Synthesis and analysis of RNA containing 6-trifluoromethylpurine ribonucleoside.

Article abstract of DOI:10.1021/ol016295i

We report the synthesis of a 5'-DMT-2'-TBDMS-protected phosphoramidite of 6-trifluoromethylpurine ribonucleoside ((TFM)P) and its use in the site-specific incorporation of 6-trifluoromethylpurine into RNA. Properties of (TFM)P-substituted RNA suggest it will be valuable in the study of RNA structure and the binding of RNA-modifying enzymes, particularly the RNA-editing adenosine deaminases. Reaction: see text.

Full text of DOI:10.1021/ol016295i

ORGANIC
LETTERS
2001
Vol. 3, No. 19
2969-2972
Synthesis and Analysis of RNA
Containing 6-Trifluoromethylpurine
Ribonucleoside
Eduardo A. Ve´liz, Olen M. Stephens, and Peter A. Beal*
Department of Chemistry, UniVersity of Utah, Salt Lake City, Utah 84112
beal@chemistry.chem.utah.edu
Received June 18, 2001
ABSTRACT
We report the synthesis of a 5′-DMT-2′-TBDMS-protected phosphoramidite of 6-trifluoromethylpurine ribonucleoside (TFMP) and its use in the
site-specific incorporation of 6-trifluoromethylpurine into RNA. Properties of TFMP-substituted RNA suggest it will be valuable in the study of
RNA structure and the binding of RNA-modifying enzymes, particularly the RNA-editing adenosine deaminases.
The enzymatic deamination of adenosine in mRNA results
in inosine at that position. Since inosine is translated as
guanosine, this reaction can lead to codon changes in mRNA,
resulting in the synthesis of proteins that are structurally and
functionally distinct from those encoded in the genome.
These RNA-editing adenosine deamination reactions are
catalyzed by the ADARs (adenosine deaminases that act on
RNA) and require that the adenosine to be modified be
present in duplex secondary structure.1 Our current under-
standing of the mechanism of the ADAR-catalyzed de-
amination of adenosine in double helical RNA is limited.
Indeed, given the lack of structural information on the
enzyme, the nature of the active site can only be speculated
upon based on sequence similarities to other enzymes.2 We
have used phosphoramidite chemistry to introduce adenosine
analogues into model substrates as probes of the ADAR
reaction mechanism.3,4 These studies indicate that ADARs
share mechanistic similarities with the well-characterized
nucleoside-modifying enzyme adenosine deaminase (ADA).
However, the hydrolytic deamination reaction catalyzed by
ADA is an imperfect model for the steps occurring in the
ADAR active site. In addition, fluorescence changes are
observed when ADAR2 binds to a 2-aminopurine-modified
RNA duplex that are consistent with a base-flipping mech-
anism wherein the nucleotide undergoing modification is
extruded from the double helix.5 This step is not required
for the ADA reaction.
In this Letter, we report the synthesis and characterization
of RNA containing an analogue of adenosine in which the
C6 amine has been replaced with a trifluoromethyl group
(6-trifluoromethylpurine ribonucleoside, TFMP). Trifluoro-
methylation of purine at C6 has been proposed to facilitate
(3) Yi-Brunozzi, H.-Y.; Easterwood, L. M.; Kamilar, G. M.; Beal, P. A.
Nucleic Acids Res. 1999, 27, 2912-2917.
(4) Easterwood, L. M.; Ve´liz, E. A.; Beal, P. A. J. Am. Chem. Soc. 2000,
122, 11537-11538.
(1) Grosjean, H.; Benne, R. Modification and Editing of RNA; ASM
Press: Washington, DC, 1998.
(2) Hough, R. F.; Bass, B. L. RNA 1997, 3, 356-370.
(5) Stephens, O. M.; Yi-Brunozzi, H.-Y.; Beal, P. A. Biochemistry 2000,
39, 12243-12251.
10.1021/ol016295i CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/18/2001

