Positively charged deoxynucleic methylthioureas: Synthesis and binding properties of pentameric thymidyl methylthiourea

Article abstract of DOI:10.1021/ja9829416

The negatively charged phosphodiester linkages in DNA {-O(PO₂^-)O-} have been replaced by a novel methylthiourea {-NHC(=SMe⁺)NH-} backbone. The backbone is positively charged, achiral, and stable and can be easily synthesiz

Full text of DOI:10.1021/ja9829416

J. Am. Chem. Soc. 1998, 120, 12419-12427
12419
Positively Charged Deoxynucleic Methylthioureas: Synthesis and
Binding Properties of Pentameric Thymidyl Methylthiourea
Dev P. Arya and Thomas C. Bruice*
Contribution from the Department of Chemistry, UniVersity of California, Santa Barbara, California 93106
ReceiVed August 17, 1998
Abstract: The negatively charged phosphodiester linkages in DNA {-O(PO2-)O-} have been replaced by a
novel methylthiourea {-NHC(dSMe+)NH-} backbone. The backbone is positively charged, achiral, and stable
and can be easily synthesized. A basic strategy for the synthesis of deoxynucleic methylthioureas (DNmt) is
described. Synthetic procedures are provided for thymidyl DNmts (1-4 linkages). Synthesis proceeds in 3′-
5′ direction and involves coupling of a protected 3′-isothiocyanate with the corresponding 5′-amine of the
growing oligo chain. Coupling reactions at room temperature are nearly quantitative, and products are easily
purified. The method of continuous variation indicates that there is an equilibrium complex with a molar ratio
of d(Tmt) to r(Ap) or d(Ap) of 2:1. Continuous variation plots carried out at temperatures from 15 to 60 °C
show a triple helical complex. Titration scans over the entire range of wavelengths (240-285 nm) confirm
binding in triple helical fashion. Thermal denaturation analyses show pronounced hysteresis with poly(dA) as
well as poly(rA). Hysteresis is more pronounced at higher ionic strengths due to a slower annealing process.
DNmt shows fidelity in binding to polynucleotides as there is little hyperchromicity observed in denaturation
of DNmt complexes to noncomplementary deoxynucleotides and ribonucleotides. The effect of ionic strength
on thermal denaturation is very pronounced, with stability greatest at low ionic strengths. Thermal denaturation
studies show melting points of >15 °C per base pair in complexes of DNmt with poly(dA) and poly(rA).
Introduction
oligonucleotides in such a manner that cellular uptake is
enhanced.12 We have previously reported the replacement of
the phosphate linkages in DNA and RNA by achiral guanido
groups that provide a new class of guanidinium (g)-linked
nucleosides which we identify as DNG.14-17 Peptide (PNA-
neutral),18,19 PHONA,20 methyl phosphonate (DNAmp-neu-
tral),21,22 phosphorothioate (DNAs-anionic),12,13,23 phosphor-
amidate,24 amido,25 MMI,26 boronated oligonucleotides,27-29
Antisense oligos with various backbone modifications1-4 have
attracted considerable attention. The negatively charged phos-
phodiester linkages of double- and triple-stranded DNA and
RNA reside side by side causing considerable charge-charge
electrostatic repulsion. This is particularly so at low (physi-
ological) ionic strength. This feature, as well as the susceptibility
of DNA and RNA to nuclease activity, limits the usefulness of
RNA and DNA as antisense or antigene drugs.5-8 Among other
requirements, successful development of antisense therapeutics
presupposes9,10 the oligos to (a) be stable in vivo, (b) have
improved permeability and cellular uptake, and (c) have greater
binding affinity with high specificity.11 The desirability of
replacing the phosphodiester linkage by other linkages that are
either neutral or positively charged and resistant toward nuclease
degradation in order to provide more effective antigene/antisense
agents has previously been discussed in detail.8,12,13 Also
considered has been the desirability in future studies to modify
(13) Morvan, F.; Porumb, H.; Degols, G.; Lefebvre, I.; Pompon, A.;
Sproat, B. S.; Rayner, B.; Malvy, C.; Lebleu, B.; Imbach, J.-L. J. Med.
Chem. 1993, 36, 280.
(14) Dempcy, R. O.; Almarsson, O.; Bruice, T. C. Proc. Natl. Acad.
Sci. U.S.A. 1994, 91, 7864.
(15) Dempcy, R. O.; Browne, K.; Bruice, T. C. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92.
(16) Dempcy, R. O.; Browne, K.; Bruice, T. C. J. Am. Chem. Soc. 1995,
117, 6140.
(17) Dempcy, R. O.; Luo, J.; Bruice, T. C. Proc. Natl. Acad. Sci. U.S.A.
1996, 93, 4326.
(18) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J. Am. Chem.
Soc. 1992, 114, 1895.
(1) Bennett, C. F. Biochem. Pharmacol. 1998, 55, 9.
(2) Alama, A.; Barbieri, F.; Cagnoli, M.; Schettini, G. Pharmacol. Res.
1997, 36, 171.
(3) Manoharan, M. Designer Antisense oligonucleotides: conjugation
chemistry and functionality placement; CRC Press: Boca Raton, FL, 1993.
(4) Mesmaeker, A. D.; Altmann, K.-H.; Wendeborn, S.; Wolf, R. M.
Pure Appl. Chem. 1997, 69, 437.
(5) Temsamani, J.; Guinot, P. Biotechnol. Appl. Biochem. 1997, 26, 65.
(6) Uhlmann, E.; Peyman, A. Chem. ReV. 1990, 90, 543.
(7) Crooke, R. M. Anticancer Drug Des. 1991, 6, 609.
(8) Crooke, S. T. Ann. ReV. Pharmacol. Toxicol. 1992, 32, 329.
(9) Miller, P.; Ts'o, P. Anticancer Drug Des. 1987, 2, 117.
(10) Milligan, J. F.; Matteucci, M. D.; Martin, J. C. J. Med. Chem. 1993,
36, 1923.
(19) Nielsen, P. E.; Haaima, G. Chem. Soc. ReV. 1997, 73.
(20) Peyman, A.; Uhlmann, E.; Wagner, K.; Augustin, S.; Breipohl, G.;
Will, D. W.; Schafer, A.; Wallmeier, H. Angew. Chem., Int. Ed. Engl. 1996,
35, 2636.
(21) Stein, C. A.; Cheng, Y.-C. Science 1993, 261, 1004.
(22) Y.Tseng, B.; Ts'o, P. O. P. Antisense Res. DeV. 1995, 5, 251.
(23) Marshall, W. S.; Caruthers, M. H. Science 1993, 259, 1564.
(24) Gryaznov, S.; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143.
(25) Mesmaeker, A. D.; Lesueur, C.; Bevierre, M.-O.; Waldner, A.;
Fritsch, V.; Wolf, R. M. Angew. Chem., Int. Ed. Engl. 1996, 35, 2790.
(26) Sanghvi, Y. S.; Swayze, E. E.; Peoc’h, D.; Bhat, B.; Dimock, S.
Nucleosides Nucleotides 1997, 16, 907.
(27) Sood, A.; Spielvogel, B.; Shaw, B. J. Am. Chem. Soc. 1989, 111,
9234.
(11) Mesmaeker, A. D.; Altmann, K.-H.; Waldner, A.; Wendeborn, S.
Curr Opin. Struct Biol. 1995, 343.
(12) Cook, P. D. In Antisense Research and Applications; Lebleu, S. T.
C. B., Ed.; CRC Press: Boca Raton, FL, 1993; p 149.
(28) Sood, A.; Shaw, B.; Spielvogel, B. J. Am. Chem. Soc. 1990, 112,
9000.
