Design, synthesis, and spectroscopic properties of extended and fused pyrrolo-dC and pyrrolo-C analogs

Article abstract of DOI:10.1021/ol3012327

The syntheses of four fluorescent nucleoside analogs, related to pyrrolo-C (PyC) and pyrrolo-dC (PydC) through the conjugation or fusion of a thiophene moiety, are described. A thorough photophysical analysis of the nucleosides, in comparison to PyC, is reported.

Full text of DOI:10.1021/ol3012327

ORGANIC
LETTERS
2012
Vol. 14, No. 12
3150–3153
Design, Synthesis, and Spectroscopic
Properties of Extended and Fused
Pyrrolo-dC and Pyrrolo-C Analogs
ꢀ
Mary S. Noe, Andro C. Rıos, and Yitzhak Tor*
´
Department of Chemistry and Biochemistry, University of California, San Diego,
9500 Gilman Drive, La Jolla, California 92093-0358, United States
ytor@ucsd.edu
Received May 5, 2012
ABSTRACT
The syntheses of four fluorescent nucleoside analogs, related to pyrrolo-C (PyC) and pyrrolo-dC (PydC) through the conjugation or fusion of a
thiophene moiety, are described. A thorough photophysical analysis of the nucleosides, in comparison to PyC, is reported.
Fluorescence spectroscopy is a powerful tool for the
detailed investigation of nucleic acids and their diverse
biomolecular interactions, due to its sensitivity and mole-
cular specificity.1 These features become accessible when
suitable fluorescent probes exist. The insignificant emis-
sion of the native nucleobases2 presents unique opportu-
nities for the design of nonperturbing fluorophores.
Creative modifications with minimal structural perturba-
tions have resulted in the development of isomorphic
fluorescent nucleoside analogs.3 These fluorophores have
been successfully employed in the monitoring of real-
time biochemical events such as drug binding,4 RNA
folding and cleavage,5 and RNAꢀprotein interactions.6
The specific utility of a fluorescent nucleoside depends on
its photophysical behavior under the desired assay
conditions.3 Analogsthat displaysensitivity to their micro-
environment, through wavelength shifts or changes in
intensity, can provide information about local parameters
such as polarity,7 viscosity,8 and pH.9 Equally, analogs
with minimal sensitivity to the microenvironment exhibit-
ing desired absorption and emission bands may be utilized
in a FRET-based system.3b,4d,4e,6b,10 The photophysical
properties of anygivenanalogcannot bepredictedthrough
simple structural analysis of the fluorophore. Only upon
the synthesis of a probe and analysis of its photophysical
characteristics can its properties, such as quantum yield,
Stokes shift, brightness, and sensitivity to polarity, be
determined to indicate its most apt implementation.
(1) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd. ed.;
Springer: New York, 2006.
(2) Daniels, M.; Hauswirth, W. Science 1971, 171, 675.
(3) (a) Sinkeldam, R. W.; Greco, N. J.; Tor, Y. Chem. Rev. 2010, 110,
2579. (b) Wilhelmsson, L. M. Q. Rev. Biophys. 2010, 43, 159.
(4) (a) Kaul, M.; Barbieri, C. M.; Pilch, D. S. J. Am. Chem. Soc. 2004,
126, 3447. (b) Shandrick, S.; Zhao, Q.; Han, Q.; Ayida, B. K.; Takahashi,
M.; Winters, G. C.; Simonsen, K. B.; Vourloumis, D.; Hermann, T.
Angew. Chem., Int. Ed. 2004, 43, 3177. (c) Srivatsan, S. G.; Tor, Y. J. Am.
Chem. Soc. 2007, 129, 2044. (d) Xie, Y.; Dix, A. V.; Tor, Y. J. Am. Chem.
Soc. 2009, 131, 17605. (e) Xie, Y.; Dix, A. V.; Tor, Y. Chem. Commun.
2010, 46, 5542.
(7) Sinkeldam, R. W.; Greco, N. J.; Tor, Y. ChemBioChem 2008, 9,
706.
(8) Sinkeldam, R. W.; Wheat, A. J.; Boyaci, H.; Tor, Y. Chem-
PhysChem 2011, 12, 567.
(9) Sinkeldam, R. W.; Marcus, P.; Uchenik, D.; Tor, Y. Chem-
(5) (a) Kirk, S. R.; Luedtke, N. W.; Tor, Y. Bioorg. Med. Chem. 2001,
9, 2295. (b) Tinsley, R. A.; Walter, N. G. RNA 2006, 12, 522.
