Stereochemistry of hydrogen removal from the 'unactivated' C-3 position of 4-hydroxybutyryl-CoA catalysed by 4-hydroxybutyryl-CoA dehydratase.

Article abstract of DOI:10.1039/b402322f

(R)- and (S)-gamma-[3-(2)H(1)]butyrolactones have been synthesised from (R)- and (S)-glycidol, respectively, and used to demonstrate that it is the pro-(S) hydrogen atom that is stereospecifically abstracted from C-3 of 4-hydroxybutyryl-CoA by 4-hydroxybu

Full text of DOI:10.1039/b402322f

Stereochemistry of hydrogen removal from the ‘unactivated’ C-3
position of 4-hydroxybutyryl-CoA catalysed by 4-hydroxybutyryl-CoA
dehydratase
Richard Scott,a Ulrike Näser,a Peter Friedrich,a Thorsten Selmer,b Wolfgang Buckelb and Bernard T.
Golding*a
a School of Natural Sciences – Chemistry, University of Newcastle upon Tyne, Bedson Building, Newcastle
upon Tyne, UK NE1 7RU. E-mail: b.t.golding@ncl.ac.uk
b Laboratorium für Mikrobiologie, Fachbereich Biologie, Phillips-Universität, Marburg D-35032, Germany
Received (in Cambridge, UK) 16th February 2004, Accepted 22nd March 2004
First published as an Advance Article on the web 23rd April 2004
(R)- and (S)-g-[3-2H1]butyrolactones have been synthesised
from (R)- and (S)-glycidol, respectively, and used to demon-
strate that it is the pro-(S) hydrogen atom that is stereospecif-
ically abstracted from C-3 of 4-hydroxybutyryl-CoA by 4-hy-
droxybutyryl-CoA dehydratase, and that this atom is not
returned to C-4.
The sequence of reactions described is highly unusual in its
requirement for the mobilisation of a hydrogen atom at each carbon
of a C3 unit. To investigate the nature of the hydrogen removal at
C-3, which is the most remarkable of all the steps described, and to
aid the detection of intermediates, samples of 3-2H1-labelled
4-hydroxybutyric acid have been synthesised in the form of labelled
g-butyrolactones. Initially, an attempt was made to prepare both
enantiomers of g-[3-2H1]butyrolactone from (S)-solketal (4) via a
single chiral intermediate (5), which was activated as a tri-
fluoromethanesulfonate ester. Compound 5 was to be reduced with
a deuteride reagent (or hydride reagent whilst the chemistry was
being optimised) to 6, which would be orthogonally deprotected for
further transformations. However, it proved impossible to reduce
intermediate 5 in the desired manner with either lithium aluminium
hydride or lithium triethylborohydride4 (Super Hydride™). This
could be attributed to the oxygen atom on the leaving group
coordinating to the adjacent silicon atom in the triisopropylsilyl
protecting group and thus promoting nucleophilic attack of hydride
on the sulfur atom with regeneration of alcohol 7 (Scheme 2).
Isolation from the reaction mixture of alcohol 7, which had been the
immediate precursor to triflate 5, supported this hypothesis.
Alternatively, the silyl group could merely suppress the rate of
substitution at the carbon bearing the triflate by steric shielding.
To avoid the problem described above, the silyloxy group of 5
was replaced by the non-coordinating vinyl group (Scheme 3).
Accordingly, the hydroxyl group of (R)-glycidol (8) was protected
as a p-methoxybenzyl ether (9). The epoxide ring of 9 was opened
with vinyl magnesium bromide to afford alcohol 10. The newly
formed hydroxyl group was activated as a methanesulfonate ester,
which was displaced with lithium aluminium deuteride to give
compound 11. It has been shown that hydride or deuteride reduction
of sulfonate esters occurs with complete inversion of ster-
eochemistry.5 Direct oxidative cleavage (by e.g. ozonolysis or
4-Hydroxybutyryl-CoA dehydratase catalyses the reversible dehy-
dration of 4-hydroxybutyryl-CoA (1) to crotonyl-CoA (2). The
enzyme is homotetrameric and contains one [4Fe-4S] cluster and
one flavin adenine dinucleotide (FAD) per subunit.1 The catalytic
reaction requires the removal of a hydrogen atom from the least
activated position of the butyryl chain of 4-hydroxybutyryl-CoA
and is the mechanistically most demanding step in the fermentation
of g-aminobutyrate (GABA) by the anaerobic bacterium Clostrid-
ium aminobutyricum. The proposed mechanism2 involves genera-
tion of ketyl radical anion 3 from the substrate, 1, by a
deprotonation at C-2, followed by one-electron oxidation of the
enolate to give an enoxy radical that suffers deprotonation at C-3.
The point of the initial deprotonation/oxidation at C-2 is therefore
to generate a species that is sufficiently acidic at C-3 (pKa ~ 14)3 to
be deprotonated. The intermediate 3 eliminates a hydroxyl group
and yields 2 after one-electron reduction to a dienolate that
undergoes protonation at C-4 (Scheme 1).
Scheme 1 Proposed mechanism for the dehydration of 4-hydroxybutyryl-
CoA to crotonyl-CoA by Clostridium aminobutyricum.
Scheme 2 Attempted reduction of a solketal-derived trifluoromethane-
sulfonate.
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C h e m . C o m m u n . , 2 0 0 4 , 1 2 1 0 – 1 2 1 1
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