(29) Spielvogel, B.; Sood, A.; Shaw, B.; Hall, I. Pure Appl. Chem. 1991,
63, 415.
10.1021/ja9829416 CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/19/1998

12420 J. Am. Chem. Soc., Vol. 120, No. 48, 1998
Arya and Bruice
Scheme 1. Structures of Some Backbone-Modified
Oligonucleotides
as DNmt (Scheme 1). In this publication we report on the
synthesis of thymidyl methylthioureas (Tmts) and the nature
of equilibrium complexes of pentameric Tmt with poly(dA) and
poly(rA).
Materials and Methods
Materials. The concentrations of nucleotide solutions were deter-
mined using the extinction coefficients (per mole of nucleotide)
calculated according to the nearest neighboring effects.46 For d(Tmt)5
we used E268 ) 8700 M-1 cm-1. All experiments were conducted in
either (a) 0.015 M phosphate buffer (pH 7-7.5) or (b) 0.008 M
phosphate buffer (pH 6.85), and the ionic strength, µ, was adjusted
with KCl and are presented with the corresponding concentration of
KCl. The concentrations of nucleosides, expressed in M/base, were 2.1
× 10-5 to 6.3 × 10-5 M, and the ionic strength µ ) 0.06-0.6. The
concentration is referred to the limiting component forming the triplex
(e.g., a concentration of 2.1 × 10-5 M/base in the reaction of A + 2T
means [A] ) 2.1 × 10-5 M/base and [T] ) 4.2 × 10-5 M/base). All
stock solutions were kept at 4 °C between experiments.
Sample Preparation. Five magnetically stirred screw-cap cuvettes
of 1-cm path length were used for data collection: four with samples
to be measured and one for the temperature monitoring. The measure-
ment chamber was purged continuously with dry nitrogen to prevent
condensation of water vapor at lower temperatures. Annealing and
melting were followed spectrophotometrically at the given wavelength.
UV Spectroscopy and Data Collection. A Cary 1E UV/vis
spectrophotometer equipped with temperature programming and regula-
tion and a thermal melting software package were used for data
collection at λ ) 260 nm. Spectrophotometer stability and λ alignment
were checked prior to initiation of each melting point experiment. For
the Tm determinations hypochromicity was used. Data were recorded
every 1.0°. The samples were heated from 25 to 95 °C at 5 deg/min
(Scheme 1), the annealing (95-10 °C) and the melting (10-95 °C)
were conducted at 0.13 deg/min, and the samples were brought back
to 25 °C at a rate of 5 deg/min. The reaction solutions were equilibrated
for 15 min at the highest and lowest temperatures.47
Synthesis. General Procedures. All reactions were performed under
a positive atmosphere of dry nitrogen. 1H NMR spectra were obtained
at 400 MHz unless indicated otherwise and 13C NMR spectra at 125
MHz; chemical shifts (δ) are relative to internal TMS, and coupling
constants (J) are in hertz. Splitting patterns are designated singlet (s),
doublet (d), triplet (t), quartet (q), multiplet (m), or broad (b). IR spectra
were taken in KBr pellets using Perkin-Elmer 1300 spectrophotometer.
TLC was carried out on silica gel (kieselgel 60 F254) and 0.25-mm
coated commercial silica plates and visualized by UV light or
p-anisaldehyde in ethanol/sulfuric acid. Flash column chromatography
employed E. Merck silica gel (kieselgel 60, 200-400 mesh) as the
stationary phase. Individual mobile-phase systems are described in the
Experimental Section. TtT refers to thymidyl dinucleotide with a
thiourea linkage, TmtT refers to a thymidyl dinucleotide with a
methylated thiourea linkage.
ethylmorpholino and dimethylamino phosphoramidates,30,31
aminomethyl phosphonates,32 and guanido (Scheme 1) are,
among others,33-40 some examples of backbone-modified oligos
with different electrostatic attractions. Small positively charged
oligos (DNG) show unprecedented binding to nucleic acids with
retention of specificity.15,16,41 The nonionic oligo DNAmp
exhibits the ability to be transported into cells by passive
diffusion/fluid-phase endocytosis and is more resistant to
degradation than DNA.12 Both DNAmp and DNAs, however,
have individual limited drawbacks of stereoisomeric complex-
ity,32 solubility (DNAmp), and toxicity (DNAs).13,42 These
findings have led recently to the development of mixed
backbone oligonucleotides (MBOs)13,42,43 where the phospho-
rothioates and methyl phosphonates have been alternated in an
oligo backbone to produce improved antisense properties. To
investigate the effects on binding and specificity of a positively
charged backbone with hydrophobicity, we have recently
designed the polycationic nucleotide linkage with methyl-
isothiouronium salts,44 or methylated thioureas,45 abbreviated
(30) Jung, P. M.; Letsinger, R. L.; Histand, G. Nucleosides Nucleotides
1994, 13, 1597.
(31) Letsinger, R. L.; Singman, C. N.; Histand, G.; Salunkhe, M. J. Am.
Chem. Soc. 1988, 110, 4470.
(32) Huang, Q.; Syi, J.-L.; Delaney, W.; Cook, A. F. Bioconjugate Chem.
1994, 5, 47.
(33) Vasseur, J.-J.; Debart, F.; Sanghvi, Y. S.; Cook, P. D. J. Am. Chem.
Soc. 1992, 114, 4006.
(34) Blattler, M. O.; Wenz, C.; Pingoud, A.; Benner, S. A. J. Am. Chem.
Soc. 1991, 120, 2674.
(35) James, K. D.; Kiedrowski, G.v.; Ellington, A. D. Nucleosides
Nucleotides 1997, 16, 1821.
(36) Jones, R. J.; Lin, K.-Y.; Milligan, J. F.; Wadwani, S.; Matteucci,
M. D. J. Org. Chem. 1993, 58, 2983.
(37) Mesmaeker, A. D.; Haner, R.; Martin, P.; Moser, H. Acc. Chem.
Res. 1995, 28, 366.
(38) Rao, T. S.; Jayaraman, K.; Durland, R. H.; Revankar, G. R.
Nucleosides Nucleotides 1997, 13, 255.
3′-Isothiocyanato-5′-O-trityl-3′-deoxythymidine (4). To a solution
of 300 mg (0.6192 mmol) of 3′-amino-5′-O-trityldeoxythymidine in
20 mL of dichloromethane was added 150 mg (0.64 mmol) of
thiocarbonylpyridone, and the resulting solution was stirred at room
temperature for 6 h. TLC analysis in 80% EtOAc:20% hexanes shows
complete disappearance of the amine (Rf ) 0.1). The product has a Rf
value of 0.8 and pyridone of 0.45. The solvent was rotovaporated and
the product chromatographed in EtOAC:hexanes (1:1) to give 290 mg
1
of the product (88%). H NMR (200 MHz, CDCl3): δ 9.636 (1H, s,
NH), 7.493 (1H, s, 6-H), 7.493-7.279 (15H, m, trityl-H), 6.2899 (1H,
t, J ) 6.18 Hz, 1′-H), 4.606 (1H, q, J ) 7.32 Hz, 1.82 Hz, 3′-H),
4.139 (1H, q, J ) 7.32 Hz, 5.12 Hz, 4′-H), 3.60 (1H, dd, J ) 8.18 Hz,
(39) Stirchak, E. P.; Summerton, J. E. J. Org. Chem. 1987, 52, 4202.
(40) Thibon, J.; Latxague, L.; Deleris, G. J. Org. Chem. 1997, 62, 4635.
