Efficient conjugation and preferential DNA binding of oligonucleotides containing 2′-O-(2-oxoethyl)arabinouridine

Article abstract of DOI:10.1016/j.tetlet.2004.08.006

Oligodeoxyribonucleotides were synthesized that contain a novel nucleoside, 2′-O-(2-oxoethyl)arabinouridine. Whereas such oligonucleotides showed only a slight reduction in the T_M values of their complexes with complementary DNA, a significant

Full text of DOI:10.1016/j.tetlet.2004.08.006

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 7327–7330
Efficient conjugation and preferential DNA binding of
oligonucleotides containing 20-O-(2-oxoethyl)arabinouridine
Timofei S. Zatsepin,a Yulia M. Ivanova,a Dmitry A. Stetsenko,b Michael J. Gaitb and
Tatiana S. Oretskayaa,*
aDepartment of Chemistry, M. V. Lomonosov Moscow State University, Leninskie gory, Moscow 119992, Russia
bMRC, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
Received1 June 2004; revised16 July 2004; accepted2 August 2004
Available online 21 August 2004
Abstract—Oligodeoxyribonucleotides were synthesized that contain a novel nucleoside, 20-O-(2-oxoethyl)arabinouridine. Whereas
such oligonucleotides showed only a slight reduction in the TM values of their complexes with complementary DNA, a significant
0
0
destabilization was observed in the case of duplexes formed with RNA. This may be explained by the C2 -endo conformation of 2 -
O-(2,3-dihydroxypropyl)arabinouridine as demonstrated by NMR experiments in D2O. The modified oligonucleotides were used to
synthesize a number of conjugates with dyes, biotin and a N-modified laminin peptide, by hydrazone and oxime formation. We sug-
gest that the 20-arabinoaldehyde-containing DNA duplexes may be valuable tools for affinity modification of DNA-binding
proteins.
Ó 2004 Published by Elsevier Ltd.
Recent advances in the chemical synthesis of modified
oligonucleotides have led to their wide application as
diagnostic agents, and as potential therapeutics (anti-
sense, antigene, aptamer or RNAi strategies).1,2 Today,
various oligonucleotide-based conjugates with specific
properties are under development. Ligands attached to
oligonucleotides are used as reporter groups (fluores-
cent, electrochemical or spin labels), for stabilization
of double or triple helical structures (intercalators and
minor groove binders) and for increasing cellular uptake
(peptides, hydrophobic molecules and carbohydrates).
Aldehyde–nucleophile coupling is used in peptide chem-
istry,3 glycochemistry4 andnucleic acidchemistry, 5,6 and
seems to be a good alternative to traditional amide,
disulfide or thioether conjugation, especially in the case
of complex biomolecules. Oligonucleotides with reactive
groups are effective tools for affinity modification of
DNA-binding proteins.7 Such reactive groups can be
incorporatedinto heterocyclic base, phosphate or sugar
moieties. The use of nucleosides with modified sugar res-
idues is particularly general. The C20-modification is
especially appropriate since this leads to minimal distor-
tions in oligonucleotide structure in general and is rela-
tively straightforwardto achieve chemically. It also
provides an opportunity to adjust the sugar conforma-
0
0
tion towards either C2 -exo or C2 -endo depending on
the type andconfiguration of the substituent, andto Ôdi-
rectÕ a 20-functionality to the major or minor groove.8
The presence of a 20-O-alkyl group causes local changes
from the B-DNA to the A-DNA-type conformation and
the substituent is placedin the minor groove. 9,10 On the
other hand, it was shown that a 20-O-methyl arabino
substituent is directed into the major groove and does
not significantly influence the B-like helix structure of
the duplex.11–13 This feature is particularly important
in our research, since we have demonstrated that oligo-
nucleotides containing 20-O-(2-oxoethyl)uridine can be
usedfor affinity moifidcation of DNA-biinndg
proteins.14,15
Here we report the preparation of 20-O-(2,3-dihydroxy-
propyl)arabinouridine phosphoramidite and its incorpo-
ration into oligonucleotides. 2D COSY and NOESY
NMR experiments show that the desired nucleoside
0
adopts the C2 -endo conformation in D2O. We also de-
Keywords: Modified oligonucleotides; Conjugation; Aldehyde; Oxime;
Hydrazone.
scribe an efficient conjugation of the 20-arabinoaldehyde
oligonucleotides with a number of fluorescent markers
anda N-modified laminin peptide.
