Solid-phase synthesis of oligomeric deoxynucleic-thiourea (DNT) and deoxynucleic S-methylthiourea (DNmt): A neutral/polycationic analogue of DNA

Article abstract of DOI:10.1016/S0960-894X(00)00085-8

A solid-phase synthesis for oligomeric DNmt is reported. Synthesis proceeds in 3'-5' direction and involves coupling of a protected 3'-isothiocyanate with the corresponding 5'-amine of the growing oligo chain. The difference in oligomeric thiourea/S-methy

Full text of DOI:10.1016/S0960-894X(00)00085-8

Bioorganic & Medicinal Chemistry Letters 10 (2000) 691±693
Solid-Phase Synthesis of Oligomeric Deoxynucleic-Thiourea
(DNT) and Deoxynucleic S-Methylthiourea (DNmt):
a Neutral/Polycationic Analogue of DNA
Dev P. Aryay and Thomas C. Bruice*
Department of Chemistry, University of California, Santa Barbara, CA 93106, USA
Received 9 December 1999; accepted 14 January 2000
AbstractÐA solid-phase synthesis for oligomeric DNmt is reported. Synthesis proceeds in 30±50 direction and involves coupling of a
protected 30-isothiocyanate with the corresponding 50-amine of the growing oligo chain. The di€erence in oligomeric thiourea/
S-methylthiourea binding to DNA is investigated. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
In recent years, much interest has been expressed in
antisense agents for the regulation of gene expression in
living cells.1,2 Among other requirements, successful
development of antisense therapeutics presupposes the
oligos to (a) be stable in vivo, (b) have improved per-
meability and cellular uptake and (c) have greater bind-
ing anity with high speci®city.3 5 We have previously
reported the replacement of the phosphate linkages in
DNA and RNA by achiral guanido and S-methyl-
thiourea groups which we identify as DNG6 8 and
DNmt respectively.9 11 A 5 unit oligomer of thymidyl
DNmt (NH+3 -(Tmt)4-T-OH) binds to complementary
poly(rA) with a melting temperature (Tm) for dissocia-
tion of the double helix estimated to be close to 85 ꢀC
and yet no base speci®c H-bonding (Watson±Crick) is
observed with poly(rC), (rG), (U), (dT) or (I).9 Due to
this strong binding, speci®city and resistance to nuclea-
Synthesis
ses, DNmt represents an extremely interesting putative
genetic regulatory agent. The stepwise synthesis of oli-
gomeric DNmt strands in solution has been reported by
us recently.9 In order to facilitate the development of
longer sequences of DNmt, a synthesis that involved
stepwise construction on a solid support was needed. In
this paper, we report the ®rst solid-phase synthesis of a
DNmt oligomer.
The monomer 6 (Scheme 1) for the SPS of oligothym-
idyl DNmts is obtained by protecting the 50-NH2, group
of 50-amino-50-deoxythymidine (1) with monomethoxy-
trityl chloride and converting the 30-OH to 30-iso-
thiocyanate in ®ve steps.8,9 The 30-OH was converted to
the mesylate 2b which upon reaction with Potassium
phthalimide in DMF followed by lithium azide gives the
azide 3 in excellent yields. Compound 3 was hydro-
genated and reacted with thiocarbonylpyridone in
CH2Cl2 to give monomer 6 in high yields9 14 (see sup-
plementary material for experimental procedures).
ControlPore Glass (CPG) resin with long chain alkyl-
amine was chosen as a convenient commercially available
*Corresponding author. Tel.: +1-805-893-2044; fax: +1-805-893-
4120; e-mail: tcbruice@bioorganic.ucsb.edu
yPresent address: Department of Chemistry, Pemson University, SC
29634.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00085-8

