Synthesis of Enantiomerically Enriched 2-Hydroxymethylalkanoic Acids by Oxidative Desymmetrisation of Achiral 1,3-Diols Mediated by Acetobacter aceti

Article abstract of DOI:10.1002/cctc.201601051

The stereoselective desymmetrisation of achiral 2-alkyl-1,3-diols is performed by oxidation of one of the two enantiotopic primary alcohol moieties by means of Acetobacter aceti MIM 2000/28 to afford the corresponding chiral 2-hydroxymethyl alkanoic acids (up to 94 % ee). The procedure, carried out in aqueous medium under mild conditions of pH, temperature and pressure, contributes to enlarge the portfolio of enzymatic oxidations available to organic chemists for the development of sustainable manufacturing processes.

Full text of DOI:10.1002/cctc.201601051

Accepted Article
Title: Synthesis of Enantiomerically Enriched 2-Hydroxymethyl
Alkanoic Acids by Oxidative Desymmetrisation of Achiral 1,3-
Diols mediated by Acetobacter aceti
Authors: Elisabetta Brenna, Flavia Cannavale, Michele Crotti, Valerio
De Vitis, Francesco G. Gatti, Gaia Migliazza, Francesco
Molinari, Fabio Parmeggiani, Diego Romano, and Sara
Santangelo
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10.1002/cctc.201601051
ChemCatChem
Synthesis of Enantiomerically Enriched 2-Hydroxymethyl Alkanoic Acids by
Oxidative Desymmetrisation of Achiral 1,3-Diols mediated by Acetobacter aceti
Elisabetta Brenna,*[a,b] Flavia Cannavale,[a] Michele Crotti,[a] Valerio De Vitis,[c] Francesco G.
Gatti,[a] Gaia Migliazza,[a] Francesco Molinari,[c] Fabio Parmeggiani,[a] Diego Romano[c] and Sara
Santangelo[a] §
§ Authors are listed in alphabetical order.
[a] Prof. E. Brenna, Miss F. Cannavale, Mr. M. Crotti, Prof. F. G. Gatti, Miss G. Migliazza, Dr. F.
Parmeggiani, Miss S. Santangelo
Dipartimento di Chimica, Materiali ed Ingegneria Chimica “Giulio Natta” Politecnico di Milano,
Via Mancinelli 7, I-20131, Milano, Italy.
E-mail: mariaelisabetta.brenna@polimi.it
[b] Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario Bianco, 9, 20131,
Milano, Italy.
[c] Mr V. De Vitis, Prof. F. Molinari, Dr. D. Romano, University of Milan, Department of Food,
Environmental and Nutritional Science (DeFENS), Via Mangiagalli 25, I-20133, Milano, Italy.
1
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Abstract
The stereoselective desymmetrisation of achiral 2-alkyl-1,3-diols is performed by oxidation of one
of the two enantiotopic primary alcohol moieties by means of Acetobacter aceti MIM 2000/28, to
afford the corresponding chiral 2-hydroxymethyl alkanoic acids (up to 94% ee). The procedure,
carried out in aqueous medium under mild conditions of pH, temperature and pressure, contributes
to enlarge the portfolio of enzymatic oxidations available to organic chemists for the development
of sustainable manufacturing processes.
2
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Introduction
The stereoselective desymmetrisation of achiral and meso substrates is a highly effective
procedure for the preparation of enantiomerically enriched or pure compounds. The technique
consists in the chemical modification of a molecule in order to remove one or more symmetry
elements precluding chirality.[1] Typically, it is achieved by using a chiral reagent or catalyst to
distinguish between two enantiotopic groups of the starting substrate, and selectively promote the
chemical transformation of only one of them. The huge potential of this procedure of optical
activation has been widely exploited on a large array of structurally diverse substrates.[2]
In the last decade, enzymes have received great attention for the optimisation of desymmetrisation
procedures:[3] e.g., biocatalysed ester hydrolyses and transesterifications have been successfully
applied to prepare enantiomerically enriched compounds starting from achiral diesters and diols.
Relevant examples involving chiral active pharmaceutical ingredients (APIs) have been recently
reported.[4]
Among biocatalysed reactions, the oxidation of alcohols and sugars mediated by acetic acid
bacteria (e.g., Acetobacter sp., Acidiphilium sp., Gluconobacter sp.) is a very powerful tool for the
development of sustainable synthetic transformations.[5] This remarkable activity is due to the
bacterial respiratory chain:[6] many types of membrane-bound dehydrogenases, located on the
periplasmatic surface of the cytoplasmatic membrane, reduce ubiquinone with concomitant
substrate oxidation, then the reduced form of ubiquinone is re-oxidized by the terminal ubiquinol
oxidases. Substrate oxidation occurs according to a two-step sequence, involving first the
conversion of the primary alcohol into aldehyde by a pyrroquinoline quinone-dependent alcohol
dehydrogenase (PPQ-ADH) and then the final transformation of the aldehyde intermediate into
carboxylic acid by an aldehyde dehydrogenase. The reaction shows exquisite chemo-, regio- and
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stereoselectivity, and it is carried out under mild conditions of temperature, pressure and pH, in
non-problematic solvents, at high turnover rates.[7] Moreover, acetic acid bacteria are acidophilic
microorganisms that thrive in acidic environments at pH values below 4.0, hence they are
particularly naturally fit to accumulate high concentrations of carboxylic acids.[8]
All these features make this oxidation suitable for the selective modification of one enantiotopic
primary alcohol moiety over another. Nonetheless, to our knowledge, the potential of this enzymatic
oxidative procedure for the desymmetrysation of achiral diols has been scarcely investigated.