*
Corresponding author. Tel.: +7 095 939 5411; fax: +7 095 939
3148; e-mail: oretskaya@belozersky.msu.ru
0040-4039/$ - see front matter Ó 2004 Publishedby Elsevier Ltd.
doi:10.1016/j.tetlet.2004.08.006

7328
T. S. Zatsepin et al. / Tetrahedron Letters 45 (2004) 7327–7330
Scheme 1. Preparation of the phosphoramidite 5. Reagents and conditions: (a) (1) TMSCl, Py, 10min; (2) 2,4-dinitrophenylsulfenylchloride, Py, 3h;
(3) NH3 aq, Py, 30min, 75%; (b) AllBr, BDDDP, MeCN, 3h, 79%; (c) OsO4 (0.05equiv), N-methylmorpholine N-oxide, THF–H2O (2:1, v/v),
overnight, 95%; (d) Lev2O, Py, DMAP, 1h, 88%; (e) Et3N(HF)3, THF, 1h, 86%; (f) DMTrCl, Py, 3h, 92%; (g) ðPri2NÞ POðCH2Þ CN,
2
2
diisopropylammonium tetrazolide, CH2Cl2, 1h, 75%; (h) K2CO3, MeOH, 3h, 90%.16
The target phosphoramidite 5 was synthesizedby a
route similar to that we publishedpreviously for the ribo
epimer17 (Scheme 1). However, we describe here the
application of protecting groups different from those
usedpreviously, which leadto a significant increase in
the overall yield. The N3-position of 30,50-O-TIPDS-pro-
nucleoside in D2O. A strong interaction was observed
0
between H-10 (d = 6.15ppm) andH-2 (d = 4.33ppm).
Furthermore, an additional cross-peak between H-20
0
andH-4 (d = 3.85ppm) confirmedthe arabino configu-
0
ration of nucleoside 6. A predominant South (C2 -endo)
conformation was supportedby the 1H, 1H coupling
tectedarabinouriidne
118 was blockedby a 2,4-id-
constants (i.e., J1 ,2 = 5.1Hz, J3 ,4 = 0Hz). Further
transformations of 4 were carriedout to provide the
building block 522 suitable for oligonucleotide synthesis.
The modified oligonucleotides were prepared by the
phosphoramidite approach. The sequences synthesized
are shown in Table 1.
0
0
0
0
nitrophenylsulfenyl protecting group, cf. Alvarez et al.19
to prevent side alkylation during the next step. 20-O-All-
yl ether 2 was synthesizedas describedearlier by Grøtli
et al.,20 then the 20-allyloxy group was convertedinto
the blocked cis-diol group by osmium tetroxide/N-meth-
ylmorpholine N-oxide oxidation followed by O-levulina-
tion. The formation of a diastereoisomeric mixture
(65:35) was observed. The levulinyl protecting group is
well-known in oligonucleotide synthesis, since it can be
selectively removedby hydrazinium acetate treatment
on a solidsupport. This methodcan also be appliedto
Generation of the 20-aldehyde group was carried out
by use of sodium periodate as described earlier
(Scheme 2).10 However the reaction time was prolonged
up to 1.5h due to steric hindrance. Conjugation of the
20-arabinoaldehyde oligonucleotides with a N-amino-
oxy-modified peptide, an aromatic hydrazine and two
hydrazides was achieved under previously reported con-
ditions10 in goodyielsd ( Table 2). The hydrazone
formed from 9-hydrazinoacridine was stable under the
analysis conditions but in the case of hydrazides, cya-
21
the solidphase synthesis of oligonucleotide conjugates.
