Appl Biochem Biotechnol (2008) 151 :21 28
`DOI l 0.1007 /s 120 I 0-008-8152-0
`
`Highly Efficient Regioselective Synthesis
`of 5'-0-lauroyl-5-azacytidine Catalyzed
`by Candida antarctica Lipase B
`
`Xi-Yu Chen • Min-Hua Zong • Wen-Yong Lou •
`Hong Wu
`
`Received: 7 November 2007 / Accepted: IO January 2008 /
`Published online: 16 Febrnary 2008
`'.c Humana Press Inc. 2008
`
`Abstract Enzymatic regioselective acylation of 5-azacytidine with vinyl laurate was
`successfolly conducted with an immobilized lipase from Candida antarctica type B (i.e.,
`Novozym 435) fr>r the first time. The acylation of 5-azacytidine took place at its primary
`hydroxyl group and the desired product 5'-O-lauroyl-5-azacytidine could be prepared with
`high reaction rate, high conversion, and excellent regioselectivity. The influences of several
`key variables on the enzymatic acylation were also systematically examined. Pyridine was
`found to be the best reaction medium. The optimum initial water activity. the molar ratio of
`vinyl laurate to 5-azacytidine and reaction temperature were 0.07, 30: L and 50 °C,
`respectively. Under the optimized conditions described above, the initial reaction rate, the
`substrate conversion, and the regioselectivity were as high as 0.58 mM/min, 95.5%,
`and >99%1, respectively, after a reaction time of around 5 h.
`
`Keywords 5-Azacytidine · Novozym 435 · Organic solvent• Regioselective acylation •
`Vinyl laurate
`
`Introduction
`
`5-Azacytidine, an analogue of the natural pyrimidine nucleoside cytidine, is employed for
`the treatment ofmyelodysplastic syndrome (MDS) [l]. However, it has some disadvantages
`in clinical application, such as difficulty to traverse biological membranes and skin layers
`due to poor lipophilicity [2], spontaneous hydrolysis in aqueous solutions and rapid
`deamination by cytidine deaminase, etc. (l, 3-6]. In order to overcome these problems, 5'(cid:173)
`monoester of 5-azacytidine could be used, which is primarily based on the fact that the
`molecules containing hydroxyl or carboxyl groups can be converted into the corresponding
`esters with the desired lipophilicity by the selection of an appropriate ester side chain [7].
`
`X.-Y. Chen · M.-H. Zong ('.81) · W.-Y. Lou · H. WL1
`Lab of Applied Biocatalysis, South China University of Technology. Guangzhou 510640, China
`e-mail: btmhzonma/scut.edu.cn
`
`CELGENE 2010
`APOTEX v. CELGENE
`IPR2023-00512
`
`