hydration at this position, creating a mimic of the transition
state for hydrolytic deamination of adenosine (Figure 1).6
Scheme 1a
Figure 1. Hydration of TFMP across the C6-N1 double bond
generates a structure similar to the transition state proposed for the
adenosine deamination reaction catalyzed by ADA.
a (a) FSO2CF2CO2Me, CuI, HMPA, DMF, 70 °C, 91% or CF3I,
Zn, CuI, DMF, 70 °C, 96%; (b) NH3/MeOH, 97%; (c) (i) DMTCl,
pyridine, AgNO3, THF, 98%; (ii) TBDMSCl, TEA, AgNO3, THF,
56%; (iii) â-cyanoethyldiisopropylphosphoramidochloridite, DIPEA,
THF, 80%.
Such a structure, in the appropriate double helical RNA
context, is a good candidate for high-affinity binding at the
ADAR active site. Furthermore, the presence of the tri-
fluoromethyl group creates a sensitive NMR probe of the
electronic environment of this nucleotide in the RNA. 19F
NMR analysis of the binding of 5-fluorodeoxycytidine-
substituted DNA to a cytosine DNA methyltransferase
allowed for delineation of features of its base-flipping
mechanism.7 This approach would be valuable at further
defining the mechanism by which ADARs base flip if the
appropriately fluorine-labeled RNA were available.
6-Trifluoromethylpurine ribonucleoside had been previ-
ously synthesized from a sugar-protected 6-chloropurine
ribonucleoside and trifluoromethyl copper (CF3Cu).8 How-
ever, the reaction time (60 h) and yield (29%) led us to
investigate other methods to obtain this compound. Given
the precedent of increased yields in copper-mediated coupling
reactions with bromides over chlorides, we attempted copper-
mediated trifluoromethylation with tri-O-acetyl-6-bromo-
purine ribonucleoside (1) (Scheme 1).
of manipulation of liquid MFSDA vs the gaseous CF3I makes
the former condition more desirable. To the best of our
knowledge, this constitutes the first reported use of the
MFSDA reagent in nucleoside synthesis. The acetyl protect-
ing groups on ribose were removed in MeOH saturated with
NH3 to give TFMP (3) in greater than 80% yield in two steps
from 1. Recently, two alternative strategies for obtaining 3
were reported.12,13 Although the trifluoromethylation step in
each route is high yielding, the overall yield reported for
each procedure is lower than that reported here.
Synthesis of a monomer useful for automated RNA
synthesis proceeded initially by protection of the 5′-hydroxyl
as the dimethoxytrityl (DMT) ether. Using the conditions
of Ogilvie, the 2′-hydroxyl was silylated regioselectively with
tert-butyldimethylsilyl chloride and AgNO3.14 Finally, the
3′-phosphoramidite (4) was generated via reaction of 2-cyano-
ethyl diisopropylphosphoramidochlorodite in the presence of
Hu¨nig’s base. Using this phosphoramidite and standard
automated RNA synthesis procedures, TFMP was incorporated
into RNA. On-line trityl monitoring during automated RNA
synthesis indicated quantitative coupling of the phosphor-
amidite (4).
Bromopurine derivative 1 is readily available from inosine
via our recently reported procedure.9 Two different tri-
fluoromethylation reactions with 1 were investigated. Both
FSO2CF2CO2Me (MFSDA)/CuI/HMPA/DMF10 (91%) and
CF3I/Zn/CuI/DMF11 (96%) lead to a high yield of 6-tri-
fluoromethylpurine ribonucleoside (2). However, the ease
The RNA was deprotected in a standard two-step sequence
using NH4OH/EtOH followed by TEA‚3HF. After depro-
tection and purification of the oligonucleotides, ESI and
MALDI mass spectrometric analysis confirmed that the TFM
P
(6) Erion, M. D.; Reddy, M. R. J. Am. Chem. Soc. 1998, 120, 3295-
3304.
(7) Klimasauskas, S.; Szyperski, T.; Serva, S.; Wu¨thrich, K. EMBO J.
1998, 17, 317-324.
nucleotide was incorporated into the RNA unaltered (Figure
2).
(8) Kobayashi, Y.; Yamamoto, K.; Asai, T.; Nakano, M.; Kumadaki, I.
J. Chem. Soc., Perkin Trans. 1 1980, 2755-2761.
(9) Ve´liz, E. A.; Beal, P. A. Tetrahedron Lett. 2000, 41, 1695-97.
(10) Qing, F.-L.; Fan, J.; Sun, H.-B.; Yue, X.-J. J. Chem. Soc., Perkin
Trans. 1 1997, 3053-3058.
(11) Prasad, A. S.; Stevenson, T. M.; Citineni, J. R.; Nyzam, V.; Knochel,
P. Tetrahedron 1997, 53, 7237-7254.
(12) Hockova´, D.; Hocek, M.; Dvora´kova´, H.; Votruba, I. Tetrahedron
1999, 55, 11109-11118.
(13) Hocek, M.; Holy, A. Collect. Czech. Chem. Commun. 1999, 64,
229-241.
(14) Hakimelahi, G. H.; Proba, Z. A.; Ogilvie, K. K. Can. J. Chem. 1982,
60, 1106-1113.
2970
Org. Lett., Vol. 3, No. 19, 2001