The compound 3 was treatedwith triethylamine tri-
hydrofluoride in THF to afford 4. O-Deprotection gave
us the nucleoside 6 to carry out D NMR experiments
2
(COSY, NOESY) on the conformation of the desired
Table 1. MALDI-TOF MS analysis of the 20-modified oligonucleotides synthesized and thermal stability of the duplexes formed by the 20-modified
oligonucleotides with complementary DNA and RNA targets
Oligonucleotide
MALDI-TOF MS
calcd/founda [M+H]+
TM values of duplexes formed by 20-modified oligonucleotides with
complementary DNA or RNAb, °C
d(TGAGCCTGGAG)
TM 0.5, °C
DTM/modc, °C
r(UGAGCCUGGAG)
TM 0.5, °C DTM/mod, °C
I
CTCCCAGGCTCA
CTCCCAGGCUCA
CUCCCAGGCTCA
CUCCCAGGCUCA
––
56.0
––
58.6
––
II
3643.4/3645.1
3643.4/3641.3
3719.4/3718.3
52.9a (53.1)d
54.3a (55.2)d
49.1a (47.0)d
49.5a (48.7)d
50.3a (49.5)d
42.9a (41.0)d
III
IV
À3.9a (À3.2)d
À11.0a (À9.1)d
U––20-O-(2,3-Dihydroxypropyl)arabinouridine or 20-O-(2-oxoethyl)arabinouridine.
a For the 20-diol modification.
b Buffer: 100mM NaCl, 10mM NaHPO4, 1mM EDTA, pH7.0.
P
c DTM/mod=
{[TM(n) À TM(I)]/M}/k, where M is the number of modified nucleotides in a duplex, k is the number of the studied duplexes.
d For the 20-aldehyde modification.

T. S. Zatsepin et al. / Tetrahedron Letters 45 (2004) 7327–7330
7329
oxyribonucleotide had only a slightly destabilizing effect
on the TM values of the complexes with DNA (Table 1).
It shouldbe notedthat here we have usedthe corre-
spondingly modified uridine derivative and that replace-
ment of thymidine by 20-deoxyuridine itself would be
expectedto decrease the TM by ca. 0.5°C.24 Nonethe-
less, the destabilization was significantly stronger in
the case of a complementary RNA (Table 1). Selective
stabilization of the duplexes with a DNA target as com-
paredto an RNA target may be explainedby the S-con-
formation of the nucleoside and thus preferential B-type
helix formation by the modified oligonucleotide.
Since incorporation of the modified nucleoside into oligo-
deoxyribonucleotides has only a marginal effect on the
TM values of their complexes with DNA targets, we
believe these reactive oligomers may be useful for affin-
ity modification of DNA-binding proteins. The investi-
gation of cross-linking of the 20-arabinoaldehyde
oligonucleotides to transcription factor NF-kB is in pro-
gress andwill be reportedin due course.
Acknowledgements
This work was supportedby Wellcome Trust CRIG
069419 andpartly by RFBR Grant No. 03-04-48957.
References and notes
Scheme 2. Preparation of the 20-aldehyde-containing oligonucleotides
andtheir conjugates. Reagents andconditions: (a) automatedoligo-
nucleotide synthesis followed by aq NH3, 55°C, overnight; (b) 80%
AcOH, 30min; (c) NaIO4, 0.2M AcONa, pH5.0, 15min; (d) R1NH2
(1.5–2equiv), 0.4M AcONa, pH4.5, DMSO (50% v/v), 3h; (e)
R2CONHNH2 (1.5–2equiv), 0.4M AcONa, pH4.5, DMSO (50%
v/v), 3h; then NaBH3CN, 30min.