Fig. 1 MALDI-TOF mass spectra for the products from (S)- (a) and (R)-
Scheme 3 Synthesis of (R)-g-[3-2H1]butyrolactone (13). Reagents and
conditions: (i) PMB–Cl, NaH, Bu4NI, DMF, 0 ? 25 °C, N2, 2 h; (ii)
CH2CHMgBr, Li2CuCl4, THF, 278 ? 25 °C, N2, 18 h; (iii) CH3SO2Cl,
Et3N, CH2Cl2, 0 °C, 30 min; (iv) LiAlD4, THF, reflux, N2, 48 h; (v)
Hg(OAc)2, THF, H2O, 25 °C, 2 h, then NaBH4, NaOH, H2O, 5 ? 25 °C, 1
h; (vi) Dess–Martin periodinane, CH2Cl2, 25 °C, 3 h; (vii) NaOH, Br2, H2O,
dioxane, 15 °C, 2 h; (viii) DDQ, CH2Cl2, H2O, 0 °C, 3 h.
4-hydroxy[3-2H1]butyryl-CoA (b).
Fig. 2 Stereospecific loss of the pro-(S) hydrogen atom from 4-hydrox-
ybutyryl-CoA.
perruthenate oxidation) of the vinyl group to acid 12 proved
problematic in the presence of the p-methoxybenzyl group, but was
eventually achieved efficiently by an indirect route involving
oxymercuration,6 Dess–Martin oxidation7 and bromoform8 reac-
tion. On deprotection of the hydroxyl group, spontaneous cyclisa-
tion occurred to afford (R)-g-[3-2H1]butyrolactone (13), which is a
convenient form of 4-hydroxybutyric acid for purification and
analysis. The (S)-enantiomer of g-[3-2H1]butyrolactone was ob-
tained by a similar route, starting with (S)-glycidol. For the
enzymatic experiments, the g-[3-2H1]butyrolactones were con-
verted to 4-hydroxy[3-2H1]butyrates with 1 M NaOH solution. The
synthesis of 4-hydroxybutyryl-CoA esters was performed using
100 mM labelled 4-hydroxybutyrate in 100 mM potassium
phosphate pH 7.4, 2.5 mM acetyl-CoA and 4-hydroxybutyrate
CoA-transferase (3 U ml21) in a total volume of 0.5 ml. After
incubation at ambient temperature for 10 min, the completeness of
the conversion was checked in an assay using 5,5A-dithiobis(2-
nitrobenzoate) (DTNB), oxaloacetate and citrate synthase; subse-
quently, acetate and 4-hydroxybutyrate CoA-transferase were
added.9 The CoA esters were purified using a C18 cartridge, eluting
with 50% aqueous acetonitrile containing 0.1% TFA. The CoA
esters from (R)- and (S)-4-hydroxy[2-2H]butyrates showed one
major peak by MALDI-TOF mass spectrometry with the same mass
of 855 Da.
The CoA esters were incubated with 4-hydroxybutyryl-CoA
dehydratase for 15 min at 37 °C and the CoA-containing products
separated with a C18 cartridge as described above. MALDI-TOF
mass spectrometry was used to analyse the products, an equili-
brated mixture of 4-hydroxybutyryl-CoA and crotonyl-CoA. The
spectra show masses of 854 and 836, respectively, for the (S)-
enantiomer and 855 and 837, respectively, for the (R)-enantiomer
(Fig. 1). The (R)-isomer therefore lost its 1H (proton at C-3),
whereas the (S)-isomer lost its 2H (deuteron at C-3).
It has thus been demonstrated that the dehydration of 4-hydrox-
ybutyryl-CoA proceeds with the stereospecific removal of the pro-
(S) hydrogen atom from the C-3 position of the substrate (Fig. 2).
The hydrogen (deuterium) removed is not returned to C-4,
indicating that the base that removes H(D) from C-3 either does not
return the abstracted atom to C-4 or only does so after exchange
with solvent. Work is in progress to determine the stereochemistry
of hydrogen removal/addition at C-2 and C-4. These results will aid
the positioning of the substrate/product in the active site of
4-hydroxybutyryl-CoA dehydratase, whose crystal structure is
currently under investigation.10 Initial results show that the
structure is very similar to that of medium chain acyl-CoA
dehydrogenase,11 even though there is only 16% sequence identity.
Interestingly, acyl-CoA dehydrogenases show the same ster-
eospecificity at C-3 as 4-hydroxybutyryl-CoA dehydratase.12
We thank the Deutsche Forschungsgemeinschaft and the Euro-
pean Community Human Potential Programme (Contract HPRN-
CT-2002-00195) for support of this research.
Notes and references
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4 H. C. Brown and S. Krishnamurthy, J. Org. Chem., 1980, 45, 849.
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7 D. B. Dess and J. C. Martin, J. Org. Chem., 1983, 48, 4155.
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9 U. Scherf and W. Buckel, Appl. Environ. Microbiol., 1991, 57, 2699.
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11 J. J. Kim and J. Wu, Proc. Natl. Acad. Sci. U. S. A., 1988, 85, 6677.
12 L. Bücklers, A. Umani-Ronchi, J. Rétey and D. Arigoni, Experientia,
1970, 26, 931; J. F. Biellmann and C. G. Hirth, FEBS Lett., 1970, 9, 335;
H. J. LaRoche, M. Kellner, H. Günther and H. Simon, Hoppe Seyler’s
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C h e m . C o m m u n . , 2 0 0 4 , 1 2 1 0 – 1 2 1 1
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