Efficient asymmetric synthesis of lamivudine via enzymatic dynamic kinetic resolution

Article abstract of DOI:10.1039/c3cc45551c

The anti-HIV nucleoside lamivudine was asymmetrically synthesized in only three steps via a novel surfactant-treated subtilisin Carlsberg-catalyzed dynamic kinetic resolution protocol. The enantiomer of lamivudine could also be accessed using the same protocol catalyzed by Candida antarctica lipase B.

Full text of DOI:10.1039/c3cc45551c

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Efficient asymmetric synthesis of lamivudine via
enzymatic dynamic kinetic resolution†
Cite this: Chem. Commun., 2013,
49, 10376
Lei Hu, Fredrik Schaufelberger, Yan Zhang and Olof Ramstrom*
¨
Received 22nd July 2013,
Accepted 9th September 2013
DOI: 10.1039/c3cc45551c
www.rsc.org/chemcomm
The anti-HIV nucleoside lamivudine was asymmetrically synthesized in resolution (DKR) method for the lamivudine synthesis,12 however
only three steps via a novel surfactant-treated subtilisin Carlsberg- requiring not only seven steps of synthesis, and undesirable
catalyzed dynamic kinetic resolution protocol. The enantiomer of reagents such as SOCl2, but also the use of chiral auxiliary-derived
lamivudine could also be accessed using the same protocol catalyzed starting materials.
by Candida antarctica lipase B.
Compared with other commonly used catalysts for asymmetric
synthesis, such as chiral transition-metal complexes and organo-
In the last decade, lamivudine (3TC, 1a) (Fig. 1) has proven to catalysts, biocatalysis is now becoming a highly potent alternative
be one of the most successful agents for the treatment of HIV as in both academia and the pharmaceutical industry.13–16 This is in
well as chronic Hepatitis B.1 The compound inhibits both type part due to the high stereoselectivities that can be obtained, the
1 and type 2 of the human HIV reverse transcriptase and also the potential to modify/optimize the performances through directed
reverse transcriptase of hepatitis B in vitro.2–6 As a permanent cure evolution protocols, the inherent environmentally benign (green)
for HIV has remained elusive to date, efficient access to bulk nature of the catalysts, and the ease of recycling processes.17–25 In
quantities of lamivudine is synthetically very valuable as contin- this study, we report a highly efficient three-step asymmetric
uous demand is expected. There are several reported methods to synthesis of lamivudine and its enantiomer through a novel
synthesize isomerically pure lamivudine. Most of the previous enzyme-catalyzed DKR protocol based on reversible formation
reports introduced the chiral 1,3-oxathiolane motif by either of the intermediate stereoisomers. The key enantioenriched
crystallizing the correct isomer from a racemic mixture,7,8 or by 1,3-oxathiolane structure was obtained in good yield and enantio-
enzymatic hydrolysis/acetylation of the other stereoisomers.9–11 purity from two achiral starting materials through an enzyme-
For example, Liotta and Koszalka developed an efficient six-step catalyzed cascade addition–cyclization–acetylation reaction, leading
pathway to racemic nucleosides and utilized a late-stage enzymatic directly to a suitable substrate for the subsequent Vorbru¨ggen
kinetic resolution with a series of lipases to esterify the undesired coupling reaction that is most frequently used for nucleoside
enantiomer. Lamivudine was then obtained in good enantiomeric synthesis. The advantage of this strategy is obvious, as the for-
excess but moderate yield.10 The major disadvantage of these mation of the hemiacetal and its transformation into a better
methods is the great loss of yield due to the nature of the kinetic leaving group for the subsequent coupling are performed in one
resolution process, as no more than 50% yield could be theoreti- pot. In addition, by using different types of enzymes, the stereo-
cally achieved while maintaining high enantiomeric purity. In 2005, chemical configuration of the oxathiolane intermediate could be
Goodyear et al. employed a crystallization-induced dynamic kinetic easily controlled, dynamically yielding the precursor of lamivudine
or its enantiomer (Fig. 1), respectively. This sets up a useful example
for the construction of highly enantioenriched oxathiolane-based
nucleosides with access to both enantiomers in a short synthetic
route. The method represents an improvement to the previous
lamivudine syntheses with respect to both efficiency and environ-
mental friendliness.
