Multienzymatic cascade synthesis of an enantiopure (2R,5R)-1,3-oxathiolane anti-HIV agent precursor

Article abstract of DOI:10.1016/j.mcat.2019.02.013

An enantiopure (2R,5R)-1,3-oxathiolane was obtained using a multienzymatic cascade protocol. By employing a combination of surfactant-treated subtilisin Carlsberg and Candida antarctica lipase B, the absolute configuration of the resulting 1,3-oxathiolane ring was efficiently controlled, resulting in an excellent enantiomeric excess (>99%). This enantiopure 1,3-oxathiolane derivative is a key precursor to anti-HIV agents, such as lamivudine, through subsequent N-glycosylation.

Full text of DOI:10.1016/j.mcat.2019.02.013

MolecularCatalysis468(2019)52–56
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.elsevier.com/locate/mcat
Multienzymatic cascade synthesis of an enantiopure (2R,5R)-1,3-
oxathiolane anti-HIV agent precursor
⁎
Yansong Rena, Lei Hua,b, Olof Ramströma,c,d,
a Department of Chemistry, Royal Institute of Technology, Teknikringen 36, S-10044, Stockholm, Sweden
b School of Pharmacy, Jiangsu University, 301 Xuefu Road, Zhenjiang, China
c Department of Chemistry, University of Massachusetts Lowell, One University Ave., Lowell, MA, 01854, USA
d Department of Chemistry and Biomedical Sciences, Linnaeus University, SE-39182, Kalmar, Sweden
A R T I C L E I N F O
A B S T R A C T
Keywords:
Enzyme
Subtilisin Carlsberg
Candida antarctica lipase B
HIV
An enantiopure (2R,5R)-1,3-oxathiolane was obtained using a multienzymatic cascade protocol. By employing a
combination of surfactant-treated subtilisin Carlsberg and Candida antarctica lipase B, the absolute configuration
of the resulting 1,3-oxathiolane ring was efficiently controlled, resulting in an excellent enantiomeric excess
(> 99%). This enantiopure 1,3-oxathiolane derivative is a key precursor to anti-HIV agents, such as lamivudine,
through subsequent N-glycosylation.
1,3-Oxathiolane
Introduction
potentially circumvented by applying dynamic kinetic resolution (DKR)
strategies. In a typical DKR protocol, the starting materials are thus
Enzymatic catalysis has drawn considerable attention as an im-
portant tool in asymmetric synthesis over the past few decades. As
environmentally benign catalysts, enzymes generally exhibit high en-
antio- and regioselectivity, resulting in economically efficient catalytic
processes [1–6]. Of the rich repertoire of useful procedures described,
the development of multienzyme-catalyzed cascade reactions is a
challenge of particular interest [7–14]. When operating under similar
reaction conditions, different enzymes can thus be concurrently or se-
quentially combined to yield new, efficient synthesis strategies. These
methods can circumvent typical drawbacks of multistep synthesis, such
as time- and solvent-consumption, as well as yield-reducing isolation
and purification schemes. For these reasons, multienzymatic synthesis
can be an efficient tool for access to important intermediates with high
enantiopurity, e.g., for the synthesis of pharmaceutically active com-
pounds [15–18].
allowed to undergo continuous racemization during the resolution
process, leading to the enrichment of one enantiomer and a high overall
yield. Nevertheless, reported enzyme-catalyzed DKR processes for the
synthesis of 1,3-oxathiolane motifs are limited [17,27].
Herein, we have addressed this challenge, and describe an efficient
multienzymatic protocol for the asymmetric synthesis of en-
antiomerically pure (2R, 5R)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl
acetate. Surfactant-treated subtilisin Carlsberg (STS) was thus used in
combination with Candida antarctica lipase B (CAL B) to yield the en-
antiopure product in THF and PBS buffer. These enzymes generally
show opposite stereoselectivity in catalytic acylation of acyclic sec-
ondary alcohols, where the S-isomer is typically favored by CAL B and
the R-isomer by subtilisin Carlsberg [28–30].