As for 1,5-diols, only the conversion of the 3-methyl-substituted derivative into the corresponding
enantiomerically enriched -lactone was investigated, either by using Gluconobacter roseus[9] or by
a two-step oxidation mediated by horse liver alcohol dehydrogenase. [10]
The oxidative desymmetrisations of 1,3-diols reported so far are limited to those of 2-methyl and 2-
butyl derivatives catalyzed by acetic acid bacteria. The microbial oxidation of 2-methyl-1,3-
propanediol leading to (R)-3-hydroxy-2-methylpropionic acid (an important building block for the
synthesis of the ACE inhibitor captopril) was described in 2003, reporting 97% enantiomeric excess
and 100% molar conversion of 5 g/L within 2 h, by using Acetobacter pasteurianus DSM 8937.[11]
More recently, A. pasteurianus IAM 12073 was found to produce the (S)-enantiomer of 2-
(hydroxymethyl)hexanoic acid from 2-butyl-1,3-diol with 89% enantiomeric excess and 82%
conversion after 24 h of incubation.[12]
The few available data on the use of acetic acid bacteria in desymmetrisation reactions
prompted us to investigate more systematically their synthetic potential for the conversion of achiral
1,3-diols into chiral hydroxyalkanoic acids according to this procedure, in order to enlarge the
portfolio of enzymatic reactions available to organic chemists for the development of sustainable
manufacturing processes. The results of our research are herein reported.
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Results and discussion
2-Alkyl and 2-aryl-1,3-diols 1a-i reported in Scheme 1 were selected as starting achiral substrates,
not only to investigate in a systematic way the effect of the substitution at position 2, but also
because of the relevance of the corresponding oxidation products, i.e. chiral -
(hydroxymethyl)alkanoic acids 2, in the synthesis of chiral APIs. As a matter of fact, most of the
known enantioselective syntheses of the resulting hydroxy acids 2 are described in patents filed by
pharmaceutical companies, as we will discuss in the following paragraphs.
Scheme 1. Microbial desymmetrisation of achiral 2-substituted 1,3-diols.
Compounds 1a, h and i were commercially available, whereas the remaining diols were prepared by
LiAlH4 treatment of the corresponding 2-substituted malonates, obtained by monoalkylation of
dimethyl malonate with the suitable alkyl bromide (Scheme 2).
Scheme 2. Synthesis of 1,3-diol substrates 1b-g: i) NaH, THF, RBr; ii) LiAlH4, THF, reflux.
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2-(Hydroxymethyl)alkanoic acids 2a and e had been already obtained by microbial oxidation with
acetic acid bacteria of the corresponding diols,[11, 12] thus these biotransformation were chosen as
benchmark for testing the activity of the strains used in this work.
Preliminary investigations were performed with three different acetic acid bacteria, namely
Acetobacter aceti MIM 2000/28,[7] Asaia bogorensis SF2.1,[13] and Gluconobacter oxydans DSM
2343,[14] known for their oxidative activity on structurally different primary alcohols. Efficient
oxidation was achieved only with A. aceti, whereas the other two strains showed either scarce or no
activity towards substrates 1a-i.
Diols 1a-i were submitted to A. aceti treatment in water at 32°C with a starting substrate
concentration of 1 g/L. The presence of either DMSO or ethanol as a cosolvent (at a concentration
as low as 1% v/v) was found to inhibit the complete conversion of the diol, thus the oxidative
desymmetrisations were carried out by adding the neat diols to the buffer, taking advantage of their
partial solubility in aqueous medium. The product distributions and the enantiomeric excess values
of the final compounds are reported in Table 1.
Diols 1f and 1h were recovered unreacted from the reaction medium: the presence either of a
branched alkyl chain or of an aromatic ring directly linked to the carbon atom in position 2 seems to
sterically hinder this enzymatic activity of A. aceti. Nearly quantitative conversions were obtained
with diols 1a and 1g, showing a methyl and a n-pentyl substituent in position 2, respectively. The
ethyl substituted diol 1b afforded the lowest conversion values.
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Table 1. Biocatalysed desymmetrisation of achiral 2-substituted 1,3-diols 1a-i with A. aceti.

Substrate
Residual
Hydroxymethyl
-Methylenic
alkanoic acid(3)
-Methyl
alkanoic acid(4)
diol (1)
alkanoic acid (2)
c[a]
(%)
95
33
62
yield[b] ee [c]
c[a] (%)
c[a] (%) ee [c] (%)
c[a] (%)
(%)
89
(%)
94 (R)
54 (R)
68 (R)
50 (S)
80 (S)
–
1a, R = Me
1b, R = Et
<1
<1
18
12
<1
>99
<1
>99
10
<1
20
17
<1
<1
–
<1
–
5
<1
47
3
3
12
–
n.d.
n.d
n.d.
n.d.
n.d.
–
25
51
64
71
–
1c, R = n-Pr
1d, R = allyl
1e, R = n-Bu
1f, R = i-Bu
1g, R = n-Pent
1h, R = Ph
85
88
n.r.[d]
98
85
–
88 (S)
–
<1
–
n.d.
–
n.r. [d]
60
1i, R = Bn
48
92 (R)
25
58 (R)
[a] Conversions calculated by GC analysis of the crude mixture after 72 h reaction time after treatment
with CH2N2;
[b] Isolation yield;
[c] Enantiomeric excesses calculated by GC analysis on a chiral stationary phase after treatment with
CH2N2.
[d] n.r.: no reaction product was observed.
The effect of substrate loading was investigated for the oxidation of compound 1g. After 72 h,
conversion was quantitative when the substrate concentration was 1.0 g L–1, while it decreased to
77% and 64% starting from 2.0 and 3.0 g L–1 of compound 1g, respectively.