`

`Appl Biochem Biotechnol (2008) 151:21-28
`
`5' -Monoester of 5-azacytidinc could be synthesized through regioselective acylation of 5-
`azacytidine. On the other hand, the regioselectivc acylation of nuclcoside is one of the
`important ways of introducing protecting groups as well as obtaining valuable nucleoside
`derivatives, and therefore will find wide applications in nucleoside chemistry.
`Several strategies for regioselective acylation of nucleosides have been reported using
`conventional chemical methods [8], but their applications are somewhat hampered due to
`the relatively low regioselectivity, the lack of easy access to some important intcm1cdiates,
`the tedious product isolation, and the environmental concerns of the process.
`To date, enzymatic acylation ofnucleosides in organic media has emerged as a promising
`procedure, due to its advantageous properties including high regioselectivity, mild reaction
`conditions, and environmental benign [9]. Besides, the use of organic solvents is especially
`advantageous when substrates or products are unstable in water. Furthermore, at a low
`water activity, many other water-dependent side-reactions can be prevented [IO]. Ferrero
`and Gotor [ 11] have reviewed the utility of biocatalysts for the modification of nucleosides.
`Various kinds of enzymes have been proven to be capable of catalyzing the acylation of
`nucleosides with desirable regioselectivity. Among them, Novozym 435, a commercially
`available lipase from Candida a11tarctica type B (CAL-B) immobilized on a macroporous resin
`of poly-(methyl methacrylate) (Lewatit VP OC 1600), is well recognized for its extraordinary
`ability to catalyze the esterification of nucleosides with substantially high regioselectivity [ 12,
`13]. For example, Novozym 435 has been shown in our previous work to be highly active and
`regioselective for the enzymatic acylation of 1-/i-o-arabinofuranosylcytosine [14].
`Generally, fatty acid vinyl esters are preferable acyl donors in acyl transfer reactions
`[ 15]. In the course of our ongoing investigation, it was found that a great amount of
`undesired by-products were produced when short-chain fatty acid vinyl esters were used as
`acyl donors for the acylation of 5-azacytidine, while the use of long-chain fatty acid vinyl
`esters such as vinyl laurate yielded little by-products. Therefore, vinyl laurate, a typical
`long-chain fatty acid vinyl ester, is here adopted as an acyl donor for the enzymatic
`acylation of 5-azacytidine.
`As an extension of our ongoing research program on efficient synthesis of various
`valuable nucleoside derivatives via enzymatic acylation. we herein for the first time report
`the successful regioselective acylation of 5-azacytidine with vinyl laurate catalyzed by
`Novozym 435 (Scheme l) in organic solvents. The enzymatic acylation process might
`become a new route to the preparation of 5'-O-lauroyl-5-azacytidine, which is more
`lipophilic and might be more bio-available than 5-azacytidine. Also, the effects of several
`crncial factors on the enzymatic acylation are described in this paper.
`
`r N;(
`l I
`!=o
`l-d
`
`N~O
`
`H
`
`H
`OH
`
`(CH2)10
`
`H
`
`H
`
`OH
`
`Novozym 435
`Organic solvents
`
`5-Azacytidine
`Scheme l Novozym 435-catalyzcd rcgioselective acylation of 5-azacytidine with vinyl laurate in organic
`solvents
`
`5'-0-Lauroyl-5-azacytidine
`
`

`

`Appl Biochern Biotechnol (2008) I 51 :21 28
`
`23
`
`Materials and Methods
`
`Biological and Chemical Materials
`
`Novozym 435 (an immobilized lipase from Candida antarctica, type B, I 0,000 U g- 1
`) was
`kindly donated by Novozymes (Denmark). 5-Azacytidine and vinyl laurate were purchased
`from Fluka (Germany). All other chemicals were from commercial sources and were of the
`highest purity available.
`
`Control of the lnitial Water Activity
`
`The reaction media, the substrate, and the enzyme were equilibrated to fixed initial water
`activities ( cvw) over saturated salt solutions in closed containers at 25 °C separately [ 16-20).
`The following salts were used: LiBr (ctw=0.07), LiCI (uw=0.11), CH3COOK (n\v=0.23),
`MgCh (<xw=0.33). Molecular sieve was used to generate the nearly anhydrous reaction
`medium (nw ~ 0).
`
`General Procedure for Enzymatic Reaction
`
`In a typical experiment, 2 ml of pyridine containing 0.02 mmol 5-azacytidine, 0.6 mmol
`vinyl laurate, and 1,000 U Novozym 435 was incubated in a IO ml Erlenmeyer shaking(cid:173)
`flask capped with a septum at 200 rpm and 40 °C. Aliquots (20 ~d) were withdrawn at
`specified time intervals from the reaction mixture, and then diluted by 50 times with a co(cid:173)
`solvent mixture of water and methanol prior to HPLC analysis. To obtain larger amounts of
`product for its structural characterization, the synthesis was scaled up ( ~ 25 mg 5-
`azacytidine and 520 µl vinyl laurate). Upon the completion of the reaction, the reaction
`mixture was filtered to remove the immobilized enzyme and was evaporated under vacuum.
`The crude product was then purified by silica gel chromatography with the mixture of
`methanol and chlorofonn (25/75, vlv) as an eluant. After crystallization from ethanol, the
`product was obtained as a white powder (yield >90%).
`
`HPLC Analysis
`
`The reaction mixture was analyzed by RP-HP LC on a 4.6 x 250 mm (5 µm) Zorbax SB-C 18
`column (Agilent Technologies Industries Co., USA) using an Agilent G 1311 A pump and a
`UV detector at 241 nm. The mobile phase was a mixture of ammonium acetate buffer
`(0.01 M, pH 4.27) and methanol (22/78, v!v) at a flow rate of 0.9 ml min- 1
`• The retention
`times for 5-azacytidine and 5'-O-lauroyl-5-azacytidine were 2.6 and 11.7 min, respectively.
`Regioselectivity was defined as the ratio of the HPLC peak area corresponding to the
`indicated product to that of all the products formed upon a certain reaction time according
`to the literature [21]. The initial rate ( ViJ) and the substrate conversion (c) were calculated
`from the HPLC date. The average error for this assay is less than 0.7%>. All reported data
`are averages of experiments performed at least in duplicate.
`
`Structure Detennination
`
`Mass spectrometric analysis in the negative ion mode was performed on an ion trap analyzer
`(Bruker HCTplus, Bruker Co., Ge1many). The capillary voltage was set at -I 13.5 V. ESI
`temperature and ion trap analyzer voltage were 300 °C and -40.0 V, respectively. The product
`
`

