Prochiral selectivity and deuterium kinetic isotope effect in the oxidation of benzyl alcohol catalyzed by chloroperoxidase

Article abstract of DOI:10.1039/a904327f

The chloroperoxidase-catalyzed oxidation of benzyl alcohol exhibits a very high prochiral selectivity, involving only the cleavage of the pro-S C-H bond.

Full text of DOI:10.1039/a904327f

Prochiral selectivity and deuterium kinetic isotope effect in the oxidation of
benzyl alcohol catalyzed by chloroperoxidase
a
b
a
Enrico Baciocchi,* Osvaldo Lanzalunga and Laura Manduchi
a
Dipartimento di Chimica, Università ‘La Sapienza’, Rome, Italy. E-mail: baciocchi@axcasp.caspur.it
Dipartimento di Chimica and Centro CNR di Studio sui Meccanismi di Reazione, Università ‘La Sapienza’, Rome,
Italy
b
Received (in Liverpool, UK) 26th May 1999, Accepted 23rd July 1999
The chloroperoxidase-catalyzed oxidation of benzyl alcohol
exhibits a very high prochiral selectivity, involving only the
cleavage of the pro-S C–H bond.
By looking at the data in Table 1, a very remarkable
observation is that from (S)-1-d practically only PhCHO is
produced, whereas the oxidation of its enantiomer (R)-1-d leads
almost exclusively to PhCDO. Comparable amounts of deut-
erated and undeuterated benzaldehydes form instead from the
racemic alcohol (±)-1-d.
These results clearly indicate that in the oxidation of (S)-1-d
the bond broken is almost exclusively the C-D bond, whereas
the oxidation of (R)-1-d involves almost exclusively the
cleavage of the C–H bond. Thus, the ratio between the kcat
values for (R)-1-d (C–H bond cleavage) and (S)-1-d (C–D bond
cleavage), reported in Table 2, should provide us with a lower
The mechanistic aspects of the reactions catalyzed by chloro-
peroxidase (CPO), a hemoprotein isolated from the fungus
Caldariomyces fumago, are attracting continuous interest as this
2 2
enzyme is able to catalyze the H O -promoted oxidation of a
1
large variety of organic compounds. There is general con-
sensus that in all cases the active oxidant is an iron(iv) oxo
+
·
IV
complex porphyrin IX (P) radical cation [P –Fe NO], also
indicated as compound I, formed by reaction of the resting
d,2
enzyme with H
O
2 2
[eqn. (1)].1
limit for the intrinsic kinetic deuterium isotope effect of the
3
H
reaction (k /k
D
). A value of 2.8 is obtained which is slightly
P–FeIII + H
O
? P –Fe NO + H
+·
IV
2
2
2
O
(1)
smaller than those (3.3, 3.5) found in the benzylic hydroxylation
of p-methylanisole.1i It therefore seems possible to suggest a
similar mechanism for the two processes, i.e. the oxidation of
benzyl alcohol catalyzed by CPO should involve the transfer of
a hydrogen atom to the ferryl oxygen of the iron oxo complex
Compound I
Here we report on a kinetic and products study of the oxidation
of benzyl alcohol 1 and the enantiomeric and racemic forms of
a-monodeuterated benzyl alcohol (1-d). The results obtained
have shown that the process is characterized by an extremely
high prochiral selectivity, providing us with useful information
5
(Scheme 1, path a). An a-hydroxy carbon radical and the iron
IV
hydroxy complex P–Fe –OH form. They may lead to the
about the reaction mechanism.
