Biocatalysis in Polymer Science - American Chemical Society

Chapter 7

Chemoenzymatic Synthesis of NucleosideBranched Poly(vinyl alcohol) 1,*

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Downloaded by UNIV OF MELBOURNE on April 4, 2017 | http://pubs.acs.org Publication Date: December 10, 2002 | doi: 10.1021/bk-2003-0840.ch007

Y. Tokiwa , H. Fan1, T. Raku , and M . Kitagawa

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1

Research Institute of Biological Resources, National Institute of Bioscience and Human-Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan Konan Chemical Ind. Company, Ltd., Osaka 569-0066, Japan Polymer Research Department, Toyobo Research Center Company, 1-1, Katata 2-Chome, Otsu, Shiga 520-0292, Japan *Correspondingauthor: email: [email protected] 2

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Transesterification reactions of hydroxyl group of ribose moiety in nucleosides with divinyladipate were catalyzed by alkaline proteases to give vinyl nucleosides, and poly(vinyl alcohol) containing nucleoside branches could be obtained by theirfree-radicalpolymerization.

Synthetic polymers containing nucleobases have attracted considerable interest. They have been a focus of intensive research as functional materials (i). For example, polymers containing nucleobases are useful as resins for purification of biomolecules. Although there have been several reports on polymers having nucleobases, few studies have examined polymers having nucleosides as shown in Figure 1. Hatanaka et ah (2) reported that polystyrene containing uridine showed high inhibition against sugar transferase. Inhibition activity of uridine polymer greatly increased compared with the corresponding monomer. Furthermore, Hoshino et ai. reported the synthesis of poly(JV-acryroyl piperidine) with NAD . The polymer showed reversibly soluble-insoluble property against water depending on temperature and was useful in the affinity precipitation method for purifying biochemical products (3). Wang et al. developed a new method of synthesizing nucleoside based polymers, which used phenolic group as a polymerizable group calalyzed by peroxidase (4). Thus nucleoside polymers have recently attractive increasing interest in the biochemical field. +

© 2003 American Chemical Society

Gross and Cheng; Biocatalysis in Polymer Science ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

93


OH

OH

f°

Figure 1. Previously reported polymers containing nucleoside

+

I

2

φ fH ç=o

OH Necleoside branched polyphenol

NHCH2CONH(CH2teS~[-CH2-CI

Poly(N-acriiOyI piperidine) with NAD

Uridine containing polymer

OH OH

-


ν©


95 In our previous studies, we investigated synthesis of biodegradable sugar-branched polymers consisting of sugar, fatty acid and poly(vinyl alcohol) by a chemoenzymatic method (5). By use of the same method, we examined the enzymatic synthesis of polymerizable nucleosides havingribosemoiety such as vinyl uridine ester, vinyl cytidine ester, vinyl adenosine ester and vinyl guanosine ester and the polymerization of these vinyl esters (Figure 2). Vinyl nucleoside esters were synthesized as follows: die transesterifications between nucleoside and divinyladipate were carried out by adding alkaline proteasefromStreptomyces sp. (ALP-101, Toyobo Co., Ltd., Osaka, Japan) or from Bacillus subtilis (Bioprase SP-10, Nagase Biochemicals Co., Ltd., Kyoto, Japan) into DMF or DMF/ DMSO (4:1 v/v) containing nucleoside, agitation for 7 days at 30°C and termination of the reaction by filtering off the enzyme. After solvents evaporation, the products were chromatographed on silica gel column with a solvent consisting chloroform / methanol as eluent, and colorless powders were obtained. The concentration of each nucleoside in the reaction mixture was measured by a high performance liquid chromatograph (HPLC) with a refractive index detector. TSK gel Amide-80 (TOSOH) was used with a mobile phase of acetonitrile/water. From the change in the value of nucleoside concentration before and after reaction, the conversion rates of each nucleoside to ester were calculated. A thin layer chromatograph (TLC) was used for qualitative analysis of nucleoside ester on silica gel plate 60F plate from Merck with a solvent consisting of ethyl acetate/methanol/water (17:3:1.5, v/v/v). The spots were developed by spraying with H S 0 and heating. The position of esterification in the enzymatic synthesis reaction was established by C NMR. The transesterifications were catalyzed by the two kinds of alkaline protease. We found that the transesterification of the individual nucleosides differed in the disparity of chemical base structure, although all nucleosides contained ribose (Table I). Uridine showed high conversion rate by alkaline proteasefromBacillus subtilis. The conversion rate of uridine rapidly rose to more than 90% in 4 days. Guanosine and adenosine do not readily dissolve in DMF. Hence DMSO was added to the solvent. By addition of DMSO, the conversion rates of these nucleosides to vinyl esters were were not so low as without addition, and interestingly the only products were only 3'-esters. We found that Streptomyces sp. protease efficiently catalyzed a one-step acylation of secondary hydroxyl group inribosemoiety of adenosine and guanosine similarly to the synthesis of galactose whose secondary hydroxyl group of C-2 position was esterified in solvent containing 20% v/v DMSO (9). This positional specificity of protease compares favorably with classical chemical synthesis. The polymerization of vinyl nucleosides were carried out in DMF and DMSO by use of azo-initiator, azobisisobutyronitrile (AIBN), and the corresponding polymers were obtained. The polymerization of vinyl nucleosides proceeded easier in DMSO than in DMF. 2

