Replacement of the phosphodiester linkage in dna with sulfamide and 3'- N-sulfamate groups

Article abstract of DOI:10.1039/b001586p

Replacing phosphodiester linkages in DNA with neutral 3'-N-sulfamate groups has little effect on the binding affinity for complementary DNA or RNA, whereas incorporation of sulfamide groups into DNA results in considerable destabilisation of isosequential

Full text of DOI:10.1039/b001586p

Replacement of the phosphodiester linkage in DNA with sulfamide and
3A-N-sulfamate groups†
Kevin J. Fettes,a Nigel Howard,a David T. Hickman,a Steven A. Adah,b Mark R. Player,b Paul F. Torrencec
and Jason Micklefield*d
a Department of Chemistry, Birkbeck College, University of London, 29 Gordon Square, London, UK WC1H 0PP
b Laboratory of Medicinal Chemistry, NIDDK, NIH Bethesda, MD, 20892–0805, USA
c
Department of Chemistry, Northern Arizona University, Flagstaff, AZ, 86011-5698, USA
d Department of Chemistry, Faraday Building, UMIST, PO Box 88, Manchester, UK M60 1QD.
E-mail: jason.micklefield@umist.ac.uk
Received (in Liverpool, UK) 25th February 2000, Accepted 27th March 2000
Replacing phosphodiester linkages in DNA with neutral 3A-
N-sulfamate groups has little effect on the binding affinity
for complementary DNA or RNA, whereas incorporation of
sulfamide groups into DNA results in considerable destabili-
sation of isosequential duplexes.
The sulfamate dinucleoside d(TnsoT) 2 was also prepared
and its conformation compared with d(TnsnT) 1, and the native
dinucleoside phosphate d(TpT) using NMR spectroscopy. As
expected the sum of the J1A2A and J1A2B vicinal coupling constants
(S1A) for the 5A-terminal ribose ring of d(TnsoT) revealed a
higher proportion of the C3A-endo, or northern (N), sugar
conformation (69% N at 30 °C) than the native d(TpT) which
exists predominantly in the C2A-endo, southern, conformation
(36% N).7 This can be attributed to the lower electronegativity
of the 3A–amino substituent of d(TnsoT) and a reduced gauche
effect between this substituent and the deoxyribose O4A atom.
Furthermore, the shift in the % of N-conformer with change in
temperature for both deoxyribose rings of d(TnsoT) was
virtually the same as that observed for d(TnsnT),7 with the
largest change occurring in the 5A-terminal ring (63% N at 70 °C
increasing to 78% at 10 °C). The native d(TpT) shows little or
no variation in sugar pucker with change in temperature. To
investigate if this effect is due, in part, to an increase in base
stacking of the modified dimers circular dichroism (CD) spectra
of d(TnsoT) d(TnsnT) and d(TpT) were recorded at various
temperatures. All spectra revealed the same basic characteristics
Modified oligonucleotides with neutral linkages replacing the
native phosphodiester groups are of interest in the development
of improved antisense and antigene agents.1 Methylphospho-
nates, one of the earliest examples of this class, can only be
