Supporting Information

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1 Supporting Information Wiley-VCH Weinheim, Germany

2 Synthesis and DNA Polymerase Incorporation of Colored 4-Selenothymidine Triphosphate for Polymerase recognition and DNA Visualization Julianne Caton-Williams, Zhen Huang* Department of Chemistry, Georgia State University, Atlanta, GA General Section Most solvents and reagents were purchased from Sigma, Fluka, or Aldrich (p.a.) and used without purification unless mentioned otherwise. Triethylamine (TEA) was dried over KOH (s) and distilled under argon. When necessary, solid reagents were dried under high vacuum. Reactions with compounds sensitive to air or moisture were performed under argon. Solvent mixtures are indicated as volume/volume ratios. Thin layer chromatography (TLC) was run on Merck 60 F 254 plates (0.25 mm thick; R f values in the text are for the title products), and visualized under UV-light or by a Ce-Mo staining solution (phosphomolybdate, 25 g; Ce(SO 4 ) 2.4H 2 O, 10 g; H 2 SO 4, 60 ml, conc.; H 2 O, 940 ml) with heating. Preparative TLC was performed using Merck 60 F 254 pre-coated plates (2 mm thick). Flash chromatography was performed using Fluka silica gel 60 (mesh size mm) using a silica gel:crude compound weight ratio of ca. 30:1 (or using Al 2 O 3 ). 1 H and 13 C-NMR spectra were recorded using Bruker-300 or 400 (300 or 400 MHz). All chemical shifts (δ) are in ppm relative to tetramethylsilane and all coupling constants (J) are in Hz. High resolution mass spectrum (HRMS) analysis was performed at Georgia State University and Scripps Center for Mass Spectrometry, California. The materials [including Klenow and Klenow (exo-), Epicentre] used in the polymerase extension reactions were purchased from Epicentre, TriLink Biotechnologies, and PerkinElner Life and Analytical Sciences. Oligonucleotides were purified on Microcon Ym-3 spin columns (Fisher Scientific) or on 15% polyacrylamide gel electrophoresis. quantification were preformed using a Fuji PhosphoImager. Gel imaging and S- 1

3 Synthesis of 4-(2-cyanoethyl)seleno-thymidine (3) The synthesis of the target compound (3) was accomplished by simultaneously deprotecting both the 5 -DMTr and 3 -TMS protecting groups of the intermediate (2) using 80% HOAc (Scheme 1 in the main text). Compound 2 (the 4-Se-thymidine derivative) was synthesized using the procedure described in (Salon, 2007). Compound 2 (740 mg, 1 mmol) was treated with 80% acetic acid (4 ml) and stirred at room temperature for 2 h to remove both the 3 -TMS and 5 - DMTr-protecting groups affording 3. Compound 3 containing the protected selenium functionality was purified on TLC plate (7.5% MeOH in CH 2 Cl 2, R f =0.27), yielding Compound 3 (0.26 g, 72%). We have converted Compound 3 directly to the Se-protected triphosphate, followed by the deprotection of the cyanoethyl protecting group with 50 mm K 2 CO 3 in MeOH. On the other hand, synthesis of Se TTP also worked by removing the cyanoethyl protecting group prior to the triphosphorylation, which is described later. 1 H NMR (400 MHz, CD 3 OD) δ: 2.11 (s, 3H, CH 3 ), 2.19 and 2.50 (m, 2H, H-2 ), 3.05 (m, 2H, CH 2 Se), 3.44 (m, 2H, CH 2 CN), 3.82 (m, 2H, H-5 ), 4.01 (m, 1H, H-3 ), 4.39 (m, 1H, H-4 ), 6.19 (t, 6.4 Hz, 1H, H-1 ), 8.16 (s, H-6, 1H). 13 C NMR (100 MHz, CD 3 OD) δ: (CH 3 ), 17.66, (CH 2 ), (C-2 ), (C-5 ), (C-3 ), (C-1 ), (C-4 ), (C-5), (CN), (C-6), (C-2), (C-4). HR-MS (ESI-TOF) of 3 (positive ion mode): molecular formula, C 13 H 17 N 3 O 4 Se; [M+H] + : (calc ). UV in methanol: (λ max = 314 nm) Synthesis of 4-seleno-thymidine Compound 3 (36.00 mg, 0.1 mmol) was subjected to mild base treatment using 0.05 M K 2 CO 3 in MeOH (2 ml, 1eq), which deprotected the protected selenium functionality and generated the free nucleoside (4-seleno-thymidine) in one hour. The progress of the reaction was monitored by UV and analytical TLC (7.5% MeOH in CH 2 Cl 2 ). When the reaction was completed (indicated by disappearance of the peak at 314 nm and by appearance and no further increase of the peak at 369 nm), the reaction was neutralized with diluted HCl to ph 7.5, followed by evaporation of the solvents and drying on high vacuum. Without further purification, the crude product (92% yield) S- 2

