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, 21000 West Ten Mile Rd, Southfield, MI, USA 48075 David.Jakeman@dal.ca Content Synthetic routes for 1-8.... 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
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
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
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
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, 321.4 GDP-2FMan 1P 606.1, 424.2 dtdp-2fman 1P 565.0 GDP-2FMan 1P 606.1, 423.9 dtdp-2fglc 1P 565.0 GDP-2FGlc 1P 606.1, 423.9 dtdp-2,2difarap 1P 583.0, 457.0, 383.3, 342.0 GDP-2,2diFArap 1P 624.2, 424.3, 341.2 dtdp-3fglc 1P 565.1, 321.4 GDP-3FGlc 1P 605.8, 362.2 dtdp-4fglc 1P 565.1, 321.9 GDP-4FGlc 1P 605.8, 362.4 dtdp-4fgal1p 565.1, 321.9 GDP-4FGal 1P 605.8, 362.4 ddp-6fglc 1P 565.1, 321.4 GDP-6FGlc 1P 606.0, 362.5 S5
HPLC for substrates used in the enzyme assay with Cps2L HPLC for substrates used in the enzyme assay with GDP-ManPP S6
Kinetic assays Michealis-Menten and Lineweaver-Burk plots with Cps2L 120 0.06 100 V (mau min -1 ) 80 60 40 Parameter Value Std. Error 1 / V (mau -1 min) 0.04 0.02 20 Vmax 177.6394 7.9107 Km 267.0554 22.5580 0 0 200 400 600 [1] (µm) 0 0 0.01 0.02 0.03 0.04 1 / [1] (µm -1 ) 80 0.1 60 0.08 V (mau min -1 ) 40 1 / V (mau -1 min) 0.06 0.04 20 0 Parameter Value Std. Error Vmax 145.4594 11.1904 Km 243.5852 30.0043 0 40 80 120 160 200 240 [2] (µm) 0.02 0 0 0.01 0.02 0.03 0.04 0.05 1 / [2] (µm -1 ) S7
40 0.16 0.12 V (mau min -1 ) 20 1 / V (mau -1 min) 0.08 Parameter Value Std. Error 0.04 Vmax 44.4213 3.0760 Km 96.6650 14.9911 0 0 40 80 120 160 200 240 [3] (µm) 0 0 0.008 0.016 0.024 0.032 0.04 0.048 1/[3] (µm -1 ) 25 0.2 20 0.16 V (mau min -1 ) 15 10 1 / V (mau -1 min) 0.12 0.08 5 Parameter Value Std. Error Vmax 31.1307 5.6578 Km 299.8383 113.2223 0.04 0 0 200 400 600 [4] (µm) 0 0 0.004 0.008 0.012 0.016 1/[4] (µm) 80 0.06 60 V (mau min -1 ) 40 20 Parameter Value Std. Error 1 / V (mau -1 min) 0.04 0.02 Vmax 80.1475 7.5148 Km 130.0319 28.8933 0 0 200 400 600 [5](µM) 0 0 0.004 0.008 0.012 0.016 0.02 0.024 1/[5] (µm -1 ) S8
0.2 20 0.15 V (mau min -1 ) 10 1 / V (mau -1 min) 0.1 Parameter Value Std. Error 0.05 Vmax 25.5845 2.6773 Km 296.7101 85.5190 0 0 200 400 600 800 1000 [7] (µm) 0 0 0.002 0.004 0.006 0.008 0.01 1/[7] (µm -1 ) 30 0.16 V (mau min -1 ) 20 10 1 / V (mau -1 min) 0.12 0.08 Parameter Value Std. Error Vmax 28.5283 1.5946 Km 51.8756 9.4192 0.04 0 0 100 200 300 400 [8] (µm) 0 0 0.02 0.04 0.06 1/[8] (µm -1 ) 20 0.16 15 0.12 V (mau min -1 ) 10 1 / V (mau -1 min) 0.08 5 Parameter Value Std. Error 0.04 Vmax 25.5539 1.9594 Km 631.9092 107.5448 0 0 200 400 600 800 1000 1200 1400 [Man 1P] (µm) 0 0 0.002 0.004 1 /[Man 1P] (µm -1 ) S9
