Synthesis and biophysical studies of oligonucleotides containing hydroxamate linkages

Article abstract of DOI:10.1016/S0040-4039(00)00661-4

Novel oligonucleotide dimers containing hydroxamate linkages (15 and 16) were synthesized, incorporated into oligonucleotide sequences and studied for their hybridization properties with complementary DNA and RNA targets. The modified oligonucleotides showed similar binding properties and enhanced resistance to exonucleases compared to natural oligonucleotides. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00661-4

Tetrahedron Letters 41 (2000) 4317±4321
Synthesis and biophysical studies of oligonucleotides
containing hydroxamate linkages
Kanda S. Ramasamy,* Liyan He, Vesna Stoisavljevic,
Brent Harpham and Wilfried Seifert
Medicinal Chemistry Division, ICN Pharmaceuticals, Inc., 3300 Hyland Avenue, Costa Mesa, CA 92626, USA
Received 29 February 2000; revised 30 March 2000; accepted 31 March 2000
Abstract
Novel oligonucleotide dimers containing hydroxamate linkages (15 and 16) were synthesized, incorporated
into oligonucleotide sequences and studied for their hybridization properties with complementary DNA
and RNA targets. The modi®ed oligonucleotides showed similar binding properties and enhanced resistance
to exonucleases compared to natural oligonucleotides. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: oligonucleotides; hydroxamate; backbone modi®cation.
Inhibition of gene expression with antisense oligonucleotides by binding to mRNA in a sequence
speci®c manner has become an attractive method for the treatment of viral diseases, cancer, and
for the study of genetic disorders.1 3 As drug candidates, they should inhibit the translation
process via duplex formation and/or able to activate RNase H to cleave the target message after
hybridization. In addition, antisense oligonucleotides should possess in vivo nuclease stability,
adequate binding anity to the target mRNA, and the ability to reach target cells and tissues. So
far, chemical modi®cations of oligonucleotides have resulted in increased solubility, nuclease
stability, cellular uptake, binding properties and RNase H activation.4 However, the quest for
new and novel modi®ed oligonucleotides with improved properties is growing.
Our interest to design novel class of oligonucleotides has led us to envision hydroxamate
nucleic acids (HONA)5 (i.e. oligonucleotides having hydroxamate internucleotide linkages, Fig. 1,
2). Hydroxamate nucleic acids o€er several advantages over previously reported modi®cations.6
The hydroxamate unit (-CO-NOH-) is stable under physiological conditions and chelates with
ferric ion, which can generate hydroxyl radicals.7 Thus, in addition to potential RNase-H clea-
vage process, oligonucleotides having hydroxamate linkages might cleave the target through
radical reactions. Furthermore, the solubility of oligonucleotides with hydroxamate linkages
* Corresponding author.
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00661-4

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might be increased by coordination with metal ions and also the charge would be reduced, which
should facilitate penetration through negatively charged cell membranes. As a prototype, herein
we report our preliminary data on the syntheses of novel thymidine dimers 15 and 16, their
incorporation into oligonucleotide sequences and measurement of hybridization of the duplex
formed between HONA and natural DNA/RNA.
Figure 1.
The synthetic route to the hydroxamate dimers 15 and 16 is shown in Scheme 1. The known
aldehyde8 4 was heated with O-methylhydroxylamine hydrochloride in the presence of pyridine at
100ꢀC for 12 h to give oxime 5 in 99% yield. Interestingly, 4 did not react with O-methyl-
hydroxylamine hydrochloride at room temperature under di€erent conditions. Reduction of 5
with sodium cyanoborohydride in dry methanol containing acetic acid (pH below 6) for 6 h at
room temperature provided one of the building blocks 6. For the building block 12, aldehyde 79
was oxidized to the corresponding carboxylic acid 8 with sodium chlorite using 2-methyl-2-butene
as HOCl scavenger.10 Esteri®cation of 8 with benzyl alcohol using water soluble carbodiimide
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDC)11 a€orded benzyl ester 9
in 87% yield. Desilylation of 9 followed by dimethoxytritylation12 and base hydrolysis provided
second building block 12. Carboxylic acid 12 was activated with O-(1H-benzotriaol-1-yl)-
N,N,N0,N0-tetramethyluronium hexa¯uoroborate (HBTU) and N-hydroxybenzotriazole13 and
then coupled with hydroxylamine 6 at room temperature to give a dimer 13 in 63% yield.
Removal of the silyl protective group from 13 with tetra-n-butylammonium ¯uoride and subsequent
phosphitylation14 gave the target phosphoramidite dimer 15.15 The second dimer 16 was also
prepared by using the same methodology depicted in Scheme 1 (60% overall yield) and substituting
O-benzylhydroxylamine hydrochloride for O-methylhydroxylamine hydrochloride during the
formation of 5.
Incorporation of the dimers 15 and 1615 into oligonucleotide sequences was accomplished
using ABI 394 DNA synthesizer and protocol,16 and the coupling eciency was found to be
higher than 95%. The binding behavior of the modi®ed oligonucleotides was assayed by examining
their ultraviolet (UV) absorbance versus temperature pro®les. An 18-mer oligonucleotide
(50-TTCCTGCTTGATGGCTTC-30) was modi®ed with 15 and 16 at di€erent locations and
hybridized to complementary DNA or RNA. Melting temperatures of the duplexes formed between
oligonucleotides containing the hydroxamate linkages and their DNA and RNA complementary

