Chemoenzymatic Preparation of Enantiomerically Enriched (R)-(–)-Mandelic Acid Derivatives: Application in the Synthesis of the Active Agent Pemoline

Article abstract of DOI:10.1002/ejoc.201700161

The enantioselective resolution of several racemic derivatives of mandelic acid methyl ester catalyzed by lipases from Pseudomonas fluorescens (Amano AK) or Burkholderia cepacia (Amano PS-C II and Amano PS-IM) has been achieved. A gram-scale lipase-mediated kinetic resolution approach has been developed that allows the facile synthesis of the corresponding methyl (R)-(–)-mandelates with excellent enantiomeric excesses (up to >99 % ee) and reaction enantioselectivity (E values up to >200). The dopaminergic agent pemoline, used in the treatment of attention-deficit hyperactivity disorder (ADHD) and narcolepsy, was synthesized with 98 % ee in a straightforward route by condensing the prepared methyl (R)-(–)-mandelate with guanidine hydrochloride under basic conditions. The desired (R)-(+)-pemoline in optically pure form (>99 % ee) was obtained after two recrystallizations from ethanol. However, it was confirmed by chiral HPLC that optically active pemoline undergoes racemization in methanol solution.

Full text of DOI:10.1002/ejoc.201700161

A Journal of
Accepted Article
Title: Chemoenzymatic preparation of enantiomerically enriched (R)-
(-)-mandelic acid derivatives: application in the synthesis towards
the active agent pemoline
Authors: Paweł Borowiecki, Marcin Poterała, and Maciej Dranka
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201700161
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10.1002/ejoc.201700161
European Journal of Organic Chemistry
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Chemoenzymatic preparation of enantiomerically enriched
(R)-(–)-mandelic acid derivatives: application in the synthesis
towards the active agent pemoline
‡
‡
Marcin Poterała,*[a] Maciej Dranka,[a] and Paweł Borowiecki*[a]
Dedicated to Prof. Jan Plenkiewicz in honor of his scientific achievements within academic career.
Abstract: The enantioselective resolutions of a several racemic
derivatives of mandelic acid methyl ester catalyzed by lipases from
Pseudomonas fluorescens (Amano AK) or Burkholderia cepacia
(Amano PS-C II and Amano PS-IM) are demonstrated. A gram-scale
lipase-mediated kinetic resolution approach have been developed,
allowing facile synthesis of the corresponding (R)-(–)-methyl
mandelates with excellent enantiomeric excesses (up to >99% ee)
and the reactions enantioselectivity (E-value up to >200). The
dopaminergic agent pemoline – used in the treatment of attention-
deficit hyperactivity disorder (ADHD) and narcolepsy
– was
synthesized with 98% ee in a straightforward route by condensing
the prepared methyl (R)-(–)-mandelate with guanidine hydrochloride
under basic conditions. The desired (R)-(+)-pemoline in an optically
pure form (>99% ee) was obtained after two recrystallizations from
ethanol. However, it was confirmed by chiral HPLC that optically
active pemoline undergoes racemization in methanol solution.
Introduction
Figure 1. Examples of biologically active compounds obtainable via (R)-
mandelic acid (RMA).
The single enantiomers of mandelic acid (MA), mainly (R)-
mandelic acid (RMA) and its analogous, constitute a valuable
intermediates for the synthesis of biologically active compounds
of high therapeutic importance and economic impact. Exemplary,
the RMA structural motif is the core of many pharmacologically
relevant compounds I–VIII (Fig. 1), such as semi-synthetic
antibiotics including modified cephalosporins (cefamandole[1] I)
and penicillins (MA-6-APA II)[2] as well as anti-cholinergic
medications (oxybutynin[3] III and homatropine[4] IV). Moreover,
derivatives of (R)-mandelic acid are also used as chiral synthons
in the preparation of anti-platelet/anti-thrombotic (clopidogrel[5]
V), vasodilator (cyclandelate[6] VI), anti-tumor {complex of cis-
[Pt(2-α-hydroxybenzylbenzimidazole)2Cl2] VII}[7] and anti-obesity
agents VIII.[8] Due to the importance of enantiomerically pure
mandelic acid, tremendous efforts have been made to establish
enantioselective routes for its production in the past few
decades. In this purpose, a large number of asymmetric
methods using the respective organic catalysts or metal-based
complexes have been developed to obtain optically active MA.
However, traditional synthetic routes towards mandelic acid
single enantiomers require harsh reaction conditions including
high pressure and temperature as well as costly, toxic and
difficult to remove catalysts, and thus reliable and efficient
‘purely-synthetic’ procedures remain still impaired. Therefore,
simultaneously to those methods, a plethora of practical and
environmentally friendly enzymatic approaches toward the
preparation of enantiomerically pure MA have been developed.
Among them noteworthy are: (i) stereoselective hydrolysis of
mandelonitrile to RMA by means of various nitrilases from i.e.
Alcaligenes faecalis ZJUTB10,[9] Alcaligenes faecalis ATCC
8750,[10] Alcaligenes sp. ECU0401,[11] Alcaligenes sp. MTCC
10675,[12] Pseudomonas putida MTCC 5110,[13] Burkholderia
cenocepacia J2315,[14] Microbacterium paraoxydans and
Microbacterium liquefaciens;[15] (ii) enantioselective microbial
degradation of SMA using growing cells of i.e. Alcaligenes
bronchisepticus,[16] Pseudomonas polycolor,[17] Gibberella
fujikuroi,[18] and Pseudomonas sp. MA02[19] or the respective
[a]
Faculty of Chemistry, Warsaw University of Technology.
Address: Koszykowa St. 3, 00–664 Warsaw, Poland.
E-mail: mpotera@ch.pw.edu.pl (Dr. M. Poterała – Department of
Organic Chemistry) and pawel_borowiecki@onet.eu or
pborowiecki@ch.pw.edu.pl (Dr. P. Borowiecki – Department of
Drugs Technology and Biotechnology).
Homepage: http://www.ch.pw.edu.pl.
These authors contributed equally to the work.
[‡]
degradation of RMA by means of
a bacterial strain of
Supporting information for this article is given via a link at the end of
the document.
Pseudomonas putida ECU1009;[20] (iii) stereoselective
bioreduction of benzoyl formate [phenylglyoxylic acid (PGA)] by
Saccharomyces cerevisiae FD11b,[21] benzoylformate reductase
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10.1002/ejoc.201700161
European Journal of Organic Chemistry
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extracted from cells of Streptococcus faecalis IFO 12964,[22] by a
D-(−)-mandelic acid dehydrogenase from Lactobacillus
curvatus[23] or reduction of PGA esters via actively fermenting
Saccharomyces cerevisiae yeast,[24] NADPH-dependent benzil
reductase (KRED1-Pglu) from the Pichia glucozyma CBS 5766
yeast,[25] NADP-dependent alcohol dehydrogenase from
Clostridium acetobutylicum (CaADH),[26] and the resting cells of
Bacillus pumilus Phe-C3;[27] and finally (iv) stereoselective α-
hydroxylation of methyl 2-phenylacetate to (S)-methyl mandelate
can be found in many biologically active molecules (Fig. 1),
surprisingly, it has not been used toward synthesis of
enantiomerically pure pemoline until now. Pemoline (2-amino-5-
phenyl-4(5H)-oxazolone,
2-imino-5-phenyl-4-oxazolidinone,
phenylisohydantoin; marketed under trade names: Betanamin;
Ceractiv; Cylert; Tradon) is non-narcotic, mild and long-acting
central nervous system (CNS) stimulant of dopaminergic activity,
likely acting via inducing the release of dopamine and
norepinephrine in brain tissue, being a surrogate for dopamine,
although not affecting endogenous intracellular dopamine. In
view of the above mentioned properties, this medication was
administrated for decades to treat attention-deficit hyperactivity
disorder (ADHD)[47] and narcolepsy.[48] Albeit pemoline is
currently withdrawn from market as it is considered to be
hepatotoxic,[49] it would be a great challenge to find if ‘an old
drug could not serve as a new again’ ? In this regard, it is
strongly recommended to perform pharmacological evaluations
on both enantiomers of pemoline in order to estimate, which of
its antipode is responsible for positive biological response useful
for management of behavioral disorders in children, and which
cause unwanted fatal liver toxicity. Therefore, straightforward
and preparative synthetic method of the preparation of both
homochiral forms of pemoline is desirable. Based on the above
considerations and to alleviate the desire of better recognition of
the mechanism of pemoline toxic liver damage in future, we
developed an integrated lipase-catalyzed bioprocess for the
synthesis of the RMA and its application towards the active
agent (R)-pemoline.
by the monooxygenase from
Helminthosporium sp.
CIOC3.3316[28] or with engineered cytochrome P450 BM-3,[29]
respectively. Interestingly, enantiomerically pure (S)-mandelic
acid was also synthesized from benzaldehyde by sequential
hydrocyanation and hydrolysis in a bienzymatic conversion—
combined (catalytic) procedure composed of a cross-linked-
enzyme aggregate (combi-CLEA) of the (S)-selective
oxynitrilase from Manihot esculenta and the non-selective
nitrilase from Pseudomonas fluorescens EBC 191[30] as well as
by
a whole-cell recombinant Escherichia coli biocatalyst
expressing simultaneously different variants of both those
enzymes.[31] In turn, other bi-enzyme system, in which a FMN-
dependent (S)-mandelate dehydrogenase (SMDH) from
Pseudomonas aeruginosa and laccase from Agaricus bisporus
were employed as biocatalysts, afforded enantiomerically pure
(R)-mandelic acid[32] in high yield. Less studied in the literature,
but also very promising in terms of efficiency of MA enantiomers
preparation seems to be highly active nitrile hydratase/amidase
enzyme system present in whole cells of Rhodococcus
erythropolis NCIMB 11540.[33]
From the view point of the frequency of the usage in
biotechnological processes toward asymmetric synthesis of MA
enantiomers, the isolated enzymes constitute predominant
source of the catalysts. Except few examples of utilization of
Results and Discussion
In this study, we aimed at developing a simple and efficient
chemoenzymatic synthesis of (R)-(–)-mandelic acid derivatives,
and at using one of them (RMA) as a chiral synthon for the
preparation of active pharmaceutical ingredient – pemoline 5.
Our approach focuses exclusively on lipase-catalyzed
enantioselective transesterification reactions of racemic methyl
mandelates rac-3a-h carried out under kinetically–controlled
conditions. In this context, the required for enzymatic
transformations racemic methyl esters of MA rac-3a-h and their
respective diesters rac-4a-h (used as an analytical standards for
reaction’s progress monitoring and enantiomeric excess
measurements) were prepared according to Scheme 1.
carbonic
anhydrase,[34]
mandelate
dehydrogenases,[35]
esterases,[36] mandelate racemase,[37] and D-lactate oxidase
(GO-LOX),[38] it is undoubtedly the lipases that focus the largest
considerable interest on this field. In this context, many
investigations have been performed over the years on the use of
lipases as biocatalysts for MA enantiomers synthesis. So far, the
most typical existing lipase-based protocol being predominant
among these efforts is (i) enantioselective transesterification.[39]
In reference to it, two various dynamic kinetic resolution (DKR)
approaches based on tandem metal–enzyme protocol[40] and a
lipase-mandelate
racemase
two-enzyme
system
in
aqueous/organic two-phase system[41] or in sole ionic liquids[42]
both applying racemization-esterification methodology are
reported. Other approaches performed in enzymatic kinetic
resolution (EKR) manner, such as (ii) enantioselective hydrolysis
of MA esters,[43] (iii) asymmetric esterification of racemic MA with
aliphatic alcohols,[44] (iv) direct enantioselective amidation of MA
with ammonia,[45] and (v) aminolysis of racemic methyl
mandelate with n-butylamine[46] have also been successfully
used.
