Stereochemistry and fate of hydrogen atoms in the diol-dehydratase- catalyzed dehydration of meso-butane-2,3-diol

Article abstract of DOI:10.1002/(sici)1522-2675(19981111)81:11<2005::aid-hlca2005>3.0.co;2-2

The transformation of meso-butane-2,3-diol into butan-2-ol by a strain of Lactobacillus brevis occurs through a diol-dehydratase-catalyzed conversion of the diol into butan-2-one, which is then reduced to the secondary alcohol by dehydrogenases. Experiments performed with deuterated meso-butane-2,3-diols showed that the dehydration reaction brings about an inversion of configuration at the (R)-configured C-atom of mesobutane-2,3- diol as a consequence of the substitution of the OH group by a H-atom; at the same time, the H-atom already bound to the (R) C-atom is retained in the resulting methylene group. The H-atom replacing the OH group was assessed to come from the medium since the H-atom at the (S)-configured C-atom was completely lost. By contrast, in the case of the conversion of (RS)-propane- 1,2-diol into propan-1-ol under the same fermentation conditions, an extensive H-transfer (ca. 80%) from the primary-alcohol function to the adjacent C-atom was observed. This fact is taken as an indication of different modes in which the two substrates are processed by the enzyme (despite the same stereochemical outcome). A speculative hypothesis is presented to interpret such a dissimilarity.

Full text of DOI:10.1002/(sici)1522-2675(19981111)81:11<2005::aid-hlca2005>3.0.co;2-2

Helvetica Chimica Acta ± Vol. 81 (1998)
2005
Stereochemistry and Fate of Hydrogen Atoms in the Diol-Dehydratase-
Catalyzed Dehydration of meso-Butane-2,3-diol
by Paolo Manittoa)*, Giovanna Speranzaa)*, Gabriele Fontanaa), and Antonietta Gallib)
a
Á
) Dipartimento di Chimica Organica e Industriale, Universita di Milano, e Centro di Studio sulle Sostanze
Organiche Naturali, CNR, via Venezian 21, I-20133 Milano
b
Á
) Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universita di Milano, via Celoria 2,
I-20133 Milano
The transformation of meso-butane-2,3-diol into butan-2-ol by a strain of Lactobacillus brevis occurs
through a diol-dehydratase-catalyzed conversion of the diol into butan-2-one, which is then reduced to the
secondary alcohol by dehydrogenases. Experiments performed with deuterated meso-butane-2,3-diols showed
that the dehydration reaction brings about an inversion of configuration at the (R)-configured C-atom of meso-
butane-2,3-diol as a consequence of the substitution of the OH group by a H-atom; at the same time, the H-atom
already bound to the (R) C-atom is retained in the resulting methylene group. The H-atom replacing the OH
group was assessed to come from the medium since the H-atom at the (S)-configured C-atom was completely
lost. By contrast, in the case of the conversion of (RS)-propane-1,2-diol into propan-1-ol under the same
fermentation conditions, an extensive H-transfer (ca. 80%) from the primary-alcohol function to the adjacent C-
atom was observed. This fact is taken as an indication of different modes in which the two substrates are
processed by the enzyme (despite the same stereochemical outcome). A speculative hypothesis is presented to
interpret such a dissimilarity.
1. Introduction. ± About a dozen of adenosylcobalamin-dependent enzymatic
rearrangements are known, all consisting of an interchange of a H-atom and a variable
group between adjacent C-atoms [1][2]. Among them, the diol-dehydratase-catalyzed
conversion of vicinal diols 1 into the corresponding aldehydes or ketones 3 with a
CH2(a) group via a geminal diol intermediate 2 [3][4] (Scheme 1) has attracted much
Scheme 1

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attention for its stereochemical implications (vide infra) [4][5]. In addition, a lot of
interest is devoted to the enzyme-controlled reaction mechanism, which continues to
be subject of controversy and speculation [6][7].
(RS)-Propane-1,2-diol hydro-lyase (EC 4.2.1.28) [8] converting both enantiomers
of propane-1,2-diol (1b) into propanal (3b) was first isolated in 1963 from Klebsiella
pneumoniae ATCC 8724 (formerly Aerobacter aerogenes) [9] and shown to be present in
other strains of Klebsiella ssp. as well as in bacteria of Enterobacteriaceae and
Propionibacteriaceae [10]. A very similar enzyme, glycerol hydro-lyase (EC 4.2.1.30)
was found to be produced by certain strains of Klebsiella pneumoniae [11] and by Lacto-
bacillus ssp. [12] which were able to transform glycerol (1c) into 3-hydroxypropanal (3c).
In 1986, Radler and Zorg [13] purified and characterized a cobalamin-dependent
dehydratase that showed a significant activity on meso-butane-2,3-diol (meso-1d ˆ 4).
This enzyme was obtained from a strain of Lactobacillus brevis (B18) which furnished,
when grown anaerobically in a medium containing meso-butane-2,3-diol, the highest
amount of butan-2-ol among several strains of heterofermentative lactic-acid bacteria
originally isolated from spoiled wines [14]. In fact, butan-2-ol (6) occurring in distillates
[15] results from diol-dehydratase-catalyzed conversion of meso-butane-2,3-diol (4)
into butan-2-one (5) followed by enzymatic reduction of the ketone to the secondary
alcohol [16] (Scheme 2).
Scheme 2
The three enzymes are distinguishable by separation and immunochemical
techniques [17] and are different for their kinetic parameters [3b][13]. However, they
have many features in common besides the prosthetic group, e.g., the activity on the
same substrates (1a ± d), the irreversible inactivation during the reaction with diols
other than ethanediol and propane-1,2-diols [18][19], and, presumably, the reaction
mechanism following the C Co bond homolysis of the adenosylcobalamin [2][7b].
Recently, we found that the conversion of meso-butane-2,3-diol (4) into butan-2-ol
(6) by a strain of Lactobacillus brevis (LB19) of our collection occurred with complete
discrimination between the two enantiomorphic-enantiotopic 1-hydroxyethyl groups
[20]. It was shown that the (R)-MeCHOH group of 4 was transformed by the diol-
dehydratase-catalyzed rearrangement into the MeCH2 and the (S)-counterpart of the
molecule into the MeCO group. This finding can be interpreted in the light of the three-
point-attachment hypothesis [4][7a][21], as schematized in Fig. 1 for (R)- and (S)-
propane-1,2-diol, by assuming a complete preference for the (R)-binding mode when
the meso-butane-2,3-diol molecule (Me in place of HB in Fig. 1,a) interacts with the
enzyme active site1).
1
)
It can be noted that a >90% preference for the (R)-binding mode was reported to occur in the enzymatic
dehydration of glycerol [23].