4 was used for the triphosphorylation reaction. A small portion of the product was purified on TLC plate (7.5% MeOH in CH 2 Cl 2, R f = 0.25), and its characterization was performed. 1 H NMR (400 MHz, D 2 O) δ: 2.08 (s, CH 3 ), 2.37 and 2.45 (m, 2H, H-2 ), 3.74 (m, 2H, H-5 ), 3.98 (m, 1H, H-3 ), 4.35 (m, 1H, H-4 ), 6.12 (t, 6.2 Hz, 1H, H-1 ), 7.55 (s, 1H, H-6). 13 C NMR (100 MHz, D 2 O) δ: 19.8 (CH3), (C-2 ), (C-5 ), (C-3 ), (C-1 ), (C-4 ), (C-5), (C-6), (C-2), (C-4). HR-MS (ESI-TOF) of 4-Se-thymidine (positive ion mode): molecular formula, C 10 H 14 N 2 O 4 Se; [M+Na] + : (calc ) UV in water: (λ max = 369 nm) 10 [M-H] - Intensity, counts mass (m/z) Figure 1S. MS analyses of 4-Se TTP. High resolution mass spectrometry (HRMS) of 4-Se TTP (C 10 H 17 N 2 O 13 P 3 Se). Observed mass [M-H] - : (Calculated mass [M-H] - : ). Synthesis, analysis and purification of 4-selenothymidine 5 -triphosphate (4) 5 -Triphosphate 4 was synthesized by a modification of Yoshikawa s procedure (Yoshikawa, 1967). 4-Se-thymidine (18.00 mg, 0.06 mmol) was dried on high vacuum overnight and dissolved in trimethyl phosphate (1 ml). After the solution was cooled to 0 o C under argon for 5 min., phosphorus oxychloride (0.02 ml, 0.22 mmol, 3.70 eq), dissolved in trimethyl phosphate (0.50 ml), was added dropwise over 5 min to the solution while stirring. The reaction mixture was stirred for 1 h at 0 o C before a dry DMF solution (1 ml) containing tributylammonium pyrophosphate (54.7 mg, 0.12 mm, 2 eq) and tributylamine (0.09 ml, 0.40 mmol, 6.5 eq) was added. The reaction solution was stirred for 5 min, followed by quenching with water (2 ml). If S- 3

5 it is necessary, little more base can be added to keep the solution basic (ph ). The reaction was stirred at room temperature for another 1.5 h. The volume of the resulting solution was measured, and NaCl aqueous solution (3 M) was added to the solution and to bring the NaCl concentration to 0.3 M. The crude triphosphates product was precipitated with three volume of ethanol thoroughly purged with argon. The suspension was placed at -20ºC for 30 min before centrifugation (10 min at 4000 rpm). The supernatant was removed and the precipitate (crude 4) was dried to remove ethanol. The crude product was re-dissolved in water (1 ml) and filtered prior to HPLC purification. It was analyzed by RP-HPLC using ZORBAX RX-C18 column (4.6 mm x 250 mm) before HPLC purification using a larger column (21.2 mm x 250 mm). Samples were eluded (1.0 ml/min, for analysis) with a linear gradient from 100 % buffer A [10 mm triethylammonium acetate (TEAA), ph 6.5] to 25% buffer B (50% acetonitrile, 10 mm TEAAc, ph 6.5) in 20 min. HPLC analysis and monitoring of the nucleoside triphosphates (4-Se-TTP and natural TTP) was performed at 369 nm and 267 nm, respectively. The purified product was analyzed by HR-MS (Fig. 1S) and by HPLC, comparing with natural thymidine 5 -triphosphate (Fig. 2S). 4-Se-thymidine 5 -triphosphate (negative ion mode): molecular formula, C 10 H 17 N 2 O 13 P 3 Se; [M-H] - : (calc ); UV in water: (λ max = 369 nm, ε= 1.8x10 4 M -1 cm -1 ) Time (min) Figure 2S. HPLC profile of Se TTP and TTP on ZORBAX RX-C18 column (4.6 mm x 250 mm). Samples were eluted (1 ml/min) with a linear gradient from buffer A (10 mm triethylammonium acetate (TEAAc, ph 6.5) to 25% buffer B (50% acetonitrile in water, 10 mm TEAAc, ph 6.5) in 20 min. 1: TTP, monitored at 267 nm (retention time 12.5 min); 2: Se TTP, monitored at 369 nm (retention time 15.8 min); 3: Se TTP, monitored at 267 nm. S- 4

