Supporting Information. 8. Real-time qpcr using a Ds-containing primer and fluorophor-dpxtps (Figures S1-S3).

Transcription

1 Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry Supporting Information 1. Chemical syntheses of Cy3- and Cy5-dPxTPs MR spectrum of Cy3-dPxTP P MR spectrum of Cy3-dPxTP. 4. Mass spectrum of Cy3-dPxTP MR spectrum of Cy5-dPxTP P MR spectrum of Cy5-dPxTP. 7. Mass spectrum of Cy5-dPxTP. 8. Real-time qpcr using a Ds-containing primer and fluorophor-dpxtps (Figures S1-S3). 1

2 Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry 1. Chemical syntheses of Cy3- and Cy5-dPxTPs. S 3 - S S S + P P P (a) P P P or P P P dpxtp Cy3-dPxTP Cy5-dPxTP Conditions: (a) 2 -dpxtp in 100 mm ac 3, a 2 C 3 buffer (p 8.5), Cy3 or Cy5 Mono S ester in DMF, r.t., 12h. General methods and materials Reagents and solvents were purchased from standard suppliers and used without further purification. 1 MR and 31 P MR spectra were recorded on a Bruker (300-AVM) magnetic resonance spectrometer. The triphosphate derivatives were purified with a DEAE-Sephadex A-25 column ( mm) and a C18 column (Shiseido). Electrospray ionization mass spectra (ESI-MS) and ESI-TF MS spectra were recorded on a Waters Micromass ZMD 4000 system equipped with a Waters 2690 LC system and a Micromass (Waters) Q-Tof Ultima global mass spectrometer, respectively. Cy3 TM and Cy5 TM Mono S esters were purchased from GE ealthcare. 1-(2-Deoxy-β-D-ribofuranosyl)-4-[3-(Cy3-carboxamidohexanamido)-1-propynyl] -2-nitropyrrole 5 -triphosphoric acid (Cy3-dPxTP). A 0.1 M ac 3 -a 2 C 3 buffer solution (p 8.6, 500 μl) of 1-(2-deoxy-β-D-ribofuranosyl)-4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole 2

3 Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry 5 -triphosphoric acid ( 2 -dpxtp) (8.4 μmol) was reacted with Cy3-Mono S ester (6.0 mg, 7.6 μmol) in DMF (300 μl) in the dark at room temperature. After 12 h, 50 mm TEAB (3.0 ml) was added to the reaction mixture. The product (2.7 μmol, 35%) was purified by DEAE Sephadex A-25 column chromatography (1.5 cm x 30 cm, eluted by a linear gradient from 50 mm to 1 M TEAB) and C18 PLC (eluted by a linear gradient of C 3 C in 100 mm TEAA, p 7.0). 1 MR (300 Mz, D 2 ) δ 8.55 (t, 1, J = 13.6 z), 7.90 (t, 2, J = 1.7 z), 7.85 (dd, 2, J = 1.2, 8.4 z), 7.78 (d, 1, J = 2.1 z), 7.39 (dd, 2, J = 1.9, 8.5 z), 7.19 (d, 1, J = 2.1 z), 6.64 (t, 1, J = 5.9 z), 6.39 (dd, 2, J = 2.8, 13.5 z), 4.59 (m, 1), (m, 9), 3.20 (q, 32, J = 7.3 z), 3.07 (t, 2, J = 6.5 z), 2.59 (dt, 1, J = 6.1, 13.3 z), 2.38 (dt, 1, J = 6.2, 13.8 z), (m, 4), 1.86 (m, 2), 1.77 (s, 12), (m, 4), (m, 56). 31 P MR (121 Mz, D 2 ) δ (bs, 1P), (d, 1P, J = 19.7 z), (t, 1P, J = 20.4 z). ESI-MS for C P 3 S 2 Calcd (M+) +, Found (M+) +, Calcd (M+/TEA) +, Found (M+/TEA) +, Calcd (M+/2TEA) +, Found (M+/2TEA) +, Calcd (M-) -, Found (M-) -. UV (10 mm sodium phosphate buffer, p 7.0) ε 293 nm = 18,400, ε 366 nm = 11,600, ε 550 nm = 180, (2-Deoxy-β-D-ribofuranosyl)-4-[3-(Cy5-carboxamidohexanamido)-1-propynyl] -2-nitropyrrole 5 -triphosphoric acid (Cy5-dPxTP). In a 0.1 M ac 3 -a 2 C 3 buffer solution (p 8.6, 500 μl), 1-(2-deoxy-β-D-ribofuranosyl)-4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole 5 -triphosphoric acid ( 2 -dpxtp) (7.6 μmol) was reacted with Cy5-Mono S ester (5.0 mg, 6.3 μmol) in DMF (300 μl) in the dark at room temperature. After 12 h, 50 mm TEAB (3.0 ml) was added to the reaction mixture. The product (3.3 μmol, 52%) was purified by DEAE Sephadex A-25 column chromatography (1.5 cm x 30 cm, eluted by a linear gradient from 50 3