Results and discussion
One such group of active compounds is constituted by nucleoside
analog reverse-transcriptase inhibitors (NRTIs, Fig. 1), such as lami-
vudine (1a) and emtricitabine (1c). Key intermediates in the synthesis
of these structures are chiral 1,3-oxathiolane derivatives, which can be
obtained through a variety of approaches. Most of these procedures
involve kinetic resolution (KR) processes of racemic mixtures, using
either selective crystallization mediated by resolving agents, or enzy-
matic esterification/hydrolysis [19–26]. In these protocols, the low
yields caused by the KR process is a major limitation, however
Recently, dynamic hemithoacetal reactions were employed for the
asymmetric formation of 1,3-oxathiolan-5-ones by lipase-catalyzed γ-
lactonization reactions [31–33]. Following an analogous strategy, the
asymmetric synthesis of 1,3-oxathiolane derivatives was undertaken
using STS or CAL B (Scheme 1). Both enzyme preparations favored the
formation of the trans-isomers, although resulting in opposite en-
antiomers. Thus, in the presence of an acyl donor in THF, glycolalde-
hyde dimer 2 and 1,4-dithiane-2,5-diol 3 were converted to the
⁎ Corresponding author at: Department of Chemistry, University of Massachusetts Lowell, One University Ave., Lowell, MA, 01854, USA.
E-mail address: olof_ramstrom@uml.edu (O. Ramström).
https://doi.org/10.1016/j.mcat.2019.02.013
Received 9 October 2018; Received in revised form 12 February 2019; Accepted 14 February 2019
2468-8231/©2019ElsevierB.V.Allrightsreserved.

Y. Ren, et al.
MolecularCatalysis468(2019)52–56
Fig. 1. Structures of lamivudine (1a), ent-lamivudine (1b)
and emtricitabine (1c).
catalyzed hydrolysis reactions [44,45], but also due to the benzoyl
group in the primary position. However, selective formation of primary
alcohols can be observed with cyclic substrates, including carbohy-
drates and nucleosides [46] (Fig. 2).
To potentially achieve higher yields in the second step, we returned
to enantioenriched isomer 4a, obtained in the STS-catalyzed DKR
process when the reaction was carried out in the presence of phenyl
acetate and triethylamine at 4 °C. Compound 4 was in this case obtained
as a mixture, consisting of two pairs of diastereomers, in which the
major isomer 4a exhibited 50% ee (Scheme 3). As shown in Fig. S2, the
ratio of the trans- and cis-isomers was 84:16, compared to a ratio of
approximately 2:1 in the reaction not catalyzed by enzyme [34]. The
enzyme preparation thus further favored formation of the trans isomers.
CAL B was subsequently used to catalyze the hydrolysis of mixture 4 in
a PBS/THF (1:1) system. The results were conspicuous, and selective
hydrolysis of the acetate group near the C2 position was observed, re-
sulting in pure primary alcohol 7 (Scheme 3, Fig. S3). According to 1H
NMR spectroscopy and chiral HPLC analysis in form of acetylated
compound 4a, an excellent diastereomeric ratio (dr) (33:1) and an ex-
cellent ee (> 99%) were determined for compound 7. Combined with
NOE-NMR spectroscopy and chiral HPLC studies [34], the absolute
configuration of compound 7 could be elucidated as 2R,5R. The results
indicate that CAL B catalyzes the regio- and stereoselective hydrolysis
of the C-2(R)-ester as well as the C-5(S)-ester, resulting in the exclusive
formation of isomer 7.
With access to the enantiopure isomer, different parameters were
next examined for optimization of the STS-CAL B-mediated enzymatic
protocol. In the first step, when toluene was used as solvent, both the ee
and the yield decreased significantly compared with THF. Moreover,
prolonging the reaction time from 2 d to 4 d could slightly improve the
yield, however at the cost of reducing the ee from 57% to 42%. In ad-
dition, increasing the STS loading did not influence the reaction sig-
nificantly (Table 1). In the cascade hydrolysis step, THF was proven to
be the optimal solvent compared with TBME and toluene [34].