hydrated form of benzaldehyde directly (oxygen rebound,
Scheme 1, path b) or stepwise with formation of the inter-
mediate a-hydroxybenzyl cation (Scheme 1, path c and d). A
similar sequence has also been proposed for the oxidation of
benzyl alcohol catalyzed by cytochrome P-450.6
2
The racemic [a- H
1
]benzyl alcohol [(±)-1-d)] and the two
2
enantiomers (R)- and (S)-[a- H
1
]benzyl alcohol [(R)-1-d and
S)-1-d, respectively] were reacted with H in H O at pH 6
0.1 M phosphate buffer), in the presence of CPO (2 nmol for 10
). In each case, the molar ratio between PhCDO
(
(
O
2 2
2
mmol of H
2
O
2
The possibility of an electron transfer mechanism facilitated
by the aromatic ring seems unlikely since the value of k /k is
and PhCHO produced in the reaction was measured by GC-MS
H
D
1
and/or H NMR, the two methods giving very similar results.
significantly higher than that (1.8) determined in the oxidation
The results are reported in Table 1. The kinetic parameters K
m
of racemic 1-d induced by the genuine one-electron oxidant
2
. 7
and kcat for the CPO-catalyzed oxidation of deuterated and
undeuterated benzyl alcohols were obtained, as usual, by
measuring the initial rates of formation of benzaldehyde at
different concentrations of substrate (Lineweaver–Burk plots).
These values are collected in Table 2.
SO4 .
The data presented in Table 1 also allow some speculation
concerning the orientation of the substrate in the enzyme active
site. In particular, it can be suggested that the substrate binds to
the enzyme orienting the C–D bond of the S enantiomer, and the
2
Table 2. Steady-state kinetic constants for the chloroperoxidase catalyzed
oxidation of benzyl alcohols by H O
2 2
Table 1 Oxidations of [a- H
1
]benzyl alcohols with H
O
2 2
catalyzed by
a
CPO
Yields (%)b
PhCDO/PhCHOc
Substrate
K
m
/mMa
k
cat/s21a
Substrate
2
1
1.3 ± 0.1
1.1 ± 0.1
1.6 ± 0.1
17.2 ± 0.7
6.6 ± 0.4
18.7 ± 0.9
(
(
(
S)-[a- H]benzyl alcohol
58
68
47
< 0.1
> 30
2.0
2
(S)-1-d
(R)-1-d
R)-[a- H]benzyl alcohol
2
±)-[a- H]benzyl alcohol
a
a
Obtained from a Lineweaver–Burk plot. The initial rates for the formation
of benzaldehyde were determined spectrophotometrically at 25 °C and pH
following the increase of absorbance at 280 nm.
The alcohol (0.1 ml of a solution 0.5 M in CD
3
CN) and CPO (Sigma) (2
nmol) were magnetically stirred in 5 ml of 0.1 M phosphate buffer pH 6.0
at 25 °C, H (200 ml, 0.5 M) was then slowly added in 1 h via a syringe
pump. After extraction with CDCl
NMR, GC and GC-MS. Benzaldehyde and [a- H ]benzaldehyde were the
1
only products formed. Yields are with respect to the starting material.
6
2 2
O
1
3
reaction mixtures were analyzed by H
2
b
c
Average of at least three determinations, the error is ±10%. Molar ratio
measured via GC-MS by the ratio between the corrected signal intensities of
the two molecular ions at m/z 107 and 106. Average of at least three
1
determinations, the error is ±10%. Similar values were obtained by H NMR
considering that the signal of the benzylic protons ortho to the carbonyl is
due to the sum of the two products (PhCHO + PhCDO) and that of the
aldehydic proton derives from PhCHO.
Scheme 1
Chem. Commun., 1999, 1715–1716
1715