6 Enzymatic incorporation of the 4-selenothymidine triphosphate Oligonucleotide synthesis and purification DNA oligodeoxyribonucleotides were synthesized on an Applied BioSystem 394 Synthesizer employing standard ß-cyanoethylphosphoramidite chemistry (1 µmol scale). Oligonucleotides were synthesized with DMTr-off mode and their average coupling efficiencies were greater than 99%. Purification of the oligonucleotides was conducted on 15% polyacrylamide gel electrophoresis. The gel was visualized under UV light (254 nm), crushed and soaked by water. The oligonucleotides in water solutions were recovered by NaCl (0.3 M) /EtOH (3 volume) precipitation. Oligonucleotides were quantified by UV-vis absorbance at 260 nm and characterized by MALDI-TOF MS. Primers (P1 and P2) were labeled at the 5 -termini with [γ- 32 P]-ATP (PerkinElner Life and Analytical Sciences) and T4 polynucleotide kinase (Epicentre). A list of oligonucleotide templates and primers used for the polymerase incorporation is presented in Table 1. Table 1. Sequences of DNA primers and templates used for the Se TTP incorporation. Primers: P1 (14mer) 5 -d-tag CGG GTT GCT GG-3 P2 (21mer) 5 -d-gcg TAA TAC GAC TCA CTA TAG-3 Templates: T1 (21mer) 3 -d-atc GCC CAA CGA CCA CCT TGG-5 T2 (55mer) 3 -d-cgc ATT ATG CTG AGT GAT ATC CGT TGG ACT ACT CCG GCT TTC CGG CTT TGC ATG T-5 The italic sequences of the templates are the primer-binding regions of the templates, and the underlined sequences are the template sites for the incorporation of Se TTP or TTP. template 5-32 P-dTAGCGGGTTGCTGG 3'-dATCGCCCAACGACCACCTTGG-5 Klenow TTP or Se TTP 5-32 P-dTAGCGGGTTGCTGGT 3 -datcgcccaacgaccaccttgg Extended DNA Primer Figure 3S. Enzymatic incorporation of a single nucleotide ( Se TTP or TTP) by Klenow exo(-) on T1 template. P1 primer was 5-32 P-labeled using T4 polynucleotide kinase and [γ- 32 P]-ATP. The DNA polymerization was performed with primer/template (5 µm), TTP or 4-Se TTP (0.1 mm each), and Klenow DNA polymerase (2.5 U/µL) at 37 o C for 1 h. Reactions were analyzed by 19% polyacrylamide gel electrophoresis. Lanes (1-4) contains P1 and T1. Lane 1: TTP, but no Klenow exo(-); lane 2: Klenow exo(-), but no TTP; lane 3: Se TTP, and Klenow exo(-); lane 4 (positive control): TTP, and Klenow exo(-). S- 5

7 Primer extension utilizing TTP or 4-Se TTP on a short DNA template The primer (P1) was 5 -end labeled with [γ- 32 P]-ATP. In brief, in a buffer of 20 mm Tris-HCl (ph 7.5), 5 mm MgCl 2 and 0.5 mm dithiothreitol (DTT), primer extension reactions were performed using equimolar ratios of the primer and template (5 µm each), equal concentration (0.1 mm each) of TTP (or Se TTP), and Klenow (exo-, final conc. 2.5 U/µL). In more details, a reaction cocktail, containing P1 (5-32 P-dTAGCGGGTTGCTGG-3 )/T1 (3 - datcgcccaacgaccaccttgg-5 ) and the reaction buffer, was prepared. The cocktail was equally distributed into four tubes, including the negative and positive controls, and the Se TTP reaction. The negative controls did not contain TTP or the polymerase, and the TTP reaction with the polymerase was used as the positive control. The polymerase (final concentration 2.5 U/µL) was added into each of these three tubes to initiate the reactions. All of the reaction mixtures were incubated at 37 o C for 1 h. The reactions were quenched by the addition of the gel loading dye solution (5 µl, containing 100 mm EDTA, saturated urea, 0.05% bromophenol blue, 0.05% xylene cyanol) and the analysis was performed on 19% polyacrylamide gel electrophoresis (PAGE). Its autoradiograph is presented in Figure 3S. Klenow worked in the incorporation as well as Klenow (exo-); data not shown. A. B. C Mass (m/z) Mass (m/z) Mass (m/z) Figure 4S. MALDI-TOF MS analysis of the Se TTP incorporation into DNA. A: MS spectrum of primer P1; its molecular formula (C 138 H 173 N 54 O 85 P 13 ); the observed mass of [M-H] - : 4349 (calc ). B: MS spectrum of the TTP-extended DNA (O-15mer); 4662, 4815 and 4966 [the O-15mer and its complexes with the matrix (2',4',6'- trihydroxyacetophenone, dehydrated, 150 Dalton)], and 6345 (T1 template). C: MS spectrum of the Se TTP-extended DNA (Se-15mer); 4726, 4876 and 5028 [the Se-15mer and its complexes with the matrix (dehydrated, 150 Dalton)], and 6344 (T1 template). The mass differences in B and C: the mass differences of 4662 and 4726, 4815 and 4876, and 4966 and 5028 Dalton are 64, 61 and 62 Dalton, respectively. These mass differences (64, 61 and 62; average 62.3) reflect the incorporation of the Se-modified thymidine vs natural thymidine (Se O = 79 16= 63). S- 6