From these results, THF was considered the optimal solvent for both
processes, establishing the cascade enzyme-mediated synthesis of en-
antiopure 1,3-oxathiolane derivatives. Compounds 2 and 3 were first
allowed to react at 4 °C in the presence of STS for 2 d, at which time the
solid was removed by filtration. PBS buffer and CAL B were subse-
quently added to the filtrate, and the reaction proceeded at ambient
condition overnight. Following this procedure, compound 7 could be
obtained in 50% yield and > 99% ee after purification by column
chromatography. This DKR-KR protocol for the formation of en-
antiomerically pure (2R,5R)-1,3-oxathiolane derivatives thus display
advantages in, for example, reducing the number of synthetic steps and
increasing the final yields in comparison to traditional KR processes.
Enzyme reusability is an important aspect when considering prac-
tical applications, not only for lowering the costs, but also for avoiding
unnecessary environmental impact. In order to examine the reusability
of the STS preparation, experiments were performed under the same
reaction conditions as used for the results in Table 1 (entry 2). After
each cycle, the enzyme was separated by filtration and subsequently
added to the new reaction vial for the next cycle. At least five con-
secutive cycles were carried out, and STS proved highly reusable in the
addition-cyclization-acetylation reaction without any significant loss of
activity (Fig. 3).
Scheme 1. Enzyme-catalyzed asymmetric formation of (2R,5R)-5-acetoxy-1,3-
oxathiolan-2-yl)methyl acetate (4a), (2S,5S)-5-acetoxy-1,3-oxathiolan-2-yl)
methyl acetate (4b).
enantioenriched (2R,5R)-1,3-oxathiolane (4a) catalyzed by STS,
whereas the (2S,5S)-isomer (4b) was the main product in the CAL B-
mediated process [17,34]. Furthermore, replacing compound 2 with the
benzoyl-protected aldehyde 5 led to a higher enantiomeric excess of
(2R)-1,3-oxathiolane derivative 5a (82% ee) in the STS-catalyzed DKR
process.
For further use in, e.g., NRTI synthesis, the enantiopure (2R)-isomer
is desired. For this, we envisaged that applying a resolution protocol to
the enantioenriched isomer 5a would result in the enantiomerically
pure compound. Since KR processes focus on resolving racemic mix-
tures involving compounds with single stereocenters, thereby yielding a
maximum of 50% yield, expanding the scope to resolve a racemate
containing two chiral centers would at most provide 25% overall yield.
Therefore, the present resolution design for the enantioenriched isomer
was expected to result in better yields compared to the conventional KR
process for racemic mixtures. As the most frequently used catalysts for
selective hydrolysis of esters [35–38], lipases were chosen in current
case.
First, racemic mixture 5 was applied to hydrolysis in a biphasic
phosphate-buffered saline (PBS)/THF system in the presence of lipases,
and the reaction was followed by chiral HPLC chromatography. Lipase
preparations from Candida rugosa (CRL), Burkholderia cepacia (PS-C I)
and Candida antarctica (CAL B) were evaluated, of which CAL B resulted
in formation of isomer 5a, indicating that selective hydrolysis of either
the benzoyl- or the acetyl-groups occurred. Furthermore, the en-
antioenriched isomer 5a (C-5-configuration unknown) was prepared
[17], and subjected to hydrolysis in the presence of CAL B in a PBS/THF
mixture. Chiral HPLC analysis showed that the enantiomer of 5a was
completely eliminated, giving rise to an excellent ee of > 99%. How-
ever, a diastereomeric ratio (5a/5b) of 4:1 was recorded (Fig. S1). To
identify the configuration of minor isomer 5b, the 5a/5b mixture was
converted to the nucleoside by conventional Vorbrüggen coupling and
deprotection (cf. Scheme 2). By comparing the chiral HPLC results of
the product with lamivudine (reference), it was observed that the
product consisted of lamivudine 1a and ent-lamivudine 1b in a ratio of
4:1. This indicates that isomer 5b possessed the 2S configuration,
thereby not being suitable for lamivudine synthesis. According to the
results in Scheme 1, the configuration of isomer 5a, prepared using the
STS-system, could be deduced to be the (2R,5R)-isomer, in which case
diastereomer 5b would have a (2S,5R) configuration. Therefore, rather
than acting upon the primary benzoyl ester in the 1,3-oxathiolane, CAL
B appeared to selectively hydrolyze the secondary acetyl ester group in
the 5S-position [39–43]. This was expected, in part since secondary
alcohols have been deemed more favorable products in most enzyme-
53