Although many approaches towards preparation of (R)-
mandelic acid have been proposed, in our opinion satisfactory
results are still demanding. Moreover, although optically active
MA scaffold is a privileged structure in medicinal chemistry, and
Synthesis of the racemic starting materials
According to the literature, the most convenient method of
mandelic acid and its derivatives preparation is the three-step
procedure: synthesis of sodium benzaldehyde-bisulfate adduct,
subsequent cyanohydrin formation and final acidic hydrolysis of
the obtained mandelonitrile.[50] However, this standard procedure
assumes usage of extremely poisonous and flammable
hydrogen cyanide or at least highly toxic sodium/potassium
cyanide, which upon standing hydrolyze to release
hydrocyanic acid as well.
a
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leads to a much complex mixtures formation, presumably
because of increased incorporation of the trichloromethyl anion
–
(CCl3 ) or consecutively formed dichlorocarbene (:CCl2) into an
aromatic ring system instead of their addition to a carbonyl
–
group by either direct attack of the nucleophilic CCl3 on the
partially positive carbon atom or insertion of ambident :CCl2 into
a
carbon-oxygen double bond. Since dihalocarbenes are
generated strictly in the organic phase via the trihalomethyl
anion, and more, the corresponding formation of
dibromocarbene from bromoform especially in homogeneous
systems where water is present is generally poor from
bromoform, Method B seems to be particularly attractive
procedure as the reactions with benzaldehydes proceed
exclusively through the nucleophilic attack by the anion on the
carbonyl group.
Scheme 1. Chemoenzymatic preparation of enantiomerically enriched
mandelic acid derivatives (R)-(–)-3a-h. Reagents and conditions: (i) TEBACl
(cat.), CHCl3, 50% NaOH, 56±2 °C, then 5 h at RT, then 18% HCl (pH 1); (ii)
CHBr3 (1 equiv), LiCl (1.9 equiv), KOH (3.9 equiv), fine-crushed ice-cubes,
1,4-dioxane, 36 h at 0–5 °C, then 24 h at 35 °C; (iii) SOCl2 (1.1 equiv), dry
MeOH, at -30 °C over 30 min, then 4 h at RT; (iv) Ac2O (1.0 equiv), dry Py (1.1
equiv), DMAP (cat.), dry CH2Cl2, 6 h at RT; (v) vinyl acetate, lipase (30% w/w),
TBME, RT, 250 rpm (laboratory shaker).
Table 1. Synthesis of the racemic mandelic acid derivatives rac-2b-h.
Mandelic acid derivatives might also be obtained from the
respective acetophenones, using chlorine in order to form
dichloromethyl aryl ketones, which easily undergo hydrolysis to
desired MAs in the presence of base.[51] Unfortunately, this two-
steps procedure requires application of chlorine, which is highly
corrosive and toxic reagent, and in addition the
dichloroacetophenone formed in the first step is a harmful heavy
lachrymatory oil. From the obvious reasons, we decided to use
much safer approach based on the transformation of
commercially available and cheap arylaldehydes 1b-h under
phase-transfer catalysis (PTC) conditions using the 50%
NaOH/CHCl3 liquid/liquid system in the presence of
benzyltriethylammonium chloride (TEBACl) at elevated
temperature (Method A)[52] or alternatively, the reactions of 1b-h
with CHBr3 performed in the presence of KOH and LiCl in a
cooled to 0–5 °C mixture of 1,4-dioxane/H2O (Method B)[53]
(Table 1). Both methods require additional basic hydrolysis of
the formed intermediate adducts, which is rapidly afforded in situ
under reaction conditions.
Entry
Product
R1
Method[a]
Yield[b] (%)
1
2
3
4
5
6
7
8
9
10
rac-2b
4-CH3C6H4-
A
B
A
B
A
B
B
B
B
B
70
75
54
71
41
69
57
73
75
68[c]
rac-2c
rac-2d
3-CH3C6H4-
4-i-PrC6H4-
rac-2e
rac-2f
rac-2g
rac-2h
4-tert-BuC6H4-
4-CH3OC6H4-
2-Naphthyl-
1-Naphthyl-
At first, to estimate the utility of these methods we decided
to compare their efficiency toward three model compounds 1b-d
selected out of the set of the available substrates 1b-h. After
series of experiments it turned out that reactions conducted via
Method B were superior over the Method A. Apart from the
substantially simplified reaction procedure, Method B outclasses
the PTC synthetic methodology in the fields of overall yield and
mildness. The desired rac-2b-d prepared by means of Method B
were obtained with yields in range of 69–75% (Table 1, entries 1,
3, and 5), whilst in the case of Method A the products rac-2b-d
were isolated in 41–70% yields (Table 1, entries 2, 4, and 6).
The differences in the yields are due to the fact that in Method A
the temperature regime must be strictly controlled, otherwise
complicated side reactions remain difficult to avoid. Exceeding
even ±2–3 °C threatens drastic decline in efficiency of the
reaction. Another serious drawback of Method A is it’s limited
accessibility towards aldehydes possessing benzene ring
substituted by electron withdrawing groups. This electronic effect
[a] Method A: 1b-d (0.1 mol), TEBACl (1.23 g, 5 mmol), CHCl3 160 mL, 50%
NaOH 25 mL, 56±2 °C, then 5 h at RT, then 18% HCl (pH 1); Method B: 1b-
h (0.13 mol), CHBr3 (31.6 g, 0.13 mol), LiCl (10.6 g, 0.25 mol), KOH (28.1 g,
0.50 mol), fine-crushed ice-cubes 100 g, 1,4-dioxane 100 mL, 36 h at 0–
5 °C, then 24 h at 35 °C. [b] Isolated yield after recrystallization from n-
hexane/AcOEt or n-heptane/AcOEt mixtures, respectively. [c] Isolated as a
crude product.
In turn, although Method B proved to be adequate toward
the employed benzaldehydes 1e-h affording products rac-2e-h
in moderate 57% (Table 1, entry 7) to high 68–75% yields
(Table 1, entries 8–10), certain modifications had to be
performed. Worthy of mention is that during the optimization
procedure (not shown herein) we found that decrease of the pH
value below 12, drastically dropped the yield of rac-2b-h,
probably due to suppression of the replacement of the bromide
atoms with hydroxyl anions in an intermediate adducts.
Therefore, maintaining the pH of the reaction medium above 12
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by successive KOH addition was required. We also found that
an addition of small portion of grounded NaOH during work-up
has a beneficial influence on the extraction procedure since very
stable emulsions were avoided. Simple addition of NaOH gave
by far better results than various physical methods applied by us
in the course of the experiments. In example, the formed
emulsions were tedious to breakdown by convenient filtering of
the crude multi-phasic extraction mixture over the Celite pad.
In the next step, mandelic acid methyl esters rac-3a-h
were readily prepared starting from the respective acids rac-2a-
h by their treatment with thionyl chloride used in 1.1 molar ratio
in the presence of anhydrous methanol. The previously
established protocol,[54] which was adopted from the literature[55]
and slightly modified by us (the temperature was decreased
from -10 °C to -30 °C), allowed the reaction to proceed smoothly,
affording the expected pure products rac-3a-h in high 87–95%
yields and excellent >99% purity according to gas
chromatography (GC) indications (Table 2).
KR of rac-3a-h using lipase-catalyzed transesterification
Although biotransformations involving lipases from different
organisms have become broadly investigated for the preparation
of optically active mandelic acid derivatives, the extension of
substrate scope in this group is still appealing. Moreover, the
search for lipases that are capable to transform MAs with
excellent enantioselectivities, which can work under harsh
industrial conditions persists as well. The previously reported
lipase-catalyzed enantioresolutions of racemic MA via
kinetically-controlled transesterification approach gave the
results of enantiomeric excess for the obtained RMA from poor
(<80% ee),[39i] moderate (80–90% ee)[39m] to high (91–98%
ee),[39a,39d,39j] and an excellent values (≥99% ee).[39c,39e,39l,40]
Strikingly, the most efficient acetylative EKR protocol,[39k] which
as the only one reached the theoretical boundary with a
conversion yield of 50% and an ee-values of >99.9% for both
enantiomeric forms of MA comprises the usage of an
extracellular lipase isolated from solvent-tolerant bacterium
Burkholderia ambifaria YCJ01. However, although this lipase
demonstrated excellent enantioselective transesterification
toward racemic MA, it’s exploitability is rather limited as it
requires cultivation broth composed of soil samples
contaminated by unknown crude oil and undefined chemicals as
well as thorough knowledge concerning isolation, purification,
and cloning techniques in order to prepare it. Thus, it is highly
desirable not only to extend the synthetic utilities of novel MA
derivatives by exploring their EKR reactions, but also to prove
that the chiral discrimination based on lipase-catalyzed acylation
protocol can be significantly improved in terms of
enantioselectivity by using biocatalysts that are easily available
on the market.
Table 2. Synthesis of the racemic mandelate methyl esters rac-3a-h.
Entry
Product[a]
rac-3a
rac-3b
rac-3c
rac-3d
rac-3e
rac-3f
R1
Yield[b] (%)
1
2
3
4
5
6
7
8
C6H4-
93
90
91
90
95
87
92
89
4-CH3C6H4-
3-CH3C6H4-
4-i-PrC6H4-
4-tert-BuC6H4-
4-CH3OC6H4-
2-Naphthyl-
1-Naphthyl-
On the basis of literature rapports on lipase-catalyzed
transesterification of MA esters and our previous experience in
the field of enzyme catalysis, we decided to exclude multivariate
investigation of experimental factors including medium
engineering, influence of the reaction medium temperature,
different enzyme loading as well as effect of the acyl group
donors on stereochemical outcome, and thus limit our studies
toward comparing the efficiency of the most promising,
according to literature, lipase catalysts [namely, Pseudomonas
fluorescens lipase (PFL) and Burkholderia cepacia lipase (BCL)].
In this regard, to determine suitable reaction conditions for the
enantioselective transesterification of MA derivatives rac-3a-h
under kinetically-controlled conditions, we initially investigated
the reaction systems with vinyl acetate as the source of an acyl
group in the presence of 30% weight/weight (w/w) loading of the
respective lipase preparation with respect to the employed
substrates rac-3a-h at room temperature (Table 3). Moreover, it
is well-established that the highest enantioselectivity for methyl
mandelates are observed in solvents with moderate to low
polarity, such as diisopropyl ether (DIPE) and/or cyclohexane,
respectively.
rac-3g
rac-3h
[a] rac-2a-h (0.1 mol), dry MeOH 180 mL, SOCl2 (13.1 g, 0.11 mol, 8.1 mL)
at 30 °C, then 4 h at RT. [b] Isolated yield after recrystallization from n-
hexane/Et2O or n-heptane/AcOEt, respectively.
In turn, mandelic acid diesters rac-4a-h, destined to be
applied for the analytical purposes, were obtained in high 79-
90% yields in convenient manner by using 1.0 equiv of acetic
anhydride and 1.1 equiv of dry pyridine in the presence of 4-
dimethylaminopyridine (DMAP) as the catalyst. The esterification
reactions were carried out in dry dichloromethane at room
temperature for 6 h. Yields of diesters rac-4a-h (79–90%) are
collected in Experimental Section.
