pH, concentrations) yielded (S)-2e
in 69% ee (Table 1, entry 5) with complete conversion of
the epoxide. No NaCl was added to the reaction mixture
because the initial amount of Cl- was sufficient to cause
racemization. Besides, by increasing the concentration of Cl-,
the rate of (S)-2e formation significantly decreased, as OCN-
competes with Cl-. The low product enantiopurity can be
assigned to the modest enantioselectivity of HheC toward
1e. The low product yields we attribute partially to hydrolytic
instability of substrate 1e but mostly to formation of
polymeric material.
Because 1c and 1d are homologues of 1e, a dynamic
kinetic resolution could also be performed with these
substrates. HheC rapidly racemizes the slower reacting
enantiomer (R)-1d. Surprisingly, in the presence of OCN-,
racemization of (R)-1d was inhibited. Because of this
inhibition of racemization of (R)-1d, the conversion of rac-
1d presented in Table 1 predominantly has the character of
a kinetic resolution. The dynamic character of the resolution
improved by adding halide.
Absolute configurations of the faster-reacting epoxide
enantiomers were determined using chiral GC by comparing
the retention times with standards. The faster reacting
enantiomers all have the same relative configuration, but
because of the priority switch,23 the (R) enantiomer was the
faster reacting one in the case of substrates 1a and 1b, and
the (S) enantiomer was in the case of 1c-1e. These results
and our earlier observations18,19 suggest that the stereochem-
ical preference of the HheC can be described by a general
substrate model (Figure 3).
and (S)-2e.13 Additionally, the configuration of oxazolidinone
(S)-2e was confirmed by crystal structure determination.24
The tentative assignments of absolute stereochemistry were
made by analogy within a related series.
A surprising observation that emerged from this study was
the narrow epoxide substrate tolerance of HheC when cyanate
was used as the nucleophile as compared to when other ring
opening anions were used (N3 , NO2 , CN-). Because the
epoxide-binding catalytic residues, the anion-binding site,
and the place where the R-group binds to the enzyme
(Scheme 1) can be clearly distinguished in the structure, there
is no obvious explanation for this phenomenon. It was also
observed that the cyanate anion can cause inhibition of ring
opening. One possible explanation could be that cyanate
induces an unproductive binding mode similar to the one
observed with the nonpreferred (S) enantiomer of styrene
oxide.25 Here, the epoxide oxygen is positioned toward the
nucleophile instead of the terminal carbon atom of the
oxirane ring.
-
-
In conclusion, we have found that HheC is a sufficiently
active and very enantioselective catalyst for the addition of
cyanate to some small terminal epoxides. In this way,
5-substituted 2-oxazolidinones were prepared for the first
time by biocatalytic kinetic resolution of epoxides with
NaOCN. Preparative scale syntheses could be performed at
15-20 g/L substrate concentrations due to the high substrate
concentration tolerance and good enzyme stability. The
results further extend the range of nucleophiles that can be
used in enzymatic epoxide ring opening reactions, which also
includes azide, cyanide and nitrite. Once again, this highly
promiscuous halohydrin dehalogenase proves to be valuable
new tool for biocatalysis.
Acknowledgment. This work was supported by the
Croatian Ministry of Science, Education and Sports (project
0982933-2908).
Supporting Information Available: Biocatalytic proce-
dures, analytical methods, chemical synthesis, crystallo-
graphic data. This material is available free of charge via
the Internet at http://pubs.acs.org.
OL800698T
Figure 3. Substrate model for HheC.
(23) Cahn-Ingold-Prelog rule.
(24) For more details, see Supporting Information.
(25) de Jong, R. M.; Tiesinga, J. J.; Villa, A.; Tang, L.; Janssen, D. B.;
Dijkstra, B. W. J. Am. Chem. Soc. 2005, 127, 13338–13343.
A comparison of optical rotation values with literature data
revealed the absolute configuration of the products (R)-2a16
2420
Org. Lett., Vol. 10, No. 12, 2008