Y. Ren, et al.
MolecularCatalysis468(2019)52–56
Scheme 2. STS-CAL B-catalyzed synthesis of lamivudine and ent-lamivudine from compounds 3 and 6; (i) phenyl acetate, STS, Et3N, THF, 4 °C; (ii) CAL B, PBS/THF,
rt, 5a:5b (4:1); (iii) silylated N4-acetylcytosine, TMSI, MeCN, 0 °C; (iv) K2CO3, MeOH, rt, 45% for two steps, 1a:1b (4:1).
oxathiolane intermediate was generated by STS-catalyzed addition-cy-
clization-acetylation and subsequently resolved by adding CAL B to
selectively hydrolyze the desired isomer. By employing a protease (STS)
and a lipase (CAL B) as catalysts, straightforward control of the absolute
configuration of the product formed could be achieved. Two achiral
starting materials were used for the formation of 1,3-oxathiolane ring
and consecutively selecting correct isomer. Furthermore, STS displayed
high reusability properties, which is crucial for practical applications.
Compared with previous lamivudine syntheses carried out in multiple
steps and involving repeated purifications, the key, enantiomerically
pure compound could be readily obtained by just one column chro-
matography step, which is time- and solvent-saving. This straightfor-
ward approach represents obvious advantages with respect to both high
catalytic efficiency and environmental friendliness. In addition, since
1,3-oxathiolane derivatives can be converted to lamivudine through N-
glycosylation reactions, our study provides efficient access to com-
pounds that can be smoothly converted to enantiopure lamivudine as
well as other nucleosides, for instance emtricitabine and apricitabine
Fig. 2. Structures of (5-acetoxy-1,3-oxathiolan-2-yl)methyl benzoate (5), ((2R)-
5-acetoxy-1,3-oxathiolan-2-yl)methyl benzoate (5a) and ((2S)-5-acetoxy-1,3-
oxathiolan-2-yl)methyl benzoate (5b).
Experimental
General methods
All commercially available starting materials were of reagent grade
and used as received. Subtilisin Carlsberg (Art. No: P5380) and Candida
antarctica lipase B (Art. No: L4777) were purchased from Sigma-
Aldrich. 1H NMR and 13C NMR data were recorded on a Bruker Avance
DMX 400 instrumentation at 400 MHz. Chemical shifts are reported as δ
values (ppm) with CHCl3/CDCl3 (1H NMR δ 7.26, 13C NMR δ 77.16) as
internal references. Thin layer chromatography (TLC) was performed
on precoated Cromatofolios AL Silica gel 60 F254 (Merck, Darmstadt,
Germany). Detection was performed using UV light, KMnO4 staining, or
PMA stain, and subsequent heating. Flash column chromatography was
performed on silica gel 60, 0.040 - 0.063 mm (SDS). Analytical high
performance liquid chromatography (HPLC) with chiral stationary
phases was performed using an HP-Agilent 1100 series controller,
equipped with Daicel chiralpak OJ (4.6 × 250 mm, 20 μm) and Reprosil
Chiral-JM (4.6 × 250 mm, 10 μm) columns. Solvents for HPLC use were
of spectrometric grade.
