Synthesis of aminoglycoside-modified oligonucleotides

Article abstract of DOI:10.1021/ol005797d

(matrix presented) To study the structural requirements of aminoglycoside binding to nucleic acids, compound 1 - an analogue of the naturally occurring nucleoside J - was synthesized. When incorporated into oligodeoxynucleotides, 1 leads to thermal stabil

Full text of DOI:10.1021/ol005797d

ORGANIC
LETTERS
2000
Vol. 2, No. 12
1693-1696
Synthesis of Aminoglycoside-Modified
Oligonucleotides
Rolf Tona, Reto Bertolini, and Ju1rg Hunziker*
Department of Chemistry and Biochemistry, UniVersity of Bern, Freiestrasse 3,
3012 Bern, Switzerland
juerg.hunziker@ioc.unibe.ch
Received March 13, 2000
ABSTRACT
To study the structural requirements of aminoglycoside binding to nucleic acids, compound 1san analogue of the naturally occurring nucleoside
Jswas synthesized. When incorporated into oligodeoxynucleotides, 1 leads to thermal stabilization of the resulting duplexes. The increase in
pairing affinity is stronger with complementary RNA than with DNA.
Structural modifications to DNA bases, apart from degra-
dative damage, are rarely found in nature.1 This contrasts
the vast abundance of altered nucleobases found in RNA.2
The most prominent DNA base modification in eukaryotes
is cytosine methylation for the silencing of genes on homo-
logous chromosomes.3 Less conservative changes, such as
the glycosylated nucleosides 2 or 3, are found in the DNA
of certain pathogens. Escherichia coli bacteriophages of the
T-even series exclusively contain 5-hydroxymethyldeoxy-
3
cytidine instead of deoxycytidine in their DNA.1 About /4
of these 5-hydroxymethylcytosine residues are â-D-gluco-
enzymes6 which helps the pathogen to escape degradation
by host restriction enzymes. In the X-ray crystal structure5
of a short self-complementary duplex, the glucose is arranged
such that the primary hydroxy group is pointing toward the
sugar-phosphate backbone of the complementary strand.
sylated. A similar modification is found in the genome of
Trypanosoma bruceisa protozoan causing African sleeping
sicknesssas well as in phages. Here, the glucose is linked
to 5-hydroxymethyldeoxyuridine.4 This unusual nucleoside
replaces thymidines within the long telomeric repeats in T.
brucei.4b
(4) This modification is also referred to as nucleoside J. (a) Gommers-
Ampt, J. H.; van Leeuwen, F.; de Beer, A. L. J.; Vliegenthart, J. F. G.;
Dizdaroglu, M.; Kowalak, J. A.; Crain, P. F.; Borst, P. Cell 1993, 75, 1129.
(b) van Leeuwen, F.; Wijsman, E. R.; Kuyl-Yeheskiely, E.; van der Marel,
G. A.; van Boom, J. H.; Borst, P. Nucleic Acids Res. 1996, 24, 2476.
(5) Gao, Y.; Robinson, H.; Wijsman, E. R.; van der Marel, G. A.; van
Boom, J. H.; Wang, A. H.-J. J. Am. Chem. Soc. 1997, 119, 1496.
(6) van Leeuwen, F.; de Kort, M.; van der Marel, G. A.; van Boom, J.
H.; Borst, P. Anal. Biochem. 1998, 258, 223.
The glucose residues of DNA containing 2 reside within
the major groove5 and render the DNA inaccessible for
(1) Kornberg, A.; Baker, T. A. DNA Replication; W. H. Freeman Co.:
New York, 1992.
(2) http://medlib.med.utah.edu/RNAmods/. Rozenski, J.; Crain, P. F.;
McCloskey, J. A. Nucleic Acids Res. 1999, 27, 196.
(3) Robertson, K. D.; Jones, P. A. Carcinogenesis 2000, 21, 461.
10.1021/ol005797d CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/16/2000

