Reversal of the apparent regiospecificity of NAD(P)H-dependent hydride transfer: The properties of the difluoromethylene group, a carbonyl mimic

Article abstract of DOI:10.1021/ja021487+

The hallmarks of pyridine nucleotide-dependent dehydrogenase reactions are the stereo- and regiospecific hydride transfer between the nicotinamide coenzyme and the corresponding substrate. When the hydride is delivered from NAD(P)H to reduce the keto-substrate, the site of attack is always at the carbonyl carbon. However, the apparent regioselectivity of the hydride transfer is reversed when difluoromethylene is used as a carbonyl mimic in the NADH-dependent enzyme, TDP-l-rhamnose synthase, which catalyzes the conversion of TDP-6-deoxy-l-lyxo-4-hexulose to TDP-l-rhamnose. The observed reversed regioselectivity can be explained by two mechanisms. One involves the formation of a carbene intermediate followed by a rearrangement involving 1,2-H shift. This mechanistic proposal is theoretically sound and would represent a rare example implicating the intermediacy of a carbene species in an enzyme reaction. However, our results are also consistent with a second mechanism in which the hydride addition to the difluoromethylene moiety occurs at the difluorinated end, opposite from the site predicted on the basis of the reduction of a normal keto functional group. Such a regioselectivity is well precedented in chemical models because nucleophilic addition to fluoroalkenes prefers a route in which the number of fluorines β to the electron-rich carbon in the transition state is maximized. In this mechanism, the difluoromethylene group may be regarded as a carbonyl mimic with reversed polarity in enzyme catalysis. While further experiments are needed to discriminate between these mechanistic possibilities, the results reported here suggest that the apparent regioselectivity of hydride transfer in a pyridine nucleotide-dependent enzyme can be changed by altering the electrochemical properties of the reaction center. Copyright

Full text of DOI:10.1021/ja021487+

Published on Web 05/06/2003
Reversal of the Apparent Regiospecificity of NAD(P)H-Dependent Hydride
Transfer: The Properties of the Difluoromethylene Group, A Carbonyl Mimic
Caroline Leriche, Xuemei He, Cheng-wei T. Chang, and Hung-wen Liu*
DiVision of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry,
UniVersity of Texas, Austin, Texas 78712, and Department of Chemistry, UniVersity of Minnesota,
Minneapolis, Minnesota 55455
Received December 31, 2002; E-mail: h.w.liu@mail.utexas.edu
Scheme 1
Pyridine nucleotide-dependent oxidoreduction reactions are
among the most extensively studied enzymatic reactions. A great
number of dehydrogenases of this class, which catalyze the
interconversion of a carbonyl and a hydroxyl group, are known
with a broad structural range of substrate specificities and diverse
catalytic roles.1 Factors that determine the stereoselectivity of this
class of enzymes have been extensively studied.2 However, little
consideration has been given to the related issues that govern the
regioselectivity of catalysis. This lack of attention may stem from
that nucleophilic addition to fluoroalkenes prefers a route in which
the number of fluorines â to the electron-rich carbon in the transition
state is maximized.7 Thus, an opposite regioselective addition based
solidly on chemical precedence may occur for the hydride reduction
of 3. As shown in Scheme 1, elimination of a fluoride ion from 6
would yield an electrophilic intermediate 7 which could trap an
active site nucleophilic residue, resulting in enzyme inactivation.
To test this idea, we synthesized TDP-4,6-dideoxy-4-difluorometh-
ylene-L-lyxo-hex-4-enopyranose (8) and examined its effect on the
catalysis of the purified TDP-L-rhamnose synthase (RfbD).8 This
dehydrogenase is NADH-dependent, and its catalysis involves the
hydride transfer of the 4′-HS of NADH to reduce the 4-keto group
of 1 to form TDP-L-rhamnose (2).