Michealis-Menten and Lineweaver-Burk plots with GDP-ManPP 30 0.15 0.12 20 V (mau min -1 ) 10 Parameter Value Std. Error Vmax 32.5953 1.6505 Km 523.3077 73.3632 1/V (mau -1 min) 0.09 0.06 0.03 0 0 1000 2000 3000 [1] (µm) 0 0 0.001 0.002 0.003 0.004 0.005 1/[1] (µm -1 ) 20 0.4 15 0.3 V (mau min -1 ) 10 1/V (mau -1 min) 0.2 5 Parameter Value Std. Error Vmax 20.9900 0.7978 Km 188.5627 19.6295 0.1 0 0 200 400 600 800 [2] (µm) 0 0 0.004 0.008 0.012 0.016 0.02 0.024 1/[2] (µm -1 ) S10
20 0.4 15 0.3 V (mau min -1 ) 10 1/V (mau -1 min) 0.2 5 Parameter Value Std. Error Vmax 20.9900 0.7978 Km 188.5627 19.6295 0.1 0 20 0 200 400 600 800 [2] (µm) Enzyme Kinetics Data 0.3 0 0 0.004 0.008 0.012 0.016 0.02 0.024 1/[2] (µm -1 ) 15 v (mau min -1 ) 10 1 / v (mau -1 min) 0.2 0.1 5 Parameter Value Std. Error 0 180 Vmax 19.1562 1.0167 Km 1304.3696 154.9894 0 1000 2000 3000 4000 5000 [Glc 1P] (µm) 0 0.04 0 0.001 0.002 0.003 1 / [Glc 1P] (µm -1 ) 150 V (mau min -1 ) 120 90 60 1 / V (mau -1 min) 0.02 30 0 Parameter Value Std. Error Vmax 211.3816 22.6284 Km 164.5472 39.8448 0 200 400 600 [Man 1P] (µm) 0 0 0.01 0.02 0.03 0.04 1 / [Man 1P] (µm -1 ) S11
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
31 P { 1 H}NMR spectra of 1 10.0 p 2.7 19 F { 1 H}NMR spectra of 1 10.0 p 2.7 S13
31 P { 1 H}NMR spectra of 2 9.7 p 2.5 19 F { 1 H}NMR spectra of 2 9.7 p 2.5 S14
31 P { 1 H}NMR spectra of 3 9.7 p 2.6 Plots of chemical changes against ph S15
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pka determination of a-glc 1P by 31P NMR 4 3.5 3 2.5 2 1.5 1 0.5 0 2 4 6 8 10 ph Parameter Value Std. Error pka 1 5.4438 0.3992 Lower Limit 3.5951 0.0201 Middle Limit 2.8980 0.6404 Upper Limit -0.0047 0.0208 pka 2 6.2461 0.0979 References (1) Bucher, C.; Gilmour, R. Angew. Chem. Int. Ed. 2010, 49, 8724. (2) Francisco, C. G.; González, C. C.; Kennedy, A. R.; Paz, N. R.; Suárez, E. Chem. Eur. J. 2008, 14, 6704. (3) Danac, R.; Ball, L.; Gurr, S. J.; Fairbanks, A. J. Carbohydr. Res. 2008, 343, 1012. (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, 4734. (6) (a) Lowary, T. L.; Eichler, E.; Bundle, D. R. Can. J. Chem. 2002, 80, 1112. (b) Card, P. J. J. Org. Chem. 1983, 48, 393. S17
NMR spectra of intermediates and final compounds S18
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ppm 1 2 3 4 5 6 7 8 9 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm S39
H-1 H-3 H-2 H-4 H-6 H-5 ppm 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 ppm S40
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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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H-1 H-3 H-4 H-6 H-6 H-5 S55
H-1 C-1 (C-1, H-1), 1 J C-H = 180 Hz S56
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