692
D. P. Arya, T. C. Bruice / Bioorg. Med. Chem. Lett. 10 (2000) 691±693
Scheme 1. Synthesis of thymidyl monomer for solid phase synthesis.
(a) MmTr-C1, pyridine. DMAP(2 mol%), 89%; (b) 1.3 equiv MsCl,
pyridine, 0 ꢀC to rt, 88%; (c) KPhTh, DMF, H2O, 100 ꢀC, 20 mm; (d)
LiN3, DMF, 110 ꢀC 90%; (e) H2S, pyridine, 4 h, rt, 90%; (f) Thio-
pyridone, CH2Cl2, 4 h, 88%; (g) succinic anhydride, DMAP, pyridine;
(h) 4-Nitrophenol, DMAP, Et3N.
Scheme 2a. Solid phase synthesis of DNT: (i) pyricline, DMF,
DMAP, monomer 8; (j) coupling: pyridine, monomer 6, DMAP (2
equiv); (k) deblocking: 4% dichloroacetic acid in dichioromethane.
support with a linker that was cleavable by mild base.
The synthesis was designed to be compatible with stan-
dard DNA synthesis techniques to facilitate future
synthesis of DNmt±DNA conjugates.15 Analogous to
DNA synthesis, 30-OH of 1 was converted to the acti-
vated nitrophenyl ester 8, was loaded as the ®rst base on
the resin.16 The coupling reaction involves the addition
of 6 to a 50-aminothymidyl residue on the resin. In a
typical synthesis, 36 mg of resin was placed in 0.5 mL
Pyridine in a 3 mL reaction vial. Stock solutions of the
isothiocyanate 6 (30 mM, 1.0 mL), DMAP (12 mM) in
pyridine were added and the vial was agitated for 4 h.
The addition step was repeated twice to insure a com-
plete reaction and then the resin was washed with
copious amounts of pyridine, methanol and ether. The
resulting 50-MmTr protected oligomer was deblocked
with 4% dichioroacetic acid in CH2Cl2 and the cycle
began again (Scheme 2a). The addition/deblocking cycle
was repeated six more times to produce the seven unit
oligomer. The thiourea was methylated with 2 mL
methyl iodide for 4 h. The 50-MmTr group was removed
in 4% DCA and the resin washed with methanol, ether.
The product was cleaved from the resin by treatment
with NH4OH at room temperature. The oligomer can
be cleaved from the resin before methylation to give the
corresponding thiourea (9a±c) as well (DNT). Methyl-
ation of the tritylated thiourea can be performed in
solution as well as on the solid phase. HPLC analysis of
the crude DNmt product showed the desired product in
>90% purity with an estimated coupling yield averaged
over the seven additions of 87% (determined by UV
analysis of absorbance of monomethoxy trityl cation).
The deprotected DNmt oligomer 9 [50-NH3+-d(Tmt)7-
OH] was puri®ed on a preparative Alltech WCX cation
exchange column employing 1.50 M ammonium acetate
bu€er, pH 6.0, as the mobile phase to give the pure
oligomer (Scheme 2b). Mass spectroscopic analysis indi-
cates the expected mass for the singly charged (m/z=
2019, calculated for (M+H)+: 2019) form of the oligo-
meric DNmt 9.
We have previously reported10,11 that Thymidyl DNmt
(50-NH+3 -d(Tmt)4-T-OH) has much stronger anity for
DNA and RNA, due to electrostatic attractions, than
DNA for RNA or vice versa. In order to further evalu-
.
ate the high stability of DNmt DNA complexes, binding
of neutral DNT (deoxynucleic-thiourea) 9a to poly (dA)
was studied.17 To investigate the interaction of 9a
{50-NH+3 -Tt-(Tt)4-OH} with polynucleotides, we con-
structed UV continuous variation plots. Mixtures of
9a with poly(dA) at 10 ꢀC (Fig. 1) reach a minimum
Scheme 2b. Solid phase synthesis of DNmt: (l) MeI, EtOH; (m)
NH4OH, rt; (n) 4% dichloroacetic acid in dichloromethane.