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Table 3. Analytical-scale lipase-catalyzed KR of rac-3a-h with vinyl acetate in TBME.
Entry
Substrate
Lipase[a]
t (h)
Conv.[b] (%)
ees[c] (%)
eep[c] (%)
E[d] (%)
1
2
3
4
5
6
7
8
Amano AK
Amano PS-C II
Amano AK
Amano PS-C II
Amano AK
Amano PS-C II
Amano AK
Amano PS-C II
Amano AK
Amano PS-C II
Amano AK
Amano PS-IM
Amano AK
Amano PS-IM
Amano AK
147
38
144
23
190
72
168
29
192
45
240
46
336
96
576
456
51
54
53
48
45
55
49
51
52
51
49
60
51
54
30
63
89
84
92
88
89
89
81
90
94
85
97
73
66
89
86
N.D.[e]
53[f]
34
126
82
117
38
49
51
170
23
>200
13
24
rac-3a
rac-3b
rac-3c
rac-3d
rac-3e
rac-3f
rac-3g
rac-3h
>99
>99
95
74
>99
85
>99
79
99
71
99
9
10
11
12
13
14
15
16
93
58
69
N.D.[e]
9
>99
N.D.[e]
90[f]
Amano PS-IM
[a] Conditions: rac-3a-h 100 mg, lipase 30 mg, vinyl acetate 0.2 mL, TBME 2 mL, RT, 250 rpm (laboratory shaker). [b] Based on GC, for confirmation the %
conversion was calculated from the enantiomeric excess of the unreacted substrate (ees) and the product (eep) according to the formula conv. = ees/(ees + eep).
[c] Determined by chiral HPLC analysis by using a Chiralcel OD-H column. [d] Calculated according to Chen et al.,[56] using the equation: E = {ln[(1 – conv.)(1 –
ees)]}/{ln[(1 – conv.)(1 + ees)]}. [e] Not determined as the enzyme activity was too low. [f] Determined by correlative method using polarimetry (calculated
according to the formula: % ee = ׀[αobs]׀/׀[α]D׀ × 100%, where [αobs] is an experimental value, and [α]D is the literature value[57] given for the corresponding solution
containing only a single pure enantiomer.
Unfortunately, both of the solvents are expensive and
difficult to recover. In addition, diisopropyl ether easily forms
explosive peroxides upon standing what drastically limits it’s
usage at industrial scale as the special safety precautions are
required. Therefore, we decided to carry out all enzymatic
reactions in tert-butyl methyl ether (TBME) as the co-solvent
since it posses similar polarity to DIPE and constitutes also a
safer alternative for it. Moreover, we were curious of the results
with TBME since, surprisingly, although it is established as one
of the most common media used in lipase-catalyzed
biotransformations, until now the influence of this solvent on
EKR of racemic mandelate esters via stereoselective acylation
by means of PFL and BCL has not been examined.
All of the enzymatic reactions were monitored by GC directly
from the crude mixture, whereby the enantiomeric excesses for
the resolved products were determined by enantioselective
HPLC after isolation and chromatographic purification (HPLC
analysis conditions for various compounds are collected in
Table S2 in supplementary document). This is due to difficulty
associated with the simultaneous baseline resolution of the
mandelates rac-3a-h (enzyme substrates) and its corresponding
acetates rac-4a-h (products) in one run. Unfortunately,
enantiomers of rac-3h were not separable on the available
Chiralcel OD-H column, and thus the ee-values had to be
determined by correlative method using polarimetry.
An initial screening indicated that both immobilized lipases
isolated from Burkholderia cepacia (Amano PS-C II and Amano
PS-IM) posses broader substrate specificity in the examined
group of racemic mandelates rac-3a-h than native lipase from
Pseudomonas fluorescens (Amano AK) as the higher
enantioselectivity factors for Amano PS-C II (E-value from 117
up to >200) and for Amano PS-IM (E=9–69) were achieved for
the corresponding substrates when compared with the results
for Amano AK (E=13–82). Moreover, the rates of
transesterification reactions catalyzed by Amano AK in the
kinetic resolution of rac-3a-h were generally significantly lower
(45–53% conversion after 144–336 h) than for those with
immobilized preparations of BCL (48–60% conversion after 23–
96 h). The stereochemical outcome of EKR of rac-3h was
excluded from the above discussion since 1-naphthyl-substituted
substrate turned out to be very resistant toward biocatalytic
transformations carried out with the examined enzymatic
systems, and also because the accuracy of the optical purity
assessment is more questionable and less comparable with
In order to reduce the mechanical stress caused by the
high-speed agitation and physical contact between lipase
preparation and stirring element responsible for enzyme
leaching from the carrier (in the case of immobilized
biocatalysts), in all of our biotransformation experiments instead
of magnetic stirrer, a laboratory shaker (agitation speed set at a
moderate 250 rpm value) was used. Although this strategy
generally results in the extension of reaction time as the mass
transfer is hindered in heterogeneous biocatalytic reactions, it
helps to maintain the activity of the enzyme in time, and thus
expanding the number of cycles enzyme can be used before it
requires replacing.
As
a
starting
point,
during
the
small-scale
biotransformation studies, the influence of the acetylation rate on
the reaction efficiency was investigated. In Table 3 we have
presented the results of reactions, which gave the best
enantiomeric excess values for the RMA derivatives (R)-(–)-3a-h
or for which the conversions were as close to 50% as possible.
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10.1002/ejoc.201700161
European Journal of Organic Chemistry
FULL PAPER
Table 4. Preparative-scale lipase-catalyzed KR of rac-3a-h with vinyl acetate in TBME.
Entry
Substrate
rac-3a
rac-3b
rac-3c
rac-3d
rac-3e
rac-3f
Lipase[a]
t (h)
1.5
6
Conv.[b] (%)
ees[c] (%)/conf.[d]/Yield[e] (%)
>99/(R)/40
eep[c] (%)/conf.[d]/Yield[e] (%)
E[f] (%)
75
1
2
3
4
5
6
7
8
Amano PS-C II
Amano AK
53
53
57
52
50
61
54
50
87/(S)/51
88/(S)/53
76/(S)/58
90/(S)/50
98/(S)/48
62/(S)/54
85/(S)/48
86[g]/(S)/55
>99/(R)/40
82
Amano PS-C II
Amano PS-C II
Amano PS-C II
Amano PS-IM
Amano PS-IM
Amano PS-IM
3
>99/(R)/29
37
1
>99/(R)/37
99
2
99/(R)/33
>>200
23
2
99/(R)/33
rac-3g
rac-3h
4
98/(R)/31
56
19
86[g]/(R)/31
37
[a] Conditions: rac-3a-h 3.5 g, lipase 1.05 g, vinyl acetate 7 mL, TBME 70 mL, RT, 250 rpm (laboratory shaker). [b] Based on GC, for confirmation the %
conversion was calculated from the enantiomeric excess of the unreacted substrate (ees) and the product (eep) according to the formula conv. = ees/(ees + eep).
[c] Determined by chiral HPLC analysis by using a Chiralcel OD-H column. [d] Assigned by comparison of the specific rotation sign values and/or HPLC retention
times with the data reported in the literature; the absolute configurations of the non-reported compounds [(R)-(–)-3d-e, (S)-(+)-4c-e, and (S)-(+)-4g] have been
assumed by analogy with the results of the other transformations. [e]Isolated yield after purification by silica-gel chromatography (n-hexane/AcOEt). [f] Calculated
according to Chen et al.,[56] using the equation: E = {ln[(1 – conv.)(1 – ees)]}/{ln[(1 – conv.)(1 + ees)]}. [g] The % ee-value is calculated from the equation: % ee =
׀[αobs]׀/׀[α]D׀ × 100%, where [αobs] is the experimental value of specific rotation, and [α]D is the literature value of specific rotation given for a solution containing only
a single pure enantiomer.[58]
those obtained from chiral HPLC for ee. In this case, we
found that the reactions proceeded very sluggishly even
catalyzed by Amano PS-IM lipase (63% conversion after 456 h),
and with poor enantioselectivity (E=9) since slower reacting
enantiomer (R)-(–)-3h was afforded with moderate 90% ee, and
the formed optically active diester (S)-(+)-4h with 53% ee.
Nevertheless, it is worth to mention that only Amano PS-IM
could catalyze acetylation of rac-3h on reasonable time-scale.
The results summarized in Table 3 clearly indicate that the best
analytical-scale lipase-catalyzed KRs of rac-3a-h in terms of
enantioselectivity, optical purity of desired products (R)-(–)-3a-h,
and the reaction rates were obtained by employing Amano PS-C
II and/or Amano PS-IM as the catalyst. Among these attempts,
the most enantioselective assay (E>200) was achieved by
Amano PS-C II for MA derivative substituted with bulky tert-butyl
group situated in para-position of benzene ring rac-3e. In this
case, when the reaction was arrested at 51% conversion, the
generated acetate (S)-(+)-4e and the remaining alcohol (R)-(–)-
3e were both obtained with very high enantiomeric purity
reaching 97% ee and 99% ee, respectively (Table 3, entry 10).
Amano PS-C II lipase was also superior towards other
substrates, including rac-3a and rac-3c-d, toward which kinetic
resolution resulted in almost homochiral alcohols (R)-(–)-3a and
(R)-(–)-3c-d (≥99% ee) if the proper conversion degrees were
retained (Table 3, entries 2, 6, and 8). In turn, although PFL is
less efficient catalyst for the tested group of racemates, the
(Amano AK)-catalyzed KR of rac-3b resulted in 88% ee for (S)-
(+)-4b, whereby the remaining enantiomer (R)-(–)-3b was
recovered in enantiomerically pure form (>99% ee) when the
reaction was carried out for at least 144 h until 53% conversion
was achieved (Table 3, entry 3). Taking into account the results
of analytical-scale studies, we decided to abandon further
investigations with Amano AK due to its moderate selectivity and
activity, however, with the exception of the substrate possessing
methyl group in the para-position of benzene ring rac-3b, within
which reaction proceeded with a industrially acceptable E-value
larger than 80, and led to furnished enantiomerically pure (R)-(–
)-3b (>99% ee).
The results of lipase-catalyzed kinetic resolutions of para-
substituted mandelates rac-3b and rac-3d-e, with the exception
of para-methoxyphenyl derivative rac-3f, showed an increase in
enantioselectivity (E-value from 117 up to >200). In general, for
comparable conversions BCL lipases display the highest activity
with the less bulky para-substituted substrates rac-3a-b and rac-
3d-f, intermediate activity with meta-substituted compound rac-
3c, and low activity toward both naphthalene derivatives rac-3g-
h. In addition, the great differences in reaction rates and
enantioselectivity values observed for PFL and BCL lipases
revealed that immobilized preparations of BCL were more
adequate for the chosen set of the racemic mandelates rac-3a-h,
although literature data report that the efficiency of both should
be more or less the same. This suggests that the reaction
environment must have some impact on their catalytic activity,
and that TBME strongly promotes transformations catalyzed by
BCL preparations, and not by PFL.