efficiently prepared at present as a mixture of diastereoisomers
at the phosphorus atom and have low affinity for com-
plementary nucleic acids.2 More recently neutral methylene-
(methylimino) (MMI),3 amide1 and 3A-thioformacetal4 mod-
ified oligonucleotides have been developed which exhibit
improved affinity for complementary nucleic acids. Fur-
thermore, neutral formacetal5 and dimethylenesulfone6 linkages
have been used effectively in the development of modified
DNA aptamers5 and dsDNA transition state analogues6 that can
bind to specific protein targets.
Earlier we reported the synthesis of dinucleotide analogues in
which the phosphodiester linkage is exchanged for a neutral
sulfamide group.7 Here, the effects of the sulfamide 1 and 3A-N-
sulfamate 2 (Scheme 1) internucleoside linkages on nucleic acid
conformation and duplex stability are described. It was
envisaged that the block synthesis approach could be used to
synthesise chimeric oligodeoxynucleotides in which one or
more of the phosphodiester groups are replaced by the neutral
linkages. With the sulfamide phosphoramidite 10 in hand7 a
route to the 3A-N-sulfamate dinucleosides was required. Al-
though isomeric 5A-N-sulfamate dinucleosides have been pre-
pared before, the synthesis uses explosive sulfuryl chloride
azide (ClSO2N3) and the overall yields are low.8 Therefore, the
development of an alternative method was necessary. Initially
the coupling of 2–hydroxyphenyl sulfamate 57 with the 5A-
alcohol 8 was investigated. However, compound 5 proved
insufficiently reactive and failed to give any of the sulfamate
dinucleoside 11 despite the earlier observation that 5 reacts
smoothly, albeit at high temperature, with the 5A-amine 7 to give
the sulfamide dinucleoside 9. In light of this, the more reactive
4–nitrophenyl sulfamate 6 was prepared by treating the 3A-
amine 3 with 4–nitrophenyl chlorosulfate 4.9 Coupling of 6 with
5A-alcohol 8 was successful resulting in the sulfamate dinucleo-
side 11 in excellent yield at room temperature. Similarly the
sulfamide dinucleoside 9 could also be prepared more effec-
tively at room temperature from 6 and the 5A-amine 7.
Desilylation of dinucleoside 11 and 3A-phosphitylation gave the
required phosphoramidite 12.
Scheme 1 Reagents and conditions: i, 4–nitrophenyl chlorosulfate 4,9
4–nitrophenol, Et3N, CH2Cl2; ii, alcohol 8 (2 equiv.), Et3N, 4Å molecular
sieves, CH2Cl2; iii, TBAF, THF; iv, 2–cyanoethyl–N,N,NA,NA–tetra-
isopropylphosphorodiamidite, 4,5–dicyanoimidazole, CH2Cl2; v, 2%
CHCl2CO2H, CH2Cl2. 4,4A–Dimethoxytrityl is abbreviated to DMTr.
† Electronic supplementary information (ESI) available: Experimental
details for oligonucleotide synthesis and duplex denaturation experiments.
See http://www.rsc.org/suppdata/cc/b0/b001586p/
DOI: 10.1039/b001586p
Chem. Commun., 2000, 765–766
This journal is © The Royal Society of Chemistry 2000
765