`

`24
`
`Fig. I R.:prcsentativc LC-MS/
`MS spectra of the main product
`with negative-ion mod.:
`
`Appl Biochem Biotechnol (2008) 151 :21 28
`
`425.2
`
`4
`
`2
`
`700 mlZ
`
`structure was determined by 13C NMR (Bruker DRX-400 NMR Spectrometer, Bruker Co.) at
`100 MHz. DMSO-d6 was used as a solvent and chemical shifts were expressed in ppm shift.
`
`Results and Discussion
`
`Product Characterization
`
`As can be seen in Fig. 1, the molecular weight detected is around 425.2, which indicates
`that the product obtained is identical with mono lauroyl ester of 5-azacytidine (MW 426).
`The ability of Novozym 435 to catalyze regioselective transfomiation has been exploited
`in the modification of polyhydroxy compounds [21, 22]. According to the published
`literature by Yoshimoto et al. [23], the acylation of a hydroxyl group of sugar results in a
`downfield shift of the peak corresponding to the O-acylated carbon atom and an upfield
`shift of the peak corresponding to the neighboring carbon atom. As evident from the data
`listed in Table I, the 13C NMR spectrum of the product shows a shift of 3.01 ppm on C5'
`towards the lower fields as compared to the same carbon atom in the unmodified 5-
`azacytidine. Also, the directly neighboring carbon atom (C4') gave a shift of about
`3 .2 7 ppm towards the higher fields due to the acylation of the hydroxyl group of C5'. In
`addition, 12 sharp peaks of -CH 3 , -CH2 and C=O appeared with the determinate chemical
`
`Table I uC NMR spectral data
`for 5-azacytidine and its acylated
`derivative (Ii, ppm)".
`
`"All samples were measured in
`DMSO-c/6 .
`
`C.irbon numbers
`
`5-Azacytidine
`
`5'-0-Lauroyl-5-azacytidinc
`
`l5Hi7
`166.18
`156.69
`
`89.64
`74.26
`69.30
`84.65
`60.47
`
`Base moiety
`2
`4
`6
`Sugar moiety
`I'
`2'
`.3'
`4'
`5'
`Acyl moiety
`C=O
`CH,
`~CH2
`
`153.29
`166.10
`156.44
`
`90.39
`73.79
`69.76
`8 l.38
`63.48
`
`169.54
`13.97
`22.54-33.91
`
`