Helvetica Chimica Acta ± Vol. 81 (1998)
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Fig. 1. Schematic picture of the binding of propane-1,2-diol at the active site of diol dehydratase according to a
three-point attachment hypothesis [4][7a][21]. Protein-substrate binding sites are represented by circled
numbers. HA(R) ˆ HA, pro-R, HB(S) ˆ HB, pro-S
In principle, meso-butane-2,3-diol could also have been handled by the enzyme as
an (S)-propane-1,2-diol molecule in which a Me group replaces the (pro-R) H-atom
(i.e., HA in Fig. 1,b) since the (RS)-propane-1,2-diol dehydratase has been shown to
remove the (pro-R) H-atom of the primary-alcohol function in the (R)-enantiomeric
form of propane-1,2-diol and the (pro-S) one in the (S)-enantiomer [5][21][22]. The
finding that both the optically active butanediols behave as purely competitive
inhibitors [19] is understandable in the light of the above stereochemical demand for H-
abstraction.
The apparent resemblance of (R)-propane-1,2-diol and meso-butane-2,3-diol in
their interaction with the biocatalyst prompted us to extend the comparison between
these two substrates even to another cryptostereochemical aspect, namely the formal
substitution of the OH group by a H-atom at the (R) C-atom, as well as to the fate of
the mobile H-atom (which is linked to the (S) C-atom in 4). This investigation, whose

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results are reported here, was performed using the LB19 strain of L. brevis of our
collection as the source of the diol dehydratase.
2. Results and Discussion. ± 2.1 Stereochemistry of the Substitution at the (R) C-
Atom of meso-Butane-2,3-diol in the Diol-Dehydratase Reaction. In an earlier study
[16], we found that Lactobacilli reduce butan-2-one (5) to an enantiomer mixture of
butan-2-ol (6) whose composition depends on the strain and the concentration of the
substrate (the ketone or its precursor, i.e., meso-butane-2,3-diol (4)). The most
abundant stereoisomer of the secondary alcohol and its enantiomeric excess can be
accurately determined by bidimensional chiral gas chromatography [15]. Thus, it
appeared that the unequivocal relationship between the two enantiotopic H-atoms
of butan-2-one (5) and those of meso-butane-2,3-diol (4) (or of the medium) could
1
2
be established by H(or H)-NMR spectroscopy, provided a certain assignment of
chemical shift was made for each of the diastereotopic protons in butan-2-ol (6) or in a
suitable derivative, e.g., 7.
When analyzed by 1H-NMR spectroscopy (300 MHz, C6D6), 1-methylpropyl
phenylcarbamate (7, see Fig. 2) showed two separate m centered at d 1.31 and 1.44. The
assignments of the former resonance to the anti-H2) and of the latter to the syn-H were
established by a comparison of the 1H-NMR spectrum of 7 with those of its
monodeuterated analogs, i.e. 8 (syn-D) and 9 (anti-D) (Fig. 2). These analogs were
prepared by LiAlD4 reduction of the epoxides of cis- and trans-but-2-ene, respectively,
followed by treatment of the resulting alcohols with phenyl isocyanate.
(2R,3S)-(1,1,1,2-2H4)butane-2,3-diol (12) was then synthesized as shown in
Scheme 3 from (S)-2-(benzyloxy)(1-2H)propanal (11) by a procedure we previously
described [20]. After fermentation of compound 12 by Lactobacillus brevis LB19 under
anaerobic conditions, an optically active butan-2-ol was isolated whose (R)/(S) ratio
was found to be 68(Æ 2): 32(Æ 2) by means of chiral gas chromatography. Inspection of
the 2H-NMR spectrum (C6D6) of the product resulting from treatment of this butan-2-
ol mixture with phenyl isocyanate revealed the presence of broad ss at d 0.79 (CD3),
1.44 (syn-D), and 1.31 (anti-D) in an intensity ratio 3 : 0.7 : 0.3. The fact that the ratios
between the abundances of the (R)- and (S)-isomers in butan-2-ol and between the
syn-D and anti-D resonance intensities in the corresponding 1-methylpropyl phenyl-
carbamate were practically identical, clearly indicated the existence of only two
deuterated species in each mixture examined, i.e., 13/14 in the alcohol and 15/16 in its
O-carbamoyl derivative (Scheme 3).
By inference, one can conclude that in the diol-dehydratase-catalyzed conversion of
meso-butane-2,3-diol into butan-2-one, the H-atom at the (R) C-atom is retained, and
the replacement of the OH group occurs with inversion of configuration. It must be
pointed out that the same outcome was observed in the case of the action of (RS)-
propane-1,2-diol hydro-lyase from Klebsiella pneumoniae on both (R)- and (S)-
propane-1,2-diols [4][5][21] (see Fig. 1).
2
)
Throughout this paper, the methylene H-atoms of 1-methylpropyl phenylcarbamate will be called syn-H
(or syn-D) and anti-H (or anti-D) according to their position relative to the (phenylcarbamoyl)oxy group
in a staggered conformation having the two Me groups in anti-periplanar orientation (as in formula 7 of
Fig. 2).