8 MS analysis of the primer extension with Se TTP on a DNA template The primer extension reaction was carried out in a total reaction volume of 50 µl, and equimolar ratios (5 µm) of primer-template (P1/T1) were used. A cocktail, containing P1/T1, the polymerase buffer and the polymerase, was divided equally into two tubes containing TTP (the positive control) and Se TTP (final concentration 0.1 mm). The two reaction mixtures were incubated at 37 o C for 30 min and subsequently purified using membrane spin columns (Microcon Ym-3 spin columns, 3000 Dalton cutoff). MS analysis (MALDI-TOF, Fig. 4S) was performed at the Scripps Research Center (Mass Spectrometry Facility). Primer P1 was consumed in both primer-extension reactions using TTP or Se TTP, indicated by the disappearance of the primer mass peak and the appearance of the extended DNA peaks. The mass peak of the DNA template did not change. The mass difference between the natural TTPextended DNA and the Se TTP-extended DNA is 63, reflecting the incorporation of the Semodified thymidine vs natural thymidine (Se O = = 63). Primer extension utilizing TTP (or 4-Se TTP) and the other dntps on a short DNA template In brief, in a buffer of 20 mm Tris-HCl (ph 7.5), 5 mm MgCl 2 and 0.5 mm DTT, primer extension reactions were performed using equimolar ratios of the primer and template (5 µm), equal concentrations of TTP (or 4-Se TTP) and the other natural dntps (0.1 mm each), and Klenow (exo-, final conc. 2.5 U/ µl). In more detail, a reaction cocktail, containing the 5-32 P- labeled P1 (5 -dtagcgggttgctgg-3 )/template T1 (3 -datcgcccaacgaccacct- TGG-5 ), the polymerase reaction buffer and equimolar concentrations of TTP (or 4-Se TTP) and the other natural dntps (datp, dctp and dgtp), was prepared. The cocktail was equally distributed into four tubes, including the negative and positive controls, and the Se TTP reaction. The negative controls did not contain TTP or the polymerase, and the TTP reaction with the polymerase was used as the positive control. The polymerase (final concentration 2.5 U/µL) was added into each of these three tubes to initiate the reactions. All of the reaction mixtures were incubated at 37 o C for 1 h. The reactions were quenched by the addition of the gel loading dye solution (5 µl, containing 100 mm EDTA, saturated urea, 0.05% bromophenol blue, 0.05% xylene cyanol) and the analysis was performed on 19% polyacrylamide gel electrophoresis S- 7