Fig. 1 Lamivudine (1a) and its enantiomer (1b).
Taking advantage of the dynamic formation of hemithio-
acetals,26,27 we recently reported that Candida antarctica lipase B
(CAL B) catalyzes the cyclization of the intermediate generated
from the reversible nucleophilic addition of sulfanylacetate 2 to
Royal Institute of Technology, Department of Chemistry, Teknikringen 30,
Stockholm, Sweden. E-mail: ramstrom@kth.se; Fax: +46 8 7912333
aldehyde 3, asymmetrically forming 1,3-oxathiolan-5-one derivative
4 (Scheme 1).19 Since the core structures of compounds 1 and 4 are
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3cc45551c
c
10376 Chem. Commun., 2013, 49, 10376--10378
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Carlsberg is known to have the opposite selectivity for acyclic
secondary alcohols compared to the selectivities of many
lipases.31–34 The commercially available subtilisin Carlsberg
was thus treated with octyl b-D-glucopyranoside and Brij 56 to
enhance its activity and stability in organic solvents.35 The
surfactant-treated subtilisin Carlsberg (STS) was applied to
the same reaction conditions as in Scheme 2. Upon comparing
the intermediate with compound 7a, it is clear that STS selected
enantiomer 7b (Scheme 3), which would lead to the asymmetric
synthesis of lamivudine. However, unsatisfactory enantiomeric
purities (up to 64% ee) were obtained, despite extensive screening
of the reaction conditions.
Scheme 1 CAL B-catalyzed synthesis of 1,3-oxathiolan-5-ones.
very similar, the possibility of applying the same strategy to the
enzyme-catalyzed asymmetric synthesis of lamivudine appeared
to be natural.
Initially, compounds 5 and 6 were mixed with triethylamine
(TEA) as the base and phenyl acetate as the acyl donor in the
presence of CAL B in toluene (Scheme 2). The intention was
that compounds 5 and 6 would react to form compound 7b (2R)
(Scheme 3) in a one-pot cyclization–diol acetylation reaction.
According to the 1H NMR and chiral HPLC analyses, an
enantioenriched intermediate was obtained in 88% yield, with
6.8 : 1 dr, and 83% ee after two days. To elucidate the absolute
configuration of precursor 7, the asymmetric nucleoside was
synthesized through a modified Vorbru¨ggen coupling and
deacylation procedure.28–30 The overall yield for this three-step
asymmetric synthesis was 37%. Chiral HPLC was then used to
identify the selectivity of CAL B by analyzing samples from both
the CAL B-catalyzed nucleoside synthesis and commercial
lamivudine. The result showed that the amplified isomer was
compound 1b; therefore it could be deduced that CAL B
selectively enriched the undesired intermediate stereoisomer
7a (2S). Nine different lipases were subsequently screened: the
lipases from Candida rugosa, Rhizopus niveus, Rhizopus arrhizus,
porcine pancreas, Penicillium camemberti, Aspergillus niger,
Pseudomonas fluorescens, Burkholderia (Pseudomonas) cepacia,
and Candida antarctica lipase A. Of these, the lipases from
Burkholderia cepacia and Pseudomonas fluorescens were also able
to catalyze the formation of the cyclized intermediate, showing
similar stereochemical preferences to CAL B.
In order to obtain higher ees from the enzymatic DKR
reaction, benzoyl protected aldehyde 8 was chosen instead of
glycolaldehyde dimer 5 to react with 1,4-dithiane-2,5-diol 6 in
the presence of STS (Scheme 4), assuming that the larger
structure would fit the enzyme active site more rigidly. This
substrate was subjected to similar reaction conditions as
depicted in Scheme 2 for two days. The isolated intermediate
9a was obtained with an enantiomeric purity of 45% ee, which
was promising for the further optimization. After Vorbru¨ggen
coupling and deprotection, the final product (40% overall yield
for three steps) was again compared with the standard sample
using chiral HPLC, and the result showed that lamivudine (1a)
was still amplified over its enantiomer under these conditions.