greater than that of a methyl group.15 The lack of any pairing
preference for TFMP may indicate that this nucleotide does
not base pair in duplex RNA and already exists in a flipped
out conformation. If so, RNA containing TFMP may bind
tightly to ADARs as it would not be necessary to overcome
the energetic barrier to disrupt base-pairing interactions.
However, this may limit the use of TFMP as a probe of the
early steps in the flipping mechanism (see below).
19F NMR analysis of fluorine-substituted nucleic acids has
proven useful in the study of protein-nucleic acid interac-
tions, including the binding of base-flipping enzymes.7,16 To
determine if 19F NMR could be used to probe the electronic
environment of the trifluoromethyl group of TFMP in oligo-
ribonucleotides, we obtained 19F NMR spectra for two
different RNA trimers, 5′-ATFMPG-3′ and 5′-CTFMPU-3′. Each
trimer gave rise to a single peak near -67 ppm relative to
CFCl3 (Figures 4A and 4B). The spectrum for a 1:1 mixture
Figure 2. The incorporation of TFMP into RNA in the sequence
5′-ATFMPG-3′was confirmed by ESI and MALDI MS (shown
above).
The effect TFMP substitution has on duplex stability was
investigated using thermal denaturation (Tm) studies of a 12
base pair RNA duplex (Figure 3). These experiments were
Figure 4. Analysis by 19F NMR indicates that differences in the
structural context of TFMP can be detected: (A) 400 µM 5′-CTFMPU-
3′ (-67.12 ppm); (B) 400 µM 5′-ATFMPG-3′ (-67.25 ppm); (C)
1:1 mixture of 200 µM each 5′-CTFMPU-3′ and 5′-ATFMPG-3′
(-67.12 and -67.25 ppm).
of the two trimers clearly shows two well-resolved peaks
corresponding to the two different TFMP nucleotides present
(Figure 4C).
Figure 3. The effect TFMP substitution has on duplex stability was
tested in the context of the RNA shown above. TFMP was found to
have a destabilizing effect on the same order as an A‚A mismatch.
Thus, as has been observed with 5-fluorouridine-substi-
tuted RNA, the fluorine-substituted nucleotide gives rise to
a unique 19F NMR signal whose chemical shift is dependent
on its structural context.17 This result indicates that 19F NMR
could be used to monitor structural changes in TFMP-
substituted RNA that significantly alter the electronic
carried out to provide one measure of how effectively TFM
P
can mimic adenosine in the context of RNA. The nucleoside
analogue was incorporated into the duplex opposite each of
the four common bases (adenine (A), guanine (G), cytosine
(C), and uracil (U)), and the Tm for each duplex was
determined. The observed Tm values were compared to the
Tm’s of duplexes with A opposite each of the four common
bases. Under the conditions of the denaturation experiment,
the duplex containing an A‚U base pair at the variable
position had a Tm ) 52 °C whereas an A‚A mismatch caused
the Tm to decrease to 40 °C. Each of the four duplexes
containing TFMP had a Tm near 40 °C, with only 3 degrees
separating the four Tm’s measured. Therefore, the TFMP
nucleotide destabilizes duplex RNA to a degree similar to
that of a purine-purine mismatch with little pairing prefer-
ence for any of the four common bases. A likely source to
this destabilizing effect is the steric demand of the tri-
fluoromethyl group, which has been suggested to be 2.5 times
environment of the TFM
P nucleotide, such as a base-flipping
conformational change induced by an ADAR.
In summary, we have synthesized a phosphormadite of
TFMP and incorporated this novel nucleoside into RNA. The
TFMP modification is destabilizing to an RNA duplex equal
to that of an A‚A mismatch. The sensitivity of the 19F
chemical shift of TFMP to its structural context indicates it
will be valuable in the study of RNA structure and the
binding of RNA binding proteins, particularly the base-
flipping RNA-editing adenosine deaminases. Furthermore,
the electron-withdrawing properties of the trifluoromethyl
group will increase the reactivity of the purine to covalent
(15) Seebach, D. Angew. Chem., Int. Ed. Engl. 1990, 29, 1320-1367.
(16) Fraydoon, R.; Evilia, C.; Lu, P. Methods Enzymol. 1995, 261, 560-
575.
(17) Hardin, C. C.; Horowitz, J. J. Mol. Biol. 1987, 197, 555-569.
Org. Lett., Vol. 3, No. 19, 2001
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hydration. This latter property suggests that TFMP-containing
RNA could inhibit RNA-editing adenosine deaminases by
transition state mimicry. Experiments to test these hypotheses
are currently underway in our laboratories.
Supporting Information Available: Synthetic procedures
and spectral data for all new compounds and procedures for
mass spectral and thermal denaturation analyses. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment. This research was funded in part by
a grant from the National Institutes of Health to P.A.B. (GM
61115).
OL016295I
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