(41) Browne, K. A.; Dempcy, R. O.; Bruice, T. C. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 7051.
(42) Agrawal, S.; Jiang, Z.; Zhao, Q.; Shaw, D.; Sun, D.; Saxinger, C.
Nucleosides Nucleotides 1997, 16, 927.
(44) Arya, D. P.; Bruice, T. C. J. Am. Chem. Soc. 1998, 120, 6619.
(45) The methyl isothiouronium salts could be drawn with the positive
charge on nitrogen.
(46) Blasko, A.; Dempcy, R. O.; Minyat, E. E.; Bruice, T. C. J. Am.
Chem. Soc. 1996, 118, 7892.
(43) Iyer, R. P.; Yu, D.; Jiang, Z.; Agrawal, S. Tetrahedron 1996, 52,
14419.
(47) Blasko, A.; Dempcy, R. O.; Minyat, E. E.; Bruice, T. C. Biochem-
istry 1997, 36, 7821.

PositiVely Charged Deoxynucleic Methylthioureas
J. Am. Chem. Soc., Vol. 120, No. 48, 1998 12421
5.12 Hz, 5′-H), 2.858 (2H, m, 2′-H), 1.5612 (3H, s, Me). δ13C (d6-
DMSO + CDCl3, 75 MHz): 149.794, 146.854, 142.249, 134.811,
127.558, 127.185, 126.628, 125.812, 109.767, 84.4025, 83.395, 80.2512,
54.410, 11.657. IR (KBr pellet): 3190, 3050 (aromatic C-H), 2083,
2047 (NdCdS), 1689 (CdO), 1458, 1273, 1099, 1063. m/z: 526.2 (M
+ H)+. Anal. Calcd: C, 68.6%; H, 5.2%. Found: C, 68.5%; H, 5.3%.
5′-Azido-3′-O-trityl-5′-deoxythymidine (2). To a solution of 4 g
(14.96 mmol) of 5′-azidodeoxythymidine (1) in 100 mL of pyridine
was added 50 mg (0.4098 mmol) of (dimethylamino)pyridine followed
by 10 g (0.035 mol) of triphenylmethyl chloride. The resulting solution
was stirred at 100 °C for 48 h. TLC analysis in EtOAc shows complete
disappearance of the starting material (Rf ) 0.1). The product has a Rf
value of 0.4. The solvent was rotovaporated and the resulting
concentrate chromatographed in EtOAC:hexanes (1:1) followed by
elution with 100% EtOAc to give 7 g of the product (91.9 %). 1H NMR
(400 MHz, CDCl3): δ 8.651 (1H, s, NH), 7.90-7.25 (15Η, m, trityl-
H), 7.173 (1H, d, J ) 1.2 Hz, 6-H), 6.33 (1H, q, J ) 5.6 Hz, 3.6 Hz,
1′-H), 4.226 (1H, h, J ) 5.6 Hz, 3.6 Hz, 3.2 Hz, 3′-H), 3.910 (1H, q,
J ) 3.2 Hz, 2.8 Hz, 4′-H), 3.329 (1H, dd, J ) 9.6 Hz, 2.8 Hz, 5′-H),
2.740 (1H, dd, J ) 9.6 Hz, 3.2 Hz, 5′-H), 1.990 (1H, m, 2′-H), 1.860
(3H, d, J ) 1.2 Hz, Me), 1.699 (2H, m, 2′-H). δ13C (CDCl3, 125
MHz): 163.209, 150.055, 146.777, 134.474, 128. 885, 128.048, 127.
479, 111.245, 87.933, 84.549, 52.134, 39.079, 12.520. IR (KBr
pellet): 3051 (aromatic C-H), 2928 (aliphatic C-H), 2099 (NdNd
N), 1686 (CdO), 1481, 1442, 1274, 1029. m/z: FAB: 510 (M + H)+.
Anal. Calcd: C, 71.4%; H, 6.0%. Found: C, 71.56%; H, 6.05%.
5′-Amino-3′-O-trityl-5′-deoxythymidine (3). To a solution of 1.5
g (2.94 mmol) of 5′-azido-3′-O-trityl-5′-deoxythymidine (2) in 100 mL
of ethanol was added 50 mg of palladium on carbon catalyst (10%).
The resulting solution was hydrogenated at 45 psi for 1 h. TLC analysis
in EtOAc showed complete disappearance of the starting material (Rf
) 0.4). The product has a Rf value of 0.1. The solution was filtered
over Celite and the solvent evaporated under pressure. Chromatography
in 15% MeOH:85% EtOAc gave 1.1 g of the pure product (77.4%).
1H NMR (400 MHz, d6-DMSO): δ 7.646 (1Η, d, J ) 1.2 Hz, NH),
7.446-7.264 (15H, m, trityl-H), 6.121 (1H, q, J ) 5.2 Hz, 4.0 Hz,
1′-H), 4.20 (1H, d, J ) 5.6 Hz, 2′-H), 3.782 (1H, p, J ) 5.2 Hz, 3.2
Hz, 4′-H), 3.347 (2H, br, NH2), 2.455 (1H, dd, J ) 4.8 Hz, 5.2 Hz,
5′-H), 2.359 (1H, dd, J ) 4.8 Hz, 5.2 Hz, 5′-H), 1.719 (3H, d, J ) 1.2
Hz, Me), 1.410 (1H, d, J ) 5.6 Hz, 2′-H), 1.380 (1H, d, J ) 6 Hz,
2′-H). δ13C (d6-DMSO, 125 MHz): 163.573, 150.419, 144.198,
136.111, 128.510, 128.047, 127.266, 109.620, 87.058, 86.527, 83.561,
74.882, 43.179, 37.520, 12.083. IR (KBr pellet): 3388, 3310 (N-H),
3054 (aromatic C-H), 2948, 2917 (aliphatic C-H), 1655 (CdO), 1447,
1273, 1023. m/z: 484 (M + H)+. HRMS (FAB): 484.22312, calcd for
C29H30N3O4 484.22363.
5′-OH-TmtT-3′-OH (6). To a solution of 30 mg (0.029 mmol) of
5′-O-trityl-TtT-3′-O-trityl (5) in 20 mL of ethanol was added 10 mL
of methyl iodide, and the resulting solution was stirred at room
temperature for 2 h, followed by heating to 35 °C for 20 min. TLC
analysis in EtOAc showed complete disappearance of the starting
material (Rf ) 0.6). The product has a Rf value of 0.5. The solvents
were then evaporated under pressure, and the residue was washed with
hexanes and dried to give 29 mg of a 5′-O-trityl-TmtT-3′-O-trityl as a
yellowish solid (97.6%). m/z: 1024 (M + H)+. HRMS (FAB):
1023.4103.
To a solution of 20 mg (0.0195 mmol) of 5′-O-trityl-Tmt-T-3′-O-
trityl in 10 mL of dichloromethane was added 10 mL of trifluoroacetic
acid. The resulting solution was stirred at room temperature for 1 h.
