The components of xRNA: Synthesis and fluorescence of a full genetic set of size-expanded ribonucleosides

Article abstract of DOI:10.1021/ol102915f

The synthesis and properties of a full set of four benzo-expanded ribonucleosides (xRNA), analogous to A, G, C, and U RNA monomers, are described. The nucleosides are efficient fluorophores with emission maxima of 369-411 nm. The compounds are expected to

Full text of DOI:10.1021/ol102915f

ORGANIC
LETTERS
2011
Vol. 13, No. 4
676–679
The Components of xRNA: Synthesis
and Fluorescence of a Full Genetic Set of
Size-Expanded Ribonucleosides
ꢀ
Armando R. Hernandez and Eric T. Kool*
Department of Chemistry, Stanford University, Stanford, California 94305-5080,
United States
kool@stanford.edu
Received December 2, 2010
ABSTRACT
The synthesis and properties of a full set of four benzo-expanded ribonucleosides (xRNA), analogous to A, G, C, and U RNA monomers, are
described. The nucleosides are efficient fluorophores with emission maxima of 369-411 nm. The compounds are expected to be useful as RNA
pathway probes and as components of an unnatural ribopolymer.
Ribonucleosides and ribonucleotides with unnatural
nucleobases have found widespread use as chemical tools
to study the functional roles,1a,b hydrogen bonding
requirements,1c structure/activity relationships,1d and me-
chanistic details1e of RNAs in biological systems. In
particular, the use of fluorescent ribonucleotides as bio-
logical probes and sensors has received much attention.2 In
an effort to minimally perturb the recognition of RNAs by
natural biomolecules, most fluorescent ribonucleotides are
designed to retain some of the original canonical Watson-
Crick base-pairing recognition groups found in natural
RNA.3
Previously, Leonard et al. described the synthesis of
fluorescent ribonucleoside analogs of adenosine (1, Figure 1)
and guanosine (2) with a benzene ring incorporated within
4,5
˚
the purine scaffold that expanded their size by 2.4 A.
Among other features, these analogs retained their canon-
ical Watson-Crick base-pairing groups. Nucleoside tri-
phosphate derivatives of 1 and 2 have been prepared
(1) (a) Lu, J.; Li, N.-S.; Koo, S. C.; Piccirilli, J. A. J. Org. Chem. 2009,
74, 8021. (b) Cho, H. D.; Oyelere, A. K.; Strobel, S. A.; Weiner, A. M. RNA
2003, 9, 970. (c) Somoza, A.; Chelliserrykattil, J.; Kool, E. T. Angew. Chem.,
Int. Ed. 2006, 45, 4994. (d) Pokharel, S.; Jayalath, P.; Maydanovych, O.;
Goodman, R. A.; Wang, S. C.; Tantillo, D. J.; Beal, P. A. J. Am. Chem. Soc.
2009, 131, 11882. (e) Araki, L.; Morita, K.; Yamaguchi, M.; Zhao, Z.-Y.;
Wilson, T. J.; Lilley, D. M. J.; Harusawa, S. J. Org. Chem. 2009, 74, 2350.
(2) (a) Stengel, G.; Urban, M.; Purse, B. W.; Kuchta, R. D. Anal.
Chem. 2010, 82, 1082. (b) Srivatsan, S. G.; Tor, Y. Nat. Protoc. 2007, 2,
1547. (c) Kimoto, M.; Mitsui, T.; Harada, Y.; Sato, A.; Yokoyama, S.; Hirao,
I. Nucleic Acids Res. 2007, 35, 5360.
(3) Zhang, C.-M.; Liu, C.; Christian, T.; Gamper, H.; Rozenski, J.;
Pan, D.; Randolph, J. B.; Wickstrom, E.; Cooperman, B. S.; Hou, Y.-M.
RNA 2008, 14, 2245.
Figure 1. The xRNA genetic set.
r
10.1021/ol102915f
Published on Web 01/07/2011
2011 American Chemical Society