1. Opalinska, J. B.; Gewirtz, A. M. Nat. Rev. Drug Discov.
2002, 1, 503–514.
2. Heller, M. J. Annu. Rev. Biomed. Eng. 2002, 4, 129–153.
3. Tam, J. P.; Xu, J.; Eom, K. D. Biopolymers 2001, 60, 194–
205.
4. Pari, F.; Nicotra, F. Chem. Commun. 2004, 623–627.
5. Zatsepin, T. S.; Stetsenko, D. A.; Arzumanov, A. A.;
Romanova, E. A.; Gait, M. J.; Oretskaya, T. S. Bioconj.
Chem. 2002, 13, 822–830.
noborohydride reduction was needed. These results are
in accordance with published data for hydrazone stabil-
ity.23 The conjugates were characterizedby MALDI
TOF MS. In all cases, the experimentally determined
molecular masses were in goodagreement with the cal-
culatedvalues ( Table 2).
6. Forget, D.; Boturyn, D.; Defrancq, E.; Lhomme, J.;
Dumy, P. Chem. Eur. J. 2001, 7, 3976–3984.
7. Meisenheimer, K. M.; Koch, T. H. Crit. Rev. Biochem.
Mol. Biol. 1997, 32, 101–140.
8. Manoharan, M. Biochim. Biophys. Acta 1999, 1489, 117–
130.
9. Lubini, P.; Zurcher, W.; Egli, M. Chem. Biol. 1994, 1, 39–
45.
Binding of the modified oligonucleotides containing
20-O-(2,3-dihydroxypropyl)arabinouridine to the com-
plementary DNA andRNA sequences was investigated
by their UV absorbance melting behaviour. Incorpora-
tion of 20-O-(2-oxoethyl)arabinouridine into an oligode-
10. Teplova, M.; Minasov, G.; Tereshko, V.; Inamati, G. B.;
Cook, P. D.; Manoharan, M.; Egli, M. Nat. Struct. Biol.
1999, 6, 535–539.
11. Gotfredsen, C. H.; Jacobsen, J. P.; Wengel, J. Tetrahedron
Lett. 1994, 35, 6941–6944.
Table 2. Isolatedyields andMALDI-TOF MS analysis of the conjugates of the 2 0-modified oligonucleotides
Conjugatedmolecule
Oligonucleotide
III
II
IV
Biotin hydrazide
74a (3853.7/3853.9)b
82a (3897.3/3893.7)b
84a (3802.6/3800.9)b
65a (4528.3/4531.0)b
79a (3853.7/3855.1)b
––
85a (3802.6/3803.0)b
––
69a (4140.0/4142.1)b
74a (4228.1/4223.5)b
62a (4036.8/4038.1)b
50a (5490.3/5489.2)b
Pyrenebutyric hydrazide
9-Hydrazinoacridine
H2NOCH2CO-DPGYIGSR-NH2
a Isolatedyieldof the conjugate.
b MALDI-TOF MS: calcd/found [M+H]+.

7330
T. S. Zatsepin et al. / Tetrahedron Letters 45 (2004) 7327–7330
12. Gotfredsen, C. H.; Jacobsen, J. P.; Wengel, J. Bioorg.
Med. Chem. 1995, 4, 1217–1225.
13. Gotfredsen, C. H.; Spielman, H. P.; Wengel, J.; Jacobsen,
J. P. Bioconj. Chem. 1995, 7, 680–688.
14. Turutin, D. V.; Zatsepin, T. S.; Timchenko, M. A.;
Kubareva, E. A.; Oretskaia, T. S. Mol. Biol. (Russ.) 2002,
36, 877–879.
15. VorobÕeva, O. V.; Zatsepin, T. S.; Timchenko, A. A.;
Karyagina, A. S.; Yamskova, O. V.; Bartunik, H. D;
Oretskaya, T. S., Kubareva, E. A. Biol. Chem. (in press).