`

`Appl Biochem Biotechnol (2008) 151 :21 -28
`
`25
`
`Table 2 Novozyrn 435-cat:ilyzed regioselective acylation of 5-az3cytidine with vinyl lm1rnte in different
`organic solvents".
`
`Media
`
`Solubility of 5-azacytidine (mM)"
`
`lgP
`
`Vii (mM min- 1
`
`) C' (%)
`
`Regiosclectivity ( ?,;,)
`
`DMSO
`DMr
`Pyridine
`
`254.0
`112.9
`21.8
`
`1.35
`-1.01
`0.71
`
`()
`
`()
`0.30
`
`0
`()
`67.0
`
`()
`0
`>99
`
`"The reactions were carried out in 2 ml of different organic solvents (nw••0. 11) containing 0.02 mmol 5-
`azacytidinc, 0.4 mmol vinyl laurate and 1000 U Novozym 435 at 200 qnn and 40 °C.
`b The solubility of 5-azacytidine in each reaction medium was detennined by HPLC analysis of the saturated
`solution at 30 °C.
`"Maximum substrate conversion
`
`shifts. So the product was proved to be 5'-0-lauroyl-5-azacytidine. And Novozym 435 was
`proved to display a startling regioselectivity up to 99% towards the 5'-hydroxyl group of 5-
`azacytidine.
`It has been reported that Candida a11tarctica lipase B has a rather narrow and deep
`channel leading to an open active site [24]. The 5'-0H of the sugar moiety of 5-azacytidine
`may have an easier access to the active site of CAL-B to attack the acyl-enzyme
`intermediate than other -OH groups at C-3' and C-2' due to less steric hindrance, thus
`resulting in preferential acylation of the 5'-0H of 5-azacytidine.
`
`Effect of Reaction Medium
`
`One of the most troublesome limitations in the acy lation of hydrophilic nucleosides is their
`poor solubility in most organic solvents. In fact, only polar organic solvents, such as
`pyridine and DMF, have been commonly used to solve the problem [25]. However, polar
`organic solvents usually strip the essential water off the enzyme molecules and then
`inactivate the biocatalyst, which greatly limits the application of enzymatic procedures in
`this area [l OJ. A less polar solvent does not inactivate the enzyme as much as a more polar
`one. As shown in Table 2, no reaction occurred in DMSO and DMF, although 5-azacytidine
`showed high solubility in these solvents. Only in pyridine could the lipase-catalyzed
`acylation of 5-azacytidine be efficiently carried out. Thus, pyridine was selected as the most
`suitable solvent for the reaction.
`
`Table 3 Effect of initial water activity on Novozym 435-catalyzed regiosclectivc acylation of 5-azacytidinc
`in pyridine".
`
`Initial water activity (nw)
`
`1';1 (rnM min 1
`)
`
`C" (%,)
`
`Regioselcctivity (%)
`
`:::O
`0.07
`0.11
`0.23
`0.33
`
`0.34
`0.39
`0.30
`0.14
`ll.09
`
`80.7
`84.4
`67.0
`14.2
`9.3
`
`>99
`>99
`>99
`>99
`>99
`
`" The reactions were pe1ic11mcd in 2 ml of pyridine with different initial water activity containing 0.02 mrnol
`5-azacytidine. 0.4 mmol vinyl laurate mid I 000 U Novozym 435 at 200 rpm and 40 °C.
`h Maximum substrate conversion
`
`