Furthermore, the usefulness of developed enzymatic
protocol was demonstrated by the investigation into the 3.5 g-
scale enantioselective acetylation of racemic mandelates rac-3a-
h (Table 4). Under kinetically-controlled conditions the reaction
times were adjusted for each substrate independently. However,
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10.1002/ejoc.201700161
European Journal of Organic Chemistry
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over-reaction (>50% of conversion) was mandatory to ensure
the desired optical purity (≥98% ee) for slower-reacting
enantiomers (R)-(–)-3a-h. Extending by 35-fold the KRs scale
gave very similar results when compared to (100 mg)-scale
biotransformations in terms of reaction rates and
enantioselectivity. However, except for the substrates rac-3b,
rac-3f and rac-3g, for which selectivity remained unaffected,
some insignificant differences can be noticed as in the case of
the reactions performed with rac-3a, rac-3c, and rac-3d the E-
values slightly dropped, whereas in case of the compounds
possessing naphthalene ring rac-3g-h enantioselectivity was
improved. In turn, a remarkable increase of the enantiomer
selectivity was obtained for rac-3e in the presence of Amano
PS-C II as the catalyst. These have some visible consequences,
and it is worth noting that whilst the (Amano AK)- and (Amano
PS-IM)-mediated kinetic resolution gave moderate (Table 4,
entries 6–8, E=23–56) to good (Table 4, entry 2, E=82) results
with respect to enantioselectivity, the acetylation catalyzed by
Amano PS-C II proved to be superior, especially in the reaction
with para-substituted tert-butyl phenyl derivative rac-3e (Table 4,
entry 5, E>>200). The results of optical purity assessment from
the conducted experiments clearly indicate that in almost all
cases, except 1-substituted naphthalene derivative rac-3h, the
ee-values of (R)-mandelic acid methyl esters were above 98%.
In addition, the yields of the enantioselective transesterifications
shown in Table 4 were in 29-40% range for slower-reacting
enantiomers (R)-(–)-3a-h, and in 48-58% range for faster-
reacting isomers (S)-(+)-4a-h. However, it should be noted that
the above-mentioned values indicate isolated yields after column
chromatography, and were calculated on the basis of the
theoretical number of moles, relative to theoretical amount
arising from conversion rate established by HPLC. This means,
that i.e. when 50% conversion is reached, up to the half of the
acetate could be obtained.
experimental data were compared with those reported in the
literature for the respective solutions (in the case of polarimetry),
and the employed chiral column (for HPLC).[59,69] The detailed
data for optical properties evaluations, including literature
references, are provided in Experimental Section. From the
stereochemical course of the performed reactions it was obvious
that in transesterification catalyzed by PFL and BCL the (S)-form
of the methyl mandelates reacted preferentially over (R)-
counterpart. However, six out of sixteen of the prepared by us
optically active alcohols (R)-(–)-3a-h and acetates (S)-(+)-4a-h,
including (R)-(–)-3d-e and (S)-(+)-4c-e, (S)-(+)-4g, have not
been reported, thereby their absolute configuration of the chiral
center could be deduced only on the basis of the optical rotation
signs with the assumption that they structurally belong to the
same homological series, and so the chemical correlation can
be applied accordingly to an empirical Tschúgaeff’s rule.[60]
Although the above enunciated empirical rule of configuration
assignment is ‘stereochemically adequate’ and sufficiently
reliable within compounds possessing single asymmetric atom,
however, we aimed to unambiguously confirm this hypothesis by
derivatization
of
methyl
(2R)-(4-tert-
butylphenyl)(hydroxy)ethanoate (R)-(–)-3e toward product,
which efficient crystallization of proper single crystal would be
feasible, and thus XRD analysis could be performed. In this
regard, we decided to transform (R)-(–)-3e into corresponding
pyridinium hexafluorophosphate salt (R)-(–)-11 which X-ray
crystal structure is presented in Fig. 2.
As it was already stated above, the enantiorecognition of
methyl
mandelates
by
means
of
lipase-catalyzed
enantioselective acylation in traditional media are reported with
already good-to-excellent enantioselectivity, hence, there was
not much margin for improvement. Nonetheless, the
enantiomeric ratio of some of these resolutions improved
considerably when the reaction was performed with immobilized
lipases from Burkholderia cepacia suspended in TBME.
Moreover, the preparation of novel RMAs in almost
enantiomerically pure form and in multi-gram quantities in easily
accessible manner showed feasibility of our protocol for large-
scale production of potentially valuable building blocks.
Figure 2. An ORTEP plot representing molecular structure of optically active
(R)-(–)-11. Thermal ORTEP-ellipsoids were drawn at 50% probability level (C
black, H gray, N blue, O red, F green, P orange). The following crystal
structure has been deposited at the Cambridge Crystallographic Data Centre
and allocated the deposition number CCDC-1527174.
Assignment of the stereochemistry of the EKRs’ products
During the course of the present study, the absolute
configurations of the resolution products were assigned mainly
on the basis of optical rotation measurements and HPLC
analysis. The signs of the optical rotation as well as the retention
times of the respective picks of HPLC analyses collected from
This task was accomplished by using straightforward
approach consisting of five-step reaction sequence as shown in
Scheme 2.
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10.1002/ejoc.201700161
European Journal of Organic Chemistry
FULL PAPER
experiment it was clear that the elevated temperature was not
responsible for deterioration in the ee-values. Therefore, the
next step was to eliminate an excess of the employed guanidine
since the presence of such a strong base could deprotonate the
formed (R)-(+)-5, and thus promote deleterious for
stereochemical outcome product racemization. This attempt
gave successful result not only in term of optical purity of the
obtained non-racemic pemoline (R)-(+)-5 (98% ee), but also in
increased isolated yield (40%) (Table 5, entry 3).
Simultaneously, we decided to remove sodium chloride, since
an inorganic salt formed during the liberation of guanidine by
means of NaOH might have been responsible for the low yields.
Albeit this procedure has not improved neither the yield (38%)
nor enantiomeric purity of the isolated pemoline (R)-(+)-5 (85%
ee) (Table 5, entry 4). Finally, with a highly enantioenriched
product (R)-(+)-5 (98% ee) in hand, we turned our efforts to
increase the optical purity of the titled API. This was achieved by
two recrystallizations from ethanol, which yielded (R)-pemoline
in homochiral form with >99% ee (Table 5, entry 5).
During the studies, a curious phenomenon was observed,
namely, that the enantiomeric purity of optically pure (R)-
pemoline (>99% ee) gradually declined accompanying the aging
of it’s methanolic solution. After one day of storage at ambient
temperature, ee-value dropped to 96% ee, whereas after four
days it decreased to 30% ee. Finally, an almost complete
racemization of the analyzed sample of (R)-(+)-5 (8% ee)
occurred after 8 days. This is a crucial information, not only for
designing an appropriate conditions for crystal growth, but
especially from the view-point of pharmaceutical and other
industries, which need to know how to store analytical reference
standards of the drug samples. In addition, awareness of
racemization progress of non-racemic pemoline in a solution in
time may also be helpful for the investigations of biological
activity, as this API may also be prone to racemization in the
body fluids. However, the evaluation of this issue in other
solutions was not our main task and exceed the frame of this
article, thus was deliberately abandoned.
Scheme 2. Synthesis of optically active (R)-(-)-11. Reagents and conditions:
(vi) dry pyridine (1 equiv), 72 h at 50 ºC, low-light conditions; (vii) KPF6 (1.9
equiv), dry CH3CN, 24 h at reflux; (viii) Ag2O (1.18 equiv), roasted CaSO4 (1.18
equiv), CH3I (4.88 equiv), 36 h at reflux, low-light conditions; (ix) LiOH (4
equiv), MeOH, 24 h at RT, then 36% HCl at 0–5 °C; (x) 8 (0.9 equiv), DCC
(0.9 equiv), DMAP (cat.), dry CH3CN, 48 h at RT.
Synthesis of the optically active pemoline
At first, racemic imino-oxazoline CNS stimulant pemoline rac-5
was obtained according to the method reported by Howell and
co-workers.[61] In the original approach ethyl mandelate was
condensed with guanidine free base (generated from the
respective hydrochloride salt by addition of equimolar amounts
of NaOH) used in a 2-fold molar excess under reflux for 1 h, thus
affording desired API in high 68% yield. In our investigation we
used racemic methyl ester rac-3a instead of ethyl ester and
followed the procedure given by Howell. Surprisingly, only trace
of the product rac-5 was formed after indicated time period. At
elongated to 60 h reaction time rac-5 was obtained with
moderate 35% yield. Moreover, after repeating the reaction with
optically active precursor (R)-(–)-3a (>99% ee), the enantiomeric
purity of (R)-(+)-5 was very poor, barely reaching 13% ee (Table
5, entry 1).
Table 5. Synthesis of (R)-pemoline (R)-(+)-5.
ees[b]
(%)
Yield[c]
(%)
Conclusions
[d]
Entry
GuanidineHCl[a]
Temp.
[α]D
1
2
2 equiv
2 equiv
Reflux
RT
13
11
35
35
N.D.[e]
N.D.[e]
In summary,
a
convenient approach towards highly
enantiomerically enriched (R)-(–)-mandelic acid derivatives was
developed. The enzyme-catalyzed kinetic resolution of the
corresponding racemic mixtures was achieved by using a simple
protocol that involved an enantioselective transesterification step
in the presence of various lipase preparations as catalysts and
vinyl acetate as an acyl group donor. An analytical-scale
investigations gave the rates of enzymatic reactions and
enantiomeric purity comparable to that obtained by the methods
published previously. The best rates and enantioselectivity
factors were predominantly obtained with immobilized lipases
from Burkholderia cepacia (Amano PS-C II and Amano PS-IM)
suspended in TBME. The scale-up of the enzymatic process
was carried out to verify its potential in an industrial application.
Eight compounds were resolved on a 3.5 gram-scale by using
optimal reaction conditions. For the majority of examples
RT
+110 (c 0.20,
24.1°C)
+95 (c 0.20,
24.3°C)
+108 (c 0.20,
27.6°C)
3
1 equiv
1 equiv
2 equiv
98
40
RT
RT
4
5
85[f]
38
>99[g]
N.D.[e]
[a] (R)-(–)-3a (3.13 mmol, >99% ee), dry EtOH 7.5 mL, NaOH (125 mg, 3.13
mmol), 60 h. [b] Determined by chiral HPLC analysis by using a Chiralcel OD-
H column. [c] Isolated yield. [d] Specific rotation, c solution in methanol. [e] Not
determined. [f] The precipitated NaCl was filtered off the reaction mixture. [g]
After additional two recrystallizations from EtOH.
To overcome this drawback, at first we have lowered the
reaction temperature. Unfortunately, enantiomeric excess of (R)-
(+)-5 obtained in room temperature reaction mixture was even
worst as it decreased to 11% (Table 5, entry 2). From this
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10.1002/ejoc.201700161
European Journal of Organic Chemistry
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conditions. The enantiomeric excesses (% ee) of kinetic resolution
products were determined by HPLC analysis performed on Shimadzu
CTO-10ASV chromatograph equipped with STD-20A UV detector and
Chiralcel OD-H (Diacel) chiral column using mixtures of n-hexane/i-PrOH
as mobile phase in the appropriate ratios given in further paragraphs of
Experimental Section [the wavelength of UV detection and both the
mobile phase composition as well as the flow rate were fine tuned for
each analysis (see Table S2 placed in supplementary document)]; the
HPLC analyses were executed in an isocratic and isothermal (30 °C)
manner. UV spectra were measured with Cary 3 spectrometer; samples
were dissolved in absolute EtOH. Optical rotations ([α]) were measured
with a PolAAr 32 polarimeter in a 2 dm long cuvette using the sodium D
line (λ=589 nm); the units of the specific rotation are: (deg×mL)/(g×dm).
