A New Method for the Preparation of Modified Oligonucleotides

Article abstract of DOI:10.1021/ol025643t

(Equation Presented) N-Nitrothymidine can be transformed into a phosphoramidite building block suitable for oligonucleotide synthesis using the standard phosphite triester solid-phase approach. The N-nitrothymidine residues remain stable during the elongation cycles and react smoothly with primary amines, furnishing oligonucleotides containing N3-modified thymidines. A number of N3-substituted oligonucleotides have been prepared using this methodology, some of them incorporating aminoalkyl or hydroxyalkyl groups.

Full text of DOI:10.1021/ol025643t

ORGANIC
LETTERS
2002
Vol. 4, No. 11
1827-1830
A New Method for the Preparation of
Modified Oligonucleotides
Olga Gorchs, Marta Herna´ndez, Lourdes Garriga, Enrique Pedroso,
,†
Anna Grandas,* and Jaume Farra`s*
Departament de Qu´ımica Orga`nica, UniVersitat de Barcelona, Mart´ı i Franque`s 1,
08028 Barcelona, Spain
jfarras@qo.ub.es
Received January 30, 2002
ABSTRACT
N-Nitrothymidine can be transformed into a phosphoramidite building block suitable for oligonucleotide synthesis using the standard phosphite
triester solid-phase approach. The N-nitrothymidine residues remain stable during the elongation cycles and react smoothly with primary
amines, furnishing oligonucleotides containing N3-modified thymidines. A number of N3-substituted oligonucleotides have been prepared
using this methodology, some of them incorporating aminoalkyl or hydroxyalkyl groups.
Progress in solid-phase synthesis methodology has provided
efficient tools for the preparation of either unmodified or
modified oligonucleotides.1 The synthesis of DNA oligomers
containing modified nucleobases, in particular, may be
carried out using two different alternatives (Scheme 1). The
first strategy for incorporating modified units requires the
synthesis of a specific phosphoramidite building block for
each of the nonnatural bases to be included. In this case,
depending on the functional groups present in these units, a
special protection scheme must be developed to guarantee
their chemical compatibility with the protocols used during
the solid-phase assembly of the DNA chain. In the second
strategy, the oligonucleotide chain is elongated using a
building block that is a precursor of the target nucleoside, a
so-called “convertible nucleoside”.2 This precursor may be
transformed into a range of differently modified nucleosides
in the final steps of the synthesis (see Scheme 1).3
Recently, it was reported4 that the substitution of the imidic
hydrogen by a nitro group strongly activates the C4-carbonyl
group of uridines and thymidines. As shown in Scheme 2,
such activated nucleobases can then be easily modified by
treatment with primary amines to provide a variety of N3-
modified nucleosides through a ring-opening-ring-closing
(3) For recent examples related to the “convertible nucleoside” strategy,
see: (a) Hwang, J.-T.; Greenberg, M. M. J. Org. Chem. 2001, 66, 363-
369. (b) Noll, D. M.; Noronha, A. M.; Miller, P. S. J. Am. Chem. Soc.
2001, 123, 3405-3411. (c) Banuls, V.; Escudier, J.-M.; Zedde, C.; Claparols,
C.; Donnadieu, B.; Plaisancie, H. Eur. J. Org. Chem. 2001, 4693-4700.
(d) Robles, J.; Grandas, A.; Pedroso, E. Tetrahedron 2001, 57, 179-194.
(e) Komatsu, Y.; Kumagai, I.; Ohtsuka, E. Nucleic Acids Res. 1999, 27,
4314-4323. (f) Kohgo, S.; Shinozuka, K.; Ozaki, H.; Sawai, H. Tetrahedron
Lett. 1998, 39, 4067-4070. (g) Erlanson, D. A.; Wolfe, S. A.; Chen, L.;
Verdine, G. L. Tetrahedron 1997, 53, 12041-12056. (h) Haginoya, N.;
Ono, A.; Nomura, Y.; Ueno, Y.; Matsuda, A. Bioconjugate Chem. 1997, 8,
271-280.
(4) (a) Ariza, X.; Bou, V.; Vilarrasa, J.; Tereshko, V.; Campos, J. L.
Angew. Chem., Int. Ed. Engl. 1994, 33, 2454-2455. (b) Ariza, X., Bou;
V.; Vilarrasa, J. J. Am. Chem. Soc. 1995, 117, 3665-3673. (c) Ariza, X.;
Farra`s, J.; Serra, C.; Vilarrasa, J. J. Org. Chem. 1997, 62, 1547-1549. (d)
Serra, C.; Aragone`s, C.; Bessa, J.; Farra`s, J.; Vilarrasa, J. Tetrahedron Lett.
1998, 39, 7575-7578. (e) Serra, C.; Farra`s, J.; Vilarrasa, J. Tetrahedron
Lett. 1999, 40, 9111-9113. (f) Ariza, X.; Vilarrasa, J. J. Org. Chem. 2000,
65, 2827-2829.
† E-mail: agrandas@qo.ub.es.
(1) Oligonucleotides and Analogues. A Practical Approach; Eckstein,
F., Ed.; IRL Press: Oxford, 1991.
(2) See, for instance: (a) MacMillan, A. M.; Verdine, G. L. J. Org. Chem.
1990, 55, 5931-5933. (b) Allerson, C. R.; Chen, S. L.; Verdine, G. L. J.
Am. Chem. Soc. 1997, 119, 7423-7433, and references therein. (c) Xu,
Y.-Z.; Zheng, Q.; Swann, P. F. Tetrahedron 1992, 48, 1729-1740. (d) Xu,
Y.-Z. Tetrahedron 1996, 52, 10737-10750, and references therein. (d)
Harris, C. M.; Zhou, L.; Strand, E. A.; Harris, T. M. J. Am. Chem. Soc.
1991, 113, 4328-4329.
10.1021/ol025643t CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/27/2002