The solvents were then evaporated under pressure, and to the resulting
gel was added 100 mL of diethyl ether. The white precipitate obtained
was filtered off and washed with ether and dichloromethane to give 9
mg of dry product (85.7%). 1H NMR (400 MHz, d6-DMSO): δ 11.56
(2Η, ΝΗ), 9.677 (1H, br, NH), 9.33 (1H, d, J ) 6.8 Hz), 9.210 (1H,
br, NH), 7.914 (1H, s, 6-H), 7.688 (1H, s, 6-H), 6.452 (1H, t, J ) 6.8
Hz, 1′-H), 6.336 (1H, t, J ) 6.8 Hz, 1′-H), 5.661 (2H, br, OH), 4.681
(1H, br, 3′-H), 4.494 (1H, m, 3′-H), 4.292-4.054 (2H, m, 4′-H), 3.989-
3.60 (4H, m, 5′-H), 2.904 (3H, s, Me), 2.715 (2H, m, 2′-H), 2.34 (2H,
m, 2′-H), 2.00 (3H, s, Me). δ13C (d6-DMSO, 125 MHz): 168.101,
163.618, 163.375, 150.319, 150.266, 136.649, 136.140, 109.816,
109.490, 83.810, 83.408, 82.915, 70.398, 60.134, 54.717, 53.837,
46.752, 44.855, 37.640, 36.525, 14.844, 14.184. IR (KBr pellet): 3465
(O-H), 3385, 3451 (N-H), 3044 (aromatic C-H), 2959 (aliphatic
C-H), 1682 (CdO), 1545, 1457, 1267, 1063. m/z: 539 (M + H)+.
HRMS (FAB): 539.1933, calcd for C22H31N6O8S 539.19240.
5′-N-MmTr-TtT-3′-OH (9a). To a solution of 100 mg (0.180 mmol)
of 3′-isothiocyanato-5′-N-MmTr-3′,5′-dideoxythymidine (7) in 10 mL
of anhydrous pyridine was added 100 mg (0.414 mmol) of 5′-amino-
5′-deoxythymidine (8) followed by 5 mg of (dimethylamino)pyridine,
and the resulting solution was stirred at room temperature for 2 h.
Pyridine was evaporated under pressure, and 20 mL of water was added
to the residue to precipitate the product. The product was extracted
into 2 × 30 mL of chloroform, washed successively with water to
remove the excess amine. The organic extracts are dried over sodium
1
sulfate and evaporated to give 130 mg of a dry product (90.9%). H
NMR (400 MHz, d6-DMSO): δ 10.596 (1H, s, NH), 10.550 (1H, s,
NH), 7.735 (2H, NH), 7.120 (4H, d J ) 7.6 Hz, trityl-H), 7.01 (2H, d,
J ) 9.2 Hz, trityl-H), 6.89 (4H, t, J ) 7.6 Hz, trityl-H), 6.819 (2H, m,
trityl-H), 6.437 (2H, d, J ) 9.2 Hz, trityl-H), 5.88 (2H, m, 1′-H), 4.734
(2H, d, J ) 4.4 Hz), 3.897 (1H, m), 3.560 (2H, m), 3.407 (3H, s, OMe),
2.89 (1H, s), 2.22 (3H, m). 1.911 (3H, m), 1.669 (1H, s), 1.528 (3H,
s, Me), 1.416 (3H, s, Me). δ13C (CDCl3, 125 MHz): 171.136, 164.460,
157.762, 150.988, 149.577, 145.913, 140.549, 137.758, 136.043,
129.754, 128.480, 127.744,126.242, 123.731, 113.065, 111.510, 84.716,
77.206, 70.144, 55.101, 45.732, 38.115, 20.986, 12.452. IR (KBr
pellet): 3467 (O-H), 3285, 3248 (N-H), 3070 (aromatic C-H), 2950
(aliphatic C-H), 2328, 1683 (CdO), 1545, 1457, 1267, 1063. m/z:
FAB 796 (M + H)+. Anal. Calcd: C, 61.9%; H, 5.7%. Found: C,
62%; H, 5.68%.
5′-NH3+-TtT-3′-OH (10a). To a solution of 120 mg (0.150 mmol)
of 5′-N-MmTr-TtT-3′-OH (9a) in 20 mL of chloroform was added 10
mL of acetic acid. The resulting solution was stirred at room temperature
for 4 h. TLC analysis in BuOH:CH3COOH:H2O (5:2:3) shows complete
disappearance of the starting material (Rf ) 0.8). The product has a Rf
value of 0.4. The solvents were then evaporated under pressure, and to
the resulting gel was added 100 mL of diethyl ether. The white
precipitate obtained was centrifuged and washed with diethyl ether and
dichloromethane to give 75 mg of dry product (94.9%). 1H NMR (400
MHz, d4-MeOD): δ 7.350 (1H, s, NH), 7.331 (1H, s, NH), 7.081 (2H,
d, J ) 1.2 Hz, 6-H), 6.063 (1H, t, J ) 7.2 Hz, 1′-H), 5.955 (1H, t, J
) 6.4 Hz, 1′-H), 4.185 (2H, m, 3′-H), 3.88 (3H, m), 3.676 (3H, s, br,
NH3+), 3.368 (1H, d, J ) 3.2 Hz), 3.327 (1H, d, J ) 3.2 Hz), 3.157
(2H, m), 2.480 (1H, br, OH), 2.235 (1H, m), 2.105 (2H, m), 1.779
(3H, s, CH3CO2-), 1.744 (3H, d, J ) 1.2 Hz, Me), 1.733 (3H, d, J )
1.2 Hz, Me). δ13C (d4-MeOD, 125 MHz): 178.892, 166.405, 152.416,
152.340, 139.588, 138.245, 112.012, 111.944, 88.487, 86.796, 86.363,
82.676, 73.019, 56.519, 42.887, 39.905, 36.810, 23.276, 12.944, 12.648,
5′-O-Trityl-TtT-3′-O-trityl (5). To a solution of 250 mg (0.474
mmol) of 3′-isothiocyanate-5′-O-trityl-3′-deoxythymidine (4) in 20 mL
of anhydrous acetonitrile was added 240 mg (0.496 mmol) of 5′-amino-
3′-O-trityl-5′-deoxythymidine (3) followed by 5 mg of (dimethylamino)-
pyridine, and the resulting solution was stirred at room temperature. A
white precipitate began to appear after 20 min. The reaction was stirred
for 2 h and the solution then cooled to 0 °C for 30 min. The white
precipitate was then collected by filtration, washed with cold ether,
and dried to give 460 mg (96.2%) of analytically pure product. TLC
analysis in EtOAc showed complete disappearance of the amine (Rf )
0.1). The product has a Rf value of 0.4, and the isothiocyanate has a Rf
1
value of 0.85. H NMR (400 MHz, CDCl3): δ 10.371 (1H, s, NH),
10.229 (1H, s, NH), 10.135 (1H, s, NH), 9.990 (1H, s, NH), 7.606
(1H, s 6-H), 7.450-7.233 (30H, m, trityl-H), 6.932 (1H, s, 6-H), 6.537
(1H, t, J ) 6.8 Hz, 1′-H), 5.989 (1H, t, J ) 6.8 Hz, 1′-H), 5.237 (1H,
b, 3′-H), 4.287 (1H, b, 3′-H), 4.146 (1H, m, 4′-H), 3.790 (1H, m, 4′-
H), 3.65 (2H, d, J ) 9.2 Hz, 2′-H), 3.345 (2H, d, J ) 9.2 Hz, 5′-H),
2.489-2.334 (4H, m, 2′-H), 1.804 (3H, s, Me), 1.392 (3H, s, Me).
δ13C (CDCl3, 125 MHz): 173.526, 164.332, 164.036, 151.064, 150.926,
150.608, 146.785, 143.758, 143.364, 128.639, 128.101, 127.941,
127.828, 127.433, 127.213, 127.152, 111.745, 111.199, 87.910, 87.478,
85.680, 84.360, 75.097, 64.803, 55.556, 36.750, 12.240, 11.640. IR
(KBr pellet): 3395, 3451 (N-H), 3361, 3317 (N-H), 3050 (aromatic
C-H), 1692 (CdO), 1535, 1447, 1267, 1053. m/z: 1009.18 (M + H)+.
