Replacement of the negative phosphodiester linkages of DNA by positive S-methylthiourea tinkers: A novel approach to putative antisense agents

Full text of DOI:10.1021/ja980629q

J. Am. Chem. Soc. 1998, 120, 6619-6620
6619
Scheme 1
Replacement of the Negative Phosphodiester
Linkages of DNA by Positive S-Methylthiourea
Linkers: A Novel Approach to Putative Antisense
Agents
Dev P. Arya and Thomas C. Bruice*
Department of Chemistry, UniVersity of California
Santa Barbara, California 93106
ReceiVed February 25, 1998
Antisense oligonucleotides with various backbone modifications
have attracted considerable attention.1-4 Among other require-
ments, successful development of antisense therapeutics
presupposes5-7 the oligos to (a) be stable in vivo, (b) have
improved permeability and cellular uptake, and (c) have greater
binding affinity with high specificity. Peptide (PNA-neutral),8
methyl phosphonate (DNAmp-neutral),9,10 phosphorothioate (DNAs-
anionic),11,12 and guanido (DNG-positive)13-15 are, among oth-
ers,3,4,7,16 a few examples of backbone-modified oligos with
different electrostatic attractions. Small positively charged oligos
(DNGs) show unprecedented binding13-15 to nucleic acids with
retention of specificity. The nonionic oligo(DNAmp) exhibits
the ability to be transported into cells by passive diffusion/fluid-
phase endocytosis17 and is more resistant to degradation than
DNA. Both DNAmp and DNAs, however, have individual
limited drawbacks of stereoisomeric complexity,18 solubility
(DNAmp), and toxicity (DNAs).3,19 These findings have led
recently to the development of mixed-backbone oligonucleotides
(MBOs)19,20 where the phosphorothioates and methyl phospho-
nates 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 designed the polycationic nucleotide linkage
with methylisothiouronium salts,21 or methylated thioureas, ab-
breviated as DNmt (Scheme 1). This linkage, while retaining
the positive charge of DNG, also combines some structural
Scheme 2
(1) Mesmaeker, A. D.; Altmann, K.-H.; Wendeborn, S.; Wolf, R. M. Pure
Appl. Chem. 1997, 69, 437-440.
backbone features of phosphorothioate and methyl phosphonate
oligonucleotides.
(2) Mesmaeker, A. D.; Altmann, K.-H.; Waldner, A.; Wendeborn, S. Curr.
Opin. Struct. Biol. 1995, 343-355.
(3) 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, 280-287.
(4) Temsamani, J.; Guinot, P. Biotechnol. Appl. Biochem. 1997, 26, 665-
71.
(5) Crooke, R. M. Anticancer Drug Des. 1991, 6, 609-646.
(6) Crooke, S. T. Ann. ReV. Pharmacol. Toxicol. 1992, 32, 329-376.
(7) Alama, A.; Barbieri, F.; Cagnoli, M.; Schettini, G. Pharmacol. Res.
1997, 36, 171-178.
(8) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J. Am. Chem.
Soc. 1992, 114, 1895-1897.
We report herein the synthesis of the pentameric thymidyl
deoxyribonucleic methylthiourea 5c (Scheme 1), in which the
phosphodiester linkages of DNA {-O(PO2-)O-} are replaced
by methylated thioureas {-NHC(dSMe+)NH-}. Preliminary
results indicate that this DNmt model compound exhibits comple-
mentary base pair recognition toward both RNA and DNA, and
the double- and triple-helical structures composed of DNmt with
DNA demonstrate remarkable stability.
(9) Tseng, B. Y.; Ts’o, P. O. P. Antisense Res. DeV. 1995, 5, 251-260.
(10) Miller, P.; Ts’o, P. Anticancer Drug Des. 1987, 2, 117-153.
(11) Marshall, W. S.; Caruthers, M. H. Science 1993, 259, 1564-1569.
(12) Stein, C. A.; Cheng, Y.-C. Science 1993, 261, 1004-1012.
(13) Browne, K. A.; Dempcy, R. O.; Bruice, T. C. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 7051-7055.