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a
Varian Mercury 400 MHz spectrometer and 1H NMR (500 MHz) and 13C
NMR (126 MHz) spectra were recorded on a Varian NMR System 500
MHz spectrometer; 1H and 13C chemical shifts (δ) are reported in parts
per million (ppm) relative to the solvent signals {i.e. CDCl3, δH (residual
CHCl3) 7.26 ppm, δC 77.16 ppm; or acetone-d6, δH (residual acetone-d6)
2.09 ppm, δC 205.87 ppm); or DMSO-d6 δH [residual (CD3)2SO) 2.49 ppm
with HDO at 3.30 ppm, δC 40.45 ppm]; or acetone-d6, δH (residual
acetone-d6) 2.09 ppm, δC 205.87 ppm); or diaminophenyl sulfone (DDS,
for spectra in D2O)}. Chemical shifts are quoted as s (singlet), d (doublet),
dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), and br s
(broad singlet); coupling constants (J) are reported in Hertz. Infrared (IR)
spectra of samples prepared in nujol were taken on a Carl Zeiss Specord
M80 instrument; Fourier transform infrared (FTIR) spectra of neat
preparative EKR succeeded in obtaining the (R)-mandelates
with excellent enantiomeric excess (≥99%) and acceptable
isolated yields (29-58%). The enantiomeric ratio (E) reached up
to >>200 for acylation of the para-substituted tert-butyl derivative
of methyl mandelate (R)-(–)-3e catalyzed by Amano PS-C II. On
the basis of the enzymatic studies toward group of the examined
MA esters, it was obvious that stereochemical outcome of
lipase-catalyzed reactions were profoundly influenced by the
steric effect of the substituents rather than electronic properties
of the aromatic ring system. For novel non-racemic MA
derivative (R)-(–)-3e the absolute configuration was
unambiguously determined by X-ray structure analysis of a
single crystal of the prepared heavy-atom analog (R)-(-)-11
possessing hexafluorophosphate ion. Homochiral methyl (R)-(–)-
mandelate (>99% ee) was further utilized to obtain
enantiomerically enriched (R)-pemoline (98% ee). Finally, single
(R)-enantiomer of pemoline (>99% ee) was achieved upon
additional double recrystallization procedure from ethanol.
Thanks to development of this synthesis, both enantiomers of
active agent pemoline can be throughout evaluated in terms to
avoid life threatening hepatic failure in future. Furthermore,
crucial analysis for non-racemic pemoline was performed for the
first time, namely, evaluation of spontaneous racemization in a
solution of methanol determined by chiral HPLC.
samples were recorded on
a Perkin Elmer System 2000 FTIR
Spectrometer equipped with a Pike Technologies GladiATR attenuated
total reflectance (ATR) accessory with a monolithic diamond crystal stage
Experimental Section
and a pressure clamp; resolution was 2 cm−1; absorption maxima (νmax
)
are given in cm−1. Elemental analyses were performed on a Elementar
Analysensysteme GmbH-VARIO EL III (Element Analyzer: CHNS).
General Remarks: Reagents (including racemic mandelic acid rac-2a)
and solvents were purchased from various commercial sources (Sigma
Aldrich, Alfa Aesar, POCH) and were used without further purification,
except aromatic aldehydes which were distilled or recrystallized (solid
reagents) before use. High-performance liquid chromatography (HPLC)-
grade solvents were purchased from POCH (Poland). Methylene chloride,
acetonitrile, and ethanol were dried by allowing them to stand over
activated (oven-roasted in high-vacuum) 3Å molecular sieves [20%
mass/volume (m/v) loading of the desiccant] at least for 48 h before
use;[62] methanol was dried by refluxing it in the presence of magnesium
powder for 2–3 h, and subsequent fractional distillation in damp-
protected apparatus under nitrogen. All non-aqueous reactions were
carried out under oxygen-free argon-protective conditions using flame-
dried glassware. Lipase from Burkholderia cepacia (formerly
Pseudomonas cepacia) [Amano PS-IM – immobilized on diatomite,
specified activity: 500 U/g, Amano PS-C II – immobilized on ceramic,
purchased from Amano Pharmaceutical Co., Ltd.], lipase from
Pseudomonas fluorescens [Amano AK – native lipase, specified activity:
>20.000 U/g, purchased from Amano Pharmaceutical Co., Ltd.]. Melting
points, uncorrected, were determined with a commercial apparatus on
samples contained in rotating glass capillary tubes open on one side
(1.35 mm inner diam. and 80 mm length). Analytical thin-layer
chromatography was carried on TLC aluminum plates (Merck) covered
with silica gel of 0.2 mm thickness film containing a fluorescence
indicator green 254 nm (F254), and using UV light as a visualizing agent.
Preparative separations were carried out by column chromatography
using thick-walled glass columns and silica gel (230–400 mesh) with
grain size 40–63 μm or activated charcoal as stationary phase,
respectively. The chromatographic analyses (GC) were performed with a
Agilent Technologies 6890N instrument equipped with a flame ionization
detector (FID) and fitted with HP-50+ (30 m) semipolar column (50 %
phenyl–50 % methylpolysiloxane); Helium (2 mL/min) was used as
carrier gas; retention times (tR) are given in minutes under these
Preparation of the racemic mandelic acid derivatives rac-2a-h
(Method A): To a solution of the appropriate aromatic aldehyde 1b-d
(0.10 mol) and benzyltriethylammonium chloride (TEBACl, 1.23 g, 5
mmol) in CHCl3 (160 mL), a 50% aqueous NaOH solution (25 mL) was
added drop by drop (1-2 drops per minute) at 56 °C under vigorous
stirring using
a mechanical stirrer. The reaction temperature was
controlled between 54 and 58 °C during the addition of the NaOH
solution. After addition, the reaction mixture was stirred at room
temperature for a further 5 h. Next, a sufficient amount of H2O was added
in order to dissolved the precipitate formed. Water phase was separated
and acidified to pH 1 with 36% HCl solution, and subsequently extracted
with Et2O in Soxhlet apparatus for 10 h. The ethereal layer was dried
over anhydrous MgSO4, the solvent was evaporated, and the resulting
crude acid rac-2b-d (41–70%) was purified by recrystallization from n-
hexane/AcOEt mixture.
Preparation of the racemic mandelic acid derivatives rac-2a-h
(Method B): At first, in the Erlenmeyer flask a mixture of LiCl (10.6 g,
0.25 mol), KOH (28.1 g, 0.50 mol), fine-crushed ice-cubes (100 g), 1,4-
dioxane (100 mL), the appropriate aromatic aldehyde 1b-h (0.13 mol),
CHBr3 (31.6 g, 0.13 mol) was prepared. The content of the flask was
stirred with ice-cooling at 0–5 °C for 36 h. The pH value of the solution
was periodically controlled, and when its value dropped below 12,
grounded KOH (3.37 g, 60 mmol) was added. Next, the flask was placed
in a laboratory thermoshaker and mixed at 35 °C for a further 24 h. After
this time, the reaction mixture was diluted with a 0.5 M aqueous KOH
solution (300 mL). Thereafter, the contents of the flask were extracted
with Et2O (3 × 50 mL), and the water phase was acidified to pH 1 with
concentrated 36% HCl solution, and again extracted with Et2O (4 × 80
mL). The combined ether extracts were dried over anhydrous MgSO4, the
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10.1002/ejoc.201700161
European Journal of Organic Chemistry
FULL PAPER
drying agent was filtered off, and the solvent was evaporated under
reduced pressure to yield the appropriate crude acid rac-2b-h, which was
further purified by recrystallization from the mixtures of n-hexane/AcOEt,
PhCH3/AcOEt or n-heptane/AcOEt, respectively.
133.5, 136.3, 174.4; Complete spectroscopic analysis was performed for
its methyl ester rac-3h.
Preparation of the racemic mandelate esters rac-3a-h: To a stirred
and cooled to –30 °C solution of the appropriate α-hydroxy-α-arylacetic
acid rac-2a-h (0.1 mol) in dry MeOH (180 mL), thionyl chloride (13.1 g,
0.11 mol, 8.1 mL) was added dropwise at a rate that the temperature of
the reaction mixture did not rise above –30 °C. When the addition was
complete, the solution was stirred at –30 °C for further 10 min, and then
left for 4 h at room temperature. Afterwards, the content of the flask was
poured into a mixture of fine-crushed ice (75 g), saturated aqueous
Na2CO3 solution and Et2O (250 mL). Thus prepared mixture was
vigorously shaken in a separating funnel, and the combined organic layer
was washed with brine, and dried over anhydrous MgSO4. After removal
of the drying agent and evaporation of the solvent in vacuum, the
resulting appropriate crude methyl ester rac-3a-h was purified by
recrystallization from n-hexane/Et2O or n-heptane/AcOEt mixture,
respectively.
Hydroxy(phenyl)acetic acid (rac-2a): purchased from commercial
supplier.
Hydroxy(4-methylphenyl)acetic acid (rac-2b): Yield 70% (according to
Method A); white solid; mp 145 °C (n-hexane/AcOEt); Yield 75% (Method
B); mp 143–144 °C (n-hexane/AcOEt) [lit.[63] 145 °C (PhCH3)]; 1H NMR
(400 MHz, CDCl3): δ 2.35 (s, 3H), 5.21 (s, 1H), 7.18–7.20 (m, 2H), 7.31–
7.33 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 21.4, 72.6, 126.7, 129.6,
134.7, 139.0, 177.0; IR (nujol): νmax = 3406 (br s), 1705, 1290, 1055, 815,
724, 700.
Hydroxy(3-methylphenyl)acetic acid (rac-2c): Yield 54% (according to
Method A); white solid; mp 91–93 °C (n-hexane/AcOEt), Yield 71%
(according to Method B); mp 91.5–92.5 °C (n-hexane/AcOEt) [lit.[64] 93–
94 °C (benzene)]; 1H NMR [500 MHz, (CD3)2SO]: δ 2.30 (s, 3H), 4.99 (s,
1H), 5.78 (br s, 1H), 7.09–7.11 (m, 1H), 7.20–7.24 (m, 3H), 12.56 (br s,
1H); 13C NMR [126 MHz, (CD3)2SO]: δ 21.1, 72.5, 123.8, 127.3, 128.1,
128.3, 137.2, 140.2, 174.2; IR (nujol): νmax = 3460, 3170 (br s), 2520 (br
s), 1715, 1705, 1670, 1530, 1340, 1328, 1310, 1268, 1250, 1235, 1210,
1150, 1079, 871, 810, 772, 710, 690, 650.
Methyl hydroxy(phenyl)acetate (rac-3a): Yield 93%; white solid; mp
54–55 °C (n-hexane/Et2O) [lit.[69] 54–55 °C (n-heptane)]; 1H NMR (400
MHz, CDCl3): δ 3.51 (d, J = 5.6 Hz, 1H), 3.76 (s, 3H), 5.18 (d, J = 5.6 Hz,
1H), 7.32–7.44 (m, 5H); 13C NMR (100 MHz, CDCl3): δ 72.8, 126.6,
128.5, 128.6, 138.2, 174.1 (The spectral data are fully consistent with
those reported previously in lit.[70]); IR (nujol): νmax = 3430, 1740, 1050,
1065; HPLC [n-hexane-i-PrOH (90:10, v/v); f=0.8 mL/min; λ=254 nm]:
tR=8.939 (S-isomer) and 14.612 min (R-isomer).