Anal. Calcd: C, 70.2%; H, 5.6%. Found: C, 70.3%; H, 5.55%.

12422 J. Am. Chem. Soc., Vol. 120, No. 48, 1998
Arya and Bruice
12.466. IR (KBr pellet): 3500-2700 (O-H, N-H + C-H), 2342,
1688 (CdO), 1545, 1459, 1267, 1060. m/z: FAB, 525 (M + H)+.
5′-N-MmTr-TtTtTtTtT-3′-OH (9d). To a solution of 120 mg (0.088
mmol) of 5′-N-MmTr-TtTtTtT-3′-OH (9c) in 20 mL of chloroform was
added 10 mL of acetic acid. The resulting solution was stirred at room
temperature for 4 h. TLC analysis in BuOH:CH3COOH:H2O (5:2:3)
showed complete disappearance of the starting material (Rf ) 0.6). The
product has a Rf value of 0.35. The solvents were then evaporated under
pressure, and to the resulting gel was added 100 mL of diethyl ether.
The white precipitate obtained was centrifuged and washed with diethyl
ether (2 × 15 mL) and dichloromethane (2 × 15 mL) to give 100.5
mg of dry product (10c, 99.5%). m/z: 1089.20 (M + H)+.
Scheme 2
Scheme 3. Synthesis of Dimeric Thymidyl Methylthiourea 6
To a solution of 105 mg (0.189 mmol) of 3′-isothiocyanato-5′-N-
MmTr-3′,5′-dideoxythymidine (7) in 10 mL of anhydrous pyridine was
added 95 mg (0.0827 mmol) of 5′-NH3+-TtTtTtT-3′-OH (10c) followed
by 25 mg of (dimethylamino)pyridine, and the resulting solution was
stirred at room temperature for 6 h. Pyridine was evaporated under
pressure and the residue washed with 2 × 20 mL of chloroform to
precipitate the product and remove any unreacted isothiocyanate. TLC
analysis in BuOH:CH3COOH:H2O (5:2:3) showed complete disappear-
ance of the amine (Rf ) 0.35). The product has a Rf value of 0.65. The
white product obtained was filtered off, washed with 2 × 10 mL of
1
water, and dried to give 129 mg of product (95.5%). H NMR (400
MHz, d6-DMSO): 11.351 (5H, br, NH), 8.126 (1H, s, 6-H), 8.114 (1H,
s, 6-H), 8.048 (1H, br, NH), 7.667 (2H, br, NH), 7.543 (2H, br, 6-H),
7.479 (1H, s, 6-H), 7.39 (4H, d, J ) 5.6 Hz, trityl-H), 7.259 (4H, m,
trityl-H), 7.154 (2H, d, J ) 5.6 Hz, trityl-H), 6.81 (2H, d, J ) 6.8 Hz,
trityl-H), 6.68 (2H, d, J ) 5.6 Hz, trityl-H), 6.16 (5H, m, 1′-H), 4.707
(3H, m), 4.19-3.82 (12H, m), 3.70 (3H, OMe), 3.684 (2H, s), 3.567
(2H, br), 2.998 (5H, s), 2.372 (5H, m), 2.139 (5H, m), 1.9 (1H, s),
1.807 (6H, s, Me), 1.79 (3H, s, Me), 1.759 (3H, s, Me), 1.705 (3H, s,
Me). δ13C (d6-DMSO, 125 MHz): 182.684, 172.218, 170.048, 163.767,
157.420, 154.863, 150.690, 150.530, 150.483, 149.631, 146.273,
137.771, 137.581, 137.042, 136.117, 135.997, 135.895, 129.650,
128.303, 127.742, 126.125, 123.959, 113.049, 110.001, 109.950,
109.564, 107.682, 106.786, 84.439, 83.612, 83.077, 82.672, 82.268,
81.675, 80.746, 79.210, 74.570, 71.286, 69.725, 55.395, 54.983, 54.142,
46.801, 44.784, 38.936, 38.306, 36.780, 36.343, 35.258, 21.198, 20.867,
12.499, 12.313, 12.233, 12.193. IR (KBr pellet): 3464 (O-H), 3288,
3228 (N-H), 3060 (aromatic C-H), 2943 (aliphatic C-H), 1689 (Cd
O), 1542, 1455, 1266, 1063. m/z: FAB, 1643 (M + H)+. Anal. Calcd:
C, 54.1%; H, 5.3%. Found: C, 53.96%; H, 5.17%.
5′-NH3+-TmtTmtTmtTmtT-3′-OH (11d). To a solution of 20 mg
(0.012 mmol) of 5′-N-MmTr-TtTtTtTtT-3′-OH (9d) in 3 mL of
dimethylformamide was added 5 mL of ethanol and 20 mL of methyl
iodide. The resulting solution was stirred at 40 °C for 5 h. The solvents
were then evaporated under pressure, and to the resulting gel was added
20 mL of acetic acid and stirred for another 2 h. Acetic acid was then
evaporated off and the glue-like residue dissolved in methanol and
precipitated with ether. The precipitate was collected by centrifugation
and reprecipitated from methanol-ether to give 19 mg of a yellowish
product (90.47%). The product was then purified on a preparative
Alltech WCX cation exchange column employing 1.50 M ammonium
acetate buffer (pH 7.0) as the mobile phase. The purity of the sample
was further confirmed by running it on an analytical cation exchange
column with 1 M guanidine-HCl as the eluant. The HPLC chromato-
gram of compound 11d is shown in the Supporting Information and
has a retention time of 18 min, consistent with the presence of five
positive charges. IR (KBr pellet): 3275, 3239 (N-H), 3060 (aromatic
C-H), 2948 (aliphatic C-H), 1690 (CdO), 1542, 1455, 1264, 1063.
m/z: (FAB) 1427 (M + H)+, (ESI) 1427.4 (M + H)+, 714.3 (M +
2H)2+, λmax ) 266.7 nm.
temperature. Compound 3 on the other hand was synthesized
in a two-step process from 5′-azido thymidine. Protection of
the 3′-hydroxyl with triphenylmethyl chloride in pyridine
followed by reduction of the azide gave compound 3 in excellent
yields. Protection of 3′-hydroxyl with base labile groups
(phenoxyacetyl) led mostly to the rearrangement involving
nucleophilic attack by the 5′-amine.
The reaction of 3 with 4 was followed under varying
temperatures and solvents to optimize coupling reaction condi-
tions. Heating the isothiocyanates to speed up the reaction led
to slight decomposition of the isothiocyanate (reaction with
traces of moisture). The reaction proceeds to completion at room
temperature in a few hours with pyridine and acetonitrile as
the solvents of choice. Thiourea 5 was methylated with excess
methyl iodide and the product detritylated with TFA to give
DNmt dimer 6. Compound 6 was found to be analytically pure
by HPLC, NMR, and MS. The 1H NMR (1D and 2D) spectrum
of compound 6 is shown in the Supporting Information and
shows the resolution of similar protons in the two sugar rings.
The methyl peak of the thiourea is considerably deshielded and
appears at 3 ppm. While the assignments can be easily made in
a dimer, longer sequences show much greater overlap and the
compounds were then characterized by a combination of NMR,
MS, IR, and elemental analysis, and, in the case of methylated
thioureas, cation exchange HPLC, MS, and UV analysis.