Notes and references
1
(a) S. Kobayashi, M. Nakano, T. Kimura and A. P. Schaap, Biochem.
Biophys. Res. Commun., 1986, 135, 166; (b) S. Kobayashi, M. Nakano,
T. Kimura and A. P. Schaap, Biochemistry, 1987, 26, 5019; (c) P. R. Ortiz
de Montellano,Y. S. Choe, G. DePillis and C. E. Catalano, J. Biol. Chem,
1
987, 262, 11641; (d) B. W. Griffin, in Peroxidases in Chemistry and
Biology, ed. J. Everse, K. E. Everse and M. B. Grisham, CRC Press, Boca
Raton, 1991, vol. II, pp. 85–137; (e) S. Colonna, N. Gaggero, L. Casella,
G. Carrea and P. Pasta, Tetrahedron: Asymmetry, 1992, 3, 95; (f) L.
Casella, M. Gullotti, R. Ghezzi, S. Poli, T. Beringhelli, S. Colonna and G.
Carrea, Biochemistry, 1992, 31, 9451;. (g) O. Okazaki and F. P.
Guengerich, J. Biol. Chem., 1993, 268, 1546; (h) L. Casella, S. Poli, M.
Gullotti, C. Selvaggini, T. Beringhelli and A. Marchesini, Biochemistry,
Fig. 1 Proposed orientation of (R)-1-d and (S)-1-d with respect to the iron
oxo complex in the CPO active site.
C–H bond of the R enantiomer, towards the oxygen of the active
oxidant, as illustrated in Fig. 1.
1
994, 33, 6377; (i) V. P. Miller, R. A. Tschirret-Guth and P. R. Ortiz de
It follows that the oxidation of benzyl alcohol catalyzed by
CPO should be characterized by a very high prochiral
selectivity with only the pro-S hydrogen [Fig. 2(a)] involved in
the process. An additional very interesting notation is that the
same spatial orientation as that observed for the oxidation of
benzyl alcohol appears to be also required in the CPO-induced
oxidation of ethylbenzene, which accordingly has been reported
to produce (R)-1-phenylethanol,1 exclusively [Fig. 2(b).
Montellano, Arch. Biochem. Biophys., 1995, 319, 333; (j) A. Zaks and
D. R. Dodds, J. Am. Chem. Soc., 1995, 117, 10419; (k) P. H. Toy, M.
Newcomb and L. P. Hager, Chem. Res. Toxicol., 1998, 11, 816; (l) S. Hu
and L. P. Hager, J. Am. Chem. Soc., 1999, 121, 872.
2
J. H. Dawson, Science, 1988, 240, 433; M. C. R. Franssen and H. C. Van
der Plas, Adv. Appl. Microbiol., 1992, 37, 41.
R
S
3 Actually, k
cat/k
cat
is a measure of the intrinsic deuterium isotope effect
j,8
only if the rate of release of the product from the enzyme–product
complex is not kinetically significant. If this condition does not hold, the
R
S
intrinsic deuterium isotope effect is expected to be larger than k cat/k cat
(ref. 4).
4
5
Reaction Rates of Isotopic Molecules, ed. L. Melander and W. H.
Saunders, Wiley, New York, 1980, ch. 10, pp. 297–305.
However, a concerted oxygen insertion, as suggested by the use of
radical probe substrates [ref. 1(j), (k)], might also be compatible with the
H D
observed intrinsic k /k values.
6
A. D. N. Vaz and M. J. Coon, Biochemistry, 1994, 33, 6442.
Interestingly, in this paper an intramolecular kinetic isotope effect of 2.6
Fig. 2 Proposed orientation of (a) benzyl alcohol and (b) ethylbenzene with
respect to the iron oxo complex in the CPO active site.
2
was determined in the oxidation of [a- H
1
]benzyl alcohol induced by
purified rabbit liver cytochrome P450 2B4, using only the racemic
monodeuterated benzyl alcohol. In preliminary experiments we have
found significantly different values of the observed intramolecular
kinetic deuterium isotope effect in the phenobarbital induced rat liver
microsomal oxidation of (R)-1-d and (S)-1-d (1 and 4, respectively).
Thus, it would seem that in the oxidation of benzyl alcohol
the OH group does not play any specific role with respect to the
spatial orientation assumed by the substrate in the enzyme
reacting pocket. Accordingly, the orientation appears not to
change when OH is replaced by a Me group. Probably, the
interaction of the aromatic ring with the side chains of Phe 103
and/or Phe 186 plays the major role in this respect. Both Phe 103
and Phe 186 are situated at the bottom of the opening of the CPO
substrate-binding pocket and have been suggested to be of
fundamental importance in establishing hydrophobic inter-
•–
III
7
(±)-1-d was reacted with SO
) in H O at pH 6 (0.1 M phosphate buffer). From the molar ratio
between PhCDO and PhCHO produced in the reaction, measured by GC-
MS, a k /k value of 1.8 was calculated.
4
2 2 8
(from Ti /K S O or g-radiolysis/
K
2 2
S
O
8
2
H
D
8 The pro-R hydrogen in ethylbenzene corresponds to the pro-S hydrogen
in benzyl alcohol.
9 M. Sundaramoorthy, J. Terner and T. L. Poulos, Structure, 1995, 3,
1367.
9
actions with organic substrates. Theoretical calculations to test
this hypothesis are under way.
Thanks are due to the National Council of Research (CNR)
and the Ministry for the University and Scientific and
Technological Research (MURST) for financial support.
Communication 9/04327F
1716
Chem. Commun., 1999, 1715–1716