Lipase-catalyzed production of optically active (S)-flurbiprofen in aqueous phase reaction system containing chiral succinyl β-cyclodextrin

The lipase-catalyzed production of optically active (S)-flurbiprofen was carried out in a dispersion reaction-system induced by chiral succinyl β-cyclodextrin (suβ-CD). The optimal reaction conditions were 500 mM (R,S)-flurbiprofen ethyl ester ((R,S)-FEE), 600 units of Candida rugosa lipase per 1 mmol of (R,S)-FEE, and 1000 mM suβ-CD at 37 °C for 72 h. An extremely high enantiomeric excess of 0.98 and conversion yield of 0.48 were achieved in the dispersed aqueous phase reaction system containing chiral suβ-CD added as a dispenser and chiral selector. The inclusion complex formability of the immiscible substrate (S)- and (R)-form of FEE with suβ-CD was compared using a phase-solubility diagram, DSC, and ¹H NMR. (S)-Isomer formed a more stable and selective inclusion complex with chiral suβ-CD. It was hydrolyzed much more selectively by lipase from C. rugosa, due to the selective structural modification through inclusion complexation with chiral suβ-CD.     

Biocatalytic preparation of (S)-phenyl glycidyl ether using newly isolated Bacillus megaterium ECU1001

A bacterial strain (ECU1001) capable of utilizing phenyl glycidyl ether as sole carbon source and energy source was isolated from soil samples through two steps of screening and was identified as a Bacillus megaterium. The epoxide hydrolase from Bacillus megaterium ECU1001 was biosynthesized in parallel with cell growth and a maximum activity of 31.0 U/l was reached after 30 h of culture when the biomass (DCW) was 9.1 g/l. A temperature of 35°C and pH 8.0 were optimal for the bioconversion. The lyophilized whole cells of Bacillus megaterium ECU1001 could preferentially hydrolyze the (R)-enantiomer of phenyl glycidyl ether, yeilding (S)-epoxide and (R)-diol with high enantioselectivity (E=47.8). The (S)-enantiomer of the epoxide remained in the reaction mixture with >99.5% ee (enantiomeric excess) at a conversion of 55.9%. The substrate concentration could be increased up to 60 mM without affecting the ee and (S)-phenyl glycidyl ether could be obtained with an optical purity of 100% ee and 25.6% yield. Therefore, the method is potentially useful for the preparative resolution of epoxides.     

Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotorula glutinis

A recombinant yeast Pichia pastoris carrying the gene encoding epoxide hydrolase (EH) of Rhodotorula glutinis was constructed and used for producing (S)-styrene oxide by enantioselective hydrolysis of racemic mixtures of styrene oxides. The EH gene was obtained by PCR amplification of cDNA of R. glutinis and integrated into the chromosomal DNA of P. pastoris to express EH under the control of AOX promoter. The recombinant yeast has a high hydrolytic activity toward (R)-styrene oxide as 358 nmol min⁻¹ (mg cell)⁻¹, which is about 10-fold higher than that of wild type R. glutinis. When kinetic resolution was conducted by the recombinant yeast at a high initial epoxides concentration of 526 mM that constitutes an epoxide–water two-liquid phase, chiral (S)-styrene oxide with an enantiomeric excess (e.e.) higher than 98% was obtained as 36% yield (theoretical, 50%) at 16 h.     

Neolignans from Ocotea catharinensis

Bark, wood and leaves of Ocotea catharinensis contain respectively 10 (average yield 0.7%.), 15 (average yield 0.004%.) and one (yield 0.4%.) neolignans of the bicyclo[3.2.1]octanoid and the hydrobenzofuranoid structural types, including the new rel-(7S,8R,1′R,4′S,5′R,6′R)-Δ^8′-4′,6′-dihydroxy-5′-methoxy-3,4-methylenedioxy-3′-oxo-8.1′,7.5′-neolignan, (7S,8S)-Δ^{1′,3′,5′,8′}-5,3′,5′-trimethoxy-3,4-methylenedioxy-8.1′,7.O.6′,4.O.7′-neolignan, (7R,8S,1′R,3′R)-Δ^5′,8′-3,4,3′,5′-tetramethoxy-4′-oxo-8.1′,7.O.6′-neolignan and rel-(7R,8S,1′R,2′S)-Δ^4′,8′-2′-hydroxy-3,4-dimethoxy-3′-oxo-8.1′,7.O.2′-neolignan.     

