Synthesis of α-deoxymono and -Difluorohexopyranosyl. 1-Phosphates and Kinetic Evaluation with Thymidylyl- and. Guanidylyltransferases

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1 Synthesis of α-deoxymono and -Difluorohexopyranosyl 1-Phosphates and Kinetic Evaluation with Thymidylyl- and Guanidylyltransferases Jian-She Zhu, a Nicole E. McCormick, a Shannon C. Timmons, b,c David L. Jakeman a,b* a College of Pharmacy, 5968 College Street, Dalhousie University, Halifax, Nova Scotia, Canada B3H 3J5 b Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2, c Present address: Department of Natural Sciences, Lawrence Technological University, West Ten Mile Rd, Southfield, MI, USA David.Jakeman@dal.ca Content Synthetic routes for S2 Anomeric O-deacetylation, phosphorylation and global deprotection... S 3 Table 1 Anomeric O-Deacetylation of Fluorinated Substrates... S 3 Table 2 Phosphorylation and Global Deprotection... S 4 Enzymatic assays... S 5 Table 3 Sugar-nucleotide products MS-MS fragmentations... S 5 HPLC for substrates used in the enzyme assay with Cps2L... S 6 HPLC for substrates used in the enzyme assay with GDP-ManPP... S 6 Kinetic assays... S 7 Michealis-Menten and Lineweaver-Burk plots with Cps2L... S 7 Michealis-Menten and Lineweaver-Burk plots with GDP-ManPP... S 10 pk a 2 determination... S 12 Chemical shift change over ph in pk a 2 determination... S 12 Plots of chemical changes against ph... S 15 References... S 17 NMR spectra of intermediates and final compounds... S 18 S1

2 Synthetic routes for 1-8. a) NH 4 OAc, DMF, 30 C, b) nbuli, Cl(O)P(OPh) 2, THF, -78 C to rt, c) (i) PtO 2 /H 2, EtOH, (ii) Et 3 N-H 2 O-MeOH (1:3:7 v/v/v), quant., d) Selectfluor TM, CH 3 NO 2 -H 2 O (5:1 v/v), e) Ac 2 O/H 2 SO 4, rt, overnight. S2

3 Anomeric O-deacetylation, phosphorylation and global deprotection Table 1 Anomeric O-Deacetylation of Fluorinated Substrates Entry Substrate Product Yield a 1 52% 2 54% 3 56% 4 78% 5 52% 6 57% a isolated yield S3

4 Table 2 Phosphorylation and Global Deprotection Product Yield a Product Method Yield a 60% A 79% 59% A 81% 57% A 100% 64% A 100% 55% B 90% 49% A 100% 57% A 83% a isolated yield S4

5 Enzymatic assays Table 3 Sugar-nucleotide products MS-MS fragmentations NDP-sugar products EPI (m/z) dtdp-man 1P 563.1, 401.2, 383.5, GDP-2FMan 1P 606.1, dtdp-2fman 1P GDP-2FMan 1P 606.1, dtdp-2fglc 1P GDP-2FGlc 1P 606.1, dtdp-2,2difarap 1P 583.0, 457.0, 383.3, GDP-2,2diFArap 1P 624.2, 424.3, dtdp-3fglc 1P 565.1, GDP-3FGlc 1P 605.8, dtdp-4fglc 1P 565.1, GDP-4FGlc 1P 605.8, dtdp-4fgal1p 565.1, GDP-4FGal 1P 605.8, ddp-6fglc 1P 565.1, GDP-6FGlc 1P 606.0, S5

6 HPLC for substrates used in the enzyme assay with Cps2L HPLC for substrates used in the enzyme assay with GDP-ManPP S6

7 Kinetic assays Michealis-Menten and Lineweaver-Burk plots with Cps2L V (mau min -1 ) Parameter Value Std. Error 1 / V (mau -1 min) Vmax Km [1] (µm) / [1] (µm -1 ) V (mau min -1 ) 40 1 / V (mau -1 min) Parameter Value Std. Error Vmax Km [2] (µm) / [2] (µm -1 ) S7

8 V (mau min -1 ) 20 1 / V (mau -1 min) 0.08 Parameter Value Std. Error 0.04 Vmax Km [3] (µm) /[3] (µm -1 ) V (mau min -1 ) / V (mau -1 min) Parameter Value Std. Error Vmax Km [4] (µm) /[4] (µm) V (mau min -1 ) Parameter Value Std. Error 1 / V (mau -1 min) Vmax Km [5](µM) /[5] (µm -1 ) S8

