Angewandte
Chemie
Figure 1. MALDI-TOF mass spectra. A) (2R)-1 (855 Da); B) upon incu-
bation of (2R)-1 with dehydratase, loss of deuterium in the product
crotonyl-CoA (2, 836 Da) and exchange by hydrogen in 4-hydroxybu-
tyryl-CoA (1, 854 Da) are observed; C) (2S)-1 (855 Da); D) upon
equilibration with dehydratase, (2S)-1 retains the deuterium, and
[2-2H]crotonyl-CoA (837 Da) is formed.
Scheme 2. Synthesis of (2R)-g-[2-2H1]butyrolactone ((2R)-7). a) Jones
reagent; b) LiAlD4; c) oxalyl chloride, DMSO, N2; d) (R)-alpine borane;
e) p-TsCl, pyridine; f) NaCN; g) acetamide, Pd(OAc)2; h) Pd/C, H2;
i) HCl. j) Samples of 7 were oxidized to succinic acid (TEMPO, NaClO,
NaClO2). (2S)-g-[2-2H1]Butyrolactone ((2S)-7) was synthesized using
(S)-alpine borane.
The question of inversion or retention at C4 during
substitution of the hydroxy group by hydrogen was solved
with (R)- and (S)-4-hydroxy[4-2H1,4-3H]butyrates. For their
syntheses, diethyl succinate was condensed with ethyl
[2H]formate to form diethyl [2H]formylsuccinate, which was
subsequently converted into g-ethoxy[4-2H]butyrolactone.
Hydrolysis yielded 4-oxo[4-2H1]butanoic acid,[19] which was
stereospecifically reduced with NAD3H through catalysis
either by 4-hydroxybutyrate dehydrogenase (4-HBDH) from
C. aminobutyricum or horse liver alcohol dehydrogenase
(ADH). NAD3H was continuously formed with [3H]formate
and formate dehydrogenase. The product obtained with ADH
was assigned as (R)-g-[4-2H1,4-3H]butyrolactone ((4R)-7),
since it is known that ADH catalyzes the transfer of the
hydride from NADH to the Re face of an aldehyde.[20] When
we found that the much more active 4-HBDH acts by the
same stereospecificity as ADH (see below), we used only this
enzyme to generate the (S)-g-[4-2H1,4-3H]butyrolactone
((4S)-7) from 4-oxo[4-3H]butanoic acid in the presence of
[2H]formate and formate dehydrogenase.
leading to nitrile 5, which was hydrolyzed to the amide 6.[10]
Deprotection and acidification gave the stereoselectively
deuterated lactones 7. Aliquots of 7 were oxidized to succinic
acid and analyzed by CD spectroscopy: (R)-[2H1]succinic
acid,
[V]206nm,248C = (À118 Æ 11) degcm3 gÀ1 dmÀ1
;
(S)-
[2H1]succinic acid, [V]206nm,248C = (134 Æ 13) degcm3 gÀ1 dmÀ1.
The latter value agrees with that published for the S
enantiomer ([V]206nm,248C = + 150 degcm3 gÀ1 dmÀ1).[11,12] The
lactones (2R)-7 and (2S)-7 were hydrolyzed to the carbox-
ylates and converted into the corresponding CoA thioesters
((2R)-1 and (2S)-1) by incubation with acetyl-CoA and 4-
hydroxybutyrate CoA-transferase.[13] MALDI-TOF mass
spectrometry revealed that (2R)-1 and (2S)-1 (m/z 855) each
contained less than 11% unlabeled material (Figure 1A,C),
which was mainly derived from the hydrogen content (8%) of
LiAlD4.
Incubation of (2R)-1 with 4-hydroxybutyryl-CoA dehy-
dratase led to the formation of an unlabeled mixture of 1
(m/z 854) and 2 (m/z 836), whose monoisotopic mass distri-
butions corresponded to natural abundance distributions of
nuclei (Figure 1B). In contrast, when (2S)-1 was used as a
substrate, complete retention of the deuterium label in 1
(m/z 855) and 2 (m/z 837) was observed; the amount of
unlabeled 1 remained constant (Figure 1C,D). The results
described above show that, as expected from crystal structure
modeling, during dehydration of 1 to 2, the 2Re hydrogen
atom is selectively removed. Previous results have shown that
loss of the 3Si hydrogen atom occurs.[5] Therefore, the
stereochemical course at C2 and C3 can be described as anti
elimination of the two hydrogen atoms, which is identical to
that of acyl-CoA dehydrogenases.[14–18] It should be noted that
the configuration of the 3Si hydrogen atom in 4-hydroxybu-
tyryl-CoA corresponds to that of the 3Re hydrogen atom in
unsubstituted acyl-CoA.
The synthesized (4R)-7 and (4S)-7 were hydrolyzed to
carboxylates and enzymatically converted into acetates as
indicated in Scheme 3. In this one-pot system, the primary
product 2 was irreversibly hydrated to (3S)-3-hydroxybutyryl-
CoA, with subsequent oxidation with excess NAD+, cleavage
to two equivalents acetyl-CoA, and final conversion into
acetyl phosphate and acetic acid. After acetyl phosphate was
hydrolyzed under acidic conditions, the acetic acid was
isolated by steam distillation. The specific 3H activities of
the isolated acetates were determined enzymatically and by
scintillation counting to be, as expected, (50 Æ 5)% of the
starting lactones, since each mole of substrate generates one
labeled and one unlabeled mole of acetate. Hence, no
3
significant H exchange occurred during the incubation. The
configuration of the acetates was analyzed enzymatically by
using malate and fumarate.[21–23]
Angew. Chem. Int. Ed. 2008, 47, 3254 –3257ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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