enzymatically;6 however, technological limitations at the
time did not allow for the synthesis of oligoribonucleotides
using these novel RNA monomers. Furthermore, analo-
gous benzo-expanded analogs of cytidine (3) and uridine
(4) have never been reported. One example of a size-
expanded uridine analog has been reported;7 however,
the glyosidic bond is at the same N1-position as natural
pyrimidine ribonucleosides and is therefore not structurally
analogous to 1 and 2 in a size-expanded RNA genetic set.
Our laboratory has previously reported the design and
synthesis of a set of four size-expanded DNA analogs
(xDNA).8,9 After incorporation into oligonucleotides using
automated oligonucleotide synthesis, these compounds
were studied for their unusual biophysical and biochemical
properties in DNA.10,11 Herein we describe the synthesis of
the complete set of size-expanded RNA analogs (xRNA,
Figure 1) and report their photophysical properties. This
information will provide a foundation on which to design
experiments to use xRNA monomers and polymers in the
study of ribonucleotide and RNA pathways in biological
systems.
approach of using mercuric cyanide as a coupling agent.4
Although both methods are known to form the desired
β-anomers of the nucleosides, they also generate a mixture
of N3- and undesired N1-regioisomers. After chromato-
graphic purification of the N-ribosylated products, an
overall yield of 61% was obtained in a ratio of 60:40
(6a:6b). The correct regioisomer (6a) was identified by
2D-NMR (Supporting Information Figure S1). To obtain
additional amounts of the desired N3-regioisomer, 6b was
converted to 6a via a transisomerization reaction involving
catalytic amounts of p-toluenesulfonic acid.13 Leonard did
not report the overall yield and regioisomeric ratios of their
N-ribosylation reaction, so it is difficult to compare the
€
prior approach to the effectiveness of the present Vorbruggen
approach for generating the rxA precursors. A tandem
O-deacetylation/nucleophilic aromatic substitution reac-
tion of 6a with ammonia generated 1 in good yield. Using
these improved methods, compound 1 can be synthesized
in 5.1% overall yield over 8 steps or 6.0% overall yield in 9
steps (including the transisomerization of 6b).
Scheme 2. Synthesis of rxG Triol (2)
Scheme 1. Synthesis of rxA Triol (1)
The synthesis of rxG, as shown in Scheme 2, has been
previously reported by us as an intermediate in dxG
€
synthesis by reaction of 7 with rib under Vorbruggen
conditions.9 However, the N3- and N1-regioisomeric mix-
ture (8a and 8b, respectively) could only be separated in
later steps. To improve rxG synthesis, we found an eluant
mixture for column chromatography that successfully
separated 8a and 8b. The correct regioisomer (8a) was
identified using 2D-NMR (Figure S1). As was done in rxA
synthesis, we were also able to generate more of the desired
8a regioisomer by transisomerization of 8b. Mild deacyla-
tion by aminolysis of 8a generated 2 in 87% yield. Com-
pound 2 was prepared in 5.0% overall yield over 7 steps or
6.3% overall yield in 8 steps (including the transisomeriza-
tion of 8b).
The synthesesof the expandedpurine nucleosides1 (rxA)
and 2 (rxG) were similar to the syntheses of their xDNA
counterparts, dxA and dxG,8,9 and to previous approaches
to the compounds as reported by Leonard and co-
workers.4,5 The protected xA nucleobase (5, Scheme 1)
was coupled to 1,2,3,5-tetra-O-acetyl-β-D-ribose (rib) under
12
Vorbruggen conditions. This method for forming N-
€
nucleosides is a less hazardous alternative to Leonard’s
(4) Leonard, N. J.; Sprecker, M. A.; Morrice, A. G. J. Am. Chem.
Soc. 1976, 98, 3987.
(5) Keyser, G. E.; Leonard, N. J. J. Org. Chem. 1979, 44, 2989.
(6) (a) Leonard, N. J.; Keyser, G. E. Proc. Natl. Acad. Sci. U.S.A.
1979, 76, 4262. (b) Leonard, N. J.; Scopes, D. I. C.; Van Der Lijn, P.; Barrio,
J. R. Biochemistry 1978, 17, 3677.
(7) Xie, Y.; Maxson, T.; Tor, Y. J. Am. Chem. Soc. 2010, 132, 11896.
(8) Liu, H.; Gao, J.; Maynard, L.; Saito, Y. D.; Kool, E. T. J. Am.
Chem. Soc. 2004, 126, 1102.
The novel expanded pyrimidines 3 (rxC) and 4 (rxU)
were modeled after their xDNA counterparts, dxC and
dxT.8,9 A major structural difference between the xDNA
and xRNA pyrimidine sets is the presence and absence of a
methyl group on the C6 position of their nucleobases,
(9) Liu, H.; Gao, J.; Kool, E. T. J. Org. Chem. 2005, 70, 639.
(10) Gao, J.; Liu, H.; Kool, E. T. J. Am. Chem. Soc. 2004, 126, 11831.
(11) Krueger, A. T.; Kool, E. T. J. Am. Chem. Soc. 2008, 130, 3989.
(13) Boryski, J.; Golankiewicz, B. Nucleosides Nucleotides 1989,
8, 529.
€
€
(12) Vorbruggen, H.; Holfe, G. Chem. Ber. 1981, 114, 1256.
Org. Lett., Vol. 13, No. 4, 2011
677