Hydroxy[4-(propan-2-yl)phenyl]acetic acid (rac-2d): Yield 41%
(according to Method A); white solid; mp 156.5–158 °C (n-
hexane/AcOEt), Yield 69% (according to Method B); mp 156–157 °C (n-
hexane/AcOEt) [lit.[65] 159.2–160 °C (petroleum ether)]; 1H NMR (400
MHz, CDCl3): δ 1.24 (d, J = 6.8 Hz, 6H), 2.91 (sept, J = 6.8 Hz, 1H), 5.23
(s, 1H), 7.24–7.26 (m, 2H), 7.35–7.37 (m, 2H); 13C NMR (100 MHz,
CDCl3): δ 23.9, 33.9, 72.5, 126.6, 126.9, 134.9, 149.7, 175.8; IR (nujol):
νmax = 3405 (br s), 1710, 1290, 1219, 1187, 1071, 815, 691.
Methyl hydroxy(4-methylphenyl)acetate (rac-3b): Yield 90%; white
solid; mp 49–50 °C (n-hexane/Et2O) [lit.[71] 49 °C (benzene/ligroin)]; 1H
NMR (400 MHz, CDCl3): δ 2.35 (s, 3H), 3.39 (d, J = 5.6 Hz, 1H), 3.76 (s,
3H), 5.14 (d, J = 5.6 Hz, 1H) 7.17–7.19 (m, 2H), 7.29–7.31 (m, 2H); 13C
NMR (100 MHz, CDCl3): δ 21.3, 53.2, 72.9, 126.7, 129.5, 135.3, 138.5,
174.4 (The spectral data are fully consistent with those reported
previously in lit.[70]); IR (nujol): νmax = 3318, 1732, 1245, 1210, 1083, 995,
975, 780, 728; HPLC [n-hexane-i-PrOH (90:10, v/v); f=0.8 mL/min; λ=254
nm]: tR=9.185 (S-isomer) and 12.946 min (R-isomer).
(4-Tert-butylphenyl)(hydroxy)acetic acid (rac-2e): Yield 57%
(according to Method B); white solid; mp 147–149 °C (n-hexane/AcOEt)
[lit.[65] 149.5–150 °C (benzene)]; 1H NMR (400 MHz, CDCl3): δ 1.31 (s,
9H), 5.23 (s, 1H), 7.35–7.42 (m, 4H); 13C NMR (100 MHz, CDCl3): δ 31.4,
34.8, 72.6, 125.9, 126.4, 134.6, 152.1, 177.7; IR (nujol): νmax = 3430 (br
s), 1711, 1262, 1148, 1080, 1055, 814, 681.
Methyl hydroxy(3-methylphenyl)acetate (rac-3c): Yield 91%; white
solid; mp 52–53 °C (n-hexane/Et2O) [lit.[72] 52 °C (n-hexane)]; 1H NMR
(400 MHz, CDCl3): δ 2.36 (s, 3H), 3.42 (br s, 1H), 3.76 (s, 3H), 5.14 (s,
1H), 7.14–7.16 (m, 1H), 7.19–7.28 (m, 3H); 13C NMR (100 MHz, CDCl3):
δ 21.6, 53.2, 73.1, 123.9, 127.4, 128.7, 129.5, 138.3, 138.6, 174.4; IR
(nujol): νmax = 3470 (br s), 2980, 1730, 1600, 1480, 1435, 1200, 1148,
1100, 1070, 970, 775, 732, 695; HPLC [n-hexane-i-PrOH (90:10, v/v);
f=0.8 mL/min; λ=254 nm]: tR=8.939 (S-isomer) and 14.612 min (R-
isomer).
Hydroxy(4-methoxyphenyl)acetic acid (rac-2f): Yield 73% (according
to Method B); white solid; mp 104–106 °C (n-hexane/AcOEt) [lit.[66]
108 °C (no data)]; 1H NMR [500 MHz, (CD3)2SO]: δ 3.73 (s, 3H), 4.95 (s,
1H), 6.89–6.91 (m, 2H), 7.31–7.34 (m, 2H); 13C NMR [126 MHz,
(CD3)2SO] δ: 55.1, 71.9, 113.5, 127.9, 132.3, 158.8, 174.4.
Methyl hydroxy[4-(propan-2-yl)phenyl]acetate (rac-3d): Yield 90%;
white solid; mp 78–79 °C (n-heptane/AcOEt) [lit.[73] 81–82 °C (no data)];
1H NMR (400 MHz, CDCl3): δ 1.24 (d, J = 6.9 Hz, 6H), 2.91 (sept, J = 6.9
Hz, 1H), 3.36 (d, J = 5.8 Hz, 1H), 3.77 (s, 3H), 5.15 (d, J = 5.8 Hz, 1H),
7.23–7.25 (m, 2H), 7.31–7.35 (m, 2H); 13C NMR (100 MHz, CDCl3): δ
24.1, 34.0, 53.1, 72.9, 126.7, 126.9, 135.8, 149.4, 174.4; IR (nujol): νmax
= 3240–3540, 1737, 1188, 1080, 980, 902, 828, 779, 715; HPLC [n-
hexane-i-PrOH (95:5, v/v); f=0.8 mL/min; λ=254 nm]: tR=11.062 (S-
isomer) and 13.837 min (R-isomer).
Hydroxy(naphthalen-2-yl)acetic acid (rac-2g): Yield 75% (according to
Method B); white solid; mp 156–157.5 ºC (n-heptane/AcOEt) [lit.[67] 162–
163.5 °C (chloroform/ethanol)]; 1H NMR (400 MHz, CD3COCD3): δ 5.40
(s, 1H), 7.47–7.54 (m, 2H), 7.64 (dd, J = 1.6 Hz, J = 8.5 Hz, 1H), 7.87–
7.95 (m, 3H), 8.02 (s, 1H); 13C NMR (100 MHz, CD3COCD3): δ 73.6,
125.5, 126.6, 126.9, 127.1, 128.5, 128.8, 134.1, 134.2, 138.3, 174.4; IR
(nujol): νmax = 3380 (br s), 3260, 1720, 1690, 1295, 1220, 1075, 1050,
823, 810, 735, 696.
Hydroxy(naphthalen-1-yl)acetic acid (rac-2h): Yield 68% (according to
Method B); white solid; mp 123–125 ºC (PhCH3/AcOEt) [lit.[68] 124–
125 °C (benzene)]; 1H NMR [500 MHz, (CD3)2SO]: δ 5.67 (s, 1H), 7.45–
7.56 (m, 3H), 7.87–7.94 (m, 1H), 8.27–8.29 (m, 1H); 13C NMR [126 MHz,
(CD3)2SO]: δ 71.0, 124.6, 125.3, 125.7, 125.8, 126.0, 128.3, 128.4, 130.7,
Methyl (4-tert-butylphenyl)(hydroxy)acetate (rac-3e): Yield 95%; white
solid; mp 52–54 °C (n-hexane/Et2O); 1H NMR (400 MHz, CDCl3): δ 1.31
(s, 9H), 3.36 (d, J = 5.8 Hz, 1H), 3.77 (s, 3H), 5.16 (d, J = 5.8 Hz, 1H),
7.32–7.35 (m, 2H), 7.38–7.41 (m, 2H); 13C NMR (100 MHz, CDCl3): δ
31.4, 34.8, 53.2, 72.8, 125.8, 126.4, 135.4, 151.7, 174.4; IR (nujol): νmax
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201700161
European Journal of Organic Chemistry
FULL PAPER
= 3450 (br s), 1730, 1255, 1190, 1083, 972, 898, 825, 769, 698; HPLC
[n-hexane-i-PrOH (95:5, v/v); f=0.8 mL/min; λ=254 nm]: tR=10.437 (S-
isomer) and 12.668 min (R-isomer).
Methyl (acetyloxy)(4-methylphenyl)acetate (rac-4b): Yield 83%;
colorless oil; 1H NMR (400 MHz, CDCl3): δ 2.19 (s, 3H), 2.36 (s, 3H),
3.72 (s, 3H), 5.89 (s, 1H), 7.19–7.21 (m, 2H), 7.34–7.36 (m, 2H); 13C
NMR (100 MHz, CDCl3): δ 20.7, 21.2, 52.6, 74.3, 127.6, 129.5, 130.8,
139.3, 169.5, 170.4 (The spectral data are fully consistent with those
reported previously in lit.[78]); IR (nujol): νmax = 1755, 1735, 1230, 1200,
1160, 1050, 805, 780, 745; HPLC [n-hexane-i-PrOH (99:1, v/v); f=0.25
mL/min; λ=254 nm]: tR=34.677 (S-isomer) and 36.610 min (R-isomer).
Methyl hydroxy(4-methoxyphenyl)acetate (rac-3f): Yield 87%; white
solid; mp 38–39 °C (n-heptane/AcOEt) [lit.[74] 37–38 °C (H2O)]; 1H NMR
(500 MHz, CDCl3): δ 3.75 (s, 3H), 3.80 (s, 3H), 5.12 (s, 1H), 6.87–6.90
(m, 2H) 7.30–7.34 (m, 2H); 13C NMR (126 MHz, CDCl3): δ 53.1, 55.4,
72.6, 114.2, 128.0, 130.6, 159.9, 174.4 (The spectral data are fully
consistent with those reported previously in lit.[70]); IR (neat): νmax = 3429,
3008, 2968, 2917, 2842, 1726, 1608, 1583, 1510, 1441, 1449, 1384,
1328, 1301, 1248, 1213, 1182, 1169, 1112, 1078, 1027, 977, 908, 834,
818, 796, 750, 712, 573, 526, 429; HPLC [n-hexane-i-PrOH (90:10, v/v);
f=0.7 mL/min; λ=254 nm]: tR=14.707 (S-isomer) and 24.420 min (R-
isomer).
Methyl (acetyloxy)(3-methylphenyl)acetate (rac-4c): Yield 80%;
colorless oil; 1H NMR (400 MHz, CDCl3): δ 2.20 (s, 3H), 2.37 (s, 3H),
3.73 (s, 3H), 5.89 (s, 1H), 7.19–7.21 (m, 1H), 7.23–7.30 (m, 3H); 13C
NMR (100 MHz, CDCl3): δ 20.9, 21.5, 52.8, 74.6, 124.9, 128.4, 128.9,
130.2, 133.7, 138.8, 169.6, 170.5; FTIR (neat): νmax = 2960, 2922, 2850,
1750, 1607, 1432, 1370, 1223, 1151, 1052, 780, 745, 696; HPLC [n-
hexane-i-PrOH (99:1, v/v); f=0.25 mL/min; λ=254 nm]: tR=21.782 (S-
isomer) and 23.480 min (R-isomer).
Methyl hydroxy(naphthalen-2-yl)acetate (rac-3g): Yield 92%; white
solid; mp 73–74 °C (n-hexane/Et2O) [lit.[75] 72.8–73 °C (n-
hexane/AcOEt)]; 1H NMR (500 MHz, CDCl3): δ 3.63 (br s, 1H), 3.76 (s,
3H), 5.36 (s, 1H), 7.45–7.55 (m, 3H), 7.81–7.87 (m, 3H), 7.91 (br s, 1H);
13C NMR (126 MHz, CDCl3): δ 53.2, 73.2, 124.2, 126.1, 126.5, 127.8,
128.6, 128.62, 133.3, 133.4, 135.7, 174.3 (The spectral data are fully
Methyl (acetyloxy)[4-(propan-2-yl)phenyl]acetate (rac-4d): Yield 79%;
colorless oil; 1H NMR (400 MHz, CDCl3): δ 1.25 (d, J = 6.9 Hz, 6H), 2.19
(s, 3H), 2.92 (sept, J = 6.9 Hz, 1H), 3.73 (s, 3H), 5.90 (s, 1H), 7.22–7.28
(m, 2H), 7.36–7.40 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 20.9, 24.0,
34.1, 52.7, 74.5, 127.1, 127.8, 131.2, 150.3, 169.6, 170.6; FTIR (neat):
νmax = 2960, 2922, 1750, 1510, 1432, 1368, 1225, 1170, 1050, 1017, 975,
928, 825, 775, 740, 720; HPLC [n-hexane-i-PrOH (99:1, v/v); f=0.4
mL/min; λ=254 nm]: tR=18.176 (S-isomer) and 19.740 min (R-isomer).
consistent with those reported previously in lit.[75]); FTIR (neat): νmax
=
3470, 3055, 2965, 1725, 1510, 1440, 1390, 1365, 1305, 1270, 1255,
1220, 1170, 1145, 1085, 985, 945, 925, 905, 870, 860, 830, 775, 750,
735, 665; HPLC [n-hexane-i-PrOH (90:10, v/v); f=0.8 mL/min; λ=254 nm]:
tR=14.588 (S-isomer) and 17.379 min (R-isomer).