Scheme 3. STS-CAL B-catalyzed synthesis of compound 7; (i) phenyl acetate,
STS, Et3N, THF, 4 °C; (ii) CAL B, PBS/THF, rt. aRatio of four isomers determined
by chiral HPLC.
Table 1
Optimization of STS-catalyzed DKR conditions.a
Entry
Time
STS (mg)
Solvent
drb
ee (%)c
Yield (%)d
1
2
3
4
5
6
2d
1d
2d
3d
4d
2d
15
30
15
15
15
30
Toluene
THF
THF
THF
THF
2.6:1
3.7:1
4.0:1
3.5:1
4.0:1
4.0:1
19
57
50
46
42
48
7
16
46
49
49
45
THF
a
Reaction conditions: compound 2 (0.12 mmol), compound 3 (0.16 mmol),
Et3N (0.13 mmol), phenyl acetate (0.71 mmol), 4 °C.
b
Estimated by 1H NMR spectroscopy from the isolated racemic mixture.
Estimated by chiral HPLC spectroscopy (Chiral-JM column) using
c
Immobilization of subtilisin Carlsberg
hexane:2-propanol (9:1) as mobile phase.
d
Isolated yield.
To a solution of octyl β-D-glucopyranoside (15 mg) and Brij 56
(15 mg) in phosphate buffer (0.5 M, pH 7.2, 6 mL) was added subtilisin
Carlsberg (60 mg). The mixture was rapidly frozen with liquid nitrogen
and lyophilized overnight.
Conclusions
In summary, we have implemented a multienzyme-catalyzed cas-
cade DKR-KR protocol for the synthesis of enantiopure (2R,5R)-2-(hy-
droxymethyl)-1,3-oxathiolan-5-yl acetate. The enantioenriched 1,3-
54

Y. Ren, et al.
MolecularCatalysis468(2019)52–56
[a]
Fig. 3. STS reusability. Reaction conditions: compound 2 (0.12 mmol), compound 3 (0.16 mmol), Et3N (0.13 mmol), phenyl acetate (0.71 mmol), 4 °C, 1 d;
[b]
Estimated by 1H NMR spectroscopy from isolated mixture;
Estimated by chiral HPLC.
Synthesis
Acknowledgments
(5-acetoxy-1,3-oxathiolan-2-yl)methyl acetate (4a)
This work was in part supported by the Swedish Research Council.
YR and LH thank the China Scholarship Council for special scholarship
awards.
Glycolaldehyde dimer 2 (15 mg, 0.12 mmol), 1,4-dithiane-2,5-diol 3
(24 mg, 0.16 mmol), triethylamine (17 μL, 0.13 mmol), and phenyl
acetate (90 μL, 0.71 mmol) in dry THF (1 mL) were added to a sealed-
cap vial containing surfactant-treated subtilisin Carlsberg (15 mg) to-
gether with 4 Å molecular sieves (30 mg). The reaction mixture was
cooled to 4 °C. After 2 d, the mixture was filtered and washed with
saturated NH4Cl and brine, after which the mixture was dried over
MgSO4 and the solvent removed under vacuum. The crude product was
purified by column chromatography (Hex:EtOAc = 11:1) affording
compound 4a as a clear oil (25 mg, 46%). 50% ee, determined by HPLC
analysis (Chiral JM column, λ = 210 nm, Hex:iPrOH = 9:1, 0.5 mL/
min). 1H NMR (400 MHz, CDCl3): δ = 6.68 (d, J = 4.1 Hz, 1 H), 5.54
(dd, J = 6.1, 4.0 Hz, 1 H), 4.33–4.21 (m, 2 H), 3.32 (dd, J = 11.5,
4.2 Hz, 1 H), 3.14 (d, J = 11.5 Hz, 1 H), 2.10 (s, 6 H); 13C NMR
(101 MHz, CDCl3) δ = 170.6, 169.9, 99.3, 83.3, 66.0, 37.7, 21.3, 20.9.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.mcat.2019.02.013.
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