Stereochemistry of the hydrogen abstraction from pyridoxamine phosphate catalyzed by alanine racemase of Bacillus stearothermophilus

Alanine racemase of Bacillus stearothermophilus catalyzes transamination as a side reaction. Stereospecificity for the hydrogen abstraction from C-4′ of pyridoxamine 5′-phosphate occurring in the latter half transamination was examined. Both apo-wild-type and apo-fragmentary alanine racemases abstracted approximately 20 and 80% of tritium from the stereospecifically-labeled (4′S)- and (4′R)-[4′-³H]PMP, respectively, in the presence of pyruvate. Alanine racemase catalyzes the abstraction of both 4′S- and 4′R-hydrogen like amino acid racemase with broad substrate specificity. However, R-isomer preference is a characteristic property of alanine racemase.     

Further farnesyl-homogentisic acid derivatives from Otoba parvifolia

The seeds of Otoba parvifolia contain three novel compounds apparently derived from homogentisic acid, rel-(1′R,5′R)-2-(1′-farnesyl-5′-hydroxy-2′-oxocyclohex-3′-en-1′-yl)-acetic acid and its acetate as well as rel-(1′R,4′S,5′R)-2-(1′-farnesyl-4′,5′-dihydroxy-2′-oxocyclohexan-1′-yl)-acetic acid δ-lactone. The structure of an additional isolate, previously described as 2-(1′-farnesyl-2′-hydroxy-5′-oxocyclohex-3′-en-1′-yl)-acetic acid γ-lactone was revised to rel-(1′R,5′R)-2-(1′-farnesyl-5′-hydroxy-2′-oxocyclohex-3′-en-1′-yl)-acetic acid δ-lactone.     

Fed-batch production of (S)-flurbiprofen in lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin and its extractive purification

Optically active (S)-flurbiprofen was produced fed-batch-wisely in a lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin (suβ-CD). A highly concentrated 480 mM (S)-flurbiprofen, corresponding to 117.0 g/l, with an enantiomeric excess of 0.98 and conversion yield of 0.48 was obtained. (S)-Flurbiprofen produced in an inclusion complex form with suβ-CD was extractively purified using three-step procedures: decomplexation of (S)-flurbiprofen and residual (R)-flurbiprofen ethyl ester ((R)-FEE) using the ethyl acetate, dissolution of (S)-flurbiprofen from (R)-FEE using a sodium bicarbonate solution, and selective precipitation of (S)-flurbiprofen using 2-propanol. Consequently, an extremely high concentration of 420 mM (S)-flurbiprofen with an optical purity higher than 98% was recovered after purification.     

Algal carotenoids 52; secondary carotenoids of algae 3; carotenoids in a natural bloom of Euglena sanguinea

Quantitative carotenoid analysis of a natural bloom of Euglena sanguinea Ehrenberg revealed the presence of β,β-carotene (1% of total carotenoids), monoesters of adonirubin (3%), diesters of (3S, 3′R)-adonixanthin (13%), diesters of (3S, 3′S)-astaxanthin (75%), 19-monoester of (3R, 3′R, 6R)-loroxanthin (1%), (3R, 3′R)-diatoxanthin (6%), diadinoxanthin (1%) and neoxanthin (traces). The carotenoid content amounted to 0.7% of the dry wt. Methods employed included TLC, HPLC, VIS, MS, CD and H NMR (400 and 500 MHz). The high content of ketocarotenoids is characteristic of secondary carotenoids produced under stressed growth conditions. Previously secondary carotenoids were associated with green algae (Chlorophyceae), but have now been encountered in Euglenophyceae.     