(c) Hall, K. B. Method Enzymol. 2009, 469, 269.
PhysChem 2011, 12, 2260.
ꢁ ꢀ
€
(6) (a) Pljevaljcica, G.; Millar, D. P. Method Enzymol. 2008, 450, 233.
(10) Borjesson, K.; Preus, S. r.; El-Sagheer, A. H.; Brown, T.;
(b) Xie, Y.; Maxson, T.; Tor, Y. J. Am. Chem. Soc. 2010, 132, 11896.
Albinsson, B.; Wilhelmsson, L. M. J. Am. Chem. Soc. 2009, 131, 4288.
r
10.1021/ol3012327
Published on Web 05/30/2012
2012 American Chemical Society

Pyrrolo-dC (PydC) and pyrrolo-C(PyC) arestructurally
modified fluorescent deoxycytidine and cytidine analogs,
which maintain a proper WatsonꢀCrick H-bonding face
(Figure 1).11 A nominal structural modification leads to a
fluorophore with a significant quantum yield and an
absorbance band, which is red-shifted from those of the
native nucleosides and aromatic amino acid residues. The
quantum yield of PyC decreases upon incorporation into
oligonucleotides and is quenched even further upon duplex
formation.12 Still, PyC has been used in numerous biophy-
sical assays including spectroscopic visualization of the
elongation complex of an RNA polymerase12,13 and the
monitoring of RNA-folding dynamics.5b,14 While modifi-
cations of PyC have gained popularity in recent years,3b,15
new analogs may expand the structural repertoire and
diversify the photophysical properties attainable to facil-
itate the development of novel assays.
analogs 1ꢀ4 (Figure 1). In correlation to our 5-modified
pyrimidines, analogs 1 and 2 represent a PyC core, which is
extended via conjugation to a thiophene. Analogs 3 and 4, in
contrast, represent a fused system, similar to both PyC and
our previously reported nucleoside alphabet,19 in which the
chromophore possesses no rotatable bonds. As such, deoxy-
and ribonucleosides 1ꢀ4 can be viewed as new PyC analogs.
We report the synthesis and evaluation of these analogs, as
well as compare and contrast their photophysical features
with one another and with PydC and PyC.
The syntheses of 1ꢀ4 were based upon the implementa-
tion of a Pd-mediated cross-coupling reaction followed by a
crucial intramolecular cyclization step (Schemes 1 and 2). This
approach employs native nucleosides as starting materials,
eliminating ambiguities commonly associated with glycosyla-
tion reactions regarding the isolation of the correct anomer
and regioisomer. Previously reported syntheses of PyC and its
analogs were based upon one-pot Sonogashira coupling and
ensuing cyclization through the use of Pd and Cu catalysts.
This cross-coupling reaction was performed successfully be-
tween 5-iodoridine and a variety of substituted alkynes.15b,20
The resulting furanopyrimidines were fluorescent but lacked a
proper WatsonꢀCrick H-bonding face. Following solid-
phase incorporation into oligonucleotides, ammonolysis re-
sulted in the efficient conversion of the furanopyrimidines into
cytidine analogs.21 This conversion may also be achieved by
treating the furanopyrimidines with methanolic ammonia
before incorporation into oligonucleotides.
The initial synthetic attempts to obtain extended thio-
phene analogs 1 and 2 were based upon this approach.
Unfortunately, all efforts toconvertthe furanopyrimidines
resulted in complex mixtures and low yields of the desired
cytidine analogs. An alternate synthesis was attempted
(Scheme 1) using 5-iodo-20-deoxycytidine (5) and cytidine
as starting materials. Cytidine (6) was acetylated and
iodinated using established methods,22 while compound
5 was purchased from a commercial source. Compounds 5
and 7 reacted readily with 2-ethynylthiophene under stan-
dard cross-coupling conditions, but the included copper
catalyst failed to facilitate further cyclization. Compounds
8 and 9 were, therefore, isolated, purified, and screened for
an alternate metal-based cyclization catalyst. Gold cata-
lysts activate alkyne moieties to a greater degree than other
metal ions,23 so a sodium tetrachloroaurate(III) dihydrate
was employed based upon successful cyclization of related
heterocycles.24 Although the cyclization yields were not
optimal, this concise synthesis produced 1 in an overall
yield of 41% in 2 steps and 2 in an overall yield of 12% in 4
total steps.