As for the observed enantioselectivity of the reaction, the (R)-enantiomers of compounds 2 were
obtained with decreasing ee values as the length of the alkyl chain in position 2 increased from
methyl to n-propyl. An inversion of stereoselectivity was promoted by the reduced flexibility of the
three carbon chain of the 2-allyl derivative 1d, affording (S)-2d with modest enantiomeric purity.
The same (S) selectivity was maintained for the n-butyl and n-pentyl substituted compounds with
higher ee values (80 and 88%, respectively). The presence of a benzyl group was found to promote
the oxidation of the primary alcohol affording the (R)-enantiomer of 2i with ee = 92%.
Since the reaction is known to occur according to a two-step sequence with an intermediate
aldehyde, the enantioselectivity is controlled by the PPQ-ADH acting during the first step. A
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tentative explanation of the observed inversion of enantioselectivity can be proposed by comparing
the steric hindrance of the alkyl substituent R in position 2 and that of the CH2OH which is left
unreacted. If R is small (i.e., Me, Et, n-Pr), the (R)-enantiomer of compound 2 is favored. When the
bulkiness of R increases (i.e., allyl, n-Bu, n-Pent), a switch to (S) selectivity is observed. The
presence of the aromatic ring in diol 1i induces a different orientation of the substrate in the enzyme
active site with respect to the other alkyl substituted diols.
The ee values of the desired hydroxymethyl derivatives 2 were found to be rather satisfactory,
especially compared to the values reported in the literature, as it is hereafter summarized. (R)-2a
and (S)-2e were obtained with enantiomeric excess very close to the one previously observed with
other strains of Acetobacter.[11, 12] Both enantiomers of the ethyl esters of 2b, e, g and i had been
previously obtained in enantiomerically enriched form by enzyme-mediated reductions of the
corresponding 2-formylderivatives by Kaneka corporation,[15] with the following ee values were
reported: (R)-2b 37-89%, (R)-2e 75-97%, (R)-2g 87%, and (R)-2i 72-93%. The Evans
oxazolidinones procedure had been employed by SmithKline Beecham[16] and Theravance [17] for
the synthesis of derivatives (R)-2g and (R)-2i to be used in the preparation of antibacterial APIs,
although no indication of the enantiomeric purity was provided. The same strategy had been
employed by Banyu Pharmaceutical Co. to prepare a suitable derivative of (R)-2d[18] as a single
enantiomer for the synthesis of opioid receptor like-1 antagonists. In 2004, Sibi et al.[19] had
reported on the radical alkylation of hydroxyacrylates, obtained by Baylis-Hillman reaction, with
alkyl iodides in the presence of chiral Lewis acids as catalysts to give hydroxymethyl compounds of
type 2. This procedure afforded both the enantiomers of 2c with ee values from 40% to 75%. The
direct enantioselective hydroxymethylation of aldehydes, using ,-diphenylprolinol trimethylsilyl
ether as an organocatalyst, had been described to afford -substituted--hydroxyaldehydes, which
were further converted to the corresponding hydroxy acids 2 by Pinnick oxidation.[20]According to
this method, (R)-2c with 93% ee had been obtained. BASF had described the opening of racemic
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oxetan-2-ones by alcohols in the presence of a lipase as a method to obtain enantiopure esters of
compounds 2b, c and e[21], with the disadvantage that, being a kinetic resolution, it occurred with
loss of at least 50% of the starting material.
The value of the oxidative desymmetrisation herein described is that it represents a biocatalysed
enantioselective synthesis, with creation of the stereogenic centre during the reaction under the
enzyme control, avoiding the use of external chiral auxiliaries to be introduced and then removed at
the end of the procedure.
Determination of the absolute configuration of hydroxymethylalkanoic acids
The absolute configuration of hydroxymethylalkanoic acids 2a (whose (R)-enantiomer is
commercially available), b, c, and e, and of the methyl esters of 2a and b was known (see the
experimental section for the corresponding values of optical rotatory power and literature
references). In the case of derivatives 2b-c, showing very low values of optical activity, a further
confirmation of the (R)-configuration was obtained by preparing two samples of the methyl esters
of (S)-2b-c, i.e. (S)-5b-c respectively, according to a known procedure[22] based on the reduction of
formyl derivatives 6b-c (Scheme 3) by baker’s yeast (BY) in aqueous medium, followed by
transesterification in methanol with a catalytic quantity of sulphuric acid. The HPLC analyses on a
chiral stationary phase of the 3,5-dinitrobenzoates 7b-c of compounds 5b-c, obtained from the
products of either A. aceti oxidation or BY reduction, were in agreement with the assignment of
(R)-configuration to those obtained by oxidation of the achiral diols.
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Scheme 3. Confirmation of the absolute configuration of (S)-2b-c. Reagents and conditions: i) BY,
water, glucose; ii) MeOH, cat. H2SO4; iii) 3,5-dinitrobenzoyl chloride, Et3N, CH2Cl2; iv) A. aceti,
water.
The absolute configuration of derivative (–)-2d was established by converting it into derivative (S)-
7c, by hydrogenation of the allyl chain (H2, Pd/C), esterification (MeOH, cat. H2SO4) and acylation
(3,5-dinitrobenzoylchloride, Et3N, CH2Cl2).
The absolute configuration of derivative (–)-2g was established to be (S) by chemical correlation,
by converting (–)-2g into (+)-(R)-8 according to the reaction sequence reported in Scheme 4.
Scheme 4: Assignment of the absolute configuration of (–)-2g. Reagents and conditions: i) A. aceti,
water; ii) MeOH, cat. H2SO4; iii) MsCl, Et3N, CH2Cl2; iv) LiAlH4, THF.