2-aminobenzonitriles.16 Following this, compounds 11
and 12 were synthesized in excellent yield employing
methods previously used by our laboratory.8
Scheme 3. Synthesis of rxU Triol (4)
Various metallating agents were screened in the C-ribo-
sylation reaction of 12 with the ribonolactone. We found
that 1.1 equiv of tert-butyllithium best generated the acti-
vated species for the nucleophilic addition reaction. Fol-
lowingreductionatC-10 using BF3 OEt2 and triethylsilane,
3
a single product, 13, wasformed in 65%yield. gHMBC and
ROESY studies on 13 revealed that the xU nucleobase was
attachedto the sugarin the correct C8-position and thatthe
nucleoside was the desired β-anomer (Figure S2).
Scheme 4. Synthesis of rxC Triol (3)
respectively. This difference required a substantial change
of synthetic approach. Our strategy for the synthesis of
xRNA C-nucleosides involved metallation of an aryl halide
nucleobase followed by nucleophilic addition to 2,3,5-tri-
O-benzyl-β-D-ribono-1,4,-lactone (rlac) and subsequent
reduction at C-10, as was done previously in our labo-
ratory.14 For the synthesis of rxU, we envisioned the use
of 8-bromo-2,4-dimethoxyquinazoline (12, Scheme 3) as an
aryl halide. After reduction at C-10, the nucleoside would
serve as a common intermediate between rxU and rxC
syntheses.
To the best of our knowledge, the synthesis of the
brominated heterocycle intermediate 12 has not been
previously reported. We investigated various synthetic
approaches to generate a sizable amount of 12. In the
end, we found that this could be best accomplished begin-
ning with the synthesis of 2-amino-3-bromobenzonitrile, 9.
Jiang and Lai previously reported the synthesis of 2-ami-
no-3-chlorobenzonitrile from 2,6-dichloroaniline using
copper(I) cyanide in NMP in 31% yield.15 Replacement
of the starting material with commercially available 2,6-
dibromoaniline and improving reaction conditions af-
forded 9 in 71% yield. Cyclization of 9 using carbon
dioxide gas in the presence of DBU produced 10 in 91%
yield, as was reported for the cyclization of various
At this point, we were interested in using 13 as the
branching point between rxU and rxC syntheses. It is
known that boron tribromide is an effective debenzylating
reagent.17 BBr3 also serves as a demethylating agent for
aryl methyl ethers.18 We found that by controlling the
amount of BBr3 and reaction conditions on 13, we could
generate two different and useful products. Using 10 equiv
of BBr3 beginning at -78 °C and warming to room
temperature while stirring overnight generated 4 in 75%
yield. On the other hand, using milder conditions (3 equiv
of BBr3 kept at -78 °C with stirring for 3 h) generated the
2,4-dimethoxyquinazoline triol nucleoside, 14 (Scheme 4),
in 79% yield. To obtain the desired rxC precursor, 15, we
performed an aromatic nucleophilic substitution reac-
tion on 14 using ammonia in methanol. We found that
only one monoamino substituted product was formed in
very good yield. 1H NMR data showed the presence of a
methoxy and amino group in the compound. ROESY
analysis of this product (15) revealed that the methoxy
and amino groups were in the desired 2- and 4-positions,
Table 1. Photophysical Data for xRNA Triols in Methanol
λmax
abs.a
(nm)
λmax
ex.
λmax
em.
xRNA
triol
ε260
εmax
brightnessc
τ
(ns)
(nm)
(nm)
(L/mol cm)
(L/mol cm)
Φb
(L/mol cm)
3
3
3
rxA
rxG
rxC
rxU
331
326
322
311
331
337
322
311
373
411
379
369
18 500
4900
5300
1000
10 500
4500
4500
3900
0.44
0.40
0.48
0.27
4600
1800
2200
1050
3.6
7.3
4.2
2.9
a Long λ maximum. b Error e ( 0.03. c Brightness calculated as εmax ꢀ Φ.
678
Org. Lett., Vol. 13, No. 4, 2011