Helvetica Chimica Acta ± Vol. 81 (1998)
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Fig. 2. 1H-NMR Spectra (300 MHz, C6D6, 303 K) of 1-methylpropyl phenylcarbamate (7; one enantiomer being
represented for clarity) and of its (2-2H1)-derivatives 8 and 9

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Scheme 3
a) PhCH2Br, Ag2O. b) LiAlD4, Et2O. c) Swern oxidation. d) [TiCl(iPrO)3], CD3Li, Et2O, 788, then addition of
11, 08 !r.t. e) H2, 10% Pd/C, EtOH, r.t. f) Lactobacillus brevis, 1 week. g) PhNCO, Et2O.
2.2. Fate of the H-Atom Linked to the (S) C-Atom of meso-Butane-2,3-diol. In the
case of the above mentioned enzyme, good evidence has established that the homolytic
cleavage of the Co C bond of the adenosylcob(III)alamin (AdoCbl) to give
.
cob(II)alamin and the 5'-deoxy-5'-adenosyl radical (AdoCH2 ) is an early event in
the enzymatic process [7b][24]. In addition, it was shown that the overall dehydration
of propanediols occurs without appreciable exchange of the C-linked H-atoms with the
solvent [25]. On the other hand, the H-migration did not appear to be intramolecular,
since it was shown that a T-atom from C(1) of propane-1,2-diol is transferred to C(2) of
the acetaldehyde derived from unlabeled ethylene glycol in experiments performed
.
with different mixtures of the two diols [26][27]. The participation of AdoCH2 as a H-
carrier having three equivalent H-atoms in the intermediate form (AdoCH3), and the
sequence of reactions of Scheme 4 were postulated [27], despite the unusually large kH/
kT isotope effects (up to 125) that were observed for the last H(T)-transfer step
(Step 4), i.e., when T-atom is transferred from the enzyme-bound [5'-3H]adenosylco-
balamin to the product-related radical to give acetaldehyde or propanal [28]. More
recently, the participation of a second H-transfer site of the enzyme has been suggested
as an explanation of the very high kH/kT value [24]. However, the actual mechanism has
not yet been convincingly established.
Taking these facts into account, the retention of the mobile H-atom of meso-
butane-2,3-diol (4) was tested by feeding L. brevis LB19 with a mixture of all the three
(2,3-2H2)-stereoisomers of 1d (the optically active stereoisomers being unaffected by
the diol dehydratase) [19]. This mixture was prepared by LiAlD4 reduction of butane-
2,3-dione ( ˆ diacetyl) and consisted of 64% of the meso-form and 36% of the rac-form
(dideuterated molecules 98 Æ 1% by MS). Butan-2-ol resulting from fermentation
((R)/(S) ca. 75 : 25) was found to contain 1 D-atom per molecule by MS measure-

Helvetica Chimica Acta ± Vol. 81 (1998)
Scheme 4 a)
2011
a) AdoCbl ˆ adenosylcob(III)alamin; AdoCH3 ˆ 5'-deoxyadenosine; Cbl(II) ˆ cob(II)alamin; Sub-H ˆ sub-
.
.
strate; Sub ˆ substrate-derived radical; Prod-H ˆ product; Prod ˆ product-related radical.
2
ments; in addition, the H-NMR spectrum of its O-(phenylcarbamoyl) derivative
exhibited the two signals at d 1.44 (syn-D) and at d 1.31 (anti-D) in the ratio 3 : 1. These
findings are consistent with both a loss of the H-atom linked to the (S) C-atom of meso-
butane-2,3-diol and the configuration inversion at the (R) C-atom established above
(see Scheme 3). That the H-loss was practically complete was confirmed by the
microbial transformation of (2S,3R)-(1,1,1,2-2H4)butane-2,3-diol (17) (Scheme 5),
which in turn was synthesized by the same procedure as for compound 12 (Scheme 3)
but starting from ethyl (R)-lactate. Only a signal at d 1.09 (CD3) was observed in the
2H-NMR spectrum of the resulting 1-methylpropyl phenylcarbamate 18, even at high
concentration in CHCl3.
Scheme 5
a) Lactobacillus brevis, 1 week. b) PhNCO, Et2O.
To rule out the possibility that the absence of D-atom at the 2-position of 18 was
due to a rapid interconversion between meso-butane-2,3-diol and (R)-3-hydroxybutan-
2-one (ˆ(R)-acetoin), which might have occurred prior to the dehydration reaction
[16], a fermentation experiment with 17 was interrupted at ca. 50% of the substrate
conversion. No change of D-content and D-distribution in the recovered diol 17 was
2
shown by H-NMR analysis. It must also be noted that a H-exchange of butan-2-one
with the medium, enzymatically or not, appears to be unlikely considering the D-
retention previously observed in the conversion of compound 12 into butan-2-ol 13/14.
Supplying our strain of L. brevis with (RS)-(1,1-2H2)propane-1,2-diol (prepared by
LiAlD4 reduction of ethyl lactate) resulted in the formation of propan-1-ol showing a
considerable retention of the mobile D-atom, i.e., a (2-2H)/(1-2H) ratio of 0.80 as
2
estimated by H-NMR analysis of the corresponding phenylcarbamate. In addition,
when feeding experiments were carried out with equimolar mixtures of meso-butane-

