Syntheses and binding studies of oligonucleotides containing N- hydroxycarbamate linkages: Potential DNA cleaving antisense oligomers

Article abstract of DOI:10.1016/S0040-4039(00)00662-6

An efficient synthesis of N-hydroxycarbamate containing thymidine dimer 9 was accomplished and it was incorporated into automatic DNA syntheses to make thymidine 16mers T*-1, T*-2 and T*-3. Binding abilities of T*-1, T*-2 and T*-3 with poly (dA)-16mer were assayed by melting denaturation (Tm) studies of the corresponding duplexes. Iron binding ability of a thymidine oligomer with N-hydroxycarbamate linkages was studied by MALDI-MS. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00662-6

Tetrahedron Letters 41 (2000) 4323±4327
Syntheses and binding studies of oligonucleotides containing
N-hydroxycarbamate linkages: potential DNA cleaving
antisense oligomers
Hui Li and Marvin J. Miller*
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
Received 24 March 2000; revised 17 April 2000; accepted 18 April 2000
Abstract
An ecient synthesis of N-hydroxycarbamate containing thymidine dimer 9 was accomplished and it
was incorporated into automatic DNA syntheses to make thymidine 16mers T*-1, T*-2 and T*-3. Binding
abilities of T*-1, T*-2 and T*-3 with poly (dA)-16mer were assayed by melting denaturation (Tm) studies
of the corresponding duplexes. Iron binding ability of a thymidine oligomer with N-hydroxycarbamate
linkages was studied by MALDI-MS. Nuclease stability assays showed that the modi®ed oligonucleotides
have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides. # 2000
Elsevier Science Ltd. All rights reserved.
Keywords: antisense oligonucleotides; N-hydroxycarbamate linkages; iron-binding.
The use of oligonucleotide analogs as rationally designed drugs continues to attract attention
in medicinal chemistry. In principle, antisense oligonucleotides target, in a sequence speci®c
manner, a particular gene, or mRNA to inhibit the expression of that gene.1 5 In recent years,
chemical modi®cations of antisense oligonucleotides have resulted in improved solubility, stability
towards nucleases, cellular uptake, and appropriate hybridization to target genes or mRNAs.6,7
In the antisense approach, oligonucleotides have been designed to bind to a given mRNA to
inhibit the translation process via duplex formation and/or RNase H-induced degradation of the
message. We proposed a novel backbone replacement for antisense oligonucleotides with N-
hydroxycarbamates as linkages (Fig. 1).8,9 Hydroxamic acids can e€ectively chelate Fe(III),
which can undergo Fenton chemistry to generate hydroxyl radicals.10 Therefore, in addition to
potential RNase-H cleavage processes, antisense oligonucleosides with N-hydroxycarbamate link-
ages might cleave the target mRNA through radical reactions. Moreover, the Fe(III) binding
ability of the antisense oligonucleoside would have the potential to assist in its cell permeability
* Corresponding author. Tel: 219-631-7571; fax: 219-631-6652; e-mail: marvin.j.miller.2@nd.edu
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00662-6

4324
through iron uptake systems.11 In order for the proposed Fenton chemistry to work, the oligo-
nucleotides with N-hydroxycarbamate linkages are required to bind both iron and target DNA or
mRNA strands with reasonable anity. Herein, we report the syntheses of thymidine oligomers
with N-hydroxycarbamate linkages and their binding ability toward poly(dA) and Fe(III).
Figure 1.
In a previous report,8 we described the syntheses of the building blocks of oligonucleotides with
N-hydroxycarbamate linkages. The conversion of the 50-hydroxyl group of a nucleoside into the
corresponding 50-N-hydroxylamino substituted derivatives can be achieved by Mitsunobu reactions
of N-(tert-butoxycarbonyl)-O-(benzyloxycarbonyl)hydroxylamine (BocNHOCbz) with nucleo-
sides. Scheme 1 shows the application of this methodology to thymidine for the syntheses of
thymidine oligomers with N-hydroxycarbamate linkages. In order for the Mitsunobu reaction to
work, proper protecting groups were introduced to thymidine. Starting from 30-O-tert-butyl-
dimethylsilyl-50-O-dimethoxytritylthymidine 1, a benzyl group was introduced to the 4-O-position
of the thymidine in two steps by base-aided condensation with tosyl chloride in dichloromethane,
followed by the reaction of the resulting sulfonate with benzyl alcohol, to give 2 in 88% overall yield
(Scheme 1). Removal of the dimethoxytrityl group in a dichloroacetic acid in dichloromethane
Scheme 1.

