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

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.

DNA - ethidium - fluorescence - oligonucleotide - phenanthridinium

References and Notes

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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.

17 Azhayev AV. Antopolsky ML. Tetrahedron  2001,  57:  4977 

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.

23 For the extinction coefficients of the oligonucleotides at 260 nm, see: Puglisi JD. Tinoco I. Meth. Enzymol.  1989,  180:  304 

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.

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31 Lamos ML. Turner DH. Biochemistry  1985,  24:  2819 

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.