To extend the synthesis to the formation of polymeric DNmt,
attempts were initially made to protect the 3′-hydroxyl with a
base labile protecting group so that the 3′-5′ chain extension
could (a) be carried out by deprotection of the acid labile trityl
group, (b) improve the solubility of monomer 8 and thioureas
in organic solvents, and (c) eliminate any possibility of reaction
between the 3′-hydroxyl and the 3′-isothiocyanate 7. Since initial
attempts (see discussion above) to carry out that transformation
were unsuccessful, anhydrous pyridine was used as a solvent
to couple 5′-aminodeoxythymidine 8 with 5′-N-Mm-trityl-3′-
isothiocyanato-dideoxythymidine. The reaction proceeds to
completion in few hours, and the 3′-OH has no reactivity with
the isothiocyanate. The lack of a protecting group at the 3′-OH
turned out to be a blessing in disguise because of the ease in
purification of polymeric thioureas that resulted from it. While
5′-N-tritylated thioureas are soluble in some organic solvents,
the detritylated amines are easily precipitated on addition of
Results and Discussion
Synthesis. Initial studies were focused on attempts to
synthesize (Scheme 3) dimeric thymidyl methylthiourea 6.
Coupling conditions for the formation of the thiourea linkage
were optimized using compounds 3 and 4. The isothiocyanate
4 was easily synthesized by reaction of the corresponding 3′-
amino-5′-O-trityldideoxythymidine with thiopyridone at room

PositiVely Charged Deoxynucleic Methylthioureas
Scheme 4. Synthesis Plan for T5-mt (11d)
J. Am. Chem. Soc., Vol. 120, No. 48, 1998 12423
chloroform or ether, making the whole purification process
rather trivial (see the Experimental Section).
purity of the sample was further confirmed by passing it through
an analytical cation exchange column with 1 M guanidine-HCl
as the eluant. The HPLC chromatograms of compounds 11a-d
are shown in the Supporting Information. The compounds 11b,
11c, and 11d have retention times of 9.8, 14, and 18 min,
respectively, consistent with the presence of three, four, and
five positive charges.
The synthesis of 11d (Scheme 4) was then accomplished via
a cyclic process starting with a condensation reaction between
3′-isothiocyanato-5′-N-Mm-trityl-3′,5′-deoxythymidine (7) and
5′-amino-5′-deoxythymidine (8), affording the 3′f5′ thiourea-
linked dimer 9. The synthesis of 7 was carried out by protection
of 5′-amino-3′-azido-3,′5′-dideoxythymidine14,15 with mono-
methoxytrityl chloride followed by hydrogenation of the azido
group. The reaction of the resulting 3′-amino-5′-N-Mm-trityl-
3′,5′-deoxythymidine with excess thiopyridone48 at room tem-
perature followed by flash chromatography gave compound 7
in 65% overall yield. Chain extension from the dimer followed
a cyclic two-step process involving deprotection of the 5′-amino
group with acetic acid. The resulting amine was then allowed
to condense, in near quantitative yields, with another equivalent
of 7 in pyridine for 4-8 h, depending upon the length of the
oligomer. 4-(Dimethylamino)pyridine was added in 5-10%
molar quantities to speed up the reaction. The thiourea-linked
thymidyl oligomers 10b-d were successfully converted to
compounds 11b-d by methylation of the thiourea linkages to
methylisothiouronium salts in excess methyl iodide. The reaction
can be carried out at room temperature or heated to 40 °C, if
necessary. Excess heating for over 4 h, however, results in partial
methylation of the 3′-OH. Deprotection of methylated polymers
with acetic acid was followed by a purification on a preparative
Alltech WCX cation exchange column employing 1.50 M
ammonium acetate buffer (pH 6-7.0) as the mobile phase. The
Equilibrium Complexes of Thymidyl DNmt with Poly-
(adenylic) Nucleic Acids. To investigate the interaction of 11d
{5′-NH3+-Tmt-(Tmt)4-OH} with polynucleotides, we constructed
UV continuous variation plots at different ionic strengths (µ),
wavelengths, and temperatures. The method of continuous
variation is based on the assumption that the decrease in
absorbance is proportional to the number of base pairs hydrogen
bonded between the interacting species. Mixtures of 11d with
poly(rA) at 30 °C (Figure 1) reach a minimum absorbance at a
mole fraction of ∼0.66 d(Tmt) to 0.34 r(Ap) (single phosphate-
linked riboadenosyl unit). These numbers indicate that triple-
stranded complexes are formed containing two d(Tmt) for every
r(Ap). The same results are obtained at 202 and 280 nm. The
absorbance change was much larger and the intersection of lines
much easier to define at 202 nm. At 15 °C and 60 °C the plots
(see the Supporting Information) have a minima at 0.67 mol %
of d(Tmt) at all three wavelengths. This confirms the stable
triple helical nature of the DNmt‚RNA complex suggested by
the lack of hyperchromic shifts in the thermal denaturation
studies from 15 to 60 °C.
The plot for 11d with poly(dA) at 30 °C is also centered
(48) Kim, S.; Yi, K. Y. J. Org. Chem. 1986, 51, 2613.
near a mole fraction of 0.67 d(Tmt) to 0.33 d(Ap) (Figure 1).

12424 J. Am. Chem. Soc., Vol. 120, No. 48, 1998
Arya and Bruice
Thermal Denaturation Studies. Annealing and melting
curves of complexes formed from 11d {5′-NH3+-Tmt-(Tmt)4-
OH} with DNA/RNA homooligomers exhibit hysteresis (Figure
4) at the rate of heating-cooling employed (0.13 deg/min).
Rates of heating and cooling of up to 0.5 deg/min are generally
slow enough to ensure attainment of the equilibrium in the case
of duplex to coil transitions in oligonucleotides, i.e., heating
and cooling curves coincide.47 The melting and annealing
behavior of DNmt complexes with poly(rA) and poly(dA) was
studied at different ionic strengths (µ) at a rate of 0.13 deg/
min. At low µ the hysteresis observed is abnormally large.
Examination of Figure 4a reveals the cause of this large
hysteresis. The complex rA‚d(Tmt)2 continues to melt after
reaching the terminal temperature of 95 °C; this leads to an
initial apparent increase in absorbance as the temperature descent
begins, thus causing the abnormally large hysteresis curve.
With an increase in µ (Figure 4b,c) annealing becomes slower.
Denaturation studies at high µ show larger hysteresis because
the positive and negative charges are attenuated by added salt.
As stated previously, the large hysteresis at lower µ can be
attributed to the limitation of melting points measurement to
100 °C. This behavior is clarified on studying the melting
behavior (Figure 4d) of d(Tmt)5 with dA-20 (a DNA sequence
containing 20 adenosine bases). As shown in Figure 4d, the
complex shows an ideal hysteresis curve as the triplex melting
is now well below 90 °C and the hysteresis observed at low µ
is an accurate indication of the melting and annealing processes
of DNmt with polynucleotides. In support of these observations
is the fact that after thermal melting in a solution containing
0.03 M KCl, reformation of the d(Tmt)2‚poly(dA) complex is
very slow. The melting mixture does not return to its original
absorbency even after standing for 36 h at room temperature.
The return to original absorbency is even slower at higher ionic
strengths. There is a µ above which the melting transitions for
11d complexes with poly(rA) and poly(dA) cannot be observed
or are too shallow to be accurate representations of a melting
point. Increasing the concentrations of the respective strands
does however show better transitions at these ionic strengths.
Fidelity in Binding to Polynucleotides. In the thermal
denaturation analysis of 11d {5′-NH3+-Tmt-(Tmt)4-OH} bound
Figure 1. Job plots of poly(rA) (5.3 × 10-5 M), poly(dA) (5.3 ×
10-5 M), and 11d in 15 mM K2HPO4 and 0.15 M KCl (pH 7.5) at 30
°C.