`

`26
`
`Fig. 2 Effect of the molar ratio of
`vinyl lauratc to 5-azacytidine on the
`enzymatic rcgioselcctive acylation
`of 5-anteytidine. The reactions
`were perfbnned in 2 ml of pyridine
`(nw=0.07) containing 0.02 mmol
`5-azacytidinc, 1.000 lJ Novozym
`435 and different amounts of vinyl
`lauratc at 200 rpm and 40 "C.
`Pilled triangle, initial reaction rate:
`/i lied square, conversion; filled
`circle, regiosclectivity
`
`] s::: 0.24
`....,
`
`Appl Biochem Biotechnol (2008) 151:21-28
`
`~ /
`
`100
`
`Q)
`
`Q)
`
`0
`
`---· 90
`~-· ~)2_ ?;,
`80 ? - ~
`70 § :~
`u
`-~
`60
`"' 1)
`is
`/ )
`0
`50
`'Si
`u Q)
`40
`"'
`"' 0
`30
`20
`
`10:1 15: I 20:l 25: I 30:I 35: l 40:l
`Vinyl laurate/5-azacytidine(mol/mol)
`
`Effect of Initial Water Activity
`
`Generally speaking, water activity (ow) plays a cmcial role in enzymatic reactions in non(cid:173)
`aqueous media [26-30). In the case of the enzymatic acylation of 5-azacytidine, the
`presence of water may foster the competitive hydrolysis of both the desired product 5'-O(cid:173)
`lauroyl-5-azacytidine and the acyl donor vinyl laurate. Therefore, it is of great importance
`to investigate the effect of initial water activity on the enzymatic acylation.
`As shown in Table 3, Novozym 435-mediated acylation of 5-azacytidine with vinyl
`laurate shows a clear dependence on the <tw of the reaction system. Both the initial reaction
`rate and the substrate conversion increased rapidly with increasing CYw value up to 0.07,
`beyond which further rise in <Yw value gave rise to a sharp drop in the initial reaction rate
`and the substrate conversion. This is because the presence of water in the reaction medium
`is essential for the enzyme to keep its catalytic confonnation. On the other hand, water can
`promote the hydrolysis reactions of both the product and the acyl donor. Therefore, there
`exists an optimal water activity for the enzymatic acylation. The lower water activity does
`not provide sufficient water for the buildup of the essential water shell for the enzyme, and
`the higher water activity implies excessive water and thereby the lower product yield and
`more inactivation of the enzyme caused by the acid from the competitive hydrolysis of
`vinyl laurate [31, 32). Additionally, Cl'.w showed no significant eftect on the regiosdectivity,
`which kept above 99% within the range examined. Obviously, the optimum initial water
`activity for the reaction was 0.07.
`
`Fig. 3 Effect of reaction tempera-
`turc on the enzymatic rcgiosclec-
`tivc acylation of5-azacytidine. The
`reactions were conducted in 2 ml of
`pyridine (cxw=0.07) containing
`0.02 mmol 5-azacytidine,
`0.6 mmol vinyl laurate and
`1,000 lJ Novozym 435 at 200 rpm
`and various temperatures. Filled
`triangle, initial reaction rate; filled
`square, conversion; filled circle,
`rcgioselectivity
`
`cj
`
`~ 0.7
`Cl
`§ 0.6
`~
`0.5
`2i 0.4
`"'
`Cl
`0
`(}.3
`';j
`u
`cj 0.2
`Q)
`"'
`.g
`]
`
`100
`
`90
`
`&
`
`0.1
`0.0 '---'------'----'----'----'---'40
`40
`50
`60
`30
`20
`Temperature ("C)
`
`50
`
`"' 0
`
`