The synthesis of 5c (Scheme 2) was accomplished via a cyclic
process starting with a condensation reaction between 3′-isothio-
cyano-5′-N-trityl-3′,5′-deoxythymidine (1) and 5′-amino-5′-deoxy-
thymidine (2), affording the 3′f5′ thiourea-linked dimer 3.14,15,21
The synthesis of 1 was carried out by protection of 5′-amino-3′-
azido-3′,5′-dideoxythymidine with trityl chloride followed by
hydrogenation of the azido group. The reaction of the resulting
3′-amino-5′-N-trityl-3′,5′-deoxythymidine with excess thiopyri-
done22 at room temperature followed by flash chromatography
gave compound 1 in 65% overall yield. Chain extension from
the dimer followed a cyclic two-step process involving depro-
tection of the 5′-amino group with acetic acid. The resulting
amine was then condensed, in quantitative yields, with another 1
equiv of 1 in pyridine for 4-8 h depending upon the length of
(14) Dempcy, R. O.; Almarsson, O.; Bruice, T. C. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 7864-7868.
(15) Dempcy, R. O.; Luo, J.; Bruice, T. C. Proc. Natl. Acad. Sci. U.S.A.
1996, 93, 4326-4330.
(16) Bennett, C. F. Biochem. Pharmacol. 1998, 55, 9-19.
(17) Cook, P. D. Antisense Research and Applications; Lebleu, S. T. C.
B., Ed.; CRC Press: Boca Raton, FL, 1993; pp 149-187.
(18) Huang, Q.; Syi, J.-L.; Delaney, W.; Cook, A. F. Bioconjugate Chem.
1994, 5, 47-57.
(19) Agrawal, S.; Jiang, Z.; Zhao, Q.; Shaw, D.; Sun, D.; Saxinger, C.
Nucleosides Nucleotides 1997, 16, 927-936.
(20) Iyer, R. P.; Yu, D.; Jiang, Z.; Agrawal, S. Tetrahedron 1996, 52,
14419-14436.
(21) The methyl isothiouronium salts could be drawn with the positive
charge on nitrogen.
(22) Complete synthetic details will be provided in our full paper.
S0002-7863(98)00629-5 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/18/1998

6620 J. Am. Chem. Soc., Vol. 120, No. 26, 1998
Communications to the Editor
{5c2‚poly(dA) and 5c‚poly(dA)}. Analysis of Figure 2 also
indicates that the DNmt triplex with RNA is more stable than
the corresponding DNmt‚DNA complex [triplex mp ) 61 °C for
poly(rA) vs mp ) 32 °C for poly(dA)]. Under identical
conditions, for solutions which contained 5c and either poly(dG)
or poly(dC), no hyperchromic shift at 260 nm was observed
between ca. 5-93 °C (Figure 2).25 From these preliminary results,
DNmt appears to bind with DNA and RNA with specificity in
forming hybrid duplex and triplex structures.
5c has a significantly greater affinity for poly(dA) and poly-
(rA) than does thymidyl DNA,26 and the effect of ionic strength
on duplex stability is quite pronounced. As expected,13,23,24,27 we
find that the ionic strength has an opposite effect on the Tm values
of DNmt hybrids with DNA (Figure 2) or RNA as compared to
DNA complexes with DNA or RNA since electrostatic interac-
tions are attenuated by increasing salt concentration. DNmt
compounds are stable under the acidic and basic conditions
required for DNA synthesis14,28-31 and would be expected to be
stable in vivo due to the absence of phosphodiester linkages.
In conclusion, we show in these preliminary investigations that
the attractive forces between negatively charged DNA or RNA
and positively charged DNmt contribute significantly to the
stability of heteroduplex and -triplex structures formed between
these species. DNmt, with positively charged {-NHC(dSMe+)-
NH-} linkages, has a lessened electrostatic attraction to DNA
and RNA when compared to the positively charged DNG with
guanido {-NHC(dNH2+)NH-} linkages. DNmt duplex and
triplex structures with DNA or RNA, however, are much more
stable at physiological ionic strength than the corresponding
structures with DNA and RNA as the sole components while less
stable than the guanido-linked thymidines that form even stronger
duplex and triplex structures. Further studies, involving the use
of mismatch sequences and complementary binding studies with
mixed sequences would help us define the line between the
binding enhancement due to electrostatic attractions of the
backbones vs the specificity of the sequence due to hydrogen
bonding. The increased hydrophobicity due to the alkyl group
should considerably alter the cell diffusion and uptake of these
oligos. Combined with the fact that these oligonucleotides can
be synthesized with relative ease, have an achiral backbone
linkage, would be stable to enzymatic hydrolysis due to the lack
of a phosphodiester linkage, have increased binding due to the
oppositely charged backbone and a alkyl group, which can be
altered to control the hydrophobic/electrostatic interactions,
DNmts should serve as a promising lead in the development of
new antisense therapeutics.