Methyl (acetyloxy)(4-tert-butylphenyl)acetate (rac-4e): Yield 87%;
colorless oil; 1H NMR (500 MHz, CDCl3): δ 1.32 (s, 9H), 2.19 (s, 3H),
3.73 (s, 3H), 5.91 (s, 1H), 7.38–7.42 (m, 4H); 13C NMR (126 MHz,
CDCl3): δ 20.9, 31.4, 34.8, 52.7, 74.5, 125.9, 127.6, 130.8, 152.6, 169.6,
170.5; FTIR (neat): νmax = 2960, 2863, 1750, 1570, 1512, 1430, 1367,
1262, 1225, 1170, 1102, 1050, 1015, 975, 928, 822, 770, 715; HPLC [n-
hexane-i-PrOH (99:1, v/v); f=0.4 mL/min; λ=254 nm]: tR=17.587 (S-
isomer) and 19.371 min (R-isomer).
Methyl hydroxy(naphthalen-1-yl)acetate (rac-3h): Yield 89%; white
solid; mp 78–80 °C (n-heptane/AcOEt); 1H NMR (400 MHz, CDCl3): δ
3.53 (d, J = 4.8 Hz, 1H), 3.74 (s, 3H), 5.82 (d, J = 4.8 Hz, 1H), 7.45–7.58
(m, 4H), 7.85–7.90 (m, 2H), 8.14–8.16 (m, 1H); 13C NMR (100 MHz,
CDCl3): δ 53.1, 71.4, 123.6, 125.2, 125.9, 126.0, 126.6, 128.8, 129.5,
131.0, 133.9, 134.0, 174.7 (The spectral data are fully consistent with
those reported previously in lit.[76]); FTIR (neat): νmax = 3470, 3055, 2965,
1725, 1510, 1440, 1390, 1365, 1305, 1270, 1255, 1220, 1170, 1145,
1085, 985, 945, 925, 905, 870, 860, 830, 775, 750, 735, 665; HPLC:
compound is indivisible on available Chiralcel OD-H column.
Methyl (acetyloxy)(4-methoxyphenyl)acetate (rac-4f): Yield 85%;
colorless oil; 1H NMR (400 MHz, CDCl3): δ 2.18 (s, 3H), 3.72 (s, 3H),
3.81 (s, 3H), 5.87 (s, 1H), 6.91 (m, 2H), 7.38 (m, 2H); 13C NMR (100 MHz,
CDCl3): δ 20.8, 52.6, 55.3, 74.1, 114.1, 125.7, 129.0, 160.2, 169.4,
170.2; FTIR (neat): νmax = 1754, 1735, 1605, 1508, 1315, 1320, 1300,
1250, 1220, 1200, 1168, 1110, 1052, 1025, 1000, 970, 920, 860, 830,
812, 790, 755, 710, 645; HPLC [n-hexane-i-PrOH (96:4, v/v); f=0.4
mL/min; λ=254 nm]: tR=22.440 (S-isomer) and 27.504 min (R-isomer).
Preparation of the racemic mandelate diesters rac-4a-h: The mixture
of the appropriate racemic methyl mandelate rac-3a-h (0.1 mmol), DMAP
(1 mg), Ac2O (1.0 equiv), and dry pyridine (1.1 equiv) was dissolved in
dry CH2Cl2 (0.5 mL) and stirred for 6 h at room temperature. After
completion of the reaction (according to TLC indications), content of the
flask was diluted with CH2Cl2 (0.5 mL) and quenched with 2M HCl (2 ×
0.5 mL), the water phase was extracted with Et2O (3 × 0.5 mL). The
combined organic phases were washed with brine (0.5 mL) and
saturated solution of Na2CO3 (0.5 mL), dried over anhydrous Na2SO4.
After filtration of drying agent and concentration to dryness, the crude
product was purified by column chromatography on silica gel using
gradient n-hexane/AcOEt (70:10 and 60:10 v/v) as an eluent, yielded
corresponding mandelic diester rac-4a-h as colorless oil.
Methyl (acetyloxy)(naphthalen-2-yl)acetate (rac-4g): Yield 83%;
colorless oil; 1H NMR (500 MHz, CDCl3): δ 2.24 (s, 3H), 3.74 (s, 3H),
6.12 (s, 1H), 7.50–7.54 (m, 2H), 7.56–7.58 (m, 1H), 7.84–7.89 (m, 3H),
7.95–7.96 (m, 1H); 13C NMR (126 MHz, CDCl3): δ 20.9, 52.8, 74.7, 124.8,
126.7, 126.9, 127.5, 127.9, 128.3, 128.9, 131.2, 133.2, 133.7, 169.5,
170.4; IR (neat): νmax = 1750, 1730, 1335, 1265, 1230, 1196, 1040, 920,
860, 830, 810, 752; HPLC [n-hexane-i-PrOH (98:2, v/v); f=0.2 mL/min;
λ=254 nm]: tR=55.647 (S-isomer) and 61.512 min (R-isomer).
Methyl (acetyloxy)(phenyl)acetate (rac-4a): Yield 90%; colorless oil; 1H
NMR (500 MHz, CDCl3): δ 2.20 (s, 3H), 3.72 (s, 3H), 5.94 (s, 1H), 7.38–
7.41 (m, 3H), 7.45–7.48 (m, 2H); 13C NMR (126 MHz, CDCl3): δ 20.8,
52.7, 74.6, 127.8, 128.9, 129.4, 133.9, 169.4, 170.4 (The spectral data
are fully consistent with those reported previously in lit.[77]); IR (nujol):
νmax = 1752, 1735, 1230, 1200, 1160, 1050, 805, 778, 740; HPLC [n-
hexane-i-PrOH (99:1, v/v); f=0.4 mL/min; λ=254 nm]: tR=22.997 (S-
isomer) and 24.383 min (R-isomer).
Methyl (acetyloxy)(naphthalen-1-yl)acetate (rac-4h): Yield 86%;
colorless oil; 1H NMR (500 MHz, CDCl3): δ 2.22 (s, 3H); 3.71 (s, 3H),
6.70 (s, 1H), 7.47–7.63 (m, 4H), 7.89–7.91 (m, 2H), 8.18–8.19 (m, 1H);
13C NMR (126 MHz, CDCl3): δ 20.9, 52.9, 72.6, 123.8, 125.4, 126.2,
127.1, 127.6, 129.0, 130.1, 130.3, 131.2, 134.1, 169.8, 170.5 (The
spectral data are fully consistent with those reported previously in lit.[57]);
FTIR (neat): νmax = 1756, 1744, 1729, 1434, 1369, 1218, 1203, 1167,
1087, 1051, 970, 927, 786, 773, 555, 496, 425, 415; HPLC: compound is
indivisible on available Chiralcel OD-H column.
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201700161
European Journal of Organic Chemistry
FULL PAPER
27.6
General procedure for the analytical-scale enzymatic KR of rac-3a-
h: To a solution of the appropriate racemic methyl mandelate rac-3a-h
(100 mg) in TBME (2 mL) the respective commercial lipase formulation
[30 mg, 30% w/w (catalyst/substrate)] and vinyl acetate (0.2 mL) were
added in one portion. The reaction mixture was shaken (250 rpm) at
room temperature by using a laboratory rotatory shaker, and its aliquots
were regularly analyzed by analytical chromatographic assays (GC and
HPLC) after proper filtration of the enzyme residue. In the case of KR of
rac-3h, for which direct HPLC analysis of % ee-values from reaction
mixture failed, additional elaboration was performed as follows: the
enzyme was filtered off and washed with TBME (5 × 2 mL), the solvent
was evaporated and the crude obtained was purified by column
chromatography eluting with gradient of n-hexane/AcOEt (80:10, 70:10,
60:10 v/v) mixture to afford (R)-(–)-3h and (S)-(+)-4h ready-to-analyze by
correlative method using polarimetry. The results of enzymatic KR
reactions carried out at analytical scale are collected in Table 3.
(221 mg, 40%, 98% ee). [α]D24.1 = +110 (c 0.20, MeOH, 98% ee); [α]D
=
+108 (c 0.20, MeOH, >99% ee); mp 240–240.5 °C (racemic pemoline,
EtOH, decomp.) [reported for racemic pemoline: lit.[80] 242–250 °C (no
data) or lit.[81] 256–257 °C (EtOH, decomp.)]; 1H NMR [500 MHz,
(CD3)2SO)]: δ 5.71 (s, 1H); 7.25–7.29 (m, 2H), 7.38–7.44 (m, 3H), 8.52
(br s, 1H), 8.74 (br s, 1H); 13C NMR [126 MHz, (CD3)2SO)]: δ 82.3, 126.2,
128.6, 128.7, 134.6, 176.7, 186.4 [Attention: in DMSO-d6 only imino form
of pemoline, that is 2-imino-5-phenyl-1,3-oxazolidin-4-one (R)-(+)-5b was
observed]; IR (nujol): νmax = 3264, 1656, 1565, 1505, 1492, 1445, 1282,
1224, 1136, 1035, 1016, 969, 935, 765, 700, 656; HRMS (ESI-TOF) m/z:
[M+H]+ Calcd for C9H9N2O2 177.0659, Found 177.0880; [2M+H]+
C18H17N4O4+ m/z: 353.1250, Found 353.1628; [M-H]- Calcd for C9H7N2O2
175.0513, Found 175.0641; [2M-H]- Calcd for C18H15N4O4- m/z: 351.1099,
Found 351.1701; HPLC [n-hexane-i-PrOH (90:10, v/v); f=0.8 mL/min;
λ=215 nm]: tR=32.816 and 40.669 min.