9 (PAGE). Its autoradiograph is presented in Figure 5S. Klenow worked in the incorporation as well as Klenow (exo-); data not shown. template 5-32 P-dTAGCGGGTTGCTGG 3 -datcgcccaacgaccaccttgg-5 Klenow TTP or Se TTP, and the other dntps 5-32 P-dTAGCGGGTTGCTGGTGGAACC 3 -datcgcccaacgaccaccttgg Full-length DNA Short-length DNA Primer Figure 5S. Enzymatic incorporation of 4-Se TTP by Klenow exo(-) on T1 template. P1 primer was 5-32 P-labeled using T4 polynucleotide kinase and [γ- 32 P]-ATP. The DNA polymerization was performed with primer/template (5 µm), TTP (or 4-Se TTP) and the other dntps (0.1 mm each), and Klenow DNA polymerase (2.5 U/µL) at 37 o C for 1 h. Reactions were analyzed by 19% polyacrylamide gel electrophoresis. All lanes contain P1 and T1. Lane 1: all dntps, but no Klenow exo(-); lane 2: Klenow exo(-), datp, dctp and dgtp, but no TTP; lane 3: datp, dctp and dgtp, Se TTP, and Klenow exo(-); lane 4: all dntps, and Klenow exo(-). Primer extension utilizing Se TTP and the other dntps on a longer DNA template The DNA polymerase reaction using a longer template was performed with a primer (P2) (5 µm, 5 -dgcgtaatacgactcactatag-3 and a template (T2), 3 -dcgcattatgctgagtg- ATATCCGTTGGACTACTCCGGCTTTCCGGCTTTGCATGT-5. The enzymatic reactions were prepared similarly to the previous polymerase experiments that utilized all dntps, except that a lower amount of the polymerase (such as 0.05 U/µL, the final concentration) was used. The reaction mixtures were incubated at 37 o C for 1 h, and then quenched by the addition of the gel loading dye (5 µl, containing 100 mm EDTA, saturated urea, 0.05% bromophenol blue, 0.05% xylene cyanol), and analyzed on 15% PAGE. In this experiment, 4-Se TTP, 4-S TTP (purchased from TriLink Biotechnologies) and TTP were compared with each other, and Klenow and Klenow (exo-) were also compared with one another. An autoradiograph is presented in Figure 6S. There are no significant difference between Klenow (exo-) and Klenow. S- 8

10 No Kf exo- No TTP 4-Se TTP TTP 4-S TTP No Kf No TTP Se TTP TTP 4-S TTP Figure 6S. Enzymatic incorporation of all natural dntps, 4-S TTP, and 4-Se TTP into DNA using the template (T2) and Klenow fragment exo- (Kf-) and Klenow fragment (Kf). Primer P2 was 5 -end-labeled using T4 polynucleotide kinase and [γ- 32 P]-ATP. The DNA polymerization reactions were performed with primer/template (5 µm each), equimolar ratios of TTP (or 4-S TTP and 4-Se TTP) and the other dntps, (0.1 mm each), and the polymerases [final concentration 0.05 U/µL (Kf-) or 0.02 U/µL (Kf)] at 37 o C for 1 h. Reaction was analyzed by 15% polyacrylamide gel electrophoresis. All lanes contain P2 and T2. Lane 1: all natural dntps, no polymerase; Lane 2: datp, dctp, dgtp and Kf-, no TTP; Lane 3: datp, dctp, dgtp, Se TTP and Kf-; Lane 4: datp, dctp, dgtp, TTP and Kf-; Lane 5: datp, dctp, dgtp, S TTP and Kf-. Lanes (6-10) are corresponding to Lane 1-5, with Kf instead of Kf-. Primer extension time-course experiment using TTP and 4-Se TTP on a longer DNA template A time-course experiment was conducted on the primer-template (P2/T2) system. Primer P2 was 5 -end labeled using T4 polynucleotide kinase and [γ- 32 P]-ATP. In a buffer of 20 mm Tris-HCl (ph 7.5), 5 mm MgCl 2 and 0.5 mm DTT, DNA polymerization reactions (50 µl scale for each reaction) were performed using equimolar ratios (5 µm, final concentration) of the primertemplate, equal concentrations (0.1 mm each, final concentration) of TTP (or 4-Se TTP) and the other natural dntps, and Klenow (0.02 U/µL, final concentration). A cocktail, containing equimolar ratio P2/T2, polymerase buffer, datp, dctp and dgtp, was equally divided into two tubes containing TTP (positive control) or 4-Se TTP. TTP or the polymerase was absent from the negative control reactions. Klenow (0.02 U/ µl, final concentration) was added into these tubes and to initiate the reaction. Aliquots (5 µl each) were taken at the zero time point (t = 0, no polymerase) and the other time points (0.5, 1, 2, 4, 10, 30, 60 min). To quench the reactions, these aliquots were added into the individual tubes containing the gel loading dye solution (5 µl, containing 100 mm EDTA, sat. urea, 0.05% bromophenol blue, 0.05% xylene cyanol,). These samples were analyzed by 15% PAGE and quantified on Fuji PhosphorImager (Figure 3 in the main text). S- 9

11 References in the Supporting Materials: Salon, J.; Sheng, J.; Jiang, J.; Chen, G.; Caton-Williams, J.; Huang, Z., Oxygen replacement with selenium at the thymidine 4-position for the Se-base-paring and crystal structure studies, Journal of American Chemical Society, 2007, 129, Yoshikawa, M. K., T.; Takenishi, T. A Novel Method for Phosphorylation of Nucleosides to 5'-Nucleotides, Tetrahedron Lett, 1967, 50, S- 10