With the correct isomer in hand, several parameters were
screened to improve the STS-mediated DKR protocol (Table 1).
TBME and THF were tested as enzyme-tolerable solvents
besides toluene according to the previous studies.17,19–21,23,27
The results in TBME were very similar to those obtained in
toluene, whereas increased dr and ee from 2 : 1 and 45% to
4.3 : 1 and 65%, respectively, were obtained in THF, indicating
that STS favors more polar solvents. Varying the amount of base
and enzyme loading did not significantly affect the yield and
selectivity. The performances of STS at different temperatures
were also addressed. The highest yield was obtained at 40 1C
but with very low stereoselectivity. Both the dr and ee could
however be well improved by lowering the temperature from
25 1C to 4 1C without significant loss of efficiency. Lowering the
temperature further gave even higher selectivities, although
resulting in lower STS activity, where the highest enantiomeric
purity (85% ee) could be recorded at –18 1C.
In order to obtain the correct stereoisomer, an enzyme with
different stereoselectivity was required. The protease subtilisin
Scheme 2 Synthesis of ent-lamivudine (1b) using CAL B; (i) phenyl acetate,
CAL B, TEA, toluene, rt, 92%; (ii) silylated N4-acetylcytosine, TMSI, MeCN, 0 1C;
(iii) K2CO3, MeOH, rt, 40% for two steps.
Scheme 4 Synthesis of lamivudine (1a) using STS: (i) phenyl acetate, STS, TEA,
THF, 4 1C, 89%; (ii) silylated N4-acetylcytosine, TMSI, MeCN, 0 1C, 51%; (iii) K2CO3,
MeOH, rt, 89%.
Scheme 3 Synthesis of intermediate 7b with STS; rt, 87%.
c
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Table 1 Optimization of the DKR conditionsa
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Entry Solvent
T [1C] STS [mg] drb
eec [%] Yieldd [%]
4 J. A. Coates, N. Cammack, H. J. Jenkinson, A. J. Jowett, M. I. Jowett,
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R. Mathis, J. Med. Chem., 1993, 36, 181.
1
2
3
4
5
6
7
Toluene
TBME
THF
THF
THF
THF
THF
25
25
25
25
25
4
20
20
20
10
30
20
20
2.0 : 1
2.5 : 1
4.3 : 1
4.9 : 1
4.5 : 1
4.3 : 1
4.3 : 1
45
47
67
67
68
82
85
74
69
92
81
92
89
23
6 S. L. Doong, C. H. Tsai, R. F. Schinazi, D. C. Liotta and Y. C. Cheng,
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a
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Reaction conditions: 0.1 mmol 8, 0.06 mmol 6, 0.1 mmol TEA,
0.3 mmol phenyl acetate; 2 d. Estimated by 1H NMR spectroscopy from
the isolated racemic mixture. Analyzed by chiral HPLC (Chiralpak
OJ column, l = 254 nm) using 10% of 2-propanol in hexane. Isolated
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b
c
d
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In summary, we have developed a three-step asymmetric synth-
esis of lamivudine using surfactant-treated subtilisin Carlsberg as a
green catalyst. This strategy represents a first entry to efficient high-
enantiopurity nucleoside analog synthesis, whereby application of
enzyme optimization techniques, such as rational redesign or
directed evolution protocols, could lead to enhanced selectivities.
We have also described that the stereochemistry of the target
molecules could be well controlled, as different isomers of the
nucleoside intermediates were selectively obtained using different
enzymes. This study thus offers a valuable methodology for the
asymmetric synthesis of lamivudine as well as other nucleosides
such as emtricitabine and apricitabine, where specific stereoisomers
are desired.
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10378 Chem. Commun., 2013, 49, 10376--10378
This journal is The Royal Society of Chemistry 2013