Compound 8 was prepared starting from methyl L-rhamnose and
was isolated as a mixture of R and â anomers (8r:8â ) 3:1).9
Because of the instability of 8 upon further purification and
lyophilization, this mixture was used directly in the subsequent
experiments.10 When TDP-L-rhamnose synthase (1 µM) was
incubated with excess 8 (6.1 mM of the R/â mixture), no irreversible
inactivation was detected. On the contrary, the enzyme recognizes
8 as a substrate, and catalysis follows Michaelis-Menten kinetics
with a kcat ) 12.7 ( 3.8 min-1 and a Km ) 40.1 ( 17.0 mM in 50
mM potassium phosphate buffer (pH 7.5) at 20 °C.11 Comparing
the kcat ) 258.3 ( 25.0 min-1 and Km ) 0.30 ( 0.03 mM for the
natural substrate (1) under identical conditions, this difluorometh-
ylene analogue (8) is clearly a kinetically competent, albeit very
poor, substrate.
The 19F NMR of 8â exhibits two doublets at δ -87.7 and -92.5
(J ) 41.5 Hz). The intensity of these two doublets decreased with
the concomitant appearance of a singlet at δ -119.9 when 8 (7.6
mM) was incubated with TDP-L-rhamnose synthase (1 µM) and
NADH (0.28 mM) in 50 mM potassium phosphate buffer (pH 7.5)
at room temperature. Because this new signal coincides with the
resonance of fluoride ion, the enzymatic processing of 8â must
involve C-F bond cleavage. The turnover product(s) was identified
by a previously developed GC/MS method12 in which the sugar
products, generated in the incubation with [4′S-2H]NADH,13 were
subjected to acid hydrolysis, sodium borohydride reduction, and
acetylation in sequence to yield glycidol tetraacetates whose MS
fragmentation patterns could be readily analyzed.12 On the basis
of the GC/MS (both EI and CI) results, we concluded that these
the fact that hydride addition always occurs at the carbonyl carbon
when a keto-substrate is reduced with NAD(P)H. This strict
conservation of regiospecificity is predicated by the chemical nature
of the keto group and the close proximity of the hydride donor and
acceptor.3 As a part of our ongoing efforts to develop inhibitors
for TDP-L-rhamnose synthase, which catalyzes the conversion of
TDP-6-deoxy-L-lyxo-4-hexulose (1) to TDP-L-rhamnose (2), we
examined the properties of an analogue of 1 in which the
difluoromethylene group replaces the carbonyl group. Interestingly,
we noted that the enzyme catalyzes a hydride transfer to the
difluoromethylene appendage in this analogue (8) at the difluori-
nated end, opposite from the site predicted on the basis of the
reduction of a normal keto functional group. This observation is
significant because it represents the first example showing that the
regiospecificity of hydride transfer catalyzed by a pyridine nucle-
otide-dependent enzyme can be changed by altering the electro-
chemical properties of the reaction center. The results are reported
herein.
The initial rationale for our inhibitor design is based on the
premise that a difluoromethylene group (3) is a potential isostere
of the carbonyl group due to the comparable electronegativity of
fluorine and oxygen, and the capability of both atoms to serve as
hydrogen-bond acceptors.4 As illustrated in Scheme 1, the enzyme-
catalyzed hydride reduction of 3, if it follows the regiospecificity
of normal catalysis, would lead to a carbanion intermediate (4).
However, beause of the repulsion (Iπ) between the electron pair of
the anionic center and those of the fluorines,4,5 formation of this
R-fluoro-substituted carbanion (4) is not favored. Such a repulsive
interaction can be alleviated by releasing a fluoride ion from 4.
This R-elimination may be facilitated by the H-bond networking
in the active site, and the nascent reactive carbene species (5) could
covalently modify a proximal residue and inactivate the enzyme.
In contrast, hydride addition to the terminal carbon of 3 would
generate a â-fluorinated carbanion 6 which can be stabilized
inductively by the fluorine electron-withdrawing effect,4,5 and also
by the negative hyperconjugation.6 In fact, it is well established
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10.1021/ja021487+ CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2
regioselectivity can also be affected by the electrochemical
characteristics of the reaction center. Even though the competence
of the difluoromethylene group as a carbonyl equivalent is poor,
the alternative outcome in regioselectivity of hydride reduction may
find application in enzyme catalysis.