Unlike the mixing curves with r(Ap), these curves are much
shallower, as the percent hypochromicity is much lower in
binding to polydeoxynucleotides. Equilibrium complexes are
the same from 30 to 60 °C (data not shown) and confirm the
stable triple helical nature of the complexes that DNmt forms
with polydeoxyadenosyl nucleotides. Although these results
establish that the three-stranded complexes d(Tmt)2‚r(Ap) and
d(Tmt)2‚r(Ap) form under these conditions, they do not specify
the composition of equimolar mixtures of d(Tmt) with r(Ap)
and d(Ap).49 In particular, one cannot determine from these
results whether two-stranded d(Tmt)‚r(Ap) is present in equimo-
lar mixtures or whether the equilibrium products are always
d(Tmt)2‚r(Ap) and free rA polymer. Continuous variation
experiments were carried out with the measurement of complete
spectra of each of the different mixtures (Figure 2).50 Figure 2
shows that, as more of d(Tmt) is being added (decreasing % of
polyrA), there is a lowering of absorbance at almost all
wavelengths.
to poly(dA) (Figure 5a), plots of absorbance at 260 nm (A260
)
Mixing curves with 0 to 50% Tmt are hypochromic at all
wavelengths between 235 and 285 nm and curves at any
wavelength, where a spectral change occurs, consist of two
intersecting straight lines with a single minimum at 66% Tmt
residues. The hypochromicity in wavelengths greater than 280
nm is more pronounced at 15 °C as opposed to 60 °C. Since it
is unlikely that d(Tmt)‚rA has the same absorbency per
nucleotide over this entire spectral range as the mixture rA +
rA‚d(Tmt)2, it is logical to conclude that a 1:1 complex does
not exist in appreciable amounts in solutions under these
experimental conditions and that the sole complex is rA‚d(Tmt)2.
Although it may not be very clear from the scale of the titration
scans (Figure 2), there are two isosbestic points for d(Tmt) and
poly(rA) mixtures. The first occurs at 287.4 nm in 0-66%
d(Tmt) mixtures (Figure 3) due to an equivalence of extinction
coefficients for rA and rA‚d(Tmt)2; the second occurs at 296
nm in 67-100% d(Tmt) and is due to an equivalence of
extinction coefficients for d(Tmt) and rA‚d(Tmt)2.
vs temperature exhibit two distinct inflections (Tm ) 35 °C and
65 °C, µ ) 0.3). We assign the inflection points in Figure 5a to
represent denaturation curves of triple- and double-helical
structures of 11d with ss DNA {11d2‚poly(dA)} and {11d‚poly-
(dA)}. Figure 5a also indicates that, under identical conditions,
for solutions which contained 11d and either poly(dG), poly-
(dC), or poly(dT), no hyperchromic shift at 260 nm was
observed between ca. 5-93 °C. However, in the thermal
denaturation analysis of 11d bound to poly(rA) (Figure 5b), plots
of A260 vs temperature exhibit only one inflection (Tm ) 65 °C
at µ ) 0.15 and Tm ) 85 °C at µ ) 0.03), representing the
melting points of the triple helical structure {11d2‚poly(rA)}.
As observed with polydeoxynucleotides, no hyperchromic shift
at 260 nm was observed between ca. 5 and 93 °C for solutions
which contained 11d and either poly(rG) or poly(rC). There
was a hyperchromic shift at <10 °C for the denatuaration of
poly(rU) annealed to itself.41 From these results, DNmt appears
to bind with DNA and RNA with specificity in forming hybrid
duplex and triplex structures.
(49) It is well-known that the formation of AT2 and AU2 complexes
leads to a hypochromic shift at 280 nm, whereas formation of AT and AU
complexes does not. Thus A:T is isochromic at this wavelength with respect
to A + T, and mixing curves carried out at 280 nm show no hypochromism
(see ref 50).
(50) Riley, M.; Maling, B.; Chamberlin, M. J. J. Mol. Biol. 1966, 20,
359.
Equation 1 (Scheme 5) depicts the annealing process (triplex
formation). Rates of formation of triplexes are generally slower
than those of duplex formation (k2 < k1). For example, in studies
with RNA‚RNA complexes, triplex association rates have been
found to be 100 times slower than the duplex association rates.51

PositiVely Charged Deoxynucleic Methylthioureas
J. Am. Chem. Soc., Vol. 120, No. 48, 1998 12425
Figure 2. Titration scans of poly(rA) (5.3 × 10-5 M) and 11d (80% poly(rA) to 0% poly(rA)) in 15 mM K2HPO4 and 0.15 M KCl (pH 7.5) at
15 °C.
Scheme 5. Triplex Annealing and Melting
of the backbones. Formation of triplex requires the attraction
of a positively charged Tmt to a neutral duplex (Tmt‚Ap) and
thus is expected to be slower than the first step (k2 < k1). In the
continuous variation plots (annealing, Figures 1 and 2), forma-
tion of the triplex as the major species at low temperatures is
then due to the thermodynamic stability of the positively charged
Tmt‚Ap‚Tmt complex over the neutral Ap‚Tmt duplex. In
melting the triplex below 95 °C, one can observe an intermediate
duplex at high µ such that k2′ > k1′ (eq 2).
Effect of Ionic Strength (µ) on Stability of DNmt‚DNA/
RNA Complexes. 11d {5′-NH3+-Tmt-(Tmt)4-OH} has a signifi-
cantly greater affinity for poly(dA) and poly(rA) than does
thymidyl DNA. As expected, we find that change in µ has an
opposite effect on the Tm values of DNmt hybrids with DNA
Figure 3. Titration scans of poly(rA) (5.3 × 10-5 M) and 11d (90%
poly(rA) to 40% poly(rA)) in 0.15 mM K2HPO4 and 0.15 M KCl (pH
7.5) at 15 °C, showing the inflection point at 287.4 nm.
(Figure 6) or RNA as compared to DNA complexes with DNA
Electrostatic repulsions between negatively charged phosphates
have been suggested as the determining factor in rates of
association for RNA and DNA complexes.52 These electrostatic
repulsions become more important in the triplex than in the
duplex because the third strand has to come in contact with the
duplex that has twice the negative charge of a single strand.
Similar rate effects would be expected with DNmt‚DNA and
DNmt‚RNA complexes. The association of Tmt and Ap (eq 1,
k1) is expected to be fast due to attraction of the opposite charges
or RNA. This is due to electrostatic interactions being attenuated
by increasing salt concentration. Thus, while DNA‚RNA
duplexes become more stable with increasing µ, DNmt‚RNA
complexes become more stable with decrease µ. This is in
accord with our previous experience with DNG‚RNA com-
plexes. The oppositely charged backbones of the DNmt‚RNA
complex provide stability. Increase in µ saturates the opposite
charges and destabilizes the complex. At any given µ the
d(Tmt)2‚RNA triplexes are more stable than the d(Tmt)2‚DNA
triplexes.
(51) Porshke, D.; M, E. J. Mol. Biol. 1971, 30, 291.
(52) Rougee, M.; Faucon, B.; Mergny, J. L.; Barcelo, F.; Giovannangeli,
C.; Garestier, T.; Helene, C. Biochemistry 1992, 31, 9269.
