RNA recognition by fluor-aromatic substituted

Article abstract of DOI:10.1081/NCN-200059755

RNA exhibits a higher structural diversity than DNA and is an important molecule in the 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

Full text of DOI:10.1081/NCN-200059755

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RNA RECOGNITION BY FLUOR-AROMATIC SUBSTITUTED
a
A. Zivković & J. W. Engels a
a Institute for Organic Chemistry and Chemical Biology, Johann-Wolfgang-Goethe University ,
Frankfurt a.M., Germany
Published online: 15 Nov 2011.
To cite this article: A. Zivković & J. W. Engels (2005) RNA RECOGNITION BY FLUOR-AROMATIC SUBSTITUTED, Nucleosides,
Nucleotides and Nucleic Acids, 24:5-7, 1023-1027, DOI: 10.1081/NCN-200059755
To link to this article: http://dx.doi.org/10.1081/NCN-200059755
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5--7):1023–1027, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200059755
RNA RECOGNITION BY FLUOR-AROMATIC SUBSTITUTED
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A. Zivkovic and J. W. Engels
Institute for Organic Chemistry and Chemical Biology,
Johann-Wolfgang-Goethe University, Frankfurt a.M., Germany
5
RNA exhibits a higher structural diversity than DNA and is an important molecule in the 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 different fluoromodifications of RNA building
blocks: 1’-deoxy-1’-phenyl-b-D-ribofuranose (B), 1’-deoxy-1’-(4-fluorophenyl)-b-D-ribofuranose (4 FB),
1’-deoxy-1’-(2,4-difluorophenyl)-b-D-ribofuranose (2,4 DFB), 1’-deoxy-1’-(2,4,5-trifluorophenyl)-b-
D-ribofuranose (2,4,5 TFB), 1’-deoxy-1’-(2,4,6-trifluorophenyl)-b-D-ribofuranose, 1’-deoxy-1’-(penta-
fluorophenyl)-b-D-ribofuranose(PFB), 1’-deoxy-1’-(benzimidazol-1-yl)-b-D-ribofuranose(BI), 1’-deoxy-
1’-(4-fluoro-1H-benzimidazol-1-yl)-b-D-ribofuranose (4 FBI), 1’-deoxy-1’-(6-fluoro-1H-benzimidazol-
1-yl)-b-D-ribofuranose(6FBI),1’-deoxy-1’-(4,6-difluoro-1H-benzimidazol-1-yl)-b-D-ribofuranose(4,6
DFBI), 1’-deoxy-1’-(4-trifluoromethyl-1H-benzimidazol-1-yl)-b-D-ribofuranose (4 TFM), 1’-deoxy-1’-
(5-trifluoromethyl-1H-benzimidazol-1-yl)-b-D-ribofuranose (5 TFM), and 1’-deoxy-1’-(6-trifluoro-
methyl-1H-benzimidazol-1-yl)-b-D-ribofuranose (6 TFM). These amidites were incorporated and tested
in a defined A, U-rich RNA sequence (12-mer, 5’-CUU UUC XUU CUU-3’ paired with 3’-GAA AAG
YAAGAA-5’).Onlyonepositionwasmodified,markedasXandY,respectively.UVmeltingprofilesofthose
oligonucleotides were measured.
Keywords Nucleosides, Base Stacking, Base Pairing, Hydrogen Bonding, RNA Stability
INTRODUCTION
Hydrogen bonds, base stacking, and solvatation are the three predominant
forces that are responsible for the stability of secondary structure of nucleic acids.
As those interactions are very important, and the number of compounds you can
investigate 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
Address correspondence to Prof. Dr. J. W. Engels, Institute for Organic Chemistry and Chemical Biology,
Johann-Wolfgang-Goethe University, Marie-Curie-Strasse 11, D-60439 Frankfurt a.M., Germany; E-mail:
Joachim.Engels@chemie.uni-frankfurt.de
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FIGURE 1 Synthesized modified phosphoramidites and their abreviations of the nucleoside ‘‘bases.’’
interesting to investigate fluorine due to its special properties such as high electro-
negativity, relatively small size, low polarizability and hydrogen-bond accepting.[1]
CHEMICAL SYNTHESES
The synthesis of fluorophenyl-b-D-ribofuranose modifications (13--18) start with
C glycosilation. Lithiation of fluorobenzenes (5 and 6) or bromofluorobenzenes
(1--4) was performed with t-BuLi (5 and 6) and n-BuLi (1--4) in either Et2O(3,4,6) or
THF(1,2,5) at À78°C and was followed by addition of 2,3,5-tri-O-benzyl-D-ribono-g-
lactone and gave intermediate lactols that were directly dehidroxilated with
triethylsilane and BF3Á Et2O to afford (7--12) stereoselectively in good yields
(Figure 2).[2,3] The deprotection of benzilated nucleosides (7--12) was performed
with Pd(OH)2/C and cyclohexene to afford (13--18) in high yields.[4,5]
FIGURE 2 Synthesis ofl fluorophenyl-b-D-ribofuranose modifications.