Figure 1. Job plot of poly(rA) (5.3 × 10-5 M) and 5c in 0.15 mM
K2HPO4 and 0.15 M KCl, pH 7.5.
Figure 2. Plots of A260 vs T (°C) for (5c) annealed to poly(dA) (+) at
µ ) 0.05 (KCl), poly(dA) (O) at µ ) 0.25 (KCl), poly(rA) (0), poly-
(dC) ([), and poly(dG) (×) at pH 7.5 (0.001 M K2HPO4 buffer) and an
ionic strength of 0.25 (KCl). The concentration of each of the oligo-
nucleotides was 2.17 × 10-5 M in bases. The ratio of 5c to polynucleotides
was 2:1. The change in absorbance (260 nm) with increasing temperature
was monitored using a Cary 1-E spectrophotometer.
the oligomer. The thiourea-linked thymidyl oligomers 4a-c were
successfully converted to compounds 5a-c by methylation of
the thiourea linkages to methylisothiouronium salts in excess
methyl iodide followed by deprotection with acetic acid and
purified on a preparative Alltech SCX 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 1.5
M guanidine HCl as the eluant (see the Supporting Information).
Compounds 5a, 5b, and 5c have retention times of 9.8, 14, and
18 min, respectively, consistent with the presence of three, four,
and five positive charges.
Acknowledgment. This work was supported by the National Institute
of Health and the Office of Naval Research.
To investigate the interaction of 5c with polynucleotides, we
constructed UV continuous variation plots at different ionic
strengths, wavelengths, and temperatures. The plots (Figure 1)
for interaction of 5c with poly(rA) and poly(dA) always show
maximum hypochromicity at an approximate 1:2::A:Tmt ratio
which indicates triple-helix formation in a TmtApTmt triad,
consistent with mixing curves obtained with other positively
charged oligonucleotides.13,22-24
The slow thermal melting and cooling profiles (0.2 deg/min)
of 5c bound to poly(dA) show pronounced hysteresis at pH 7.0
and at an ionic strength of 0.15 (see the Supporting Information),
further evidence of the triple-helical binding of positively charged
5c to polynucleotides.23,24 In the thermal denaturation analysis
of 5c bound to poly(dA), plots of absorbance at 260 nm (A260) vs
temperature exhibit two distinct inflections (Figure 2). On this
basis, we assign the plots in Figure 2 to represent denaturation
curves of triple- and double-helical structures of 5c with ssDNA
Supporting Information Available: Hysteresis curves of 5c and poly-
(dA) with specific conditions for melting and annealing, HPLC chro-
matogram, mass spectrum, and UV absorption spectrum of compound
5c (3 pages, print/PDF). See any current masthead page for ordering
information and Web access instructions.
JA980629Q
(25) Little hyperchromic shift was observed with polyribonucleosides when
melted with 5c under similar conditions.
(26) The duplex has a melting point of >85 °C in 10-3 M K2HPO4 with
poly(dA), whereas Tp-5 (5 mer of DNA) does not show any appreciable
binding below an ionic strength of 0.12; Tg-5 (DNG) on the other hand, has
an even higher duplex melting temperature of >95 °C.
(27) Letsinger, R. L.; Singman, C. N.; Histand, G.; Salunkhe, M. J. Am.
Chem. Soc. 1988, 110, 4470-4471.
(28) Rasmussen, C. R.; Villani, F., Jr.; Reynolds, B. E.; Plampin, J. N.;
Hood, A. R.; Hecker, L. R.; Nortey, S. O.; Hanslin, A.; Costanzo, M. J.;
Howse, R. M.; Molinari, A. J. Synthesis 1988, 460-466.
(29) Wu, T.; Ogilvie, K. K.; Pon, R. T. Nucleic Acids Res. 1989, 17 (9),
3501-3517.
(30) Dempcy, R. O.; Browne, K. A.; Bruice, T. C. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 6097-6101.
(31) Chaix, C.; Molko, D.; Teoule, R. Tetrahedron Lett. 1989, 30 (1), 71-
74.
(23) Blasko, A.; Dempcy, R. O.; Minyat, E. E.; Bruice, T. C. Biochemistry
1997, 36, 7821-7831.
(24) Blasko, A.; Dempcy, R. O.; Minyat, E. E.; Bruice, T. C. J. Am. Chem.
Soc. 1996, 118, 7892-7899.