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1 Precisely regulated galactosylation of nucleoside analogues in aqueous hydrophilic solvents

2 catalyzed by solvent-stable β-galactosidase

3 Youzhi Zhou a, Jianlin Chu a, Jinsong Zhang a, Ke Liu a, Bingfang He b,*

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5 a College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, China.

6 b School of Pharmaceutical Sciences, Nanjing Tech University, China.

7 Fax: + 86 25 5813 9935

8 Tel: + 86 25 5813 9902

9 E-mail: bingfanghe@njtech.edu.cn

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Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2016

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25 Cloning, heterologous expression and purification of BMG

26 The genomic DNA of Bacillus megaterium YZ08 was extracted using the Genomic DNA extraction

27 kit (HAOJIA, Shanghai, China). The gene sequence of BMG was then obtained using primers of

28 BMG-F (5'-GCGGGATCCGACGACGACGACAAGATGTTAAAAACCGGCAAGAAATT-3') and

29 BMG-R (5'-GCGCTCGAGTTATAGAGGTTTTAGCGTAAACG-3'). The PCR product was ligated

30 into pET28a (Novagen, Germany) vector using ClonExpress II One Step Cloning Kit (Vazyme,

31 China). The verified recombinant plasmid was introduced into E. coli JM109 (DE3) for expression.

32 Transformants were cultivated in LB medium containing kanamycin (50 μg/ml) at 37 ℃, 180 rpm till

33 OD600 ~0.6-1.0, and then induced by the addition of 0.5 mM IPTG at 20 ℃ for 6 h. The cells were

34 harvested by contrifugation and used for protein purification.

35 The recombinant BMG was purified as His-tagged protein in a Ni2+-affinity chromatography. All

36 purification procedures were followed according to the manufacturer's instructions (GE Healthcare).

37 The eluate containing recombinant BMG was incubated for 12 h at 4 ℃ in the presence of

38 recombinant enterokinase (rEK, Duly, Nanjing), which effectively cleaved the BMG after Asp4-Lys.

39 After the cleavage reaction, the reaction mixture was eluted with the binding buffer through a Ni2+-

40 affinity column to remove the His-tagged rEK. The collected eluate without rEK was dialyzed in 20

41 mM Tris-HCl buffer (pH 7.5).

42 The molecular mass of native BMG was analyzed by gel filtration chromatography on a Superdex

43 200 pg gel filtration column (GE Healthcare). Purified BMG and protein standard (Gel Filtration

44 Calibration Kits, GE Healthcare) were both eluted with 50 mM phosphate buffer (pH 7.2) containing

45 150 mM NaCl at a rate of 1 mL/min. The molecular mass of BMG was determined from the standard

46 curve based on elution volumes. Protein concentrations were determined according to Bradford

47 method with bovine serum albumin as the standard.

48 HPLC analysis

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49 The mixture of enzymatic galactosylation was analyzed by RP-HPLC on an Agilent TC-C18 column

50 (4.6 mm × 250 mm, 5 μm) with a flow rate of 1 ml/min. An elution [water/methanol: 95/5 (v/v)] was

51 used for the analysis of the mixture of enzymatic galactosylation of uridine, β-D-

52 arabinofuranosyluracil, 2'-deoxyuridine, 5-methyl-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine and

53 inosine. The retention times of monogalactoside and substrate were 7.7 and 8.2 min (uridine, Fig S2);

54 7.7 and 10.1 min (β-D-arabinofuranosyluracil, Fig S3). The retention times of digalactoside,

55 monogalactoside and substrate were 5.9, 7.9 and 11.4 min (2'-deoxyuridine, Fig S4); 12.3, 16.7 and

56 18.6 min (5-methyl-2'-deoxyuridine, Fig S5); 8.6, 11.2 and 16.7 min (5-fluoro-2'-deoxyuridine, Fig

57 S6); 8.6, 12.3 and 16.7 min (inosine, Fig S7). An elution [water/methanol: 90/10 (v/v)] was used for the

58 analysis of the mixture of enzymatic galactosylation of 2', 3'-dideoxythymidine. The retention times of

59 digalactoside, monogalactoside and 2', 3'-dideoxythymidine were 10.4, 16.8 and 31.9 min (Fig S8).

60 An elution [water/methanol: 80/20 (v/v)] was used for the analysis of the mixture of enzymatic

61 galactosylation of AZT. The retention times of digalactoside, monogalactoside and AZT were 8.1,

62 12.6 and 21.5 min (Fig S9).

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64 Fig S1 SDS-PAGE analysis of the recombinant BMG. Lane 1, marker; Lane 2, total cell protein after

65 IPTG induction; Line 3, BMG purified by Ni2+ column; Lane 4, purified BMG removed of His-tag.

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67 Fig S2 HPLC analysis of the mixture of enzymatic galactosylation of uridine in buffer (balck) and 10 %

68 DMSO (blue).

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70 Fig S3 HPLC analysis of the mixture of enzymatic galactosylation of β-D-arabinofuranosyluracil in

71 buffer (balck) and 10 % DMSO (blue).

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73 Fig S4 HPLC analysis of the mixture of enzymatic galactosylation of 2'-deoxyuridine in buffer (balck)

74 and 10 % DMSO (blue).

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76 Fig S5 HPLC analysis of the mixture of enzymatic galactosylation of 5-methyl-2'-deoxyuridine in

77 buffer (balck) and 10 % DMSO (blue).

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79 Fig S6 HPLC analysis of the mixture of enzymatic galactosylation of 5-fluoro-2'-deoxyuridine in

80 buffer (balck) and 10 % DMSO (blue).

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82 Fig S7 HPLC analysis of the mixture of enzymatic galactosylation of inosine in buffer (balck) and 10 %

83 DMSO (blue).

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85 Fig S8 HPLC analysis of the mixture of enzymatic galactosylation of 2',3'-dideoxythymidine in buffer

86 (balck) and 10 % DMSO (blue).

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88 Fig S9 HPLC analysis of the mixture of enzymatic galactosylation of AZT in buffer (balck) and 10 %

89 DMSO (blue).

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91 Fig S10 Mass spectrometry for AZT monogalactoside.

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93 Fig S11 Mass spectrometry for AZT digalactoside.

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95 Fig S12 13C for the 5'-O-β-galactosyl-3'-azido-3'-deoxythymidine.

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97 Fig S13 1H for the 5'-O-β-galactosyl-3'-azido-3'-deoxythymidin.

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99 Fig S14 Mass spectrometry for 2'-deoxyuridine monogalacroside.

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101 Fig S15 Mass spectrometry for 2'-deoxyuridine digalacroside.

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103 Fig S16 13C for the 5'-O-β-galactosyl-3'-azido-2'-deoxyuridine

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105 Fig S17 1H for the 5'-O-β-galactosyl-3'-azido-2'-deoxyuridine