The secondary hydroxy group in the 2-position is located
near a phosphodiester group on the strand containing 2
(Figure 1).
The synthesis of protected 5-hydroxymethyl-2′-deoxyuri-
dine glycosyl acceptor 12 has already been described.8,9
However, the rather low yields and high costs for starting
materials in these syntheses led us to explore a different route.
5-Hydroxymethyluracil (6), obtained in one step from
uracil,10 was coupled with tetraacetylribose 5 to give 5-hy-
droxymethyluridine (7) in 79% yield (Scheme 1).11 To
differentiate the two primary hydroxy groups, 7 was reacted
with dimethoxytrityl chloride. After removal of the acetyl
groups, the ribose was selectively protected at the 3′- and
5′-hydroxyls with TIPDS to give 10. Barton-McCombie
reduction of the 2′-hydroxy group gave deoxynucleoside 11
in high yield. The DMT protecting group could then easily
be removed to give the glycosyl acceptor 12 in 43% overall
yield from 5-hydroxymethyluracil (6).
Since we felt that the synthesis of a suitable 2,6-
diaminoglucose donor from a commercially available
monosaccharide would require eight to ten steps, we chose
instead to start from a natural product. The aminoglycoside
neomycin B (15) contains a 2,6-diamino-2,6-dideoxy-D-
glucose subunit and is readily available. The glycosidic
linkage between the ribose residue and the aminocyclohexitol
2-deoxystreptamine (2-DOS) can be cleaved selectively
under acidic conditions to give neamine hydrochloride 16.12
The amino functions in neamine were first protected as
trifluoroacetamides and then the hydroxy groups were
esterified with acetic anhydride to give 17 in 67% yield
(Scheme 2). This neamine derivative could be hydrolyzed
with HBr in acetic acid to give a mixture of the desired
glycosyl donor 18 and the 2-DOS byproduct 19.13
The coupling of glycosyl bromide 18 and uridine deriva-
tive 12 proceeded smoothly under mercury salt activation
to give diaminoglucosylated nucleoside 13 in 84% yield.
Removal of the silyl protecting groups and standard ma-
nipulations14 then provided phosphoramidite building block
14. With this, oligonucleotides 22 and 26 (Table 1) were
prepared on a 1.3 µmol scale by automated solid-phase
synthesis.15,16
Figure 1. The glucose moiety of nucleoside analogue 2 is located
within the major groove of double stranded DNA.3 By replacing
the hydroxy groups at the 2- and 6-position of the glucose by
ammonium groups, the two oligonucleotide strands could be
clamped together through electrostatic interactions.
Incorporating 2 into oligonucleotide duplexes reduces their
thermal stability. The replacement of ordered water in the
major groove by hydrogen-bonding contacts between the
glucose and nucleobases leads to a favorable entropic
contribution which, however, cannot fully compensate for
the smaller enthalpy of duplex formation.7 This seems to be
a tolerable tradeoff for the pathogens mentioned, to protect
their genome from degradation.
To improve the thermal stability and yet retain the desired
protection from nucleases, we sought to synthesize oligode-
oxynucleotides (ODNs) containing nucleoside analogues 1
and 4. The amino groups, positively charged at neutral pH,
should improve binding to a complementary strand by
electrostatic interactions (Figure 1). However, 4 when built
into ODNs behaves in a similar manner to that of 2: a
decrease in melting temperature is observed.8 Most likely,
the 2-ammonium group of 4 is located near a phosphodiester
group of the strand it is attached to and thus does not affect
duplex formation. Here, we report the synthesis of ODNs
containing 1 which show the anticipated behavior.
Replacing one thymidine residue with 2,6-diamino-â-D-
glucose-modified 1 at a central position in the reference
(7) Bertolini, R.; Hunziker, J. Unpublished data.
(8) Hunziker, J. Bioorg. Med. Chem. Lett. 1999, 9, 201.
(9) de Kort, M.; Ebrahimi, E.; Wijsman, E. R.; van der Marel, G. A.;
van Boom, J. H. Eur. J. Org. Chem. 1999, 2337.
(10) Cline, R. E.; Fink, R. M.; Fink, K. J. Am. Chem. Soc. 1959, 81,
2521.
(11) All new compounds were fully characterized by UV, IR, 1H and
13C NMR, and mass spectroscopy.
(12) Park, W. K. C.; Auer, M.; Jaksche, H.; Wong, C.-H. J. Am. Chem.
Soc. 1996, 118, 10150.
(13) The mixture of these two compounds could only be separated by
silica gel chromatography with substantial loss of 18. Nevertheless, the ease
of preparation of 18 outweighted the poor yields of this strategy.
(14) Conolly, B. A. In Oligonucletides and Analogues; Eckstein, F., Ed.;
Oxford University Press: Oxford, 1991; pp 155-183.
(15) Oligonucleotides 20-28 were prepared on a Pharmacia Gene
Assembler Plus using the standard protocol. The coupling time for 14 was
changed from 2 to 6 min. Average coupling yields for aminoglycoside-
modified nucleosides (>98%) were in line with unmodified phosphora-
midites. The oligonucleotides were purified by reverse-phase HPLC followed
by ion-exchange HPLC. The integrity of the isolated oligonucleotides 20-
28 was subsequently confirmed by MALDI-ToF mass spectrometry: m/z
(monoanion, H+-form) 22, calcd 3155.0, found 3154.9; 26, calcd 3363.3,
found 3364.4. Matrix conditions as described in the following: Pieles, U.;
Zu¨rcher, W.; Scha¨r, M.; Moser, H. E. Nucleic Acids Res. 1993, 21, 3191.
1694
Org. Lett., Vol. 2, No. 12, 2000