Acknowledgment. This work was supported in part by National
Institutes of Health Grants GM35906 and GM54346. C.L. thanks
the Singer-Polignac Foundation (France) for a Postdoctoral Fellow-
ship. We would also like to thank the reviewers of this paper for
their valuable comments on the mechanisms shown in Scheme 2.
Supporting Information Available: Scheme and synthesis (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Dolphin, D., Poulson, R., Avramovic, O., Eds. Pyridine Nucleotide
Coenzymes, Chemical, Biochemical, and Medical Aspects; Wiley-Inter-
science: New York, 1987; Parts A and B.
(2) Re´tey, J.; Robinson, J. A. Stereospecificity in Organic Chemistry and
Enzymology; Verlag Chemie: Weinheim, 1982; Chapter 3.
(3) On the basis of the theoretical transition structure of hydride transfer
involving 1,4-dihydropyridine (Wu, Y.-D.; Houk, K. N. J. Am. Chem.
Soc. 1987, 109, 2226) and the well-defined normal C-H bond length of
1.073 Å for R2CH2 (The Chemist’s Companion; Gordon, A. J., Ford, R.
A., Eds.; John-Wiley: New York, 1987), one can estimate the net distance
for an effective hydride transfer to be approximately 0.55 Å or less (see:
Kreevoy, M. M.; Ostovic, D.; Truhlar, D. G.; Garrett, B. C. J. Phys. Chem.
1986, 90, 3766).
glycidol derivatives are a mixture of 9, 10, and 11.14 Interestingly,
(4) (a) Smart, B. E. In Organofluorine Chemistry, Principles and Commercial
Applications; Banks, D. E., Smart, B. E., Tatlow, J. C., Eds.; Plenum
Press: New York, 1994; Chapter 3. (b) Zhao, Z.; Liu, H.-w. J. Org. Chem.
2001, 66, 6810.
(5) (a) Hudlicky, M.; Pavlath, A. E. Chemistry of Organic Fluorine
Compounds II; American Chemical Society: Washington, DC, 1995. (b)
Chambers, R. D. Fluorine in Organic Chemistry; John Wiley: New York,
1973.
(6) (a) Reed, A. E.; Schleyer, P. v. P. J. Am. Chem. Soc. 1990, 112, 1434.
(b) Dixon, D. A.; Fukunaga, T.; Smart, B. E. J. Am. Chem. Soc. 1986,
108, 4027. (c) Sleigh, J. H.; Stephens, R.; Tatlow, J. C. J. Fluorine Chem.
1980, 15, 411.
(7) Chambers, R. D.; Bryce, M. R. In ComprehensiVe Carbanion Chemistry;
Buncel, E., Durst, T., Eds.; Elsevier: Amsterdam, 1987; Part C, Chapter
5.
(8) The rfbD gene (Jiang, X.-M.; Neal, B.; Santiago, F.; Lee, S. J.; Romana,
L. K.; Reeves, P. R. Mol. Microbiol. 1991, 5, 695) was amplified by the
polymerase chain reaction (PCR) using the genomic DNA of Salmonella
enterica LT2 as the template and was cloned into a pUC18 vector.
Purification of the RfbD protein (TDP-L-rhamnose synthase) from the E.
coli HB101-rfbD recombinant strain involved two chromatographic
steps: DEAE-Sepharose and Mono Q.
(9) The scheme and a brief discussion of the synthesis can be found in the
Supporting Information.
(10) Because the natural substrate (1) has a â-configuration at the anomeric
carbon, only the â-isomer of 8 should be recognized and processed by
TDP-L-rhamnose synthase.
(11) A typical assay included 5 mM compound 8 (mixture of R/â isomers),
2.8 mM NADH, 1 µM of enzyme in 50 mM potassium phosphate buffer
(pH 7.5). This mixture was incubated at 25 °C for 60 min, and the products
were analyzed directly by NMR or derivatized followed by GC/MS
analysis.12
(12) (a) Weigel, T. M.; Liu, L.-d.; Liu, H.-w. Biochemistry 1992, 31, 2129.