Side reactions of the oxidative desymmetrisation
During the oxidation reactions of diols 1b-e and i the formation of the dehydrated product 3 and of
the corresponding reduced 2-methyl alkanoic acid 4 was observed (Scheme 1 and Table 1), whereas
oxidation of 1a and 1g afforded the corresponding enantiomerically enriched hydroxy acid as the
only detectable product. Mixtures of derivatives 3 and 4 were isolated from the reaction medium
after the bio-oxidations of diols 1b and 1c. The two components of each mixture were identified by
1
GC/MS and by H NMR spectroscopy. Compounds 4d and 4e could be detected only by GC/MS
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analysis of the corresponding crude residues obtained after the extraction procedure. (R)-2-Methyl-
3-phenylpropanoic acid (4i) showing an ee value of 58% could be isolated as a pure compound
from the reaction mixture when diol 1i was the starting substrate.
The formation of these 2-methylalkanoic acids was supposed to involve an enzyme-mediated
transformation, mainly on the evidence that compound 4i was obtained enriched in the (R)-
enantiomer. Carbon-oxygen bond cleavage is a very common reaction in biological systems, and
several mechanisms have evolved in nature to promote deoxygenation procedures.[23] Deoxysugars
are important examples of compounds obtained by this kind of reaction, through a multi-step
sequence that varies according to the position of the OH group to be removed.[24] For example, C-6
deoxygenation of D-hexoses is catalyzed by a NAD+-dependent nucleotidyldiphosphohexose (NDP-
hexose) 4,6-dehydratase (Eod), belonging to the short-chain dehydrogenase/reductase family, and it
consists of three sequential steps (Scheme 5a):[25] i) oxidation of the OH group in position 4 of
NDP-D-glucose to give NDP-4-ketoglucose with formation of NADH; ii) elimination of water
across C-5 and C-6; iii) final reduction of the 4-keto-Δ5,6-glucose intermediate by NADH to afford
NDP-6-deoxy-4-keto-glucose.
We hypothesised a similar pathway to explain the formation of deoxygenated compounds 3 and 4
(Scheme 5b): oxidation of the primary alcohol to the intermediate hydroxymethyl aldehyde,
dehydration to -methylenic aldehyde, reduction of the C=C double bond, and oxidation to acid 4.
The oxidation of the intermediate unsaturated aldehyde can afford derivative 3.
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Scheme 5. Mechanism of the C-6 deoxygenation of D-hexoses (a) and an analogous tentative
pathway for the explanation of the formation of deoxygenated products 3 and 4 (b).
Because of the highly oxidative character of the culture broth, and the remarkable reactivity of the
formyl group towards oxidation, the hypothesized aldehyde intermediates could not be detected
during the reactions of diols 1. In order to support this dehydration mechanism, feeding experiments
(72 h reaction time) were performed using the unsaturated and saturated aldehydes that are
precursors of acids 3b and 4b (R = Et). When 2-ethylacrolein was added to the A. aceti culture in a
concentration of 1.0 g L–1, a mixture containing 5% of saturated acid 4b and 95% of unsaturated
acid 3b was recovered. The use of a lower concentration of the aldehyde (0.1 g L–1) afforded a final
reaction mixture containing a higher amount of acid 4b (44%) besides 3b (56%). When 2-
methylbutanal was fed directly to the fermentation medium, either at 0.1 or 1.0 g L–1 concentration,
complete oxidation to the corresponding carboxylic acid 4b was observed. The alternative pathway,
in which the hydroxy acid 2b is dehydrated to 3b and then hydrogenated to 4b, was excluded on the
basis of the fact that no alkene reduction was observed when commercially available acrylic acid 3b
was added directly to the A. aceti culture.
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Conclusions
Acetic acid bacteria are non-pathogenic microorganisms, and are readily available due to their
widespread use in the food industry, which makes them ideal candidates for biocatalytic
applications. In this case, the Acetobacter-mediated desymmetrisation of achiral 1,3-diols has been
shown to be an effective procedure for the preparation of enantiomerically enriched hydroxymethyl
alkanoic acids. The interest in these derivatives increased considerably in the last years because
they can be converted into -substituted--lactone APIs,[26] currently studied as caseinolytic
protease (ClpP) inhibitors, to develop a novel therapeutic strategy to simultaneously sensitize
pathogenic bacteria to host defenses and pharmaceutical antibiotics. For this purpose, the required
hydroxymethyl acid derivative is generally obtained with the use of the Evans chiral oxazolidinones
auxiliary.
The Acetobacter-mediated oxidation occurs under mild operative conditions, in aqueous
solution at room temperature, requiring neither the use of complex purpose-made chiral catalysts,
nor the assistance of chiral auxiliaries that are to be removed after the creation of the stereocentre.
The starting 1,3-diols are readily prepared by a common synthetic sequence, that can be easily
extended to the preparation of specific compounds. An interesting change of enantioselectivity from
(S) to (R) was observed when the length of the linear alkyl chain in position 2 of the starting diol
increased from three to four carbon atoms. Very few data are reported in the literature on the
stereochemical course of the A. aceti-mediated oxidation, and most of them are referred to the
[27]
kinetic resolution of racemic secondary alcohols.
Thus, this work lays the groundwork for a
more detailed investigation of the steric and electronic effects controlling the orientation of the
starting diol in the PPQ-ADH active site, and finally influencing the stereochemical outcome of this
oxidative desymmetrisation.
Further study will be devoted to investigate the hypothesized dehydration pathway and optimise the
reaction conditions to inhibit the formation of deoxygenated compounds 3 ad 4. Preliminary
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experiments performed by using immobilized A. aceti cells have produced interesting results in
terms of increase of conversion and reduction of side products.