`

`Appl Biochem Biotechnol (2008) 151 :21 -28
`
`27
`
`Effect of the Molar Ratio of Vinyl Laurate to 5-azacytidine
`
`Thermodynamically, high molar ratio of vinyl laurate to 5-azacytidine may push the
`reaction towards the acylation of 5-azacytidine and speed up the reaction. As depicted in
`Fig. 2, the enzymatic acylation of 5-azacytidine was greatly affected by the molar ratio of
`vinyl laurate to 5-azacytidine. Remarkable enhancement in both the initial rate and the
`substrate conversion was observed with the increase of the ratio up to 30: 1, beyond which
`both the initial rate and the substrate conversion showed no appreciable improvement with
`further increase in the molar ratio. It was also worth noting that throughout the range of
`molar ratio of vinyl laurate to 5-azacytidine tested, the regioselectivity manifested no
`variation and kept above 99~~>. Therefore, 30: I was selected as the favorable molar ratio of
`vinyl laurate to 5-azacytidine for the enzymatic acylation. It is obvious that the excessive
`amount of vinyl laurate was necessary for the lipase-catalyzed acylation, which was in good
`accordance with our previous report [30]. Also, it has been proved experimentally that the
`presence of excessive amount of vinyl laurate inhibits the hydrolysis of the desired product
`(5'-O-lauroyl-5-azacytidine). In addition, the hydrolysis of vinyl laurate might consume
`considerable amount of vinyl laurate and lower the acylation rate and substrate conversion
`[32, 33].
`
`Effect of Reaction Temperature
`
`Temperature has great effect on the activity, selectivity and stability of a biocatalyst and the
`thetmodynamic equilibrium of a reaction as well [34]. As show in Fig. 3, within the range
`from 20 to 50 °C, higher temperature resulted in both higher initial rate and higher substrate
`conversion. Further rise in temperature, however, led to a drastic drop in both the initial rate
`and substrate conversion. The regioselectivity of the reaction constantly maintained above
`99% at temperatures ranging from 20 to 60 °C. The partial inactivation of the lipase in
`pyridine at a higher temperature (above 50 °C) may partly account for the drop in both the
`initial rate and the substrate conversion, which was further supported by assaying the
`residual activity of the enzyme aiter being incubated at temperatures higher than 50 °C.
`Thus, the optimum reaction temperature was shown to be 50 °C.
`
`Conclusions
`
`In summary, the regioselective acylation of 5-azacytidine with vinyl laurate could be
`successfully perfom1ed. Under the optimized conditions, the initial rate, the substrate
`conversion and the regioselectivity were as high as 0.58 mM/min, 95.5%,, and >99%,
`respectively, after a reaction time of around 5 h. The results described here further
`highlights the versatility of lipases and show that the enzymatic acylation of nucleosides is
`a promising area.
`
`Acknowledgement
`\Ve acknowledge the National Natural Science Foundalion of China (Grant No.
`20676043), Science and Technology Project of Guangdong Province (Grant No. 2006A I 0602003;
`20071301 I 000005), Science and Technology Project of Guangzhou (Grant No. 2007Z3-E4 IO I), the Natural
`Science Foundation of Guangdong Province (Grant No. 05006571 ), the Doctoral Program of Higher
`Education (Grant No. 2007056 I 080) and the Open Project Program of the State Key Laboratory of Catalysis,
`Dalian Institute of Chemical Physics, Chinese Academy of Sciences (Grant No. N-06-06) for financial
`support.
`
`