Resolution of 1,3-dioxolane derivatives using the lipase from an Acinetobacter junii SY-01

Acinetobacter junii SY-01 producing a lipase enantioselectively hydrolyzing 1,3-dioxolane derivatives was isolated from water sludge sample and the effect of solvent, acyl donor, vinyl acetate concentration, substrate concentration, operating temperature and immobilization on activity and enantioselectivity was studied for the resolution of 1,3-dioxolane derivatives through transesterification reaction using a lipase from the isolated strain. Best selectivity was obtained at lower substrate concentration (3–5 mM), higher vinyl acetate concentration (500–1000 mM) and lower temperature (30–40 °C) in the reaction mixture. Lipase immobilized onto Accurel MP-1000 (micro-porous polypropylene) gave the best results and the reactivity was about 29-fold higher than the free enzyme without the decrease of enantioselectivity. Resolution of 1,3-dioxolane derivatives was carried out in flask scale containing 100 ml solvents using the lipase immobilized onto Accurel MP-1000. In this reaction, the yield and enantiomeric excess of the remaining (2R, 4S)-alcohol were 31.2% and 98.2%, respectively. This result suggests that it can be used as an alternative method, compared to the present synthetic method, for the production of optically pure (2R, 4S)-itraconazole.     

酵母脂肪酶分子进化及催化合成(S)-1,4-苯并二口恶烷*

Objective: 1,4-Benzodioxane is an important chiral intermediate for antihypertensive (Proroxan and Doxazosin), antidepressant (MCK-242) and other drugs, and it displays a broad spectrum of applications in the pharmaceutical field. Currently, in spite of high-yield advantage of chemical synthesis, there are some problems of environmental pollution and low production safety. Using lipase to catalyze synthesis of 1,4-benzodioxane provides a new pathway of green synthesis of 1,4-benzodioxane. However, natural enzymes face the dilemma of poor enantioselectivity. Therefore, molecular evolution was performed on Candida antarctica lipase B, and a technical route for the catalytic synthesis of 1,4-benzodioxane was established. Methods: Firstly, the key amino acid residues involved in substrate binding and conversion in the active center of Candida antarctica lipase B were analyzed, and saturation mutagenesis libraries on the interaction sites were constructed. Improved mutants with high efficiency and high enantioselectivity were then obtained using HPLC detection. Furthermore, catalytic synthesis conditions of mutant D223N/A225K were systematically optimized. Results: The results indicated that the mutants mainly derived from the pairwise site D223/A225 (such as D223N/A225K and D223G/A225W) were biased towards the synthesis of (S)-isoforms, while most of the mutants derived from the pairwise site E188/I189 (such as E188D/I189M) showed a bias for the synthesis of (R)-isoforms. Compared with WT, the ee_s value of the best mutant D223N/A225K to synthesize (S)-1,4-benzodioxane was increased from 11.9% to 29.3%. After systematic optimization of the reaction conditions, an ee_s value of (93.9±0.16)% and a conversion rate of (47.5±2.33)% were achieved using mutant D223N/A225K to catalyze kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate in n-butanol/phosphate buffered saline (20∶80, V/V) biphasic solvent at 37℃ for 50 min. Conclusion: An efficient kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate was successfully achieved by molecular evolution and optimization of conditions, which provides a new example for the creation of new enzymes by protein engineering technology, and also provides a theoretical and technical foundation for the efficient synthesis of (S)-1,4-benzodioxane molecules by enzymatic methods.     

Total syntheses of (+/-)-ovalicin, C4(S *)-isomer, and its C5-analogs and anti-trypanosomal activities