+
-
Absolute configuration assignment of the EKRs’ products
General procedure for the preparative-scale enzymatic KR of rac-3a-
h: To a suspension of the appropriate racemic methyl mandelate rac-3a-
h (3.5 g) and the lipase [1.05 g, 30% w/w (catalyst/substrate)] in TBME
(70 mL), vinyl acetate (7 mL) was added. Thus composed enzymatic
reaction system was shaken at room temperature and 250 rpm on a
laboratory rotatory shaker until the appropriate conversion was reached
(according to GC and HPLC analysis). Next, the reaction was terminated
by filtering off the enzyme. After washing the enzyme with TBME (2 × 25
mL), the permeate was concentrated under reduced pressure, and the
crude residue was purified by column chromatography on silica gel using
gradient of n-hexane/AcOEt (80:10, 70:10, 60:10, 50:10 and 40:10 v/v)
mixture as an eluent thus yielding the respective resolution products [(R)-
(–)-3a-h and (S)-(+)-4a-h]. The detailed experimental conditions, yields
and the results of enzymatic KR reactions (including enantiomeric excess
data for the resolved products and values of enantioselectivity factor) are
collected in Table 4. Physical, spectroscopic and analytical data are
identical as for the corresponding racemic standard compounds rac-3a-h
and rac-4a-h. The specific rotations for the enantiomerically enriched
esters (R)-(–)-3a-h and diesters (S)-(+)-4f-h are as follows: (R)-(–)-3a:
[α]D26 = –202.40 (c 1.02, CHCl3, >99% ee) {lit.[70] [α]D30 = –108.70 (c 1.00,
CHCl3, 78% ee)}. (R)-(–)-3b: [α]D25 = –161.70 (c 1.11, CHCl3, >99% ee)
[derivatization of (R)-(–)-3e toward hexafluorophosphate salt (R)-(–)-
11]
Preparation of 1-(2-hydroxyethyl)pyridinium bromide (7): In a three-
neck round-bottom flask equipped with a thermometer, reflux condenser
attached with drying tube filled with anhydrous CaCl2, and argon gas inlet,
an equimolar amounts of freshly distilled pyridine (9.9 g, 0.13 mol) and
bromoethanol 6 (15.6 g, 0.13 mol) were stirred for 72 h at 50 ºC under
low-light conditions and a gentle argon flow. Next, the reaction mixture
was cooled to room temperature, portion of dry Et2O was added, and the
precipitate formed was washed thoroughly with Et2O (2 × 15 mL).
Purification of the crude was performed by recrystallization from 2-PrOH
to yield product 7 (19.1 g, 0.09 mol, 72%) as a white solid. Mp 96–98 °C
(2-PrOH) [lit.[82] 99–103 °C (EtOH)]; 1H NMR (400 MHz, D2O): δ 4.07 (t, J
= 4.8 Hz, 2H), 4.73 (t, J = 4.8 Hz, 2H), 8.10 (t, J = 7.0 Hz, 2H), 8.59 (t, J
= 8.0 Hz, 1H), 8.87 (d, J = 6.8 Hz, 2H) (The spectral data are fully
consistent with those reported previously in lit.[82]); 13C NMR (100 MHz,
D2O): δ 61.0, 64.1, 128.8, 145.3, 146.6 (The spectral data are fully
consistent with those reported previously in lit.[83]); Anal. Calcd for
C7H10BrNO: C, 41.20; H, 4.94; N, 6.86. Found: C, 41.30; H, 5.04; N, 6.76.
{lit.[70] [α]D = –82.60 (c 1.00, CHCl3, 90% ee)}. (R)-(–)-3c: [α]D = –
147.20 (c 1.00, CHCl3, >99% ee). (R)-(–)-3d: [α]D22 = –147.50 (c 1.28,
CHCl3, >99% ee) {lit.[58] [α]D21 = –127.20 (c 1.00, CHCl3, 96% ee)}. (R)-(–
)-3e: [α]D24 = –136.80 (c 1.00, CHCl3, 99% ee). (R)-(–)-3f: [α]D26 = –112.68
(c 1.03, acetone, 99% ee) {lit.[70] [α]D30 = –129.1 (c 1.00, CHCl3, 90% ee)
or lit.[76] [α]D24 = +93.00 (c 1.08, acetone, 66% ee) for (S)-enantiomer}.
30
24
Preparation of 1‐(2‐hydroxyethyl)pyridin‐1‐ium hexafluorophosphate
(8): A solution of 7 (0.5 g, 2.5 mmol) and KPF6 (0.9 g, 4.80 mmol) in dry
CH3CN (4 mL) was stirred for 24 h at reflux temperature. Next, the
reaction mixture was cooled to room temperature, the precipitate formed
was filtered off, and the resulting permeate was evaporated to dryness.
The remaining residue was dissolved in AcOEt (10 mL) and stirred for 1
h at room temperature. After this time, the precipitate formed was filtered
off, the solvent was evaporated under reduced pressure, and AcOEt (8
mL) was added. The content of the flask was further stirred for 1 h at
room temperature, and the precipitated KPF6 was subsequently removed
by filtration. The whole procedure was repeated twice by adding the
respective portions of AcOEt (6 mL and 4 mL). After evaporation of the
solvent residues, the desired product 8 (625 mg, 2.33 mmol, 93%) was
afforded as white solid, which was further used without purification.
23
28
(R)-(–)-3g: [α]D = –168.29 (c 1.02, CHCl3, 98% ee) {lit.[75] [α]D = –
164.00 (c 1.00, CHCl3, >99.9% ee)}. (R)-(–)-3h: [α]D24 = –157.90 (c 1.00,
CHCl3, 86% ee) {lit.[58] [α]D25 = +184.60 (c 1.00, CHCl3, >99% ee) for (S)-
enantiomer or lit.[79] [α]D28 = +157.70 (c 0.50, EtOH, >99.2% ee) for (S)-
26
enantiomer}. (S)-(+)-4f: [α]D = +118.94 (c 1.14, acetone, 62% ee)
{lit.[39m] [α]D = –7.05 (c 0.45, CHCl3, 97.1% ee)}. (S)-(+)-4g: [α]D
25
23
=
+164.67 (c 1.00, CHCl3, 85% ee). (S)-(+)-4h: [α]D25 = +194.10 (c 1.00,
CHCl3, 86% ee) {lit.[57] [α]D27 = +226.40 (c 1.00, CHCl3, >99% ee)}.
Synthesis of optically active pemoline [(R)-(+)-5]: To a solution of
guanidine hydrochloride (299 mg, 3.13 mmol) in dry EtOH (7.5 mL)
NaOH (125 mg, 3.13 mmol) was added, and the whole was stirred at
room temperature for 1 h until white precipitate was formed. Next, methyl
(2R)-hydroxy(phenyl)ethanoate (R)-(–)-3a (520 mg, 3.13 mmol, >99%
ee) was added, and thus composed reaction mixture was further stirred
for 60 h at RT. After this time, the content of the flask was partially
condensed to a 1/3 of the initial volume, and the ice-cold H2O (5 mL) was
added. The obtained suspension was neutralized by means of glacial
acetic acid. Filtration gave white solid, which was rinsed with ice-cold
H2O (4 × 5 mL), and Et2O (3 × 5 mL) to yield desired product (R)-(+)-5
Preparation of methyl (2R)‐2‐(4‐tert‐butylphenyl)‐2‐methoxyacetate
[(R)-(–)-9]: A mixture of (R)-(–)-3e (1.42 g, 6.02 mmol, 99% ee), freshly
prepared powdered Ag2O (1.7 g, 7.12 mmol), roasted CaSO4 (2.3 g, 7.12
mmol), and methyl iodide (4.2 g, 29.36 mmol, 9.5 mL) was stirred for 36
h at reflux temperature under low-light conditions. After this time, the
content of the flask was allowed to cool, the precipitate formed was
filtered off, and washed with portions of Et2O (3 × 5 mL). The permeate
was evaporated to dryness, and the residual crude product was
chromatographed on silica gel using gradient of n-hexane/AcOEt (90:10,
80:10, 70:10, and 60:10 v/v) mixture as an eluent to afford respective
product (R)-(–)-9 as an oil. The obtained product (R)-(–)-9 was used as a
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201700161
European Journal of Organic Chemistry
FULL PAPER
crude mixture in the next step. Caution: fresh Ag2O was prepared by
adding a 10% aqueous solution of NaOH to a 10% aqueous solution of
AgNO3 until precipitation stopped; next, the slurry of Ag2O formed was
filtered off, and the excess of water was evaporated under reduced
pressure (all the procedure was performed under low-light conditions). In
turn, CaSO4 was oven-roasted at 270 °C, left to cool and used
immediately.
An absolute (R)-configuration for the compound molecule was
successfully determined using anomalous dispersion effects. Flack
parameter[87] calculated from 1544 selected quotients (Parsons'
method)[88]
equals
−0.004(18).
CCDC1527174
contains
the
supplementary crystallographic data for compound (R)-(–)-11. This can
be obtained free of charge on application to CDC, 12 Union Road,
Cambridge
CB21EZ,
UK
(Fax:
(+44)1223-336-033;
email:
deposit@ccdc.cam.ac.uk).
Preparation of (2R)‐2‐(4‐tert‐butylphenyl)‐2‐methoxyacetic acid [(R)-
(–)-10]: To a stirred solution of (R)-(–)-9 (827 mg, 3.50 mmol) in MeOH
(7 mL) 1M aqueous solution of LiOH monohydrate (14 mL) was slowly
added. The reaction mixture was stirred for 24 h at RT. Afterwards,
methanol was removed under vacuum, and residue was extracted with
Et2O (3 × 10 mL). Subsequently, the aqueous layer was cooled to 0–5 °C,
acidified using concentrated 36% HCl solution until the pH 1, and back-
extracted with Et2O (3 × 10 mL). The combined organic layers were
quenched with brine (2 × 5 mL), dried over anhydrous MgSO4, and
concentrated to give (R)-(–)-10 (762 mg, 3.43 mmol, 98%) as a white
solid. Mp 118–119.5 °C (Et2O); [α]D27 = –119.40 (c 0.65, CHCl3, 99% ee);
1H NMR (400 MHz, CDCl3): δ 1.31 (s, 9H), 3.41 (s, 3H), 4.76 (s, 1H),
7.34–7.37 (m, 2H), 7.37–7.40 (m, 2H); 13C NMR (100 MHz, CDCl3): δ
31.3, 34.7, 57.3, 81.8, 125.6, 126.9, 132.0, 152.0, 175.1; FTIR (neat):
νmax = 3219 (br s), 2950, 2902, 2866, 2827, 1748, 1514, 1460, 1412,
1363, 1270, 1256, 1223, 1191, 1183, 1097, 989, 834, 772, 706, 679, 582,
542, 435; Anal. Calcd for C, 70.24; H, 8.16. Found: C, 70.27; H, 8.14.
Acknowledgements
This research was supported by the Warsaw University of
Technology, Faculty of Chemistry is appreciated. The authors
wish to gratefully acknowledge Prof. Jan Plenkiewicz for proof-
reading and critical opinions helpful for preparing this manuscript.
Keywords: mandelic acid • biocatalysis • lipases • kinetic
resolution • pemoline
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a white solid. Mp 130–133 °C (CH3CN/Et2O); [α]D22 = –25.50 (c 0.70,
acetone, 99% ee); 1H NMR (400 MHz, CD3COCD3): δ 1.31 (s, 9H), 3.29
(s, 3H), 4.75 (m, 2H), 4.84 (s, 1H), 5.12 (m, 2H), 7.26–7.28 (m, 2H),
7.40–7.42 (m, 2H), 8.16 (m, 2H), 8.71 (m, 1H), 8.96–8.98 (m, 2H); 13C
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Table of Contents
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Biocatalysis, Enantiopure drugs*
Marcin Poterała,* Maciej Dranka, and
Paweł Borowiecki*
Page No. – Page No.
Chemoenzymatic preparation of
enantiomerically enriched
(R)-(–)-mandelic acid derivatives:
application in the synthesis towards
the active agent pemoline
Chemoenzymatic synthesis of homochiral (R)-pemoline was developed. For this
purpose, methyl (R)-(–)-mandelate was prepared in an environmentally benign
route leading through cyanide-free synthesis of racemic mandelic acid, and
acetylative kinetic resolution approach using lipases as biocatalysts. Enzymatic
protocol can also be applied with excellent enantioselectivity toward novel MA
derivatives.
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