Synthesis of modified RNA-oligonucleotides for structural investigations

Article abstract of DOI:10.1081/NCN-120022827

RNA exhibits a higher structural diversity than DNA and is an important molecule in biology of life. It shows a number of secondary structures such as duplexes, hairpin loops, bulges, internal loops etc. However, in natural RNA, bases are limited to the f

Full text of DOI:10.1081/NCN-120022827

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Synthesis of Modified RNA-Oligonucleotides for
Structural Investigations
a
A. [Zbreve]ivković & J. W. Engels a b
a Institute for Organic Chemistry and Chemical Biology , Johann-Wolfgang-Goethe
University , Frankfurt a.M., Germany
b Universtät Frankfurt, Institut für Organische Chemie , Mertonviertel-Marie-Curie-Strasse
11, D-60439, Frankfurt, Germany
Published online: 31 Aug 2006.
To cite this article: A. [Zbreve]ivković & J. W. Engels (2003) Synthesis of Modified RNA-Oligonucleotides for Structural
Investigations, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 1167-1170, DOI: 10.1081/NCN-120022827
To link to this article: http://dx.doi.org/10.1081/NCN-120022827
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1167–1170, 2003
Synthesis of Modified RNA-Oligonucleotides for
Structural Investigations
˘
A. Zivkovic and J. W. Engels
*
´
Institute for Organic Chemistry and Chemical Biology, Johann-Wolfgang-Goethe
University, Frankfurt a.M., Germany
ABSTRACT
RNA exhibits a higher structural diversity than DNA and is an important mole-
cule in biology of life. It shows a number of secondary structures such as
duplexes, hairpin loops, bulges, internal loops etc. However, in natural RNA,
bases are limited to the four predominant structures U, C, A, and G and so
the number of compounds that can be used for investigation of parameters of
base stacking, base pairing and hydrogen bond, is limited. We synthesized differ-
ent fluoromodifications of RNA building blocks: 10-deoxy-10-(2,4,6-trifluorophe-
nyl)-ß-D-ribofuranose (F), 10-deoxy-10-(2,4,5-trifluorophenyl)-ß-D-ribofuranose
(M) and 10-deoxy-10-(5-trifluoromethyl-1H-benzimidazol-1-yl)-ß-D-ribofuranose
(D). Those amidites were incorporated and tested in a defined A, U- rich RNA
sequence (12-mer, 50-CUU UUC XUU CUU-30 paired with 30-GAA AAG
YAA GAA-5’) (Schweitzer, B.A.; Kool, E.T. Aromatic nonpolar nucleosides as
hydrophobic isosters of pyrimidine and purine nucleosides. J. Org. Chem. 1994,
59, 7238 pp.). Only one position was modified, marked as X and Y respectively.
UV melting profiles of those oligonucleotides were measured.
Key Words: RNA; Fluorobenzenes; Fluorobenzimidazoles; Ribonolactone;
Duplex melting curve.
*Correspondence: J. W. Engels, Universta¨t Frankfurt, Institut fur Organische Chemie,
¨
Mertonviertel-Marie-Curie-Strasse 11, D-60439 Frankfurt, Germany; Fax: þ49 697 982
9148; E-mail: joachim.engels@chemie.uni-frankfurt.de.
1167
DOI: 10.1081/NCN-120022827
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

˘
´
Zivkovic and Engels
1168
INTRODUCTION
Hydrogen bonds, base stacking and solvatation are three predominant forces
which are responsible for the stability of secondary structure of nucleic acids. As
those interactions are very important, and the number of compounds you can inves-
tigate is limited to four predominant structures (U(T), C, G and A), we decided to
synthesize some novel nucleic acid analogues where the nucleobases are replaced
by fluorobenzenes and fluorobenzimidazoles.
It is important and interesting to investigate fluorine due to it’s ‘‘special’’ proper-
ties such as high electronegativity, relatively small size, low polarizability, hydrogen-
bonding ability etc.[1]
CHEMICAL SYNTHESES
The synthesis of 10-deoxy-10-(2,4,6-trifluorophenyl)-ß-D-ribofuranose (F) and 10-
deoxy-10-(2,4,5-trifluorophenyl)-ß-D-ribofuranose (M) starts with C glycosilation
(Fig. 1). Lithiation of 1a and 1b was performed with t-BuLi and n-BuLi in either
Et2O or THF at ꢀ78ꢁC and was followed by addition of 2,3,5-tri-O-benzyl-D-
ribono-g-lactone[2] and gave intermediate lactols (3a and 3b) which were directly
dehidroxilated with triethylsilane and BF3ꢂEt2O to afford stereoselectively 4a and
4b in 52 and 65% yield.[3] The deprotection of benzilated nucleosides (4a and 4b)
was performed with Pd(OH)2=C and cyclohexene to afford 5a and 5b in 98 and
94% yield, respectively (Fig. 2).
The synthesis of 10-deoxy-10-(5-trifluoromethyl-1H-benzimidazol-1-yl)-ß-D-ribo-
furanose followed procedure of Vorbrueggen[4] (57% of 9). Deprotection was done
with 0,2 M CH3ONa in CH3OH within 3 h to afford 10 in 91% yield (Fig. 3).
Synthesis of corresponding amidites was done by usual procedures.
Figure 1. Synthesized modified phosphoramidite and the one-letter abreviations of the
nucleoside ‘‘bases’’.