Total syntheses of (±)-ovalicin, its C4(S^*)-isomer 44, and C5-side chain intermediate 46 were accomplished via an intramolecular Heck reaction of (Z)-3-(tert-butyldimethylsilyloxy)-1-iodo-1,6-heptadiene and a catalytic amount of palladium acetate. Subsequent epoxidation, dihydroxylation, methylation, and oxidation led to (3S^*,5R^*,6R^*)-5-methoxy-6-(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]octan-4-one (2), a reported intermediate. The addition of a side chain with cis-1-lithio-1,5-dimethyl-1,4-hexadiene (27) followed by oxidation afforded (±)-ovalicin. The functional group manipulation afforded a number of regio- and stereoisomers, which allow the synthesis of analogs for bioevaluation. The structure of 44 was firmly established via a single-crystal X-ray analysis. The stereochemistry at C4 generated from the addition reactions of alkenyllithium with ketones 2, 40, and 45 is dictated by C6-alkoxy functionality. Anti-trypanosomal activities of various ovalicin analogs and synthetic intermediates were evaluated, and C5-side chain analog, 46, shows the strongest activity. Compound 44 shows antiproliferative effect against HL-60 tumor cells in vitro. Compounds 46 and a precursor, (3S^*,4R^*,5R^*,6R^*)-5-methoxy-4-[(E)-(1′,5′-dimethylhexa-1′,4′-dienyl)]-6-(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]octan-4-ol (28), may be explored for the development of anti-parasitic drugs.     

Uptake and release effects of diethylpropion and its metabolites with biogenic amine transporters

Three metabolites of diethylpropion (1), (±)-2-ethylamino-1-phenyl-propan-1-one (2), (1R,2S)-(−)-N,N-diethylnorephedrine (3a) and (1S,2R)-(−)-N,N-diethylnorephedrine (3b) were synthesized. Their uptake and release effects with biogenic amine transporters were evaluated. A major finding of this study is that the in vivo activity of diethylpropion on biogenic amine transporters is most likely due to metabolite 2 as diethylpropion (1) and the metabolites 3a and 3b showed little or no effect in the assays studied. These studies also revealed that 2 acted as a substrate at the norepinephrine (IC₅₀=99 nM) and serotonin transporters (IC₅₀=2118 nM) and an uptake inhibitor at the dopamine transporter (IC₅₀=1014 nM). The potent action of 2 at the NE transporter supports the hypothesis that amphetamine-type subjective effects may be mediated in part by brain norepinephrine.     

Esterified, optical pure (3S, 3′S)-astaxanthin from flowers of Adonis annua

The quantitative carotenoid composition of the red flower petals of Adonis annua is reported. Optically pure (3S, 3′S)-astaxanthin occurs both as a diester (64% of total carotenoid) and as a monoester (11%). The optical purity was determined by hydrolysis of the natural esters in the absence of oxygen and subsequent HPLC analysis of the paren -ketol esterified with (−)-camphanic acid. All non-animal sources hitherto examined synthesize pure 3S,3′S- or 3R,3′R-isomers of astaxanthin, whereas marine animal sources contain mixtures of all three optical isomers, including the meso form.     

Alkaloids from Heimia salicifolia

Two alkaloids, 9β,2′-dihydroxy-4′′,5′′-dimethoxy-lythran-12-one or 9β-hydroxyvertine (1) and (2S,4S,10R)-4-(3-hydroxy-4-methoxyphenyl)-quinolizidin-2-acetate (2), as well as seven known alkaloids, lythrine (3), dehydrodecodine (4), lythridine (5), vertine (6), heimidine (7), lyfoline (8) and epi-lyfoline (9), were isolated from Heimia salicifolia. The structures of these compounds were elucidated by extensive spectroscopic techniques. Furthermore, the structures of 2, 3, and 6 were confirmed by X-ray crystallography, including absolute configuration determination of 2 and 6. Compounds 6 and 9 showed moderate antimalarial activity.     

Enantioselective epoxide hydrolase activity of a newly isolated microorganism, Sphingomonas echinoides EH-983, from seawater

A marine microorganism, Sphingomonas echinoides EH-983, which possesses epoxide hydrolase (EH) activity was isolated from seawater and characterized. The EH of S. echinoides EH-983 preferentially metabolized (R)-enantiomer when the racemic styrene oxides were supplied as substrates. The optimal pH and temperature for the enantioselective hydrolysis by whole-cells ofS. echinoides EH-983 were 7.0 and 20 °C, respectively. When kinetic resolution was conducted with a racemic mixture of styrene oxides at an initial concentration of 40 mM, enantiopure (S)-styrene oxide was obtained in 180 min with a yield of 21.3%. To our best knowledge, S. echinoides EH-983 is the first marine microorganism that is reported to have EH activity.     