D. P. Arya, T. C. Bruice / Bioorg. Med. Chem. Lett. 10 (2000) 691±693
693
In summary, an ecient and rapid solid-phase method
for the synthesis of thiourea and S-methylthiourea
linked DNA analogues has been successfully demon-
strated. This solid-phase synthesis technique opens the
door for the rapid synthesis of DNmt/DNT oligomers
for further binding studies, for combinatorial libraries
and the synthesis of DNmt-peptide conjugates on solid
phase. If the nucleotide bases are protected with base-
labile phenoxyacetyl groups, the synthesis can be
accomplished as detailed above and the base protecting
groups removed by subsequent ammonia treatment
after removal of the MmTr protecting groups. Attach-
ment of charged functional groups to the 50-amine of
DNT (9a±c) should give them increased solubility and
enable comparative studies of neutral thiourea with
DNmt and DNG oligos. The synthesis and binding
studies of such mixed sequences should be very inter-
esting and are currently the focus of our continuing
investigations.
Figure 1. Job plots of poly(dA) with 50-NH3+-d(Tt)4-T-OH in a con-
5
centration of 4.0Â10 M/base at 260 nm in 15 mM potassium phos-
phate (pH 7.5).
Acknowledgement
This project was supported by the National Institute of
Health (3 R37 DKO9171-3451).
References and Notes
1. Bennett, C. F. Biochem. Pharmacol. 1998, 55, 9.
2. Hawkins, J. W. Antisense and Nucleic Acid Drug Develop-
ment 1995, 5, 1.
3. Cohen, J. S. Antiviral Res. 1991, 16, 121.
4. Cook, P. D. Medicinal Chemistry Strategies for Antisense
Research; CRC Press: Boca Raton: 1993.
Figure 2. Job plots of poly(dA) with 50-NH3+-d(Tmt)4-T-OH in a
concentration of 4.0Â10 5 M/base at 260 nm in water (*) and 15 mM
potassium phosphate (&).
absorbance at a mol fraction of ꢁ0.5 d(Tmt) to 0.5
5. Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J.
Am. Chem. Soc. 1992, 114, 1895.
d(Ap) (single phosphate-linked adenosyl unit).
6. Dempcy, R. O.; Almarsson, O.; Bruice, T. C. Proc. Natl.
Acad. Sci. USA. 1994, 91, 7864.
7. Dempcy, R. O.; Browne, K.; Bruice, T. C. J. Am. Chem.
Soc. 1995, 117, 6140.
8. Dempcy, R. O.; Browne, K.; Bruice, T. C. Proc. Natl. Acad.
Sci. USA 1995, 117, 92.
9. Arya, D. P.; Bruice, T. C. J. Am. Chem. Soc. 1998, 120, 12.
10. Arya, D. P.; Bruice, T. C. J. Am. Chem. Soc. 1998, 120,
6619.
11. Arya, D. P.; Bruice, T. C. Proc. Natl. Acad. Sci. USA 1999,
96, 4384.
12. Blasko, A.; Dempcy, R. O.; Minyat, E. E.; Bruice, T. C. J.
Am. Chem. Soc. 1996, 118, 7892.
13. Blasko, A.; Dempcy, R. O.; Minyat, E. E.; Bruice, T. C.
Biochemistry 1997, 36, 7821.
14. Kim, S.; Yi, K. Y. J. Org. Chem. 1986, 51, 2613.
15. Barawkar, D. A.; Bruice, T. C. Proc. Natl. Acad. Sci. USA
1998, 95, 1104.
These numbers indicate that double stranded complexes
are formed containing one d(Tt) for every d(Ap). This
is in contrast to the 2:1 binding observed between
DNmt DNA complexes (Fig. 2) where triple helices are
.
always observed. DNmt DNA complexes always show a
minimum at 2:1 and a break at 1:1 is observed only
when mixing plots are carried out in pure water (Fig. 2).
.
This is in agreement with the fact that DNmt DNA
.
complexes are destabilized upon addition of salt, as
previously reported.9,10 Melting curves for the duplex
ꢀ
ing much weaker binding compared to the DNmt DNA
.
9a poly(dA) gave a melting point less than 15 C, show-
.
triplexes. Changing the ionic strength (up to 0.2 M KCl)
.
had no e€ect on the stability of the DNT DNA duplex.
As re¯ected in the dA260 values in Figures 1 and 2, the
.
% hypochrornicity is considerably less for DNT DNA
16. Atkinson, T.; Smith, M. In Solid-Phase Synthesis of Oligo-
deoxvribonucleotides by the Phosphitetriester Method; Gait, M.
J. Ed.; Oxford University Press: New York, 1990; pp 35.
17. 9a (after removal from CPG) was dissolved in DMSO and
diluted with 15 mM phosphate bu€er.
.
(Fig. 1) implying weaker binding for DNT DNA
duplex. Attempts to study binding of longer DNT
(9b±c) were unsuccessful due to the lack of solubility of
thiourea oligomers in aqueous solution.