Table 1 Electrospray mass spectrometry data and duplex melting temperatures for oligonucleotides 13–18
Tma (DTm/mod.)b/°C
Electrospray MS/Da
DNA d(CGCA10CGC)
RNA r(CGCA10CGC)
1.0 M [Na+] 0.1 M [Na+]
M+ (calc.) M+ (found) 0.02 M [Na+]
0.1 M [Na+]
13 d(GCGT10GCG)
14 d(GCGT4TnsnTT4GCG)
15 d(GCGTnsnTTTTnsnTTTTnsnTGCG)
16 d(GCGT4TnsoTT4GCG)
17 d(GCGTnsoTTTTnsoTTTTnsoTGCG)
4875.2
4873.3
4869.5
4874.3
4872.5
4875.3
4872.8
4869.7
4874.2
4872.5
4870.6
43.2
55.0
66.4
49.0
40.0 (23.2)b
33.7 (23.2)
43.6 (+0.4)
44.0 (+0.3)
44.8 (+0.3)
51.7 (23.3)
45.0 (23.3)
55.1 (+0.1)
55.5 (+0.2)
53.3 (–0.3)
60.9 (25.5) 45.9 (23.1)
54.6 (23.9) 41.0 (22.7)
66.0 (20.4) 47.8 (21.2)
N.D.c
N.D.c
48.0 (20.3)
46.0 (20.6)
18 d(GCGTnsoTTnsoTTnsoTTnsoTTnsoTGCG) 4870.7
a Tm values are accurate to within ± 0.5 °C. b The changes in Tm per modification (DTm/mod.) are shown in parentheses.c Tm was not determined.
of strong and weak positive bands at 280 and 225 nm,
respectively, as well as a negative band at 255 nm, suggesting
that the native and the modified dimers are adopting similar
overall conformations. Upon increasing temperature all showed
a significant decrease in the intensity of the bands at 280 and
255 nm. Notably the change in band intensity for d(TnsoT) was
twice the magnitude of that observed for d(TnsnT) and d(TpT)
which were virtually identical. This suggests that d(TnsoT) has
an higher propensity to adopt a base stacked helical conforma-
tion than d(TnsnT) and d(TpT), which base stack to a lesser
extent.10
cleotides appear to form more stable duplexes than DNA
incorporating the isomeric 5A-N-sulfamate modification, which
is reported to result in a drop in Tm of 1.5 °C per modification.8
The destabilisation of duplexes by sulfamide and 5A-N-
sulfamate modifications may be accounted for by the 5A-amino
substituent common to both. In the case of isosteric and
isoelectronic N5A?P3A phosphoramidate DNA, the presence of
a 5A-NH group completely abolishes base pairing with com-
plementary nucleic acids.12 This is attributed to poor hydration
and steric clashes between the 5A-amino hydrogen atom and
either the H2A atom of an adjacent deoxyribose ring in an A–
type conformation, or the pyrimidine H6 and deoxyribose O4A
atoms in a B-type conformation.12
The data reported here suggest that the 3A-N-sulfamate
modification may be of use in the development of improved
antisense or antigene agents. Moreover, given that the 3A-N-
sulfamate group causes minimal disruption of nucleic acid
duplexes, is sterically and electronically more similar to the
phosphodiester group than other neutral linkages so far
developed, it might also be useful as a general phosphodiester
replacement for other applications. For example, one can
envisage probing nucleic acid–protein interactions using this
modification to determine which phosphodiester groups are
involved in electrostatic contacts with protein residues. Alter-
natively 3A-N-sulfamate groups might be used to stabilise
aptamers, or hammerhead ribozymes for use in vivo.
Solid phase synthesis of 16-mer chimeric oligodeoxynucleo-
tides, incorporating 3A–N-sulfamate or sulfamide linkages, was
carried out under standard conditions using the modified dimer
phosphoramidites 10 and 12. The oligonucleotides prepared,
13–18, were characterised by electrospray mass spectrometry
and all have the same sequence GCGT10GCG11 (Table 1). The
duplex melting temperatures (Tm) of modified oligonucleotides
with complementary DNA were then determined by variable
temperature UV spectroscopy and compared with the Tm for the
native duplex. Modified oligonucleotides with one and three
central sulfamide linkages, 14 and 15, both show a similar
change in Tm per modification (DTm/mod.), compared with the
native duplex, of 23.2 °C at 0.02 and 0.1 M salt concentration
[Na+]. At 1.0 M salt concentration the modification was even
more destabilising. In contrast incorporation of one, three and
five sulfamate groups, 16, 17 and 18, has little effect on duplex
stability with complementary DNA and is even moderately
stabilising at low salt concentration with an observed increase in
Tm of 0.3 °C per modification. Upon increasing salt concentra-
tion the Tm dropped by 0.4 °C for one sulfamate modification.
It is likely that the neutral linkages reduce the electrostatic
repulsion between strands which is more evident at low salt
concentration when fewer cations are present to mask the
negatively charged phosphodiester groups. With complemen-
tary RNA at 0.1 M salt concentration, the sulfamide group had
a similar effect on duplex stability with a drop in Tm of ca.
3.0 °C per modification. However the 3A-N-sulfamate modified
oligonucleotides formed slightly less stable duplexes with RNA
than DNA with a drop in Tm of 1.2 °C for one modification and
a more moderate drop in DTm/mod of 0.3 and 0.6 °C for three
and five modifications respectively. It is probable that oligonu-
cleotides with more 3A-N-sulfamate linkages have an higher
proportion of C3A-endo sugar rings and are therefore more likely
to adopt an A–type conformation which is preferred for binding
to RNA.
We thank the EPSRC for studentships to K. J. F. and D. T. H.,
Polkit Sangvanich for assistance with electrospray mass
spectrometry and the EPSRC National Chiroptical Spectros-
copy Service.
Notes and references
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oligonucleotides hybridise with complementary nucleic acids
with similar binding affinity as native DNA, whereas the
sulfamide congeners have significantly lower affinity. It is
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oligonucleotides are more preorganised into a base stacked
helical conformation, in the single stranded state, which would
reduce the loss of entropy on binding to complementary RNA
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duplexes. Furthermore, the 3A-N-sulfamate modified oligonu-
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