Experimental Section
General methods
1H and 13C NMR spectra were recorded on a 400 or 500 MHz spectrometer in CDCl3 solution at r.t..
The chemical shift scale was based on internal tetramethylsilane.
GC/MS analyses were performed using a HP-5MS column (30 m × 0.25 mm × 0.25 µm, Agilent).
The following temperature program was employed: 60°C (1 min) / 6°C min-1 / 150°C (1 min) /
12°C min-1 / 280°C (5 min).
Chiral GC analyses were performed on a Chirasil-DEX-CB (25 m × 0.25 mm × 0.25 μm,
Chrompack) column or on a DAcTBSil.BetaCDX column (25 m × 0.25 mm × 0.25 μm, Mega),
installed on a gas chromatograph equipped with FID (carrier gas H2, constant flow 3.7 mL/min,
tinjector = 250°C, tdetector = 250°C). Chiral HPLC analyses were performed on a Chiralcel OD column
(4.6 mm × 250 mm, Daicel) installed on an HPLC instrument with UV detector (λ = 220 nm).
TLC analyses were performed on Merck Kieselgel 60 F254 plates. All the chromatographic
separations were carried out on silica gel columns.
Microorganisms, growth and biotransformation conditions
Acetobacter aceti MIM 2000/28 and Asaia bogorensis SF2.1 from the MIM collection
(Microbiologia Industriale Milano), and Gluconobacter oxydans DSM 50049 (Deutsche Sammlung
von Mikroorganismen) were employed in the preliminary screening. All strains were routinely
maintained on GYC slants (glucose 50 g L−1, yeast extract 10 g L−1, CaCO3 30 g L−1, agar 15 g L−1,
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pH 6.3) at 28°C. Strains, grown on GYC slants for 24 h at 28°C, were inoculated into a 100 mL
baffled Erlenmeyer flasks containing 20 mL of liquid GLY medium (glycerol 25 g L−1, yeast extract
10 g L−1, pH 5, distilled water). After 24 h at 28°C, 150 rpm, the entire culture was used to
inoculate 2 L baffled Erlenmeyer flasks containing 200 mL of GLY medium and incubated in the
same conditions. A. aceti MIM 2000/28 was selected for the development of the oxidative
desymmetrisation reactions.
The biotransformations were performed directly inside the culture flasks, according to the following
procedure. The suitable neat diol 1a-i (200 mg) was added to the cultural broth (200 mL), and the
flask was shaken at 150 rpm at 30°C. After 72 hours, the reaction mixture was centrifuged to
remove the bacterial cells, the supernatant was brought to pH 2.0 with aqueous HCl and extracted
with Et2O. The combined organic phases were dried over anhydrous Na2SO4. The residue was
chromatographed on a silica gel column, eluting with hexane and increasing amounts of ethyl
acetate to obtain the corresponding hydroxy acid.
Characterisation of the products obtained by oxidative desymmetrysation of diols 1a-i
(R)-(Hydroxymethyl)propanoic acid ((R)-2a)
From 1a (0.200 g, 2.22 mmol) derivative (R)-(–)-2a was obtained (0.231 g, 89 %): ee = 94%
1
(determined on the corresponding methyl ester); []D = - 11.3 (c 1.00, EtOH) lit.[29]; H NMR
(CDCl3, 300 MHz):[30] δ = 5.70 (1H, bs,), 3.75 (1H, d, J = 6.0 MHz), 2.74 (1H, m), 2.10 (1H, s,),
1.22 (3H, d, J = 7.4 MHz); 13C NMR (CDCl3, 75 MHz): 13.2, 41.6, 64.0, 180.1.
(R)-2-(Hydroxymethyl)butanoic acid ((R)-2b)
From 1b (0.200 g, 1.92 mmol) derivative (R)-(–)-2b was obtained (0.057 g, 25 %): ee = 54%
(determined on the corresponding methyl ester); []D = –2.8 (c 0.50, CHCl3); 1H NMR (CDCl3, 400
MHz):[31] δ = 3.90-3.70 (2H, m, CH2OH), 2.56 (1H, m, CHCOOH), 1.80-1.55 (2H, m, CHCH2),
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13
1.00 (3H, t, J = 7.6 Hz, CH2CH3); C NMR (CDCl3, 100.6 MHz):[31] δ = 180.4, 62.7, 49.0, 21.6,
11.8.
Methyl (R)-2-(hydroxymethyl)butanoate ((R)-5b)
Compound (R)-2b (0.050 g, 0.424 mmol) was refluxed in MeOH (4 mL) in the presence of a
catalytic quantity of H2SO4, to afford after the usual work-up the corresponding methyl ester (+)-
(R)-5b (0.053 g, 95%): ee = 54% (by chiral HPLC of the 3,5-dinitrobenzoate derivative), []D =
+2.4 (c 1.1, CHCl3), lit. [32] for (R)-5b ee = 88 % []D = +4.1 (c 1.0, CHCl3); 1H NMR (CDCl3, 400
MHz): [33] δ = 3.84-3.66 (5H, m+ s, CH2OH+OCH3), 2.52 (1H, m, CHCOOCH3), 1.75-1.50 (2H, m,
CH2CH3), 0.95 (3H, t, J = 7.4 Hz, CH2CH3); 13C NMR (CDCl3, 100.6 MHz): δ = 175.0, 63.0, 51.8,
49.1, 21.8, 11.8; GC/MS (EI) tR = 6.50 min: m/z (%) = 114 (M+–18, 8), 102 (50), 87 (100).