4

B


96

r


OH

OH

RrUradt Cytosine

c«o

I-

OH

OH

OH

CHaOH

OH

ÇHjpH

I

to (ÇH?k

OH

011

R:Adenine Guanine

ι

? CH=CH 2

Adenine

Guanine

Cytosine

Uracil

Figure 2. Enzymatic synthesis of vinyl nucleoside derivatives

Table I. Synthesis of vinyl sugar esters catalyzed by proteases from Streptomyces sp. or Bacillus subtilis a)

Nucleoside Enzyme Solvent Product Conversion(%) Ref 6 99 Uridine A 5'-ester DMF 75 6 Cytidine 5'-ester A DMF Adenosine 50 7 Β DMF/DMSO 3'-ester 6 25 Guanosine Β DMF/DMSO 3'-ester 95 8 Thymidine 5'-ester Β DMF a) Conversion rates were calculatedfromdecrease of each nuclesoside's concentration measured by HPLC. A: alkaline proteasefromBacillus subtilis (Bioprase SP-10, Nagase), B: Alkaline proteasefromStreptomyces sp. (ALP-101, Toyobo)



Figure 3. Enzymatic synthesis of vinyl thymidine and its polymerization


98 For nucleoside having deoxyribose moiety, thymidine, transesterification with divinyladipate catalyzed by Streptomyces sp. protease was examined (Figure 3). The conversion rate of thymidine increased rapidly at two days and then reached a plateau. Only one spot was detected by TLC. The product was purified by silica gel chromatography, and was characterized by C NMR analysis. Characterization of die products by C NMR revealed that thymidine ester was substituted at C-5' position of thymidine. Rich and Dordick (10) reported that effective acylation of thymidine was catalyzed by thymidine-imprinted Bacillus licheniformis protease and the acylation with vinyl butyrate could occur at 5'- and 3'-hydroxyl groups. We found that Streptomyces sp. protease selectively catalyzed acylation of 5'-hydroxyl group in thymidine and the product was only 5'-0-vinyladipoyl thymidine. The thymidine ester was polymerized with azo-initiator, AIBN, in DMSO, to give a polymer. Nucleosides are constituents not only of polymeric biomolecules such as DNA and RNA, but also of monomeric biomolecules such as ATP, NAD, UDP and so on, and play important roles in a variety of biochemical reactions. Hence these polymers having nucleoside branches would be useful as functional materials in thefieldof biochemistry. 13


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Acknowledgment. This work was supported by the New Energy and Industrial Technology Development Organization in Japan.

References 1. Inaki, Y.; Sugita, S.; Takemoto, T. J. Polym. Sci., Part A:Poly.Chem.. 1996, 24, 3201., Inaki, Y.; Ebisutani, K.; Takemoto, T. J. Polym. Sci., Part A: Poly. Chem.. 1986, 24, 3249., Cheng, C. M.; Egbe, M. I.; Grasshoff, J.M.;Guarrera, D. J.; Pai, R. P.; Warner, J.C.;Taylor, L. D. J. Polym. Sci., Part A: Poly. Chem. 1995, 33, 2515. 2. Hatanaka, K; Takeshige, H; Kannno, K; Maruyama, A; Oishi, J; Kajihara, Y; Hashimoto, H. J.Carbohydr. Chem. 1997, 16, 667. 3. Hoshino, K.; Koumoto, K.; Morohashi, S. The 5th Asia-Pacific Biochemical Engineering Conference, Thailand (1999). 4. Wang, P.; Dordick, J. S. Macromolecules 1998, 31, 941. 5. Shibatani, S.; Kitagawa, M.; Tokiwa, Y.Biotechnol.Lett. 1997, 19, 511. 6. Tokiwa, Y.; Fan, H.; Raku, T.; Kitagawa, M. ACS Polym. Prep. 2000, 41, 1818. 7. Tokiwa, Y.; Kitagawa, M.; Fan, H.; Yokochi, T.; Raku, T.; Hiraguri, Y.; Shibatani, S.; Maekawa, Y.; Kashimura, N.; Kurane, R.Biotechnol.Tech. 1999, 13, 563. 8. Kitagawa, M.; Fan, H.; Raku, T.; Kurane, R.; Tokiwa, Y.Biotechnol.Lett. 2000, 22, 883. 9. Kitagawa, M.; Fan, H.; Raku, T.; Shibatani, S.; Maekawa, Y.; Hiraguri, Y.; Kurane, R.; Tokiwa, Y.Biotechnol.Lett. 1999, 21, 355. 10. Rich, J. O.; Dordick, J. S. J. Am. Chem. Soc. 1997, 119, 3245.


Biocatalysis in Polymer Science - American Chemical Society

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