4325
solution a€orded an 87% yield of desired compound 3. The Mitsunobu reaction of 3 with N-(tert-
butoxycarbonyl)-O-(benzyl)hydroxylamine (BocNHOBn) gave desired hydroxyamino derivative
4 in good yield (Scheme 1). When 4 was exposed to 10% tri¯uoroacetic acid in dichloromethane,
both the Boc and 4-O-Bn groups were removed to give 5 in 99% yield. Use of 5 in a coupling
reaction provided the corresponding thymidine dimer with the desired N-hydroxycarbamate
linkage as described below (Scheme 2).
Scheme 2.
The p-nitrophenyl (PNP) carbonate 7 was obtained in 97% yield by reaction between 50-O-
dimethoxytritylthymidine 6 and p-nitrophenyl chloroformate. Since direct reaction of 5 with 7
was sluggish, 1-hydroxy-7-azabenzotriazole (HOAt) was used to form a more active carbonate
reaction intermediate for the coupling and generated dimer 8 in 83% yield. Compound 8 was then
treated with n-tetrabutylammonium ¯uoride (TBAF) to remove the silyl group in 95% yield. The
resulting 30-hydroxy intermediate was reacted with chlorophosphoramidite to a€ord 9 in 89%
yield (Scheme 2).
Dimer 9 was then incorporated into thymidine 16mers using phosphoramidite chemistry on
solid support according to a standard 1 mmole synthesis cycle.12 Oligonucleotides with O-benzyl-
N-hydroxycarbamate (@) internucleoside linkages (up to three) were deprotected and removed
from the solid support by treatment with concentrated aqueous ammonia solution at rt for 1 h.
Benzyl groups were then removed by hydrogenolysis in acetic acid and triethylamine bu€er
solution (pH 7.15) to give T*-1, T*-2 and T*-3 (Scheme 3).
Scheme 3.

4326
The binding of thymidine oligomers with N-hydroxycarbamate linkages to Fe(III) was studied
by the synthesis of a model heptathymidylate analog using the same methodology mentioned
above. When the heptamer T*TT*TT*TT was treated with iron(III)-acetylacetonate, Fe(acac)3,
MALDI-MS showed distinct Fe complex formation, indicating e€ective binding ability of N-
hydroxycarbamate linkages toward Fe(III).
The stability of duplexes of the thymidine 16mer analogs with poly (dA) was investigated by
thermal denaturation experiments (Tm).13 Incorporation of one, two and three modi®cations in
the middle of the sequence led to a progressive decrease in the melting temperature of the duplex
compared to the natural DNA duplex. The degree of the destabilization depended on the steric
factors of the linkages. While a more pronounced destabilization was observed with the O-benzyl-
N-hydroxycarbamate linked compounds (ÁTm/modꢀ3.7ꢁC), the anity of the deprotected
N-hydroxycarbamate linked oligonucleotides for poly (dA) was only slightly decreased (ÁTm/
modꢀ1.4ꢁC). It was also observed that T*-3 did not further destabilize the duplex compared to
T*-2 (Table 1).
Table 1
Thermal stability (Tm)a of duplexes formed between oligothymidylate analogs and poly (dA) targets and half-lives of
oligothymidylate analogs towards nuclease S1
Nuclease resistance of N-hydroxycarbamate linked oligonucleotides towards nuclease S1
(endonuclease) was investigated in comparison with the resistance of unmodi®ed d(T)16.14 Due to
the modi®ed linkage, a stabilizing e€ect was observed. The half-life of oligonucleotides increased
when more N-hydroxycarbamate linkages were incorporated into the sequence (Table 1).
N-Hydroxycarbamate linked oligonucleotides represent a new class of backbone modi®ed
DNA analogs. Thymidine 16mer analogs with alternating phosphodiester and N-hydroxy-
carbamate linkages were synthesized with one to three modi®cations and their biophysical properties
were studied. Replacement of negatively charged phosphodiester linkages by N-hydroxy-
carbamate linkages only slightly weaken duplex stability of the oligonucleotide analogs when
hybridized with poly (dA). The resistance of N-hydroxycarbamate linked oligonucleotides
towards endonuclease is improved. Binding studies indicated a modi®ed oligonucleotide with
three N-hydroxycarbamate linkages does bind with Fe(III) and also binds to the target poly (dA).
Work is currently in progress to study the ability of T*-3 to perform Fenton chemistry and cleave
the DNA sense strand sequence-speci®cally.

4327
Acknowledgements
We gratefully acknowledge the NIH (AI 30988) and Kimeragen, Inc. for the support of this
research, use of the Biosciences Core facility, Mass Spectrometry and NMR facility at the
Department of Chemistry and Biochemistry, University of Notre Dame. We thank Professor P.
Huber, C. Foss, and Dr. M. Prorok for discussions and help and also Ms. Maureen Metcalf for
assistance with the manuscript.
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