4319
a
ꢀ
.
Scheme 1. (i) MeOHN HCl/Py/100 C; (ii) NaCNBH3/ACOH/MeOH; (iii) NaClO2/2-methyl-2-butene/t-BuOH/H2O;
(iv) BnOH/EDC/N,N-dimethylaminopyridine; (v) TBAF/THF/H2O/Py; (vi) DMTCl/TEA/CH2Cl2; (vii) 1N NaOH/THF/
H2O/H+; (viii) HBTU/H-hydroxybenzotriazole/MeCN; (ix) (i-Pr)2NP(Cl)OCH2CH2CN/i-Pr2NEt/CH2Cl2 T=thymine
strands are summarized in Table 1. Compared to unmodi®ed DNA, incorporation of a hydroxamate
dimer 15 at 30-end of an oligonucleotide (Table 1, line 3) led to an increase in Tm of 0.2ꢀC against
DNA and RNA complements. Substitution of 15 at 50-end, decreased the Tm with DNA by
0.2ꢀC and the RNA duplex stability is una€ected (Table 1, line 4). Incorporation of 15 at 30-end
and 50-end formed stable duplexes with DNA/RNA complements. When 15 was substituted in
the middle of the oligonucleotide (Table 1, line 6), the Tm was decreased by 0.4ꢀC against DNA/
RNA. On the other hand, incorporation of 15 at 30-end, 50-end and in the middle (Table 1, line 7),
did not disturb duplex stability with neither DNA nor RNA. However, when 15 was substituted
at 30-end and in the middle (Table 1, line 7), the duplex stability is decreased with DNA/RNA by
a Tm of 0.2ꢀC (Table 1, line 8). A similar binding pattern (data not shown) was observed when
another hydroxamate dimer 16 is incorporated either at the 50-end or 30-end or in the middle of
the sequence. However, compared to 15, hydroxamate dimer 16 destabilized both DNA and RNA
duplexes (ÁTm of 1.0ꢀC/modi®cation). Interestingly, compared to unmodi®ed oligonucleotides,
incorporation of hydroxamate modi®ed dimer at the 30-end of the oligonucleotides, exhibited
tenfold increase in resistance to exonucleases.18
This study suggests that a smaller substitution (CH3 group) on the hydroxamate dimer 15 did not
a€ect duplex stability and is a preferred dimer over 16 for oligonucleotide backbone modi®cations.

4320
Table 1
Tm values of oligonucleotides containing O-methylhydroxamate linkagea
On the other hand, dimer 16 with a bulky substitution (benzyl) in oligonucleotide sequence
decreases its ability to form stable duplexes with complementary DNA/RNA. The introduction
of bulky group such as benzyl in the hydroxamate unit 16 is sterically demanding which may alter
the structure and the stability of the duplex formed with DNA/RNA complement. The weaker
binding of HONA containing dimer 16 is indicative that the internucleotide hydroxamate link-
ages will not tolerate larger groups. The presence of bulky groups may force the oligonucleotides
to adopt a di€erent conformation than that of oligonucleotides having unmodi®ed phosphate
backbone. Furthermore, it seems that the hydroxamate dimer 15 adopts a more favorable geo-
metry in the duplex with DNA/RNA complement than that of 16.
In summary, oligonucleotides containing hydroxamate dimers have been synthesized for the
®rst time and studied for their ability to form stable duplexes. Interestingly, oligonucleotides
containing hydroxamate dimer 15 not only displayed a similar or even slightly higher anity
for RNA target than the natural analogues, but also showed substantial resistance towards
30-exonucleases.
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