Scheme 1. Synthesis of Diaminoglucose-Modified 5-Hydroxymethyl-2′-deoxyuridine Phosphoramidite 14a
a (a) BSA, TMSOTf, CH3CN, 75 °C, 2 h; (b) DMTCl, DMAP, pyridine, rt, 3 h; (c) Na2CO3, MeOH, rt, 17 h; (d) TIPDSCl, pyridine, rt,
22 h; (e) thiocarbonyldiimidazole, CH3CN, rt, 21 h; (f) Bu3SnH, AIBN, toluene, 80 °C, 50 min; (g) Cl2HCCOOH, pyrrole, CH2Cl2, rt, 15
min; (h) 18, Hg(CN)2, molecular sieves 3 Å, rt, 16 h; (i) TBAF, AcOH, THF, rt, 30 min; (j) DMTCl, pyridine, rt, 4 h; (k)
i
(iPr2N)(NCCH2CH2O)PCl, Pr2NEt, THF, rt, 1 h. DMT ) 4,4′-dimethoxytrityl.
duplex d(T10)•d(A10) leads to a decrease in thermal stability
(22‚21 ∆Tm/mod ) -5 °C, Table 1). In a mixed sequence
context (26‚24), however, an increase of ∆Tm/mod ) +1.5
°C results. The different effects of 1 in these pairing systems
might be due to the particular structure of oligo(dA)‚oligo-
(dT) duplexes which have a wide and flat major groove.17
The same trend is seen for the analogous glucose modifica-
tion (23‚21 vs 27‚25), although a destabilization is observed
in both cases. With a complementary RNA strand, the
stabilizing effect of nucleoside 1 is even more pronounced
(26‚28 ∆Tm/mod ) +3.5 °C). Not surprisingly, the increase
in affinity due to the diaminoglucose modification is stronger
Scheme 2. Preparation of 2,6-Diaminoglucose Donor 18a
a (a) Aqueous HCl, MeOH, reflux, 18 h; (b) F3CCOOEt, MeOH, Et3N, rt, 16 h; (c) Ac2O, pyridine, rt, 2 h; (d) HBr, AcOH, rt, 16 h.
Org. Lett., Vol. 2, No. 12, 2000 1695