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FIGURE 3 Synthesis of benzimidazole nucleosides.
The synthesis of benzimidazole modifications followed procedure of
Vorbrueggen (Figure 3).[6] Deprotection was done with 0.2 M CH3ONa in
CH3OH within 3 h to afford modified nucleosides in high yields.[2,7]
Synthesis of corresponding amidites was done by usual procedures.[3]
RESULTS AND DISCUSSION
The modified nucleosides were tested in a defined RNA sequence. In the 12-
mer oligoribonucleotides (5’-CUU UUC XUU CUU-3’ paired with 3’-GAA AAG
YAA GAA-5’) only one position was modified, marked as X and Y, respectively.[8]
All measurements were done in phosphate (pH = 7) buffer containing 140 mM
NaCl, 10 mM Na2HPO4 and 10 mM NaH2PO4 (at wavelength of 260, the same
results at 274 nm). First we measured only RNA duplexes containing natural bases.
The Wobble base pair U Á G shows the highest Tm (38.6°C, Figure 4). The U Á C
FIGURE 4 Some of the synthesized double modified duplex RNA (5’-CUU UUC XUU CUU paired with 3’-
GAA AAG YAA GAA-5’) and their thermodynamic properties (Errors: Tm = 0.2°C; DG°= 2%).

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FIGURE 5 Graph 1. Pairing of some of the nucleosides with the natural bases; Graph 2: Pairing of some
modified nucleosides.
and U Á U mismatches show nearly the same stability (Tm = 30.4°C and
Tm = 30.1°C, Figure 4).
In a second series we measured oligonucleotides with modifications paired with
natural bases and some results are shown in Figure 4 and Figure 5. Comparing
the thermodynamic data obtained from UV measurements brought us to the
following conclusions:
1. All modifications show much greater stability paired in-between themselves than
paired with natural bases (U, C, G, and A).
2. 2,4,6 TFB modification shows much lower stability than 2,4 DFB and 2,4,5 TFB
modifications, although 2,4 DFB has also higher stability compared to 2,4, TFB
(0.4--1.5°C).
3. 4,6 DFBI is so far the best fluorobenzimidazole modification we synthesized;
lately, synthesised 6FBI modification can compete with it, while all TFM
nucleosides show greater decrease of Tm values probably due to the size of
trifluoromethyl group, which is rather more similar to isopropyl than methyl
group.
4. All imidazole nucleosides inhibited the primase activity with remarkably similar
potency. Changing the shape of the base from one that closely resembled
normal bases (4,6 DFBI) to ones whose shape substantially varied from that of
normal bases (5 and 6 TFM for example) did not greatly affect inhibition.[7]
Comparing the results achieved in our group on the subject of fluoro
modifications we decided to employ NMR and crystal structure studies of both
nucleosides and oligonucleotides to proof binding and behavior of modifications in
duplexes.[9]
For the first investigation we decided to use 2,4 DFB as fluorobenzene modi-
fication and 4,6 DFBI as fluorobenzimidazole modification (one opposite the other) as
they have show the best thermodinamical results from the compounds tested up to

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A. Zivkovic and J. W. Engels
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now. We based our NMR studies on the work of Varani&coworkers and decided
to use the following 20-mer sequence: 5’-GGG AXA ACU UCG GUU YUC CC-3’.
REFERENCES
1. Smart, B.E. Fluorine substituent effects (on bioactivity). J. Fluorine Chem. 2001, 109, 3–11.
2. Parsch, J.; Engels, J.W. C--F. . .H--C hydrogen bonds in ribonucleic acids. J. Am. Chem. Soc. 2002, 124,
5664–5672.
3. Parsch, J.; Engels, J.W. Synthesis of fluorobenzene and benzimidazole nucleic-acid analogues and their
influence on stability of RNA duplexes. Helv. Chim. Acta 2000, 83, 1791.
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4. Zivkovic, A.; Bats, J.W.; Engels, J.W. Crystal structures of fluoro- and chloro-modified benzene nucleosides.
Coll. Symp. Ser. 2002, 5, 396–398.
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5. Zivkovic, A.; Engels, J.W. Synthesis of modified RNA-oligonucleotides for structural investigations.
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6. Vorbru¨ggen, H.; Ho¨fle, G. On the mechanism of nucleoside synthesis. Chem. Ber. 1981, 114, 1256.
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7. Moore, C.L.; Zivkovic, A.; Engels, J.W. Human DANN primase uses watson-crick hydrogen bonds to
distinguish between correct and incorrect nucleoside triphosphates. Biochemistry 2004, 43, 12367–12374.
8. Schweitzer, B.A.; Kool, E.T. Aromatic nonpolar nucleosides as hydrophobic isosters of pyrimidines and purine
nucleosides. J. Org. Chem. 1994, 59, 7238.
9. Allain, F.; Varani, G. Divalent metal ion binding to conserved wobble pair defining the upstrime site of
cleavage of groups I self splicing introns. Nucleic Acids Res. 1995, 23(3).