Figure 1. WatsonꢀCrick hydrogen-bonding faces of C and PyC.
Extended (1 and 2) and fused (3 and 4) PyC analogs.
Our program has focused on the development of diverse
nonperturbing fluorescent nucleoside analogs via the coju-
gation or fusion of aromatic heterocycles to the native
bases, especially the pyrimidines.3a For example, placing
a furan or thiophene moiety at the 5-position of uridine
leads to sensitive nucleoside analogs, which have been
used to detect abasic sites16 and oxidatively damaged
nucleosides17 and monitor RNAꢀdrug interactions.4c
Although these useful nucleosides possess measurable
sensitivity, low fluorescence quantum yields leave room
for improvement.18 Studies have demonstrated that ham-
pering the free rotation of the furan or thiophene moieties
in viscous media dramatically increases the quantum
yield.8 This inspired the design and synthesis of nucleoside
(11) (a) Berry, D. A.; Jung, K.-Y.; Wise, D. S.; Sercel, A. D.; Pearson,
W. H.; Mackie, H.; Randolph, J. B.; Somers, R. L. Tetrahedron Lett.
2004, 45, 2457. (b) Thompson, K. C.; Miyake, N. J. Phys. Chem. B 2005,
109, 6012.
(12) Liu, C.; Martin, C. T. J. Mol. Biol. 2001, 308, 465.
(13) Liu, C.; Martin, C. T. J. Biol. Chem. 2002, 277, 2725.
(14) Buskiewicz, I. A.; Burke, J. M. RNA 2012, 18, 434.
(15) (a) Wahba, A. S.; Azizi, F.; Deleavey, G. F.; Brown, C.; Robert,
F.; Carrier, M.; Kalota, A.; Gewirtz, A. M.; Pelletier, J.; Hudson,
R. H. E.; Damha, M. J. ACS Chem. Biol. 2011, 6, 912. (b) Hudson,
R. H. E.; Ghorbani-Choghamarani, A. Synlett 2007, 2007, 0870.
(c) Wahba, A. S.; Esmaeili, A.; Damha, M. J.; Hudson, R. H. E. Nucleic
Acids Res. 2010, 38, 1048.
(19) (a) Srivatsan, S. G.; Greco, N. J.; Tor, Y. Angew. Chem., Int. Ed.
2008, 47, 6661. (b) Shin, D.; Sinkeldam, R. W.; Tor, Y. J. Am. Chem.
Soc. 2011, 133, 14912.
(20) (a) Robins, M. J.; Barr, P. J. J. Org. Chem. 1983, 48, 1854.
(b) Hudson, R. H. E.; Choghamarani, A. G. Nucleosides, Nucleotides,
Nucleic Acids 2007, 26, 533.
(21) Woo, J.; Meyer, R. B.; Gamper, H. B. Nucleic Acids Res. 1996,
24, 2470.
(16) Greco, N. J.; Tor, Y. J. Am. Chem. Soc. 2005, 127, 10784.
(17) Greco, N. J.; Sinkeldam, R. W.; Tor, Y. Org. Lett. 2009, 11,
1115.
(22) Piton, N.; Mu, Y.; Stock, G.; Prisner, T. F.; Schiemann, O.;
Engels, J. W. Nucleic Acids Res. 2007, 35, 3128.
(23) Shen, H. C. Tetrahedron 2008, 64, 7847.
(18) See, however, refs 8 and 19b.
(24) Arcadi, A.; Bianchi, G.; Marinelli, F. Synthesis 2004, 610.