`

`28
`
`References
`
`Appl Biochem Biotechnol (2008) 151:21-28
`
`I. Kaminskas. E, Farrell, A. T., Wang, Y. C., Sridhara, R., & Pazdur, R. (2005). Oncologisr. JO, 176--182.
`2. Romanova, D., & Novotny, L. (1996). Journal o/Chromarography B, 675, 9-15.
`3. Shatiee, M .. Griffon, J. F., Gosselin. G., Cambi, A., Vincenzctti, S .. Vita. A., et al. ( 1998). Bioclu:mical
`Phan1111cology, 56, 1237-1242.
`4. Matin, D., Teijeiro, C .. & Pina, J. J. (1996). Journal ofElectroanal,-rical Chemi.1·rrv, 407, 189--194.
`5. Seisler. J. A., Abbasi, M. M., Kelley, J. A., & Driscoll, J. S. ( 1977). Journal ofiv!edicinal Chemistrv. 20,
`806-812.
`6. Beisler, J. A. (1978). Journal of lv!edicinal Chemistrv. 21, 204--208.
`7. Ghosh, M. K., & Mitra. A. K. (1991). Pharmaceutical Research, 8, 771-775.
`8. Siedlecki. P., Boy, R. G., Comagic, S., Schirrmacher, R .. Wiessler, M., Zielenkiewicz, P .. et al. (2003).
`Biochemical and Biophvsical Research Communications, 306, 558--563.
`9. Li, X. F., Zong, !vi. H., Wu. H., & Lou. W. Y. (2006). Journal o/Biotechnology, 124, 552-560.
`I 0. Secundo, F., & Carrea, G. (2002). Journal of A-folecular Catalysis. B, Enzvmatic, 19--20, 93-102.
`11. Ferrero, M., & Gotor, V. (2000). 1\-fo11atshejie fiir Chemie, 13 I, 585--6 I 6.
`12. Moris, F .. & Gotor, V. (1993 ) . .Journal o( Organic Chemistrv, 58, 653--660.
`13. Mei, Y., Miller, L., Uao. W .. & Uross. R. A. (2003). Biomacromolecult's, 4, 70-74.
`14. Li, X. F., Lou, W. Y.. Smith, T. J .. Zong. M. H .. Wu, H., & Wang, J. F. (2006). Grel!n Chemistry, 8. 538-
`544.
`15. Ganske, F., & Bomscheuer, U. T. (2005). Journal of Molecular Catalvsis. B, £11::vmatic, 36, 40--42.
`16. Wehtje, E., Kaur, J., Adlercreutz. P .. Chand, S., & lvlattiasson, B. ( 1997). Enzl'me and Microhial
`Fechnolog_v, :! I, 502 51 0.
`17. Ducret, A., Trani, !vi., & Lortie, R. (1998). Enzyme and Microhial Technolog:v, .:2, 212-216.
`18. Han, J. J., & Rhee, J. S. (1998). t'nzym<' a11d lvlicrohial Technology, ]], 158-164.
`I 9. Ma, L.. Persson, M., & Adlcrcreutz, P. (2002). t'nzvme and Microhial Teclmolog:v, 31, 1024--1029.
`20. Wang, H., Zong. M. H., Wu, H., & Lou, W. Y. (2007). Journal o/ Biotechnologv. 1::9, 689 -695.
`21. Thetisod, M., & Klibanov, A. !vi. ( 1986). Journal ul the American Chemical Societv, I 08, 5638--5640.
`22. McCabe, R. W., & Taylor, A. (2004). Enzvme and Microhial Teclmologv. 35, 393-398.
`23. Wang, N., Chen, Z. C., Lu. D. S., & Lin. X. F. (2005). Bioorganic & Medicinal Chemistrv Ldters, 15,
`4064---4067.
`24. Uppenberg, J., ()hmcr. N., Norin, M., Hult, K., Kleywegt, G. J., Patkar, S., et al. ( 1995). Biochemistrv,
`3.J, 16838- 16851.
`25. Fan, H., Kitagawa, M., Raku. T., & Tokiwa. Y. (2004). Biotec/mnlogy Letters. 26, 1261-1264.
`26. Wehtje, E., Castes, D., & Adkrcreutz, P. (1997). Journal ofMolecular Catalvsis. B, Enz_pnatic, 3. 221-
`230.
`27. Halling, P. J. (1994). £11::vme and Micruhial Technology, 16, 178--206.
`28. Bell, G .. Halling, P. J., Moore, 8. D., Partridge, J., & Rees, D. G. ( I 995). T,-ends in Biofechnology, 13.
`468-473.
`29. Klibanov, A. M. (1997). Frend1· in Biotechnology, I 5, 97-101.
`30. Li, X. F .. Zong, M. H., & Yang, R. D. (2006). Journal o/iv!olecular Catalvsis. B, En::vmatic, 38. 48--53.
`31. Degn, P., & Zimmennann. W. (2001 ). Biotechnology and Bioengineering, 74, 483-491.
`32. Weber. H.K .. Weber, H., & Kazlauskas. R. J. (1999). Tetrahedron: .4sy111111etrv, JI!, 2635-2638.
`33. Moris, F .. & Uotor, V. (1993). Tetraht'drcm, 49. 10089-10098.
`34. Klibanov, A. M. (2001). Nature, 409, 241-246.
`
`