To evaluate the utility of the xRNA nucleosides to be em-
ployed as fluorescence probes, we measured the photophysical
properties of the entire genetic set (1-4) in methanol
(Table 1) and water at pH 7.0 (Table S1). Previously, we
reported the photophysical properties of the xDNA mono-
mers in methanol.11 In addition, partial photophysical data
for rxA and rxG triols have been reported by Leonard.4,5,6a
Our experiments reveal that all four nucleosides have
high quantum yields (Table 1). The data show that rxA is
the brightest of the four, owing to its higher molar absorp-
tivity. In water, the data for rxA are in good agreement
with those of Leonard.4 In methanol, the fluorescence
spectra of rxA (Figure 2A) and those of dxA are similar,
with the exception that the emission of dxA is red-shifted
by 20 nm.11
The rxG nucleoside emits furthest to the red (411 nm) of
the set. Our data for rxG in water seem to be in general
agreement with previous limited data for rxG triol in aqueous
buffer.5,6a We also find that rxG in methanol (Figure 2B)
has very similar photophysical properties to those of dxG in
the same solvent.11 Interestingly, the emission and excita-
tion wavelengths of rxG are all blue-shifted (by ca. 20 nm) in
water as compared to data taken in methanol.
We also measured the photophysical properties of the
previously unknown xRNA pyrimidines (rxC and rxU). In
comparison to the xDNA pyrimidines,11 there is a small
blue-shifting of absorption, excitation, and emission max-
ima by ∼10 nm (in each case) in the xRNA pyrimidine
nucleosides. This difference is likely due to the absence of
the C6 methyl group in rxC and rxU. We find that the two
have virtually the same properties in methanol (Figure 2C
and D) or water.
In summary, we have synthesized a complete four-letter
benzo-expanded RNA genetic set, in good yield and purity.
We have also demonstrated the photophysical properties of
the xRNA nucleosides, which we find to be efficient
fluorophores. Future work will be directed toward the use
of these compounds as nucleotide probes and in unnatural
RNA polymers.
Acknowledgment. This work was supported by the Na-
tional Institutes of Health (GM063587). We also acknowl-
edge the NIH for support of A.H. as a Ruth L. Kirschstein
NRSA predoctoral fellow (GM084680). We thank Dr.
Urvashi Sahni (Stanford University) for technical assis-
tance and Dr. Stephen R. Lynch (Stanford University) for
assistance with NMR experiments.
Supporting Information Available. Experimental pro-
cedures, NMR data, fluorescence experiment details,
UV-vis and fluorescence spectra, and fluorescence life-
time data. This material is available free of charge via the
Internet at http://pubs.acs.org.
Figure 2. Fluorescence spectra of rxA (A), rxG (B), rxC (C), and
rxU (D) triols in methanol. Excitation spectra were monitored
at the emission maxima.
(14) Silverman, A. P.; Kool, E. T. J. Am. Chem. Soc. 2007, 129, 10626.
(15) Jiang, J.; Lai, Y.-H. J. Am. Chem. Soc. 2003, 125, 14296.
(16) Mizuno, T.; Ishino, Y. Tetrahedron 2002, 58, 3155.
(17) Kundu, N. G.; Hertzberg, R. P.; Hannon, S. J. Tetrahedron Lett.
1980, 21, 1109.
respectively (Figure S2). Demethylation of 15 using
sodium iodide in acetic acid generated 3 in excellent
yield. Compound 3 was synthesized in 19.2% overall
yield over 8 steps, and compound 4 was prepared in
22.6% yield in 6 steps.
(18) Sakamoto, T.; Kondo, Y.; Sato, S.; Yamanaka, H. Tetrahedron
Lett. 1994, 35, 2919.
Org. Lett., Vol. 13, No. 4, 2011
679