Download PDF
Synlett 2007(13): 2111-2115  
DOI: 10.1055/s-2007-984899
LETTER
© Georg Thieme Verlag Stuttgart · New York

Phenanthridinium as an Artificial DNA Base: Comparison of Two Alternative Acyclic 2′-Deoxyribose Substitutes

Linda Valis, Hans-Achim Wagenknecht*
Institute for Organic Chemistry, University of Regensburg, Universitätsstr. 31, 93053 Regensburg, Germany
Fax: +49(941)94348020; e-Mail: achim.wagenknecht@chemie.uni-regensburg.de;
Further Information

Publication History

Received 18 May 2007
Publication Date:
17 July 2007 (online)

    Permissions and Reprints All articles of this category

Abstract

(S)-1-Amino-2,3-propanediol and (2S,3S)-2-amino-1,3-butanediol have been used as two different acyclic substitutes for 2′-deoxyriboside in order to synthetically incorporate the phenanthridinium chromophore of ethidium as an artificial DNA base. The comparison of the optical properties of one representative duplex bearing phenanthridinium attached to the two alternative acyclic linkers does not exhibit significant differences.

Key words

DNA - ethidium - fluorescence - oligonucleotide - phenanthridinium

14

The product 2 was co-evaporated three times with toluene and dried under high vacuum. Experimental data of 2: R f 0.60 (CH2Cl2-MeOH, 10:2). 1H NMR (250 MHz, DMSO-d 6): δ = 8.81 (d, J = 8.5 Hz, 1 H, NH), 4.70 (m, 2 H, CHOH, OH), 3.74 (m, 1 H, CHNH), 3.67-3.71 (m, 1 H, OH), 3.53-3.60 (m, 1 H, CH 2OH), 3.46-3.48 (m, 1 H, CH 2OH), 0.85 (d, J = 8.3 Hz, 3 H, Me).

15

The product 3 was purified by flash chromatography (silica gel; CH2Cl2, 0.1% pyridine, 0-2% MeOH). Experimental data of 3: R f 0.17 (CH2Cl2-MeOH, 100:0.5). 1H NMR (300 MHz, DMSO-d 6): δ = 9.25 (d, J = 8.2 Hz, 1 H, NH), 7.21-7.40, 6.86-6.89 (m, 13 H, DMT-H), 4.70 (m, 1 H, CHOH), 3.86-3.95 (m, 2 H, OH, NHCH), 3.73 (s, 6 H, OMe), 3.14-3.18 (dd, J = 3.6, 9.3 Hz, 1 H, CH 2ODMT), 2.94-2.99 (m, 1 H, CH 2ODMT), 0.93 (d, J = 6.0 Hz, 3 H, Me). 13C NMR (75 MHz, DMSO-d 6): δ = 157.9, 156.7, 156.3 (q, 2 J CF = 36 Hz), 149.5, 144.8, 136.0, 135.6, 135.4, 129.6, 127.7, 127.5, 126.5, 123.8, 117.9, 114.1 (q, 1 J CF = 288 Hz, CF3), 113.0, 85.1 (OCPh3), 64.7 (CHOH), 62.4 (CH2ODMT), 56.0 (NHCH), 54.9 (OMe), 20.0 (Me). MS (ESI): m/z (%) = 526.0(8) [M + Na]+, 303.3 (100) [DMT]+, 1028.9 (4) [2 × M + Na]+. C27H28F3NO5: 503.51.

16

The product 4 was co-evaporated twice with Et2O and dried under high vacuum. Experimental data of 4: R f 0.24 (CH2Cl2-MeOH, 20:1). 1H NMR (300 MHz, DMSO-d 6): δ = 7.19-7.41, 6.83-6.92 (m, 13 H, DMT-H), 4.43 (m, 1 H, CHOH), 3.73 (s, 6 H, OMe), 3.63 (m, 1 H, OH), 2.99-3.04 (m, 1 H, NH2CH), 2.81-2.85 (m, 1 H, CH 2ODMT), 2.58 (m, 1 H, CH 2ODMT), 0.95 (d, J = 6.3 Hz, 3 H, Me). 13C NMR (75 MHz, DMSO-d 6): δ = 157.9, 145.1, 135.9, 135.8, 129.6, 127.7, 126.4, 113.0, 85.1 (OCPh3), 66.6 (CHOH), 65.1 (CH2ODMT), 56.5 (NH2CH), 54.9 (OMe), 20.1 (Me). MS (ESI): m/z (%) = 430.1 (16) [M + Na]+, 303.3 (100) [DMT]+, 815.0 (8) [2 × M + H]+. C25H29NO4: 407.50.