Scheme 1. Strategies for the Solid-Phase Synthesis of Modified Oligonucleotides Using the Phosphite Triester Methodology
mechanism.4a,b In this Letter we describe the preparation of
oligodeoxynucleotides with thymines modified at the N3
position, making use of the 3′-[(â-cyanoethyl)-N,N-diiso-
propyl]phosphoramidite of 5′-O-(4,4′-dimethoxytrityl)-N-
The N-nitronucleoside 2 was deacetylated, and the key
phosphoramidite 4 was obtained in good yield (see Scheme
3) from the DMT-protected precursor 3 by treatment with
(â-cyanoethyl)-N,N,N′,N′-tetraisopropylphosphorodiamid-
ite in the presence of tetrazole. Compound 4 proved to be
as stable as any of the other phosphoramidite derivatives and
could be stored in the refrigerator for months without being
degraded. Finally, the oligonucleotide-resins 5a-c, bearing
nitrothymidine (4, DMT-TNO -CEP) as the key building block
2
(Scheme 3). Reaction of N-nitrothymidine-containing oli-
gonucleotide-resins with primary amines yields oligonucleo-
tides with N3-modified thymines at the positions in which
thymidine had been replaced by 3-nitrothymidine.
The strategy proposed in the present work may give access
to a variety of modified oligomers using a single building
block. To the best of our knowledge, this is the first example
of a “convertible nucleoside” that allows the N3 position of
the pyrimidine moiety to be manipulated.
one or two TNO residues, were prepared using the standard
2
phosphite triester solid-phase methodology (see Table 1). As
expected, phosphoramidite 4 could be easily incorporated
a
Scheme 3. Synthesis of DMT-TNO -CEP (4)
2
A preliminary series of experiments was carried out in
order to establish the chemical compatibility of the N-nitro
moiety with the solid-phase synthesis protocols. Thus, the
protected N-nitrothymidine derivative 2 was chosen as model
compound and was treated with all the reagents used for the
oligonucleotide assembly.5 None of those treatments pro-
duced any significant degradation of the N-nitronucleoside
moiety.
Scheme 2. Reaction of N-Nitrothymidine Residues with
Primary Amines
a (a) Ac2O, py, rt (99%); (b) CF3COONO2 (3 equiv), CH2Cl2,
0 °C, 25 min (82%); (c) HCl/MeOH 0.17 M, rt, 24 h (88%); (d)
DMT-Cl (1.2 equiv), DMAP (0.1 equiv), py, rt, 4 h (68%); (e)
[NC(CH2)2O]P(NiPr2)2 (1.2 equiv), tetrazole (0.5 equiv), ACN, rt,
1 h (83%).
1828
Org. Lett., Vol. 4, No. 11, 2002