A novel resolution of a pharmaceutically important bridged bicyclic ketone intermediate via selective enzymatic reduction with a commercially available ketoreductase

An efficient route to the pharmaceutically important (6S,9R)-11-oxo-5,6,7,8,9,10-hexahydro-6,9-methanobenzocyclooctene intermediate has been demonstrated via kinetic resolution of 11-oxo-5,6,7,8,9,10-hexahydro-6,9-methanobenzocyclooctene using a commercially available ketoreductase. Biocatalytics KRED 101 has been shown to selectively reduce the (6R,9S) enantiomer leaving behind the desired (6S,9R) enantiomer. This novel reaction is the first demonstration of a high yielding (44% versus 50% maximum theoretical yield) highly stereoselective (>99% ee) resolution of a bicyclic ketone, via enzymatic reduction using a commercially available ketoreductase, where the stereochemistry of the substrate is determined by a bridged ring system. Several challenges were overcome, including enhancing the selectivity of the enzyme by controlling temperature and increasing substrate solubility by employing a combination of cyclodextrin and organic co-solvent in the aqueous reaction system.     

Effect of (6R)- and (6S)-tetrahydrobiopterin on L-3,4-dihydroxyphenylalanine (DOPA) formation in NRK fibroblasts transfected with human tyrosine hydroxylase type 2 cDNA

-erythro-5,6,7,8-Tetrahydrobiopterin (BH₄), which is the cofactor of aromatic amino acid hydroxylases, plays an important role in the biosyntheses of monoamine neurotransmitters. BH₄ exists as natural (6R)- and unnatural (6S)-isomers. In our previous reports, only (6R)-isomer significantly stimulated cofactor activity for tyrosine, tryptophan and phenylalanine hydroxylases (TH, TPH, PAH) in whole animals or in tissue slices. In this study we have compared the in situ cofactor activity on TH between natural (6R)- and unnatural (6S)-isomers in clonal cells. We have transfected human TH type 2 cDNA into the normal rat kidney (NRK) fibroblasts. These cells expressed TH protein, but had neither DOPA decarboxylase (DDC) nor BH₄. Thus, TH activity was observed only in the presence of exogenous BH₄. We compared the difference in in situ DOPA formation by TH activity in the presence of (6R)- or (6S)-BH₄ in the human TH-transfected cells. The effect of exogenous BH₄ was also compared between (6R)- and (6S)-isomers in rat pheochromocytoma PC12h cells, which contained approximately 100 μM endogenous (6R)-BH₄. The rate of uptake of both BH₄ isomers into these cells increased in proportion to the pterin cofactor concentrations in the incubation medium up to 400 μM but was nearly saturated at 1 mM BH₄. TH-transfected NRK fibroblasts formed DOPA only in the presence of exogenously added (6R)- or (6S)-BH₄ dose-dependently and released DOPA into the medium. At a saturating concentration of 1 mM, (6R)-BH₄ was approximately three times as active as (6S)-BH₄. In contrast, in PC12h cells which contained endogenous (6R)-BH₄ (approximately 100 μM), exogenous (6R)-BH₄ activated DOPA formation maximally at 500 μM about 10-fold, while (6S)-BH₄ activated it only slightly, about 2.5-fold. These results suggest that (6S)-isomer has lower cofactor activity with TH in the cells than (6R)-isomer. This TH transfected fibroblasts should be useful to assess cofactor activities of tetrahydropteridines in the cell.     