16. TMS––trimethylsilyl, Py––pyridine, All––allyl, BDDDP––
2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-
J.; Oretskaya, T. S. In Innovation and Perspectives in Solid
Phase Synthesis and Combinatorial Libraries; Epton, R.,
Ed.; Mayflower Worldwide: Kingswinford (in press).
22. 1H NMR (CDCl3, d, ppm): 7.68 (d, 1H, H-5,
J5,6 = 7.2Hz), 7.45–7.20, 6.85–6.73 (m, 13H, DMTr),
6.23 (t, 1H, H-10, J1 ,2 = 3.0Hz), 5.58 (d, 1H, H-6), 5.32
(m, 1H, CH2CH), 4.45–4.28 (m, 2H, H-20, CHCH2a),
4.17–4.12 (m, 2H, H-40, CHCH2b) 3.89–3.79 (m, 3H, H-30,
CH2CH), 3.73 (s, 6H, DMTr), 3.52 (d, 1H, H-50a,
0
0
J5 a,5 b = 12.1Hz), 3.47 (d, 1H, H-50b), 2.78 (m, 2H,
CO2CH2), 2.66 (m, 2H, CH2CO), 2.54 (t, 2H, CH2CN),
2.15 (s, 6H, COCH3) 1.1 (s, 12H, CH3CH); 13C NMR
(CDCl3, d, ppm): 206.63 (C@O, Lev), 172.41 (OC@O,
Lev), 162.59 (C-4), 158.67 (DMTr), 150.54 (C-2), 144.43
(DMTr), 140.45 (C-6), 132.28, 130.19, 128.29, 127.96,
127.12, 117.74 (DMTr), 117.62 (CN), 113.22, 112.11
(DMTr), 103.05 (C-5), 100.04 (C-10), 87.16 (C-40), 86.47
(C-20), 84.64 (C-30), 83.04 (DMTr), 68.27 (C-20 0), 65.49 (C-
10 0), 62.27 (C-50), 62.04 (C-30 0), 59.92 (POCH2), 55.32
(DMTr), 42.79 (PNCH), 37.51 (CH2COCH3), 29.92
(CO2CH2), 29.76 (COCH3), 26.11 (CH3, Pri), 20.26
(CH2CN); 31P NMR (ppm): 150.40, 151.73; MALDI-
MS: calcd[M+H] + = 1016.1. Found1016.1.
0
0
1,3,2-diazaphosphorin,
Lev––levulinyl,
DMAP––4-
dimethylaminopyridine, DMTr––4,40-dimethoxytrityl.
17. Kachalova, A. V.; Zatsepin, T. S.; Romanova, E. A.;
Stetsenko, D. A.; Gait, M. J.; Oretskaya, T. S. Nucleo-
sides, Nucleotides Nucleic Acids 2000, 19, 1693–1707.
18. Dioubankova, N. N.; Malakhov, A. D.; Stetsenko, D. A.;
Gait, M. J.; Volynsky, P. E.; Efremov, R. G.; Korshun, V.
A. ChemBioChem. 2003, 4, 841–847.
19. Alvarez, K.; Tworkowski, I.; Vasseur, J.-J.; Imbach, J.-L.;
Rayner, B. Nucleosides Nucleotides 1998, 17, 365–378.
20. Grøtli, M.; Douglas, M.; Beijer, B.; Garcia, R. G.; Eritja,
R.; Sproat, B. J. Chem. Soc., Perkin Trans. 1 1997, 2779–
2788.
23. Sayer, J. M.; Peshkin, M.; Jencks, W. P. J. Am. Chem.
Soc. 1973, 95, 4277–4287.
21. Kachalova, A. V.; Zubin, E. M.; Zatsepin, T. S.; Agap-
kina, Yu. V.; Ivanova, Yu. M.; Stetsenko, D. A.; Gait, M.
24. Freier, S. M.; Altmann, K. H. Nucleic Acids Res. 1997, 25,
4429–4443.