Table 1. N-Nitrothymidine-Containing Oligonucleotide-Resins
Table 2. Modification of Resins 5a-c with Primary Aminesa,b
Prepareda-c
oligonucleotide-resin
2
5a
5b
5c
T-T-T-TNO -T-T-T-resin
T-TNO -T-T-T-T-T -T-resin
T-GiBu-ABz-TNO -CBz-ABz-T-resin
NO2
2
2
a TentaGel-NH2 resin (Rapp Polymere GmbH) was used as the solid
support. The first 5′-DMT-protected nucleoside was attached to the resin
by means of a standard succinyl linker, and the initial substitution degree
was in the range of 40-190 µmol/g. b Oligonucleotide elongation was
carried out at 2-5 mmol scale following standard phosphite triester
procedures: 0.1-0.15 M solutions of 4 or the commercially available
(Glen Research Corp.) 3′-phosphoramidite derivatives (5′-DMT-Nu-
P[O(CH2)2CN]NiPr2, Nu ) TNO , T, ABz, CBz, GiBu) in anhydrous ACN
2
and 0.5-0.6 M solutions of tetrazole in anhydrous ACN were used for the
coupling steps (15 min). A 0.1 M solution of tBuOOH in CH2Cl2 was used
in the oxidation steps (1.5 min). All syntheses were performed in an Applied
Biosystems 380B DNA synthesizer. c Average coupling yields, calculated
from the UV absorbance at 498 nm of the DMT cation generated in the
deprotection steps, ranged between 99.5% (5a) and 98.2% (5c).
resin
modification conditions
product
5a
5a
5a
5b
5c
5c
5c
5c
MeNH2 (33% in H2O), 1 h, rt
BnNH2 (neat), 1 h, rt
6a
6b
6c
7
8a
8b
8c
into the corresponding oligomers using exactly the same
conditions as those employed for other phosphoramidite
derivatives, with no significant difference in the coupling
yields. In addition, no evidence of degradation of the
N-nitrothymidine moiety was found during the subsequent
elongation cycles.
H2N(CH2)3NH2 (neat), 1 h, rt
MeNH2 (33% in H2O), 1 h, rt
BnNH2 (0.25 M in ACN), 4 days, rt
H2N(CH2)3NH2 (0.1 M in ACN), 24 h, rt
HO(CH2)5NH2 (0.14 M in ACN), 48 h, rt
PhNH2 (neat), 2 h, rt
After assembly of the three oligonucleotide-resins
5a-c, we turned our attention to their reaction with primary
amines. As shown in Table 2, various oligonucleotide
analogues were prepared. The modification reaction was
straightforward and could be carried out efficiently under a
variety of conditions. Either the neat amine, concentrated
aqueous solutions, or dilute solutions of the amine in
acetonitrile (ACN) afforded the target product, provided that
the reaction times were long enough so as to allow for the
completion of the reaction.
Treatment of the oligonucleotide-resins with the primary
amines to some extent also cleaved the oligonucleotides from
the resin and removed protecting groups. In any case,
concentrated aqueous ammonia was added to the reaction
mixture, and the reaction was left to proceed overnight at
55 °C, to ensure completeness of all of the deprotection
processes (cleavage of the nucleoside-resin bond and
removal of the phosphate and nucleobase protecting groups).
This additional treatment was also found to reduce the
amount of byproducts present in the final crude mixtures.
As summarized in Table 2, good results were obtained
with all of the alkylamines, but reaction of resin 5c with
aniline led to a complex reaction mixture in which no
modified nucleoside-containing product could be identified.
This result indicates that aromatic amines are not nucleophilic
enough to complete the reaction and establishes the lower
limit to the scope of the proposed methodology.
a In all cases, the analytical reverse-phase HPLC chromatograms of the
crude products showed a single major peak (80-93%), which corresponded
to the expected oligomers 6-8, and very minor peaks associated with other
nucleosidic byproducts. As expected, retention times of the modified
oligonucleotides 6-8 (R * H) were always higher than those of the
corresponding unmodified ones (R ) H). Reverse-phase HPLC analyses
were performed on either Spherisorb (ODS-2, 25×0.4 cm, 5 µm) or
Phenomenex (C18, 25×0.46 cm, 10 µm) columns using linear gradients of
0.05 M aqueous triethylammonium acetate and ACN/H2O (1:1). b Identities
of all of the major products were established either by electrospray or
MALDI-TOF (2,4,6-trihydroxyacetophenone) mass spectrometry (negative
mode) of the HPLC-purified material. Identities of 8a-c were also
established by enzymatic digestion of the HPLC-purified materials with
snake venom phosphodiesterase and alkaline phosphatase. Samples of the
modified nucleosides were used as standards.
carried one and two N-nitrothymidine residues, respectively.
These results clearly point out that several modifications can
be simultaneously introduced into oligonucleotide-resins
bearing multiple N-nitrothymidine residues by means of a
single final reaction with primary amines. Moreover, the
modification reaction is compatible with the presence of
additional free amine and hydroxyl groups in the modifica-
tion reagent. Successful isolation of the target modified
oligonucleotides 8a and 8b, after treatment of the oligo-
nucleotide-resin 5c with either 5-aminopentanol or 1,3-
propanediamine (see Table 2), illustrates the fact that
protection of the second functional group in the modification
reagent is not necessary.
In summary, our results show that the phosphoramidite
derivative 4 is a good building block for the synthesis of
oligonucleotide-resins containing N-nitrothymidine residues.
The N-nitrothymidine residues behave as “convertible nucleo-
sides” that can be transformed into a range of N3-modified
thymidines by reaction with primary alkylamines.
It is worth noting that no remarkable differences were
found in the reactions involving resins 5a and 5b, which
(5) (1) Tetrazole, ACN, 24 h, rt; (2) Ac2O/2,6-lutidine, THF, 24 h, rt;
(3) N-methylimidazole, THF, 24 h, rt; (4) I2, H2O/py/THF, 24 h, rt; (5)
t-BuOOH 3 M, toluene, 24 h, rt.
Org. Lett., Vol. 4, No. 11, 2002
1829

Acknowledgment. This work was supported by funds
from the Spanish MEC (Grants PM96-0033 and PB97-0941-
C02-01) and MCYT (Grants BQU2000-0647 and BQU2001-
3693-C02-01), as well as the Generalitat de Catalunya
(2000SGR018, 2000SGR021, 2001SGR49, 2001SGR51 and
Centre de Refere`ncia de Biotecnologia).
Supporting Information Available: Enzymatic digestion
results and NMR data of 2, 3, 4, and N-nitrothymidine. This
material is available free of charge via the Internet at
http://pubs.asc.org.
OL025643T
1830
Org. Lett., Vol. 4, No. 11, 2002