The absolute configuration was confirmed by comparing the HPLC analysis of the 3,5-
dinitrobenzoate derivative 7b prepared from (+)-(R)-5b with a sample the of 3,5-dinitrobenzoate
derivative of (S)-5b prepared by baker’s yeast reduction of ethyl 2-formylbutanoate 6b,[22]followed
by transesterification with MeOH and a catalytic quantity of H2SO4.
Chiral HPLC: Chiralcel OD (n-hexane/i-PrOH 9:1, 0.6 mL/min), tR (R)-7b = 51.30 min, tR (S)-7b =
61.32 min.
2-Methylenebutanoic acid (3b) and 2-methylbutanoic acid (4b)
From 1b (0.200 g, 1.92 mmol), beside derivative (R)-2b (25 %), a 1:1.5 (1H NMR) mixture of 3b
1
and 4b (0.097 g) was isolated: H NMR (CDCl3, 400 MHz), signals of 3b[34] δ = 6.27 (1H, br s,
CHH=C), 5.65 (1H, br s, CHH=C), 2.34 (1H, q, J = 7.2 Hz, CH2C=), 1.10 (3H, t, J = 7.2 Hz,
CH2CH3); signals of 4 [35] δ = 2.42 (1H, m, CHCOOH), 1.60 (2H, m, CH2CH), 1.18 (3H, t, J = 6.9
Hz, CHCH3), 0.89 (3H, t, J = 7.4 Hz, CH2CH3); GC/MS (EI): 3b tR = 5.46, m/z (%) = 100 (M+, 78),
85 (52), 55 (100); 4b tR = 4.99: m/z (%) = 87 (M+–15, 25), 74 (100), 57 (55).
(R)-2-(Hydroxymethyl)pentanoic acid ((R)-2c)
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From 1c (0.200 g, 1.69 mmol) derivative (R)-2c was obtained (0.114 g, 51%): ee = 68%
(determined on the corresponding methyl ester) []D = –2.3 (c 2.2, CHCl3), lit. [36] for (R)-2c ee =
92 % []D = –3.0 (c 10, CHCl3)]; 1H NMR (CDCl3, 400 MHz):[36] δ = 3.85-3.70 (2H, m, CH2OH),
13
2.62 (1H, m, CHCOOH), 1.70-1.30 (4H, m, CHCH2CH2), 0.93 (3H, t, J = 7.6 Hz, CH2CH3); C
NMR (CDCl3, 100.6 MHz):[36] δ = 180.0, 63.2, 47.5, 30.5, 20.6, 14.1.
Methyl (R)-2-(hydroxymethyl)pentanoate ((R)-5c)
Compound (R)-2c (0.090 g, 0.681 mmol) was refluxed in MeOH (4 mL) in the presence of a
catalytic quantity of H2SO4, to afford after the usual work-up the corresponding methyl ester (R)-5c
(0.096 g, 97%): ee = 68% (by chiral HPLC of the 3,5-dinitrobenzoate derivative), []D = –2.4 (c
1.5, CHCl3); 1H NMR (CDCl3, 400 MHz):[19] δ = 3.80-3.70 (5H, m+s, CH2OH+OCH3), 2.60 (1H,
m, CHCOOCH3), 1.70-1.45 (2H, m, CHCH2CH2), 1.40-1.30 (2H, m, CHCH2CH2), 0.95 (3H, t, J =
7.4 Hz, CH2CH3); 13C NMR (CDCl3, 100.6 MHz): δ = 175.8, 63.2, 51.6, 47.2, 30.6, 20.4, 13.9;
GC/MS (EI) tR = 8.53 min: m/z (%) = 128 (M+–18, 1), 116 (20), 104 (28), 87 (100).
The absolute configuration was confirmed by comparing the HPLC analysis of the 3,5-
dinitrobenzoate derivative 7c prepared from (R)-5c with a sample the of 3,5-dinitrobenzoate
derivative of (S)-5c prepared by baker’s yeast reduction of ethyl 2-formylpentanoate 6c,[22] followed
by transesterification with MeOH and a catalytic quantity of H2SO4.
Chiral HPLC: Chiralcel OD (n-hexane/i-PrOH 9:1, 0.6 mL/min), tR (S)-7c = 46.9 min, tR (R)-7c
57.2 min.
2-Methylenepentanoic acid (3c) and 2-methylpentanoic acid (4c)
From 1c (0.200 g, 1.69 mmol), beside derivative (R)-2c (51 %), a 4:1 (1H NMR) mixture of 3c and
[37]
1
4c (0.039 g) was isolated. Only the signals
of 3c are herein reported: H NMR (CDCl3, 400
MHz), δ = 6.27 (1H, br s, CHH=C), 5.63 (1H, br s, CHH=C), 2.28 (1H, t, J = 7.5 Hz, CH2C=),
1.60-1.40 2H, m, CH2CH3), 0.90 (3H, t, J = 7.2 Hz, CH2CH3); GC/MS (EI): 3c tR = 6.80: m/z (%) =
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114 (M+, 25), 99 (31), 86 (42), 69 (100); 4c tR = 6.10: m/z (%) = 115 (M+–1, 1), 101 (5), 87 (15), 74
(100).
(S)-2-(Hydroxymethyl)pent-4-enoic acid ((S)-2d)
From 1d (0.200 g, 1.72 mmol) derivative (S)-2d was obtained (0.143 g, 64%): ee = 50%
(determined by conversion into (S)-7c), []D = – 0.4 (c 2.4, CHCl3); 1H NMR (CDCl3, 400 MHz): δ
= 5.84-5.72 (1H, m, CH=C), 5.16-5.00 (2H, m, CH2=C), 3.80 (2H, d, J = 6.1 Hz, CH2OH), 2.70
(1H, quintuplet, J = 6.2 Hz, CHCOOH), 2.52 – 2.40 (1H, m, CHHC=), 2.38 – 2.26 (1H, m,
CHHC=); 13C NMR (CDCl3, 100.6 MHz): δ = 179.2, 134.6, 117.6, 62.5, 47.1, 32.5; GC/MS (EI) as
methyl ester: tR = 8.11 min: m/z (%) = 112 (M+–32, 12), 97 (68), 67 (100).