Org. Lett., Vol. 14, No. 12, 2012
3151

Scheme 1. Synthesis of Extended Analogs 1 and 2
Scheme 2. Synthesis of Fused Analogs 3 and 4
The syntheses of the fused thiophene PyC analogs 3 and
4 (Scheme 2) were initiated with the use of commercially
available 11 and known modifications of uridine to afford
13.25 Cytidine starting materials were explored, but all
reactions involving cytidine analogs were lower yielding
and more problematic to purify. As numerous methods
exist for the exchange of a C-4 carbonyl for an amine, the
conversion of a uridine to a cytidine core was left until the
penultimate step. The Pd-mediated Stille coupling reaction
was high yielding for both the unprotected 20-deoxynucleo-
side 11 and the acetylated ribonucleoside 13 and afforded
mildly fluorescent nucleosides 14 and 16.26 Installing a
halogen at the 3-position of the thiophene allowed for the
screening of catalysts capable of inducing the desired in-
tramolecular cyclization to the pyrrole moiety. Since the
5-position of the thiophene undergoes electrophilic aromatic
substitution more readily than the 3-position, an excess of
bromine was added to afford dibrominated products 17 and
18.27 Selective zinc-mediated debromination of the 5-posi-
tion yielded 19 and 20, respectively.28 Following the activa-
tion of the C-4 carbonyl, a one-pot conversion of the U to C
analog and O-deacetylation produced 21 and 22.
The intramolecular cyclization of 21 and 22 to yield the
desired fused PyC analogs proved to be the biggest syn-
thetic hurdle. BuchwaldꢀHartwig amination reactions
were first attempted through the use of various ligands
and Pd catalysts. All reactions yielded, however,
only starting material and/or the debrominated
product 5-(thiophen-2-yl)-cytidine or 5-(thiophen-2-yl)-
20-deoxycytidine. Copper catalysts with various ligands
were explored next. Of all attempted reactions, only a
single set of conditions which include copper(I) iodide,
cesium carbonate, and N,N0-dimethylethylenediamine
(N,N0-DMEDA) provided the cyclized products 3 and 4.29
Although the cyclization reactions were not as high yield-
ing as the preceding steps, they provided ample quantities
of nucleosides 3 and 4 for full analytical and photophysical
studies. Compound 3 was synthesized in an overall yield of
18% over 6 steps, while compound 4 was prepared in an
overall yield of 22% over 6 steps.
The photophysical properties of nucleosides 1ꢀ4 along
with PyC and PydC are summarized in Table 1. As
expected, the presence of a ribose or deoxyribose moiety
(1 vs 2, or 3 vs 4) had little impact on the photophysics of
the respective chromophores. The nucleosides displayed
red-shifted absorption bands at 369 nm (1), 371 nm (2),
and 357 nm (3 and 4), longer wavelengths than those of the
parent PydC (343 nm) and PyC (342 nm) chromophores in
water (Figure 2A). These bands were further red-shifted
whenthe nucleosides were dissolvedin dioxane, anaprotic,
less polar solvent. Somewhat surprisingly, in aqueous
solution, the thiophene extended analogs displayed only
slight shifts in emission maxima, 469 nm (1) and 473 nm
(2), from the parent nucleosides PydC (463 nm) and PyC
(461 nm). However, the significantly higher quantum
yields of 0.41 and 0.43 for 1 and 2, respectively, are over
nine times that of PyC (0.04) and PydC (0.05). The quantum
yields of 1 and 2 showed moderate sensitivity to solvent
polarity, exhibiting a hypsochromic shift and increase of
fluorescence in dioxane. The exceptional property of
1 and 2 is the brightness value (Φε), which is
primarily due to large extinction coefficient (ε) values of
(25) Asakura, J.; Robins, M. J. J. Org. Chem. 1990, 55, 4928.
(26) Wigerinck, P.; Pannecouque, C.; Snoeck, R.; Claes, P.; De
Clercq, E.; Herdewijn, P. J. Med. Chem. 1991, 34, 2383.
(27) Liu, J.; Aerschot, A. V.; Luyten, I.; Wigerinck, P.; Pannecouque,
C.; Balzarini, J.; De Clercq, E.; Herdewijn, P. Nucleosides Nucleotides
1995, 14, 525.
(28) Luyten, I.; Jie, L.; Vanaerschot, A.; Pannecouque, C.; Wigerinck,
P.; Rozenski, J.; Hendrix, C.; Wang, C.; Wiebe, L.; Balzarini, J.; Declercq,
E.; Herdewijn, P. Antivir. Chem. Chemother. 1995, 6, 262.
(29) Lu, H.; Yuan, X.; Zhu, S.; Sun, C.; Li, C. J. Org. Chem. 2008, 73,
8665.