9 V (mau min -1 ) 10 1 / V (mau -1 min) 0.1 Parameter Value Std. Error 0.05 Vmax Km [7] (µm) /[7] (µm -1 ) V (mau min -1 ) / V (mau -1 min) Parameter Value Std. Error Vmax Km [8] (µm) /[8] (µm -1 ) V (mau min -1 ) 10 1 / V (mau -1 min) Parameter Value Std. Error 0.04 Vmax Km [Man 1P] (µm) /[Man 1P] (µm -1 ) S9

10 Michealis-Menten and Lineweaver-Burk plots with GDP-ManPP V (mau min -1 ) 10 Parameter Value Std. Error Vmax Km /V (mau -1 min) [1] (µm) /[1] (µm -1 ) V (mau min -1 ) 10 1/V (mau -1 min) Parameter Value Std. Error Vmax Km [2] (µm) /[2] (µm -1 ) S10

11 V (mau min -1 ) 10 1/V (mau -1 min) Parameter Value Std. Error Vmax Km [2] (µm) Enzyme Kinetics Data /[2] (µm -1 ) 15 v (mau min -1 ) 10 1 / v (mau -1 min) Parameter Value Std. Error Vmax Km [Glc 1P] (µm) / [Glc 1P] (µm -1 ) 150 V (mau min -1 ) / V (mau -1 min) Parameter Value Std. Error Vmax Km [Man 1P] (µm) / [Man 1P] (µm -1 ) S11

12 pka 2 determination To a basic solution (ph > 9) of Glc 1P or 1-3 (9 mmol L 1 in 10% D 2 O) was titrated HCl (0.2 mol L 1 ). After each addition, the sample was thoroughly mixed, the ph measured, and 31 P{ 1 H} and 19 F{ 1 H} NMR spectra recorded. The obtained NMR spectra were processed in Topspin software. The chemical shifts of the observed signal in the 31 P{ 1 H} or 19 F{ 1 H} NMR spectra were determined using the manual peak picking function in Topspin. The chemical shift data was plotted against ph, and a best fit was obtained using GraFit 5 (Erithacus) nonlinear regression software. Chemical shift change over ph in pk a 2 determination 31 P { 1 H}NMR of Glc 1P 10.4 p 2.2 S12

13 31 P { 1 H}NMR spectra of p F { 1 H}NMR spectra of p 2.7 S13

14 31 P { 1 H}NMR spectra of p F { 1 H}NMR spectra of p 2.5 S14

15 31 P { 1 H}NMR spectra of p 2.6 Plots of chemical changes against ph S15

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17 pka determination of a-glc 1P by 31P NMR ph Parameter Value Std. Error pka Lower Limit Middle Limit Upper Limit pka References (1) Bucher, C.; Gilmour, R. Angew. Chem. Int. Ed. 2010, 49, (2) Francisco, C. G.; González, C. C.; Kennedy, A. R.; Paz, N. R.; Suárez, E. Chem. Eur. J. 2008, 14, (3) Danac, R.; Ball, L.; Gurr, S. J.; Fairbanks, A. J. Carbohydr. Res. 2008, 343, (4) Weiberth, F. J.; Gill, H. S.; Jiang, Y.; Lee, G. E.; Lienard, P.; Pemberton, C.; Powers, M. R.; Subotkowski, W.; Tomasik, W.; Vanasse, B. J.; Yu, Y. Org. Process Res. Dev. 2010, 14, 623. (5) Card, P. J.; Reddy, G. S. J. Org. Chem. 1983, 48, (6) (a) Lowary, T. L.; Eichler, E.; Bundle, D. R. Can. J. Chem. 2002, 80, (b) Card, P. J. J. Org. Chem. 1983, 48, 393. S17

18 NMR spectra of intermediates and final compounds S18

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39 ppm ppm S39

40 H-1 H-3 H-2 H-4 H-6 H-5 ppm ppm S40

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44 H-1 H-2 H-3 H-4 H-6 H-5 H-1 H-3 H-2 H-4 H-6 H-5 P-1 (P-1, H-1) (P-1, H-2) P-2 (P-2, H-6) (P-2, H-5) S44

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55 H-1 H-3 H-4 H-6 H-6 H-5 S55

56 H-1 C-1 (C-1, H-1), 1 J C-H = 180 Hz S56

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