2-Methylpent-4-enoic acid (4d)
Compound 4d was detected by GC/MS analysis in the crude residue, obtained after the work-up of
the reaction of diol 1d: GC/MS (EI) 4d tR = 5.50 min: m/z (%) = 114 (14), 99 (21), 69 (82), 41
(100).
(S)-2-(Hydroxymethyl)hexanoic acid ((S)-2e)
From 1e (0.200 g, 1.52 mmol) derivative (S)-2e was obtained (0.157 g, 71%): ee = 81% (chiral GC
analysis as a methyl ester after diazomethane treatment), []D = –5.1 (c 0.75, CH3OH), lit.[38] for
(R)-2e ee = 99% []D = +6.5 (c 1.0, CH3OH); 1H NMR (CDCl3, 400 MHz)[38] δ = 3.85-3.75 (2H, m,
CH2OH), 2.61 (1H, quint, J = 5.9 Hz, CHCOOH), 1.75-1.65 (1H, m, CHCHHCH2), 1.60-1.45 (1H,
m, CHCHHCH2), 1.40-1.28 (4H, m, CHCH2(CH2)2), 0.91 (3H, t, J = 6.4 Hz, CH2CH3); 13C NMR
(CDCl3, 100.6 MHz): δ = 180.1, 63.2, 47.9, 29.8, 28.4, 22.7, 14.0; GC/MS (EI) as methyl ester: tR =
10.81 min: m/z (%) = 142 (M+–18, 1), 130 (20), 104 (54), 87 (100).
Chiral GC: DAcTBSil.BetaCDX, 60°C / 0.8°C min-1 / 90°C / 30°C min-1 / 220°C (2 min), tR (R)-2e
= 29.9 min, tR (S)-2e 30.6 min.
2-Methylhexanoic acid (4e)
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Compound 4e was detected as a methyl ester by GC/MS analysis in the crude residue, obtained
after the work-up of the reaction of diol 1e: GC/MS (EI) 4e tR = 5.97 min: m/z (%) = 129 (M+–15,
2), 113, (8), 101 (25), 88 (100).
(S)-2-(Hydroxymethyl)heptanoic acid ((S)-2f)
From 1f (0.200 g, 1.37 mmol) derivative (S)-(–)-2f was obtained (0.186 g, 85%): ee = 92%
(determined on the corresponding methyl ester), []D = –2.1 (c 2.3, CHCl3); 1H NMR (CDCl3, 400
MHz): [39] δ = 3.85 – 3.70 (2H, m, CH2OH), 2.59 (1H, quint, J = 5.9 Hz, CHCOOH), 1.70-1.57 (1H,
m, CHCHHCH2), 1.55-1.42 (1H, m, CHCHHCH2), 1.40-1.20 (6H, m, CHCH2(CH2)3), 0.88 (3H, t,
J = 6.5 Hz, CH2CH3); 13C NMR (CDCl3, 100.6 MHz): δ = 179.6, 63.2, 47.9, 31.8, 28.4, 26.9, 22.5,
14.0.
Methyl (S)-2-(hydroxymethyl)heptanoate ((S)-5f)
Compound (S)-(–)-2f (0.120 g, 0.750 mmol) was refluxed in MeOH (4 mL) in the presence of a
catalytic quantity of H2SO4, to afford after the usual work-up the corresponding methyl ester (–)-5f
(0.125 g, 96%): ee = 92% (chiral HPLC of the 3,5-dinitrobenzoate derivative); [D = –8.9 (c 1.1,
1
CHCl3); H NMR (CDCl3, 400 MHz):[39] δ = 3.80-3.65 (5H, m+s, CH2OH+ COOCH3), 2.58 (1H,
m, CHCOOH), 1.70-1.57 (1H, m, CHCHHCH2), 1.56-1.47 (1H, m, CHCHHCH2), 1.40-1.20 (6H,
13
m, CHCH2(CH2)3), 0.88 (3H, t, J = 6.4 Hz, CH2CH3); C NMR (CDCl3, 100.6 MHz): δ = 176.0,
63.3, 51.8, 47.7, 31.8, 28.6, 27.0, 22.5, 14.0; GC/MS (EI): tR = 13.83 min m/z (%) = 156 (M+-18, 1),
144 (25), 104 (83), 87 (100).
Chiral HPLC: Chiralcel OD (n-hexane/i-PrOH 9:1, 0.6 mL/min), tR (R)-7f = 45.1 min, tR (S)-7f =
57.0 min.
(R)-2-Methylheptan-1-ol ((R)-8)
Compound (–)-5f (0.130 g, 0.747 mmol) was treated with mesyl chloride (0.102 g, 0.896 mmol)
and Et3N (0.113 g, 0.112 mmol) in CH2Cl2 (5 mL). After the usual work up, the mesylated
derivative was isolated and submitted without any further purification to LiAlH4 (0.042 g, 1.12 mol)
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reduction in refluxing THF (10 mL), to afford after the usual work-up compound (R)-(+)-8 (0.073
g, 75%): ee = 88% (chiral GC of the acetyl derivative), []D = +11.5 (c 1.4, CHCl3), lit. [40] for (S)-8
ee = 98% []D = –13.1 (c 1.15, CHCl3); 1H NMR (CDCl3, 400 MHz): [41] 3.50 (1H, dd J = 10.4 and
5.7 Hz, CHHOH), 3.43 (1H, dd J = 10.4 and 6.5 Hz, CHHOH), 1.61 (1H, m, CHCH3), 1.40-1.15
(8H, m, CH(CH2)4), 0.95-0.85 (6H, d+t, CHCH3+ CH2CH3); 13C NMR (CDCl3, 100.6 MHz): 68.5,
35.9, 33.3, 32.3, 30.5, 26.8, 22.8, 16.7, 14.2; GC/MS (EI): tR = 9.2 min (as acetyl derivative): m/z
(%) = 112 (M+–60, 10), 97 (5), 84 (15), 56 (60), 43 (100).