4 Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry mm to 1 M TEAB) and C18 PLC (eluted by a linear gradient of C 3 C in 100 mm TEAA, p 7.0). 1 MR (300 Mz, D 2 ) δ 8.05 (dt, 2, J = 6.1, 13.0 z), (m, 5), 7.33 (dd, 2, J = 4.9, 8.4 z), 7.20 (d, 1, J = 1.9 z), 6.64 (t, 1, J = 6.0 z), 6.56 (t, 1, J = 12.4 z), 6.30 (dd, 2, J = 13.9, 16.5 z), 4.57 (m, 1), (m, 9), 3.20 (q, 23, J = 7.3 z), 3.07 (t, 2, J = 6.5 z), 2.59 (dt, 1, J = 6.1, 13.6 z), 2.39 (dt, 1, J = 6.2, 13.6 z), 2.22 (dt, 4, J = 7.4, 16.4 z), 1.84 (m, 2), 1.70 (s, 12), 1.59 (m, 4), (m, 45). 31 P MR (121 Mz, D 2 ) δ (d, 1P, J = 19 z), (d, 1P, J = 17.2 z), (t, 1P, J = 18.3 z). ESI-TF MS for C P 3 S 2 Calcd (M+) +, Found (M+) +, Calcd (M+/TEA) +, Found (M+/TEA) +, Calcd (M+/2TEA) +, Found (M+/2TEA) +. UV (10 mm sodium phosphate buffer, p 7.0) ε 648 nm = 280,000. 4

5 Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry 2. 1 MR spectrum of Cy3-dPxTP. S S o o + n m k j l i h g f d e b c P P P Cy3-dPxTP a n 2 5 Cy3 (Ar ) 3 1 o D k 5 l 5 a TEA f 2 2 b g j C 3 d e i m c h TEA 1 MR (300 Mz, D 2 ) spectrum of Cy3-dPxTP P MR spectrum of Cy3-dPxTP. 31 P MR (121 Mz, D 2 ) spectrum of Cy3-dPxTP. 5

6 Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry 4. Mass spectrum of Cy3-dPxTP. Calcd.: (M+) + Found : (M+) + Calcd.: (M+/TEA) + Found : (M+/TEA) + Calcd.: (M+/2TEA) + Found : (M+/2TEA) + ESI-MS (positive) spectra of Cy3-dPxTP MR spectrum of Cy5-dPxTP. S S p n o l p o m + k j i h g f d e b c P P P a Cy5-dPxTP o 2 5 Cy5 (Ar ) 3 1 n p D k 5 l 5 a TEA C 3 TEA f b g 2 2 j d e i m c h 1 MR (300 Mz, D 2 ) spectrum of Cy5-dPxTP. 6

7 Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry P MR spectrum of Cy5-dPxTP. 31 P MR (121 Mz, D 2 ) spectrum of Cy5-dPxTP. 7. Mass spectrum of Cy5-dPxTP. Calcd.: (M+) + Found : (M+) + Calcd.: (M+/TEA) + Found : (M+/TEA) + Calcd.: (M+/2TEA) + Found : (M+/2TEA) + ESI-TF MS (positive) spectra of Cy5-dPxTP. 7

8 Ct RFU (x 10 3 ) -d(rfu)/dt Ct RFU -d(rfu)/dt Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry 8. Real-time qpcr using a Ds-containing primer and fluorophor-dpxtps (Figures S1-S3) A B C PCR cycles y= x R= Temperature ( C) Initial template (copy numbers) Log Starting Quantity Figure S1. Amplification plots (A), dissociation curves (B), and linear standard curve analysis (C) of a 10-fold dilution series from 3 to copies of a 98-bp double-stranded DA, using a Ds-containing primer and Cy3-dPxTP. The cycle threshold values (Ct) obtained from panel B were plotted against the log of the template copies. A B C PCR cycles Temperature ( C) y= x R= Initial template (copy numbers) Log Starting Quantity Figure S2. Amplification plots (A), dissociation curves (B), and linear standard curve analysis (C) of a 10-fold dilution series from 3 to copies of a 98-bp double-stranded DA, using a Ds-containing primer and Cy5-dPxTP. The cycle threshold values (Ct) obtained from panel B were plotted against the log of the template copies. 8

9 Ct RFU -d(rfu)/dt Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry A B C PCR cycles y= x R= Temperature ( C) Initial template (copy numbers) Log Starting Quantity Figure S3. Amplification plots (A), dissociation curves (B), and linear standard curve analysis (C) of a 10-fold dilution series from 3 to copies of a 98-bp double-stranded DA, using a Ds-containing primer and FAM-dPxTP. The cycle threshold values (Ct) obtained from panel B were plotted against the log of the template copies. 9