(b) Lonngren, J.; Svensson, S. AdV. Carbohydr. Chem. Biochem. 1974,
29, 41. (c) Rubenstein, P. A.; Strominger, J. L. J. Biol. Chem. 1974, 249,
3776.
(13) Arnold, L. J.; You, K. Methods Enzymol. 1978, 54, 223.
(14) Compound 9 was the predominant species derived mainly from the
unreacted 8r, and no 10 and 11 were detected when the incubation was
performed in the absence of enzyme.
(15) The fact that 13 could only be detected by MS analysis (to give 10) but
not by 19F NMR indicates that little of this intermediate was accumulated
during turnover.
(16) (a) Vinson, W. A.; Prickett, K. S.; Spahic, B.; Ortiz de Montellano, P. R.
J. Org. Chem. 1983, 48, 4661. (b) Ichikawa, J.; Sonoda, T.; Kobayashi,
H. Tetrahedron Lett. 1989, 30, 1641.
(17) (a) Jones, M., Jr., Moss, R. A., Eds. Carbenes; Wiley: New York, 1973;
Vol. I, Chapter 1. (b) Moss, R. A.; Ho, G.-J.; Liu, W. J. Am. Chem. Soc.
1992, 114, 959.
(18) Evenseck, J. D.; Houk, K. N. J. Phys. Chem. 1990, 94, 5518.
(19) (a) Groves, J. T.; Avaria-Neisser, G. E.; Fish, K. M.; Imachi, M.;
Kuczkowski, R. L. J. Am. Chem. Soc. 1986, 108, 3837. (b) Kluger, R.
Chem. ReV. 1987, 87, 863. (c) Kern, D.; Kern, G.; Neef, H.; Tittmann,
K.; Killenberg-Jabs, M.; Wikner, C.; Schneider, G.; Hu¨bner, G. Science
1997, 275, 67. (d) Lee, J. K.; Houk, K. N. Science 1997, 276, 942.
the mass spectra for the C-4 containing fragments in 10 and 11 are
uniformly shifted by one mass unit, and each of those fragments
in 11 also contains a M + 2 peak. Such an increment of one and/
or two mass unit(s) of these C-4 bearing fragments is indicative of
deuterium incorporation from [4′S-2H]-NADH into the turnover
products at C-4′ of the exocyclic difluoromethylene group. Such a
reversal of regioselectivity for hydride transfer has not been reported
for pyridine nucleotide-dependent enzymes.
Our results may be directly explained by a mechanism in which
the addition of hydride from NADH occurs at the difluorinated
carbon of the exocyclic methylene double bond of 8 (Scheme 2A).
The π bond can be restored when the nascent carbanion (12)
eliminates one of the â-fluorines to give 13.15 A second round of
hydride reduction will result in loss of the remaining vinylic fluoride
to afford 14.16 This mechanistic proposal is unique because the
enzyme-catalyzed hydride transfer takes place at the opposite end
of the π bond in the reduction of a difluoromethylene group from
what is observed in the reduction of a carbonyl group. An alternative
route (Scheme 2B), which does not alter the regiochemistry of the
initial hydride addition, is also conceivable. This mechanism
involves the formation of a carbene intermediate (15) followed by
a 1,2-H shift to give 13, which then undergoes a second round of
carbene formation and 1,2-H rearrangement to yield the observed
product (14). The proposed rearrangement is well documented in
carbene chemistry17 and is expected to be extremely facile.18
However, while proposed, the intermediacy of a carbene species
has not been fully substantiated in enzyme catalysis.19
Clearly, the difluoromethylene functionality in compound 8 has
assumed a role as a carbonyl mimic with an apparently reversed
regioselectivity for hydride reduction. The implication of either the
reversal of the site of hydride attack or the participation of a carbene
intermediate to account for the experimental results is provocative.
While the regiospecificity of hydride reduction in enzyme reactions
is determined by the effective binding of the substrate with a defined
orientation in the enzyme active site, our results indicate that the
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