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2,3-diol and (RS)-propane-1,2-diol, one of these species having a D-atom in place of
the labile H-atom, ca. 10% of the D-atom lost by propane-1,2-diol was found in the
(pro-S) position at C(3) of butan-2-ol, whilst no D-transfer was observed from
meso-butane-2,3-diol to propan-2-ol. Thus, it appears that the two diols are processed
by the dehydratase in a rather different way, at least with regard to the H-transfer
mechanism.
In our opinion, the loss of D-atom bound to the (S) C-atom of meso-butane-2,3-diol
and the results of the above crossover experiments may be interpreted in terms of the
general radical chain mechanism schematically represented in Scheme 6. This is an
upgraded version of that proposed by Finke [7b] involving protein-bound radical
intermediates with cob(II)alamin as a ꢀspectatorꢁ (bound-radical mechanism) [7a][29].
The main radical cycle is consistent with the large isotopic effects mentioned above
[24][28][30] and is supported by a lot of analogies between diol dehydratase and two
other adenosylcobalamin-dependent enzymes, namely ethanolamine ammonia lyase
[31] and ribonucleotide reductase [32]. In fact, evidence has been provided for the
participation of a Enz-XH site in the process catalyzed by both enzymes [33][32b]
(XH being probably the thiol group of a cysteine residue) [31b][32a] and for the
Scheme 6. Schematic Representation of the Putative Two-Cycle Radical Mechanism for Diol-Dehydratase-
Catalyzed Transformationsa). See Scheme 4 for abbreviations.
.
.
a) [± ± ±]: enzyme active site; AdoCH2/AdoCH3: coenzyme H-carrier; X /XH: protein H-carrier.

Helvetica Chimica Acta ± Vol. 81 (1998)
2013
involvement of 5'-deoxyadenosine (AdoCH3) in at most 1 turnover out of 1000 in the
case of ribonucleotide reductase [7b]. The mechanism of Scheme 6 encompasses an
additional radical cycle in which the product-related radical abstracts a H-atom from
AdoCH3, while a second substrate molecule is entering the active site of the enzyme (as
in the case of the main cycle)3). Finally, the reversibility of the H-transfer from Enz-XH
.
to AdoCH2 and of the homolytic dissociation of AdoCbl (Steps 2 and 1, resp., in
Scheme 6), may be responsible for the washout of T-atom from the coenzyme to solvent
[7b][32b][33].
One can assume that in the course of both radical cycles, no significant H-exchange
occurs between the Enz-XH group and the medium, while it is likely that it takes place
when the holoenzyme is in the resting state. By the main cycle, a C-bound H-atom is
transferred from the Enz-XH site to the adjacent C-atom of the same molecule, thus
mimicking an intramolecular 1,2-shift. On the other hand, to account for the H-transfer
from a substrate molecule to another one [26][27], we have introduced the secondary
cycle. This demands the intermediacy of the vitamin-B12 coenzyme acting as a pool of
enzyme-bound H-atoms in communication with the mobile H-atoms on different
molecules of substrate and product.
When the substrate is propane-1,2-diol, every escape route of those represented as
Steps 5, 5', and 6 in Scheme 6 can be exploited by the product-related radical, that is the
main cycle giving rise to an ꢀapparently intramolecularꢁ H-transfer or the secondary
cycle causing ꢀintermolecularꢁ H-transfer (with temporary incorporation of the mobile
H-atom into the ꢀvitamin-B12-coenzyme poolꢁ) or the termination path by which the H-
atom removed from the substrate, and stored in the Enz-XH site, will be exchanged
with the medium before a new turnover starts.
If the product-related radical arises from meso-butane-2,3-diol, only termination
steps should be allowed. The reason might be a steric hindrance to the binding of a
second substrate molecule, while the active site is occupied by reacting meso-butane-
2,3-diol. This means that the mobile H-atom of meso-butane-2,3-diol is lost in the
medium even in the presence of propane-1,2-diol as an accompanying substrate for the
enzymatic transformation. According to the above assumptions, the observed D-
transfer from (RS)-(1,1-2H2)propane-1,2-diol to the meso-butane-2,3-diol-derived
product (i.e., butan-2-ol) can be explained as due to isotopic labeling of the adenosyl
H-carrier by the former diol molecules processed through the secondary cycle.
3. Concluding Remarks. ± The stereochemistry and the fate of H-atoms in the
transformation of meso-butane-2,3-diol into butan-2-one by action of the diol
dehydratase occurring in L. brevis are summarized in Scheme 7. In addition, the loss,
the intermolecular, and the (postulated) apparently intramolecular transfer of the
mobile H-atom in diol molecules can be explained on the basis of the reactions
depicted in Scheme 6 (which have been formulated in the spirit of a working
hypothesis).
3
)
In this respect, it has been suggested that the enzyme might have at least two substrate-binding sites [7b].