Novel microbial epoxide hydrolases for biohydrolysis of glycidyl derivatives

Microbial isolates from biofilters and petroleum-polluted bioremediation sites were screened for the presence of enantioselective epoxide hydrolases active towards tert-butyl glycidyl ether, benzyl glycidyl ether, and allyl glycidyl ether. Out of 270 isolated strains, which comprised bacteria, yeasts, and filamentous fungi, four were selected based on the enantioselectivities of their epoxide hydrolases determined in biotransformation reactions. The enzyme of Aspergillus niger M200 preferentially hydrolyses (S)-tert-butyl glycidyl ether to (S)-3-tert-butoxy-1,2-propanediol with a relatively high enantioselectivity (the enantiomeric ratio E is about 30 at a reaction temperature of 28 °C). Epoxide hydrolases of Rhodotorula mucilaginosa M002 and Rhodococcus fascians M022 hydrolyse benzyl glycidyl ether with relatively low enantioselectivities, the former reacting predominantly with the (S)-enantiomer, the latter preferring the (R)-enantiomer. Enzymatic hydrolysis of allyl glycidyl ether by Cryptococcus laurentii M001 proceeds with low enantioselectivity (E = 3). (R)-tert-Butyl glycidyl ether with an enantiomeric excess (ee) of over 99%, and (S)-3-tert-butoxy-1,2-propanediol with an ee-value of 86% have been prepared on a gram-scale using whole cells of A. niger M200. An enantiomeric ratio of approximately 100 has been determined under optimised biotransformation conditions with the partially purified epoxide hydrolase from A. niger M200. The regioselectivity of this enzyme was determined to be total for both (S)-tert-butyl glycidyl ether and (R)-tert-butyl glycidyl ether.     

Adenosine receptor agonists: Synthesis and biological evaluation of the diastereoisomers of 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA

Among the recently reported 2-(ar)alkynyl derivatives of 5′-N-ethylcarboxamidoadenosine (NECA), the (R,S)-2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA [(R,S)-PHPNECA or SCH 59761] was found to be a very potent agonist at A₁ and A_2A receptor subtypes, with a K_i of 2.5 nM and 0.9 nM, respectively. Furthermore, this compound showed an inhibitory activity on platelet aggregation 16-fold higher than NECA, being the most potent anti-aggregatory nucleoside reported so far. Since this compound bears a chiral carbon in the side chain, the diastereoisomer separation was undertaken both by chiral HPLC and by a stereospecific synthetic method. Binding assays have shown that the (S)-diastereomer is about fivefold more potent and selective than the (R)-diastereomer as agonist of the A_2A receptor subtype [(S)-PHPNECA, K_iA_2A = 0.5 nM; (R)-PHPNECA, K_iA_2A = 2.6 nM]. Functional studies indicated that (S)-PHPNECA possesses marked vasodilating activity and produces a relevant decrease in heart rate. Moreover, the (S)-diastereomer proved to be about ten times more potent than the (R)-diastereomer in inducing cardiovascular effects, in in vivo hemodynamic studies. However, the greatest difference between these two enantiomers resulted in the platelet aggregation test: in fact, the (R)-diastereomer displayed an inhibitory activity similar to that of NECA, whereas the (S)-diastereomer was 37-fold more active than NECA as an inhibitor of rabbit platelet aggregation, induced by ADP. These data suggest that (S)-PHPNECA could be a useful tool to investigate the mode of binding of agonists to the platelet adenosine receptor subtype.     

Regioselective enzymatic aminoacylation of lobucavir to give an intermediate for lobucavir prodrug

Synthesis of lobucavir prodrug, L-valine, [(1S,2R,3R)-3-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (BMS 233866), requires regioselective coupling of one of the two hydroxyl groups of lobucavir (BMS 180194) with valine. Either hydroxyl group of lobucavir could be selectively aminoacylated with valine by using enzymatic reactions. N-[(Phenylmethoxy)carbonyl]-L-valine, [(1R,2R,4S)-2-(2-amino-6-oxo-1H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester (3, 82.5% yield), was obtained by selective hydrolysis of N,N′-bis[(phenylmethoxy)carbonyl]bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester (1) with lipase M, and L-valine, [(1R,2R,4S)-2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (4, 87% yield) was obtained by hydrolysis of bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester, dihydrochloride (2), with lipase from Candida cylindracea. The final intermediate for lobucavir prodrug, N-[(phenylmethoxy)carbonyl]-L-valine, [(1S,2R,4R)-3-(2-amino-6-oxo-1H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester (5), could be obtained by transesterification of lobucavir using ChiroCLEC™ BL (61% yield), or more selectively by using immobilized lipase from Pseudomonas cepacia (84% yield).