Chiral GC: Chirasil-DEX-CB, 60°C / 0.8°C min-1 / 90°C / 90°C min-1 / 180°C, tR (S)-8 = 16.48
min, (R)-8 = 16.6 min (as acetyl derivative).
(R)- 2-Benzyl-3-hydroxypropanoic acid ((R)-2i)
From 1i (0.200 g, 1.20 mmol) derivative (R)-2i was obtained (0.104 g, 48%): ee = 92% (determined
on the corresponding methyl ester), []D = +14.2 (c 2.1, CHCl3), lit. [42] for (R)-2i ee = 86.4 % []D
= +13.5 (c 3.1, CHCl3); 1H NMR (CDCl3, 400 MHz)[42] δ = 7.35-7.15 (5H. m, ArH), 3.76 (2H, m,
CH2OH), 3.12-3.04 (1H, m, CHCOOCH3), 2.94-2.84 (2H, m, CH2Ph); 13C NMR (CDCl3, 100.6
MHz):[42] δ = 178.9, 138.4, 129.1, 128.8, 126.8, 62.0, 48.8, 34.2; GC/MS (EI): tR = 19.11 min (as
methyl ester): m/z (%) = 194 (M+, 2), 176 (50) 117 (75), 91 (100).
Methyl (R)-2-benzyl-3-hydroxypropanoate ((R)-5i)
Compound (R)-2i (0.090 g, 0.500 mol) was refluxed in MeOH (4 mL) in the presence of a catalytic
quantity of H2SO4, to afford after the usual work-up the corresponding methyl ester (R)-5i (0.087 g,
90 %): ee = 92%, []D = +23.9 (c 1.1, CHCl3), lit. [43] for (S)-5i ee = 96 % []D = –25.1 (c 0.56,
CHCl3); 1H NMR (CDCl3, 400 MHz):[43] δ = 7.32-7.16 (5H. m, ArH), 3.73 (2H, m, CH2OH), 3.69
(3H, s, OCH3), 3.07-2.98 (1H, m, CHCOOCH3), 2.92-2.80 (2H, m, CH2Ph); 13C NMR (CDCl3,
100.6 MHz):[43] δ = 175.1, 138.7, 129.1, 128.7, 126.7, 62.4, 51.9, 39.6, 34.6; GC/MS (EI) tR = 19.11
min: m/z (%) = 194 (M+, 2), 176 (50) 117 (75), 91 (100)
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Chiral GC: DAcTBSil.BetaCDX, 70°C / 10°C min-1 / 110°C (60 min) / 50°C min-1 / 220°C (2 min),
(R)-5i tR = 51.80 min, (S)-5i tR = 53.9 min.
(R)-2-Methyl-3-phenylpropionic acid ((R)-4i)
From 1i (0.200 g, 1.20 mmol), beside derivative (R)-2i(48%), compound (R)-4i (0.020 g, 10%) was
isolated: ee = 58%, (by chiral GC of the methyl ester); []D = –17.8 (c 1.2, CHCl3), lit. [44] for (S)-4i
ee = 99.2 % []D = + 30.2 (c 0.82, CHCl3); 1H NMR (CDCl3, 400 MHz):[44] δ = 7.35-7.15 (5H. m,
ArH), 3.08 (1H, dd J = 13.4 and 6.3 Hz, PhCHH), 2.77 (1H, m, CHCOOH), 2.67 (1H, dd J = 13.4
13
and 8.0 Hz, PhCHH), 1.18 (3H, d J = 6.9 Hz, CH3CH); C NMR (CDCl3, 100.6 MHz): [45] δ =
182.4, 139.2, 129.1, 128.6, 126.7, 41.3, 39.5, 16.6; GC/MS (EI) tR = 16.29 min: m/z (%) = 164 (M+,
23), 118 (7), 91 (100).
Chiral GC: Chirasil DEX CB, 65°C / 0.5°C min-1 / 85°C / 50°C min-1 / 180°C (2 min), (R)-4i tR =
37.9 min, (S)-4i tR =38.8 min.
Keywords
Acetic acid bacteria – Biocatalysis – Desymmetrisation – Enantioselectivity – Oxidation
Acknowledgement
This work was supported by Fondazione Cariplo, grant n. 2014-0568. Miss Francesca Tentori is
kindly acknowledged for helpful assistance.
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A helpful discrimination between
the two enantiotopic alcoholic
moieties of 2-substituted-1,3-diols is
made by Acetobacter aceti in the
oxidation process to afford
Elisabetta Brenna,* Flavia Cannavale,
Michele Crotti, Valerio De Vitis,
Francesco G. Gatti, Gaia Migliazza,
Francesco Molinari, Fabio Parmeggiani,
Diego Romano and Sara Santangelo
enantiomerically enriched 2-
hydroxymethylalkanoic acids.
Page No. – Page No.
Synthesis of Enantiomerically
Enriched 2-Hydroxymethyl Alkanoic
Acids by Oxidative
Acetobacter aceti
Desymmetrisation of Achiral 1,3-
Diols mediated by Acetobacter aceti
26
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