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Scheme 7. Cryptostereochemical Features of the Dehydration of meso-Butane-2,3-diol by Lactobacillus brevis
We gratefully acknowledge financial support from MURST. Thanks are due to Dr. Diego Monti, CNR,
Milano, for running NMR spectra and Dr. Mauro Scarpellini, DISMA, Milano, for technical assistance in
fermentation experiments.
Experimental Part
General. TLC: silica gel 60 F254 precoated aluminum sheets (Merck); detection either by UV or spraying a
ceric sulfate/ammonium molybdate soln., followed by heating to ca. 1508; eluent, hexane/Et2O 7:3. Flash
chromatography (FC): silica gel 40 ± 63 mm (Merck). Anal. GC: Dani 3800 gas chromatograph; home-made
2 m  2 mm i.d. glass column, 20% Carbowax 20M on Chromosorb W, 60 ± 80 mesh; injector, 2108; detector,
2208; Conditions A, 4 min at 608, then to 1508 at 108/min, and 8 min at 1508; Conditions B, isothermal analysis at
2008; tR in min. For chiral GC, see [15]. GC/MS: Hewlett-Packard-CG-5972 instrument coupled with a HP-5890
MS detector, Ultra-1 (25 m  0.2 mm i.d.) column; temp. 508. M.p.: Büchi-530 melting-point apparatus;
uncorrected. Optical rotations: Perkin-Elmer-241 polarimeter; 1-dm cell. NMR Spectra: Bruker-AC-300
spectrometer at 300.13 (1H), 46.07 (2H), and 75.47 MHz (13C); d in ppm vs. solvent as internal ref.: d(H/D) 7.25,
d(C) 77.00 for C(H)DCl3; d(H/D) 7.15 for C6(H)D6; d(H/D) 2.50 for (C(H)D3)2SO; J in Hz; 13C multiplicities
from DEPT spectra.
1-Methylpropyl Phenylcarbamate (7) and 1-Propyl Phenylcarbamate. To a soln. of commercial butan-2-ol
(6; 0.2 ml, 2.2 mmol) in dry Et2O (3 ml), excess of phenyl isocyanate was added and the mixture heated under
reflux and N2 until completion (GC analysis). Usual workup and recrystallization from hexane gave pure 7.
Following the same procedure, 1-propyl phenylcarbamate was prepared from the corresponding alcohol.
Data of 7: Rf 0.34. M.p. 63 ± 648. 1H-NMR (C6D6): 0.79 (t, J ˆ 7.3, Me(3)); 1.09 (d, J ˆ 6.8, Me C(1)); 1.31
(m, H C(2)); 1.44 (m, H C(2)); 4.88 (sext. J ˆ 6.3, H C(1)); 6.01 (br. s, NH); 6.81 (t, J ˆ 7.9, 1 arom. H);
7.06 (t, J ˆ 7.8, 2 arom. H); 7.29 (d, J ˆ 8.0, 2 arom. H). 13C-NMR (CDCl3): 9.52 (q); 19.62 (q); 28.75 (t); 73.18
(d); 118.49 (d); 123.15 (d); 128.96 (d); 138.09 (s); 153.38 (s).
Data of 1-Propyl Phenylcarbamate: Rf 0.39. M.p. 578. 1H-NMR (C6D6): 0.74 (t, J ˆ 7.4, Me); 1.41
(m, 2 H C(2)); 3.97 (t, J ˆ 6.7, 2 H C(1)); 6.23 (br. s, NH); 6.82 (t, J ˆ 7.5, 1 arom. H); 7.07 (t, J ˆ 7.8,
2 arom. H); 7.30 (d, J ˆ 7.8, 2 arom. H). 13C-NMR (CDCl3): 10.29 (q); 22.28 (t); 66.83 (t); 118.70 (d); 123.31
(d); 128.99 (d); 138.03 (s); 153.76 (s).
(1RS,2RS)-1-Methyl(2-2H1)propyl Phenylcarbamate (8) and (1RS,2SR)-1-Methyl(2-2H1)propyl Phenyl-
carbamate (9). To a stirred suspension of LiAlD4 (290 mg, 6.9 mmol) in dry Et2O (15 ml), 500 mg (620 ml,
6.9 mmol) of cis-2,3-dimethyloxirane ( ˆ cis-2,3-epoxybutane, Aldrich) was added dropwise at r.t. under N2. The
mixture was heated under reflux for 2 h and then stirred at r.t. overnight. After cooling to 08, the reaction was
quenched by the sequential addition of H2O (0.5 ml), 5n NaOH (1 ml), and H2O (0.5 ml). The mixture was
stirred for additional 2 h, the resulting white precipitate filtered off and washed with Et2O, the aq. layer of the
filtrate separated, and the org. phase dried (Na2SO4) and cautiously distilled using a Vigreux column. Fractions
containing butan-2-ol (by GC analysis, Conditions A) were collected and treated with excess phenyl isocyanate
as described above to give pure 8 (690 mg, 51% overall yield).
Following the same procedure, pure 9 was obtained from trans-2,3-dimethyloxirane (56% overall yield).
Data of 8: 1H-NMR (C6D6): d as for 7, except for the absence of the m at 1.44. 2H-NMR (C6H6): 1.44
(s, 2H C(2)).
Data of 9: 1H-NMR (C6D6): d as for 7, except for the absence of the m at 1.31. 2H-NMR (C6H6): 1.31
(s, 2H C(2)).
(2R,3S)-(1,1,1,2-2H4)Butane-2,3-diol (12). (S)-2-(Benzyloxy)(1-2H) propanal (11) was prepared in 61%
yield from commercial ethyl (S)-lactate (10; 98% e.e.) by a published procedure [34] using LiAlD4 instead of
LiAlH4. An Et2O soln. (ca. 40 ml) of [Ti(CD3)(iPrO)3], prepared from [TiCl(iPrO)3] (1.9 ml, 8 mmol) and
CD3Li ´ LiI (0.5m in Et2O, 17 ml) according to [35], was treated dropwise with a soln. of 11 (1.24 g, 7.5 mmol) in
dry Et2O (4 ml) at 08 under N2, then allowed to warm to r.t., and stirred overnight. The mixture was cooled to 08,