The thermal denaturation results have been reduced to the
unit of Tm per base pair in order to be able to compare Tm values

12426 J. Am. Chem. Soc., Vol. 120, No. 48, 1998
Arya and Bruice
Figure 4. (a-c) Hysteresis curve of 11d and poly(rA) in 8.5 mM K2HPO4 and 8.5 mM Na2HPO4KCl (pH 6.85) at different ionic strengths (µ )
0.06-0.3). (d) Hysteresis curve of 11d and dA-20 in 8.5 mM K2HPO4 and 8.5 mM Na2HPO4KCl (pH 6.85) at an ionic strength of 0.03 (KCl). Data
were recorded every 0.4°. The samples were heated from 25 to 95 °C at 5 deg/min, the annealing (95-5 °C) and the melting (5-95 °C) were
conducted at 0.13 deg/min, and the reaction solutions were equilibrated for 15 min at the highest and lowest temperatures.
for DNmt bound to RNA and DNA with results of others for
DNA bound to modified nucleotides (Figure 7). The plots of
Tm/base pair of DNG‚RNA duplex and DNG‚RNA triplex15,41
as a function of µ are also presented for comparison. While
DNmt binds to poly(rA) and poly(dA) over a wide range of
ionic strengths, the binding is less strong than shown by DNG.
The effect of increasing µ on the destabilization of DNmt‚DNA
and DNmt‚RNA complexes is much more pronounced compared
to DNA‚RNA duplexes which become more stable with an
increase in µ.
lent. The effect of ionic strength on melting is more pronounced
with DNmt interacting with poly(A) and has an opposite effect
as compared to DNA complexes with DNA or RNA. This is
due, of course, to electrostatic interactions being attenuated by
increasing salt concentrations. Earlier works in this laboratory
show that the Tm of the double helix of pentameric thymidyl
DNA with poly(dA) at µ ) 0.12 is ca. 13 °C,41 whereas the
DNmt duplex with poly(dA) is estimated to be greater than 80
°C. At an ionic strength of 0.03, on the other hand, the five
bases of DNmt are estimated to dissociate from a double helix
with poly(rA) at >80 °C. A comparison with other positively
charged oligonucleotides, ethylmorpholino phosphoramidate
(Tm/bp ) 2-3) and aminomethyl phosphonate (Tm/bp ) 2-3),31
containing positively charged ammonium groups connected via
an alkyl linkage (Scheme 1, S6) to the central phosphorus atom
of the backbone) and DNG (guanido-linked backbone, Tm/bp
) 15-25) shows that the melting temperatures for DNmt
complexes are much higher than the morpholino- or amino-
methyl-linked oligos. This implies that, like DNG, DNmt
maintains its positive charge in proper alignment to maximize
its interaction with the backbone of the negatively charged
phosphates of the opposite strand. On comparison with DNG,
Conclusion
Replacement of the phosphodiester linkages of DNA with
methylthiourea linkages provides the polycation deoxyribo-
nucleic methylthiourea (DNmt). The thermal denaturation
analysis of 11d {5′-NH3+-Tmt-(Tmt)4-OH} bound to poly(rA)
and poly(dA) exhibits pronounced hysteresis. The DNG poly-
cation-d(Tg)4-T-azido15,41 has been shown to bind to poly(dA)
and poly(rA) with unprecedented affinity and with base-pair
specificity to provide both double- and triple-stranded helices.
Positively charged DNmt, similarly has a significantly greater
affinity for poly(dA) and poly(rA) than does the DNA equiva-

PositiVely Charged Deoxynucleic Methylthioureas
J. Am. Chem. Soc., Vol. 120, No. 48, 1998 12427
Figure 7. Plot of Tm per base pair vs µ in 8.5 mM K2HPO4 and 8.5
mM Na2HPO4 (pH 6.85). The plots are DNA. RNA duplex (9), DNG‚
RNA duplex (×), DNG‚DNA (+), DNmt‚RNA (b), and DNmt‚DNA
[first (O) and second transitions (0)]. The concentration of each of the
oligonucleotides was 2.17 × 10-5 M in bases. The ratio of 11d to
polynucleotides was 2:1. Ionic strengths were held constant by adding
potassium chloride. The data points at 100 °C for melting of DNG and
DNmt complexes are approximations. The melting points for DNG‚
RNA complexes would be much higher than that for DNmt‚RNA
complexes.
would conceivably cause a loss of fidelity in binding for longer
DNG sequences. The presence of an alkylated positively charged
thiourea allows us to investigate the balance between charge,
hydrophobicity, and fidelity. Conceivably, derivatives of DNmt
wherein the methyl group has been replaced with other moieties
to investigate the cell permeability and diffusion properties of
these compounds should not alter the enhancement in binding
as long as the positive charge rests on the backbone as opposed
to the linkers that are tethered to it.53
From these results, the following conclusions can be drawn
about DNmt: (a) Thymidyl DNmt has much stronger affinity
for DNA and RNA, due to electrostatic attractions, than DNA
for RNA or vice versa. (b) Thymidyl DNmt is specific for its
complementary tracts of adenine bases and does not interact
with guanylic, cytidylic, or uridylic tracts. (c) The thermal
stability of DNmt‚RNA and DNmt‚DNA structures is attenuated
by increasing salt concentrations. (d) DNmt forms triple helical
structures from 15 to 60 °C that are very stable under
physiological ionic strength conditions. (e) DNmt binding to
poly(rA) is stronger than that to poly(dA). (f) DNmt oligos can
be synthesized with relative ease, have an achiral backbone
linkage, and would be stable to enzymatic hydrolysis due to
the lack of a phosphodiester linkage.
Figure 5. (a) Plots of A260 vs T (°C) for (11d) annealed to poly(dA)
(+) at µ ) 0.03 (KCl), poly(dA) (]) at µ ) 0.3 (KCl), poly(dG) (0),
poly(dC) (×), and poly(dT) (O) in 8.5 mM K2HPO4 and 8.5 mM Na2-
HPO4 (pH 6.85) and an ionic strength of 0.15 (KCl). The concentration
of each of the oligonucleotides was 2.17 × 10-5 M in bases. The ratio
of 11d to polynucleotides was 2:1. (b) Plots of A260 vs T (°C) for (11d)
annealed to poly(rA) (0) at µ ) 0.05 (KCl), poly (rA) (O) at µ ) 0.12
(KCl), poly(rC) (+), poly(rG) (×) in 8.5 mM K2HPO4 and 8.5 mM
Na2HPO4 (pH 6.85) and an ionic strength of 0.15 (KCl). The
concentration of each of the oligonucleotides was 2.17 × 10-5 M in
bases. The ratio of 11d to polynucleotides was 2:1.
Acknowledgment. This work was supported by a grant from
the National Institutes of Health.
Supporting Information Available: Job plot of 11d and
poly(rA) at 60 °C, HPLC chromatograms of methylthiourea 6
and 11c,d, FAB and ESI mass spectrum of 11d, H NMR and
COSY spectrum of dimer 6, and text describing experimental
procedures and characterization of compounds 9b,c and 11b,c
(9 pages, print/PDF). See any current masthead page for ordering
information and Web access instructions.
Figure 6. Plot of Tm of 11d2:poly(rA)/poly(dA) triplexes as a function
of ionic strength in 8.5 mM K2HPO4 and 8.5 mM Na2HPO4 (pH 6.85).
The concentration of each of the oligonucleotides was 2.17 × 10-5 M
in bases. The ratio of 11d to polynucleotides was 2:1.
1
the DNmt complexes are found to melt at a lower temperature
(Figure 7). The fact that DNmts bind less tightly, than the
guanido compounds (DNGs), provides a measure of the strength
of control of binding with our cationic oligonucleotides.
Specifically, at low µ, a 5-mer of DNG does not even come off
poly(dA) in boiling water. Such strong electrostatic interactions
JA9829416
(53) The comparisons in melting point differences presented are however
of a qualitative nature and should not be overinterpreted (as pointed out by
the reviewer) to conclude trends in the thermodynamic stability of these
different backbone-modified oligos.