Table 1. Melting Temperatures (Tm) of Oligonucleotides Containing 1a
1 M NaCl
∆Tm/mod [°C]
0.15 M NaCl
∆Tm/mod [°C]
oligonucleotide duplex
d(T10)‚d(A10) (20‚21)
Tm [°C]b
Tm [°C]b
33
28
29
51
54
49
41
48
23
20
nd
44
49
nd
37
nd
d(TTTT1TTTTT)‚d(A10) (22‚21)
d(TTTT2TTTTT)‚d(A10) (23‚21)
-5
-4
-3
nd
d(CTGAATCGAC)‚d(GTCGATTCAG) (24‚25)
d(C1GAA1CGAC)‚d(GTCGATTCAG) (26‚25)
d(C2GAA2CGAC)‚d(GTCGATTCAG) (27‚25)
d(CTGAATCGAC)‚r(GUCGAUUCAG) (24‚28)
d(C1GAA1CGAC)‚r(GUCGAUUCAG) (26‚28)
+1.5
-1
+2.5
nd
+3.5
nd
a Oligonucleotide concentration: 4 µM in 10 mM NaH2PO4, pH 7.0, and the indicated amount of NaCl. b Absorbance detected at 260 nm. Melting
temperatures represent the mean value of three melting curves. Heating rate: 0.5 °C/min.
at low ionic strength,18 which indicates that the amino groups
are indeed protonated at pH 7.0.
strand if the duplex adopts a more RNA-like conformation.
This electrostatic attraction might actually pull the backbones
together and alter duplex conformation. In fact, the CD
spectrum of the DNA-DNA duplex 26‚24 differs more from
its unmodified counterpart than the corresponding DNA-
RNA duplex 26‚28 (data not shown).
In summary, we have successfully synthesized ODNs
containing 2,6-amino-â-D-glucose-modified nucleoside 1.
These display increased affinity toward DNA and even more
so for RNA complementary strands. More detailed structural
and synthetic characterization of this pairing system will be
reported in due course.
Aminoglycosides show a marked preference for RNA.19
The same is seen here for ODNs containing the diamino-
glucose-modified nucleoside 1. In DNA-RNA heterodu-
plexes and RNA duplexes, the major groove is deep and
narrow.17 The primary ammonium group of 1 might thus be
located closer to the phosphodiester groups of the opposing
(16) The HPLC trace of crude ODNs 22 and 26 showed that a substantial
amount of byproducts was presentspresumably not fully deprotected ODNs
which arise from the capping step of the oligo synthesis. Acetic anhydride
could acylate the trifluoroacetamide functions of the diaminoglucose residues
and these acetamides might not be fully deprotected under standard
deprotection conditions (concentrated ammonia at 55 °C for 16 h). MALDI-
ToF MS analysis of these byproducts was inconclusive.
(17) Saenger, W. Principles of Nucleic Acid Structure; Springer-
Verlag: New York, 1984.
(18) 5-Aminoalkyl- and alkynyl-modified pyrimidine oligonucleotides
also bind stronger to complementary DNA under low salt conditions and
neutral pH than their unmodified counterparts. (a) Hashimoto, H.; Nelson,
M. G.; Switzer, C. J. Am. Chem. Soc. 1993, 115, 7128. (b) Heystek, L. E.;
Zhou, H.; Dande, P.; Gold, B. J. Am. Chem. Soc. 1998, 120, 12165.
Acknowledgment. This work was supported by a re-
search grant from the Swiss National Science Foundation
(Grant 20-53692.98).
OL005797D
(19) Tor, Y. Angew. Chem., Intl. Ed. 1999, 38, 1579.
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Org. Lett., Vol. 2, No. 12, 2000