Helvetica Chimica Acta ± Vol. 81 (1998)
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quenched by addition of 2n HCl, and extracted with Et2O. The org. layer was washed with H2O, dried
(Na2SO4), and evaporated. FC (hexane/Et2O 8 : 2 !6 : 4) gave (2R,3S)-3-(benzyloxy)-(1,1,1,2-2H4)butan-2-ol
(950 mg, 69%) as a colorless oil. Rf 0.11. GC (Conditions B): tR 14.7. 1H-NMR (CDCl3): 1.16 (d, J ˆ 6.5,
Me(4)); 2.28 (br. s, OH); 3.48 (q, J ˆ 6.5, H C(3)); 4.50, 4.62 (2d, each J ˆ 11.8, PhCH2); 7.26 ± 7.35
2
(m, 5 arom. H). H-NMR (CHCl3): 1.13 (C2H3); 3.89 (2H C(2)).
(2R,3S)-3-(Benzyloxy)-(1,1,1,2-2H4)butan-2-ol (400 mg, 2.1 mmol) was hydrogenated over 10% Pd/C
(270 mg) in EtOH (25 ml) at r.t. for 2 h (GC control). After removal of the catalyst and the solvent, bulb-to bulb
distillation afforded 12 (190 mg, 96%). Colorless oil. B.p. 1108/4 Torr (ꢀKugelrohrꢁ). GC (Conditions A): tR 18.5.
2H-NMR (DMSO): 1.03 (C2H3); 3.34 (2H C(2)); ratio 3 :1.
(2S,3R)-(1,1,1,2-2H4)-Butane-2,3-diol (17). The same procedure as in the synthesis of 12 was used, starting
2
from ethyl (R)-lactate. H-NMR (DMSO): 1.03 (C2H3); 3.34 (2H C(2)); ratio 3 :1.
(2,3-2H2)Butane-2,3-diols. The published procedure [36] was used with the following modification: the
mixture (1 g of butane-2,3-dione ( ˆ diacetyl) and 0.4 g of LiAlD4 in 20 ml of Et2O) was quenched at 08 by the
sequential addition of H2O (0.5 ml), 4n NaOH (1 ml), and H2O (0.5 ml). Usual workup and bulb-to-bulb
distillation (100 ± 1058/4 Torr) afforded (2,3-2H2)butane-2,3-diols (770 mg, 72%) as a 64 : 36 mixture of the
meso- (tR 18.5) and rac-diastereoisomer (tR 17.6) by GC analysis (Conditions A). (2H2)-Species 98 Æ 1% by GC/
1
MS. H-NMR ((D6)DMSO): 0.98 (s, Me (rac)); 1.03 (s, Me (meso)); 4.24 (s, OH (meso)); 4.28 (s, OH (rac)).
2H-NMR (DMSO): 3.34 (2H C(meso)); 3.39 (2H C(rac)).
(RS)-(1,1-2H2)Propane-1,2-diol was prepared by LiAlD4 reduction of (RS)-ethyl lactate [23]. GC
(Conditions B): tR 2.1. (2H2)-Species 98 Æ 1% by GC/MS. 1H-NMR (CDCl3): 1.13 (d, J ˆ 6.0, Me); 3.19
(br. s, OH); 3.87 (br. q, J ˆ 6.0, H C(2)); no signals in the range 3.20 ± 3.70.
Fermentations. Lactobacillus brevis (LB 19 strain) was from our collection. The inoculum was prepared
from freeze-dried cells as previously described [16]. Fermentation experiments (1% inoculum) were carried out
at 308 for a week in a sterilized synthetic medium [16] under anaerobic conditions using (labeled) meso-butane-
2,3-diol (1 g/l), propane-1,2-diol (1 g/l), or a 1 :1 mixture of the two diols (1 g/l each).
Isolation of Fermentation Products. Cells of Lactobacillus brevis were separated by centrifuging, and the
broth was distilled. Fractions containing butan-2-ol and/or propan-1-ol (GC, Conditions A, tR 6.8 and 5.7, resp.)