Modified RNA-Oligonucleotides
1169
Figure 2. Synthesis of 10-deoxy-10-(2,4,6-trifluorophenyl)-ß-D-ribofuranose and 10-deoxy-10-
(2,4,5-trifluorophenyl)-ß-D-ribofuranose.
Figure 3. Synthesis of 10-deoxy-10-(5-trifluoromethyl-1H-benzimidazol-1-yl)-ß-D-ribofura-
nose.
RESULTS AND DISCUSSION
The modified nucleosides were tested in a definied RNA sequence. In the 12mer
oligoribonucleotides (50-CUU UUC XUU CUU-30 paired with 30-GAA AAG YAA
GAA-50) only one position was modified, marked as X and Y respectively.[5] All
measurements were done in phosphate (pH¼7) buffer containing 140 mM NaCl,
10 mM Na2HPO4 and 10 mM NaH2PO4(at wavelength of 260 –same results at
274 nm). First we measured only RNA duplexes containing natural bases. The
Wobble base pair UꢂG shows the highest Tm (38,6ꢁC, Table 1). The UꢂC and UꢂU
mismatches show nearly the same stability (Tm¼ 30,4ꢁC and Tm¼30,1ꢁC, Table 1).
In a second series we measured oligonucleotides with fluorobenzene modifica-
tions paired with natural bases (Fig. 4). In all cases Tm values are lower than those
for natural bases. Possible explanation for this could be failure of hydrogen bonding
Table 1. Synthesized Double Modified Duplex RNA (50-CUU UUCXUU CUU paired with
30-GAA AAG YAA GAA-50) and their thermodynamical properties (Errors: Tm ¼ 0,2ꢁC;
DGꢁ ¼ 2%).
Y¼A
DGꢁ
Y¼C
DGꢁ
Y¼G
DGꢁ
Y¼U
DGꢁ
Y¼D
Tm
DGꢁ
Tm
Tm
Tm
Tm
X
F
(ꢁC) (kcal=mol) (ꢁC) (kcal=mol) (ꢁC) (kcal=mol) (ꢁC) (kcal=mol) (ꢁC) (kcal=mol)
23,3
7,8
8,4
20,6
26,7
7,1
8,8
22,8
28,7
7,7
9,3
22,9
27,5
13
8,9
25
26,1
8,3
8,5
M 25,6

˘
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Zivkovic and Engels
1170
Figure 4. Pairing of nucleosides F and M with the natural bases and modification D in the
center of base pair RNA duplex measured by thermal melting point temperature.
between modified and natural bases and that the modified bases are less solvated by
water molecules than the natural ones. Very small differences in Tm values by pairing
for example M against purine or pyrimidine bases indicate that there are no hydro-
gen bonds. It can be noticed that M is pairing much better than F against natural
bases (Tm values higher up to 6,1ꢁC (Fig. 4)). Explanation for this is not yet clear.
F is pairing better with modified imidazole D than against the natural bases but with
modification M it is opposite (still M is pairing better against D than F).
REFERENCES
1. Smart, B.E. Fluorine substituent effects (on bioactivity). J of Fluorine Chemistry
2001, 109, 3–11.
2. Parsch, J.; Engels, J.W. Synthesis of fluorobenzene and fluorobenzimidazole
nucleic acid analogues and their influence on stability of RNA duplexes. Helv.
Chim. Acta 2000, 83, 1791 pp.
3. Parsch, J.; Engels, J.W. C-F . . . H-C Hydrogen bonds in ribonucleic acids. J.
Am. Chem. Soc. 2002, 124, 5664–5672.
4. Vorbruggen, H.; Ho¨fle, G. Nucleoside synthesis. XXIII on the mechanism of
¨
nucleoside synthesis. Chem. Ber. 1981, 114, 1256 pp.
5. Schweitzer, B.A.; Kool, E.T. Aromatic nonpolar nucleosides as hydrophobic
isosters of pyrimidine and purine nucleosides. J. Org. Chem. 1994, 59, 7238 pp.