Synthesis of 2′-modified nucleotides

Article abstract of DOI:10.3390/50500727

We show in this work the development of modified building blocks for the synthesis of nuclease resistant hammerhead ribozymes. These modified monomers retain the 2′-OH which is essential for the activity of these molecules. Our final goal is to generate a

Full text of DOI:10.3390/50500727

Molecules 2000, 5, 727-729
molecules
ISSN 1420-3049
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Synthesis of 2’-Modified Nucleotides*
Mariana Gallo1,**, Héctor Torres1 and Adolfo Iribarren1,2
1Instituto de Investigación en Ingeniería Genética y Biología Molecular, UBA - CONICET. Vuelta de
Obligado 2490 2°P (1428). Buenos Aires, Argentine
E-mail: airi@proteus.dna.uba.ar
2Centro de Estudios e Investigaciones. Universidad Nacional de Quilmes. Roque Saenz Pena 180
(1876). Prov. Buenos Aires, Argentine
* This paper missed our publication deadline and was thus omitted from the Proceedings of the 12th
National Symposium of Organic Chemistry (XII SINAQO), published in Molecules 2000, 5, 252-615.
The Editorial Board has now decided to publish it separately in this issue.
** Author to whom correspondence should be addressed.
Received: 4 May 2000 / Accepted: 10 May 2000 / Published: 15 May 2000
Abstract: We show in this work the development of modified building blocks for the syn-
thesis of nuclease resistant hammerhead ribozymes. These modified monomers retain the 2’-
OH which is essential for the activity of these molecules. Our final goal is to generate a
catalytic antisense approach with potential application in gene therapy.
Keywords: Modified nucleotides, ribozymes.
Introduction
A ribozyme is a RNA molecule with a defined sequence and structure that recognizes and hydro-
lyzes a determined phosphate bond of a specific RNA sequence (substrate). Ribozymes can act in an
intermolecular way (trans rupture) and they can also be designed to recognize a wide range of target
sequences. Hammerhead ribozyme has been minimized to a fragment of ca. 40 nucleotides long. This
structure includes the region responsible for the activity and two flanking regions which make possible
the recognition of the complementary RNA sequence.
© 2000 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.

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Molecules 2000, 5
The development of nuclease resistant ribozymes is highly desirable because of their potential ap-
plication in gene therapy. They could act based on the antisense principle and additionally offer a
catalytic mechanism, therefore allowing to reduce the necessary drug concentration.
Many studies have demonstrated that 2’-hydroxyls present in certain positions are essential for
maintaining the hydrolytic activity of these molecules [1-2]. Due to the fact that the modifications pro-
posed up to now lack this functional group [3-6], we have started the synthesis of the necessary build-
ing blocks to generate ribozymes with these characteristics. Furthermore, these building blocks could
potentially be useful in the synthesis of ribozyme inhibitors as well as in the synthesis of other RNA
sctructures where tertiary interactions are important for activity.
Results and Discussion
As shown in the scheme, the selective derivatization of the 2’-hydroxyl was achieved through the
protection of 3’- and 5’-hydroxyls by formation of a disiloxane.
O
O
O
NH
NH
NH
N
N
O
O
O
O
O
iPr
iPr
N
O
iPr
O
HO
Si
O
O
Si
i
ii
iPr
O
O
O
OH
O
Si
Si
OH OH
iPr
iPr
iPr
iPr
O
O
N
O
N
iii
NH
NH
NH
N
O
O
O
Si
iPr
O
O
iPr
iPr
iPr
iPr
O
O
Si
O
O
Si
O
v
Si
iv
iPr
OH
OTs
O
O
CH
2
O
Si
OH
O
O
OH
Si
iPr
iPr
iPr
iPr
iPr
iPr
O
O
N
i
v
O
NH
NH
NH
N
O
N
iPr
O
vii
O
O
viii
HO
O
O
Si
O
AcO
O
CH
3
iPr
CH
3
CH
3
OH
O
OH
Si
OH
OH
OAc
ix
iPr
iPr
O
O
N
O
NH
NH
NH
N
N
O
DMTO
O
O
O
AcO
HO
O
O
xi
xi i
x
CH
3
CH
CH
3
3
O
OTHP
THP
O
OAc
OH
OTHP
P
iPr2N
O
CN
i: a: TIPDSCl2, Py; b: MeOH; ii: PCC, Ac2O, CHCl2; iii:a: MePh3PBr, THF; b: Et2O, NHCl(aq);
2
4
iv: a: OsO4, N-MNO, t-BuOH, THF; b: NaHSO31M, EtOAc; v: TsCl, Py; vi: a: NaBH, DMF,
4
b: NH Cl (aq),vii: TBAF, THF; viii: Ac O, Py; ix: DHP, pTsOH, Dioxane; x: NH , MeOH;xi: DMTrCl,
4
2
3
DMAP, TEA, Py; xii: iPr NEt, CH2Cl2,[(CH ) CH]2 NP(Cl)OCH2CH2CN.
3 2
2

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Molecules 2000, 5
The regioselective oxidation with osmiun tetroxide allowed the isolation and purification of the
diol. The optimization of the 2’-hydroxyl protection was a key step. Several protecting groups were
tried in order to overcome the steric problems produced by this tertiary function [1-2]. The 5’-O-
dimethoxytrityl group as well as the 3’-O-phosphoramidite are the necessary derivatizations required
for the solid phase synthesis of the corresponding oligonucleotides.
Experimental
1
13
All the intermediates have been purified by column chromatography, affording satisfactory H, C
and 31P NMR spectra.
References and Notes
1. Pley, H.W.; Flaherty, K.M.; McKay, D.B. Nature 1994, 372, 68-74.
2. Scott, W.G.; Finch, J.T.; Klug, A. Cell 1995, 81, 991-1002.
3. Paolella, G.; Sproat, B.S.; Lamond, A.I. The EMBO Journal 1992, 11, 1913-1919.
4. Yang, J.; Usman, N.; Chartrand, P.; Cedergren, R. Biochemistry 1992, 31, 5005-5009.
5. Heidenreich, O.; Benseler, F.; Fahrenholz; Eckstein, F. J. Biol. Chem. 1994, 269, 2131-2138.
6. Beigelman, L.; McSwiggen, J.A.; Wincott, F.E.; Usman Thackray. J. Biol. Chem. 1995, 270,
25702-25708.
Samples Availability: available from the authors.
© 2000 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.