were collected and continuously extracted with Et2O. This Et2O extract, when necessary, was analyzed by chiral
GC and/or GC/MS. To prepare the O-(phenylcarbamoyl) derivatives of butan-2-ol and propan-1-ol, the Et2O
extract was cautiously concentrated using a Vigreux column and the residue treated with excess phenyl
isocyanate. After refluxing with stirring for 24 h, phenylurea was filtered off and washed with Et2O and the
combined Et2O soln. evaporated. FC (hexane/Et2O 8 :2 !7: 3) of the residue gave 1-methylpropyl and/or 1-
propyl phenylcarbamate which were characterized by comparison with authentic samples prepared from
commercial butan-2-ol and propan-1-ol.
Fermentation of (2R,3S)-(1,1,1,2-2H4)Butane-2,3-diol (12). The extracted butan-2-ol was shown (chiral GC)
to be a (2R)/(2S) mixture (13/14) in the ratio 68(Æ 2): 32(Æ 2). The 1-methyl-(2,3,3,3-2H4)propyl phenyl-
carbamate (15/16) was isolated in 38% yield. [a]D25
ˆ
7.6 (c ˆ 0.2, CHCl3) ([a]D25
ˆ
20.2 (c ˆ 1.1, CHCl3) for a
reference sample prepared from enantiomerically pure ( )-(R)-butan-2-ol). 2H-NMR (C6H6): 0.79 (C2H3(3));
1.31 (2H C(2)); 1.44 (2H C(2)); ratio 3 : 0.3 : 0.7.
Fermentation of (2,3-2H2)Butane-2,3-diols. After fermentation of meso/rac-(2,3-2H2)butane-2,3-diol (64 :36;
prepared as described above), 1-methyl(2-2H1)propyl phenylcarbamate was isolated in 31% yield (rel. to the
meso-isomer). 2H-NMR (C6H6): 1.31 (2H C(2)); 1.44 (2H C(2)); ratio 0.25 :0.75. (2H1) Species >98% by GC/
MS of the corresponding alcohol.
Fermentation of (2S,3R)-(1,1,1,2-2H4)Butane-2,3-diol (17). The 1-(2H3)methylpropyl phenylcarbamate (18)
2
was obtained in 35% yield. H-NMR (C6H6): 1.09 (C2H3(1)).
Fermentation of (RS)-(1,1-2H2)Propane-1,2-diol. The (1,2-2H2)propyl phenylcarbamate was obtained in
2
47% yield. H-NMR (C6H6): 1.41 (2H C(2)); 3.97 (2H C(1)); ratio 0.80 :1.0.
Fermentation of meso-Butane-2,3-diol/(RS)-(1,1-2H2)Propane-1,2-diol 1:1. The (1,2-2H2)Propyl phenyl-
2
carbamate was obtained in 35% yield. H-NMR (C6H6): 1.41 (2H C(2)); 3.97 (2H C(1)); ratio 0.72 :1.0.
The 1-methyl(2-2H1)propyl phenylcarbamate was obtained in 15% yield. 2H-NMR (C6H6): 1.31 (2H C(2));
1.44 (2H C(2)); ratio 0.63 :0.37.
The 2H-NMR of the 1:1 mixture of the above carbamates showed an intensity ratio 1:0.03 when referred to
the signals at 3.97 and at 1.31 and 1.44, the last two being taken together.
Fermentation of (2,3-2H4)Butane-2,3-diols and (RS)-Propane-1,2-diol (meso-(2,3-2H2)Butanediol/Propane-
diol 1:1). The 1-Methyl(2-2H1)propyl phenylcarbamate was obtained in 21% yield. 2H-NMR (C6H6): 1.31
(2H C(2)); 1.44 (2H C(2)); ratio 0.22 :0.78.
No D-atom was observed in the 2H-NMR of 1-propyl phenylcarbamate which was obtained in 34% yield.