3152
Org. Lett., Vol. 14, No. 12, 2012

Table 1. Photophysical Data for Nucleosides 1ꢀ4
Stokes
shiftd
polarity
sensitivitye
compd
solvent
λ
abs (nm)a
λ
em (nm)
εb
Φc
brightness (Φε)
PydC
H2O
342
351
343
349
369
383
371
379
357
367
357
365
461
439
463
439
469
449
473
449
474
447
478
447
4.03
5.00
5.04
5.00
11.6
13.6
12.9
15.0
2.38
3.16
2.42
3.21
0.05
0.10
0.04
0.10
0.41
0.50
0.43
0.47
0.01
0.70
0.01
0.74
7.6
5.7
7.6
6.0
5.7
3.9
5.9
4.0
7.0
4.9
7.1
5.0
0.20
0.50
0.20
0.50
4.8
71
67
70
73
75
74
dioxane
H2O
PyC
dioxane
H2O
1
2
3
4
dioxane
H2O
6.8
5.5
dioxane
H2O
7.1
0.024
2.2
dioxane
H2O
0.024
2.4
dioxane
a Long λ maximum. b 103
represent the slope of the line in the plot of Stokes shift versus solvent polarity (see SI for details).
M . .
ꢀ1cmꢀ1 c Relative quantum yields. d 103 cmꢀ1 e Polarity sensitivity values are expressed in cmꢀ1/(kcal molꢀ1) and
3
and 4 (7.1 ꢁ 103 cmꢀ1) were comparable to the parent
PydC and PyC (7.6 ꢁ 103 cmꢀ1), they were higher than 1
(5.7 ꢁ 103 cmꢀ1) and 2 (5.9 ꢁ 103 cmꢀ1). Most striking is
the extreme sensitivity that the quantum yields of 3 and 4
exhibited to solvent polarity. In the polar protic environ-
ment of water, 3 and 4 both displayed a very low quantum
yield of 0.01. However, in an aprotic and less polar
environment, the quantum yields of 3 and 4 increased
dramatically. These quantum yield values of 0.70 and
above are exceptional, considering the minimal structural
modification from the virtually nonemissive cytidine nu-
cleosides. This increase occurs concomitantly with a de-
crease in Stokes shift resulting in values of approximately
5 ꢁ 103 cmꢀ1 for both nucleosides. Nucleosides 3 and 4
have similar ε in both water (2.38 ꢁ 103 and 2.42 ꢁ 103 Mꢀ1
cmꢀ1) and dioxane (3.16 ꢁ 103 and 3.21 ꢁ 103 Mꢀ1 cmꢀ1),
which are significantly lower than those of nucleosides 1 and
2. This results in comparatively lower brightness values for 3
(0.024 in water and 2.2 in dioxane) and 4 (0.024 in water and
2.4 in dioxane). Notably, the brightness values for 3 and 4
are nearly 100-fold higher in dioxane than in water.
Figure 2. Absorption (dashed) and emission (solid) spectra of 2
(A) and 4 (B) in water (red) and dioxane (black).
In summary, the PyC family is joined by new members.
The effective syntheses of nucleosides 1ꢀ4 via novel,
metal-catalyzed cyclization reactions resulted in four nu-
cleosides displaying complementary photophysical prop-
erties. These nucleosides make a significant and diverse
addition to the growing toolbox of nonperturbing, iso-
morphic, fluorescent analogs.
11.6 ꢁ 103 and 12.9 ꢁ 103 Mꢀ1 cmꢀ1 (1 and 2, respectively, in
water). Both nucleosides show a linear correlation between
Stokes shift and solvent polarity with reasonable polarity
sensitivity values (see Supporting Information (SI)). Notably,
both the absorption and emission spectra display sensitivity
to solvent polarity for the entire nucleoside series.
Acknowledgment. We thank the NIH for their generous
support (Grant No. GM 069773) and the NSF
(Instrumentation Grant CHE-0741968).
The fused thiophene PyC analogs exhibited photophy-
sical characteristics that were distinctive from the thio-
phene extended system (see Figure 2B). In water,
nucleosides 3 and 4 exhibited the most red-shifted emission
maxima (474 and 478 nm, respectively) along with the
lowest emission intensity of this nucleoside series. In
dioxane, the increased emission intensity is dwarfing in
comparison to that in water, with maxima at 447 nm.
Supporting Information Available. Experimental pro-
cedures, NMR data, and detailed photophysical data and
calculations. This material is available free of charge via
the Internet at http://pubs.acs.org.
While the Stokes shifts of nucleosides 3 (7.0 ꢁ 103 cmꢀ1
)
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 12, 2012
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