18

The product 6 was purified by flash chromatography (silica gel; CH2Cl2-MeOH, 100:3, 0.1% pyridine; then CH2Cl2-MeOH, 10:3, 0.1% pyridine). Experimental data of 6: R f 0.58 (CH2Cl2-MeOH, 20:3). 1H NMR (300 MHz, DMSO-d 6): δ = 10.67 (s, 1 H, NH, 3-alloc), 10.38 (s, 1 H, NH, 8-alloc), 9.08 (d, 3 J = 9.3 Hz, 1 H, H-1), 9.02 (d, 3J = 9.1 Hz, 1 H, H-10), 8.57 (s, 1 H, H-4), 8.26 (m, 1 H, H-9), 8.10 (dd, 3 J = 9.1 Hz, 4 J = 1.1 Hz, 1 H, H-2), 7.82-7.71 (m, 6 H, 6-Ph, H-7), 7.39-7.20 (m, 9 H, ArH, DMT-H), 6.88 (m, 4 H, ArH, DMT-H), 5.99 (m, 2 H, CH2=CH, 3- and 8-alloc), 5.37 (m, 1 H, CH 2=CH, trans, 3-alloc), 5.30 (m, 1 H, CH 2=CH, trans, 8-alloc), 5.25 (m, 1 H, CH 2=CH, cis, 3-alloc), 5.21 (m, 1 H, CH 2=CH, cis, 8-alloc), 4.67 (d, 3 J = 5.5 Hz, 2 H, OCH2, 3-alloc), 4.57 (d, 3 J = 5.5 Hz, 2 H, OCH2, 8-alloc), 4.73 (m, 1 H, CHOH), 4.63 (m, 2 H, H-1′), 3.72 (s, 6 H, OMe), 3.08 (m, 1 H, CH 2ODMT, NHCH), 2.80 (m, 2 H, H-3′), 2.58 (m, 1 H, CH 2ODMT), 2.09 (m, 2 H, H-2′), 0.93 (d, J = 6.3 Hz, 3 H, Me). MS (ESI): m/z (%) = 901.4 (100) [M]+, 599.3 (15) [M + H - DMT]+, 303.3 (33) [DMT]+, 468.3 (35). C55H57N4O8 +: 902.06.

19

The product 7 was purified by flash chromatography (silica gel; CH2Cl2-MeOH, 100:5, 0.1% pyridine; then EtOAc-MeOH-H2O, 6:2:2, 0.1% pyridine). Experimental data of 7: R f 0.60 (EtOAc-MeOH-H2O, 6:2:2). 1H NMR (300 MHz, DMSO-d 6): δ = 8.67 (d, 3 J = 9.1 Hz, 1 H, H-1), 8.62 (d, 3 J = 9.3 Hz, 1 H, H-10), 7.67 (m, 5 H, 6-Ph), 7.51 (m, 2 H, H-9, H-4), 7.36-7.20 (m, 10 H, ArH, DMT-H, H-2), 6.86 (m, 4 H, ArH, DMT-H), 6.38 (s, 2 H, 3-NH2), 6.26 (s, 1 H, H-7), 5.96 (s, 2 H, 8-NH2), 4.50 (m, 3 H, H-1′, CHOH), 3.72 (s, 6 H, OMe), 3.27 (m, 1 H, NH2CH), 3.00 (m, 2 H, H-3′), 2.79 (m, 1 H, CH 2ODMT), 2.63 (m, 1 H, CH 2ODMT), 2.25 (m, 2 H, H-2′), 0.92 (d, J = 6.3 Hz, 3 H, Me). MS (ESI): m/z (%) = 733.4 (100) [M]+, 431.3 (13) [M + H - DMT]+, 303.3 (85) [DMT]+. C47H49N4O4 +: 733.92.

20

The product 8 was dried under high vacuum. Due to the high lability of the trifluoroacetyl groups the structure was confirmed only by MS. Experimental data of 8: MS (ESI): m/z (%) = 1021.3 (100) [M]+, 303.3 (38) [DMT]+. C53H46F9N4O7 +: 1021.94.

21

The product 9 was dried under high vacuum. Due to the observed high hydrolytic lability the structure was confirmed only by MS. Experimental data of 9: MS (ESI): m/z (%) = 1221.4 (100) [M]+, 303.3 (39) [DMT]+. C62H63F9N6O8P+: 1222.16.

22

An extended coupling time (1 h instead of 1.5 min for standard couplings), a higher phosphoramidite concentration (0.2 M instead of 0.067 M), and three coupling cycles interrupted by washing steps were necessary to achieve nearly quantitative coupling.

24

The extinction coefficients of phenanthridinium at 260 nm is 45.200 M-1cm-1.9

25

Experimental data of ssDNA1: ε260 = 200.700 M-1cm-1. MS (MALDI-TOF): m/z calcd for C181H225N64O98P16: 5359; found: 5359.

26

Experimental data of ssDNA2: ε260 = 200.700 M-1cm-1. MS (MALDI-TOF): m/z calcd for C182H228N64O98P16: 5374; found: 5374.

32

UV-VIS spectra and melting temperatures were measured on a Cary 100 (Varian) instrument. Fluorescence spectra were recorded on a Fluoromax-3 (Jobin-Yvon) equipment with a bandpass of 2 nm (excitation and emission) and correction for intensity and for Raman emission from the buffer solution. ESI-MS measurement was performed on a TSQ 7000 (Finnigan) instrument, MALDI-TOF MS on a Bruker Biflex III spectrometer (A = 50 mg/mL 3-hydroxypicolinic acid in MeCN-H2O, B = 50 mg/mL diammonium citrate, A:B = 9:1 for the matrix formation). C18-RP HPLC columns (300 Å) were from supplied by Supelco. The oligonucleotides were prepared on an Expedite 8909 DNA synthesizer (ABI) using CPG (1 µmol) and chemicals from ABI and Glen Research. The trityl-off oligonucleotides were cleaved and deprotected by treatment with concd NH4OH at 60 °C for 10 h (unmodified oligonucleotides), for 5.5 h (modified oligonucleotides), dried and purified by HPLC on RP-C5 (300 Å, Supelco) using the following conditions: A = NH4OAc buffer (50 mM), pH = 6.5; B = MeCN; gradient = 0-15% B (for the unmodified oligonucleotides) and 0-30% B (for the modified oligonucleotides) over 60 min. Duplexes were formed by heating to 90 °C (10 min), followed by slow cooling.