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Helvetica Chimica Acta ± Vol. 81 (1998)
REFERENCES
[1] a) ꢀVitamin B12 ± Proceedings of the Third European Symposium on Vitamin B12 and Intrinsic Factorꢁ, Eds.
B. Zagalak and W. Friedlich, W. de Gruyter, Berlin, 1979; b) ꢀB12ꢁ, Ed. D. Dolphin, Wiley-Interscience,
New York, 1982, Vols. I and II; c) J. Halpern, Science (Washington D.C.) 1985, 227, 869; d) B. T. Golding,
D. R. R. Rao, in ꢀEnzyme Mechanismsꢁ, Eds. M. I. Page and A. Williams, Royal Society of Chemistry,
London, 1989, p. 404.
[2] B. T. Golding, Chem. Brit. 1990, 26, 950; B. T. Golding, W. Buckel, in ꢀComprehensive Biological Catalysisꢁ,
Ed. M. L. Sinnot, Academic Press, London, 1997, p. 239.
[3] a) R. H. Abeles, in ꢀThe Enzymesꢁ, Ed. P. D. Boyer, Academic Press, New York, 1972, Vol. 5, pp. 481;
b) T. Toraya, in ꢀMetal Ions in Biological Systemsꢁ, Eds. H. Sigel and A. Sigel, Marcel Dekker, New York,
1994, Vol. 30, pp. 217 ± 254.
[4] D. Arigoni, in [1a], pp. 389 ± 410.
Â
[5] J. Retey, J. A. Robinson, ꢀStereospecificity in Organic Chemistry and Enzymologyꢁ, Verlag Chemie,
Weinheim, 1982, p. 185.
Â
[6] B. T. Golding, in [1b], Vol. I, pp. 543 ± 582; J. Retey, Angew. Chem., Int. Ed. Engl. 1990, 29, 355; P. George,
J. P. Glusker, C. W. Bock, J. Am. Chem. Soc. 1995, 117, 10131.
[7] a) R. G. Finke, D. A. Schiraldi, B. J. Mayer, Coord. Chem. Rev. 1984, 54, 1; b) R. G. Finke, in ꢀMolecular
Mechanisms in Biorganic Processesꢁ, Eds. C. Bleasdale and B. T. Golding, Royal Society of Chemistry,
London, 1990, pp. 244 ± 280.
[8] Z. Schneider, A. Stroinski, ꢀComprehensive B12ꢁ, W. de Gruyter, Berlin, 1987, p. 230.
[9] H. A. Lee, R. A. Abeles, J. Biol. Chem. 1963, 238, 2367.
[10] T. Toraya, S. Kuno, S. Fukui, J. Bacteriol. 1980, 141, 1439.
[11] Z. Schneider, E. G. Larsen, G. Jacobson, B. C. Johnson, J. Pawelkiewicz, J. Biol. Chem. 1970, 245, 3388.
[12] T. L. Talarico, W. J. Dobrogosz, Appl. Environ. Microbiol. 1990, 56, 1195.
[13] F. Radler, J. Zorg, Am. J. Enol. Vitic. 1986, 37, 206.
[14] H. Schütz, F. Radler, System. Appl. Microbiol. 1984, 5, 169.
[15] P. Manitto, F. Chialva, G. Speranza, C. Rinaldo, J. Agric. Food Chem. 1994, 42, 886.
[16] G. Speranza, S. Corti, G. Fontana, P. Manitto, A. Galli, M. Scarpellini, F. Chialva, J. Agric. Food Chem.
1997, 45, 3476.
[17] R. G. Forage, M. A. Foster, Biochim. Biophys. Acta 1979, 569, 249.
[18] T. Toraya, T. Shirakashi, T. Kosuga, S. Fukui, Biochem. Biophys. Res. Commun. 1976, 69, 475.
[19] K. W. Moore, J. H. Richards, Biochem. Biophys. Res. Commun. 1979, 87, 1052.
[20] G. Speranza, P. Manitto, G. Fontana, D. Monti, A. Galli, Tetrahedron Lett. 1996, 37, 4247.
Â
[21] J. Retey, A. Umani-Ronchi, D. Arigoni, Experientia 1966, 22, 72.
[22] B. Zagalak, P. A. Frey, G. L. Karabatos, R. H. Abeles, J. Biol. Chem. 1966, 241, 3028.
[23] W. W. Bachovchin, R. G. Eagar, K. W. Moore, J. H. Richards, Biochemistry 1977, 16, 1082.
[24] E.-I. Ochiai, in ꢀMetal Ions in Biological Systemsꢁ, Eds. H. Sigel and A. Sigel, Marcel Dekker, New York,
1994, Vol. 30, pp. 255 ± 278.
[25] A. M. Brownstein, R. H. Abeles, J. Biol. Chem. 1961, 236, 1199.
[26] R. H. Abeles, B. Zagalak, J. Biol. Chem. 1966, 241, 1245.
[27] R. H. Abeles, in [1a], p. 371.
[28] M. K. Essenberg, P. A. Frey, R. H. Abeles, J. Am. Chem. Soc. 1971, 93, 1242.
[29] B. T. Golding, L. Radom, J. Chem. Soc., Chem. Commun. 1973, 939; R. J. Anderson, S. Ashwell,
R. M. Dixon, B. T. Golding, ibid. 1990, 70.
[30] W. W. Cleland, CRC Critical Rev. Biochem. 1982, 13, 385.
[31] a) B. M. Babior, in [1b], Vol. II, pp. 263 ± 287; b) B. M. Babior, Biofactors 1988, 1, 21.
[32] a) S. Licht, G. J. Gerfen, J. Stubbe, Science (Washington D.C.) 1996, 271, 477; b) J. Stubbe, Annu. Rev.
Biochem. 1989, 58, 257; c) J. Stubbe, Biochemistry 1988, 27, 3893.
[33] R. J. OꢁBrien, J. A. Fox, M. G. Kopczynski, B. M. Babior, J. Biol. Chem. 1985, 260, 16131.
[34] K. Takai, C. H. Heathcook, J. Org. Chem. 1985, 50, 3247.
[35] M. T. Reetz, J. Westermann, R. Steibach, B. Wenderoth, R. Peter, R. Ostarek, S. Maus, Chem. Ber. 1985,
118, 1421; M. T. Reetz, K. Kesseler, S. Schmidtberger, B. Wenderoth, R. Steibach, Angew. Chem., Int. Ed.
Engl. 1983, 22, 989; Angew. Chem. Suppl. 1983, 1511.
[36] I. Plouzennec-Houe, J.-L. Lemberton, G. Perot, M. Guisnet, Synthesis 1983, 659.
Received May 27, 1998