Synthesis and solvatochromic fluorescence of biaryl pyrimidine nucleosides

Article abstract of DOI:10.1021/ol400930s

Fluorescent pyrimidine analogs containing a fused biphenyl unit were prepared in high yields using stereoselective N-glycosylation and Suzuki-Miyaura cross-coupling reactions. The resulting push-pull fluorophores exhibit highly solvatochromic emissions from twisted intramolecular charge-transfer (TICT) states.

Full text of DOI:10.1021/ol400930s

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
Synthesis and Solvatochromic
Fluorescence of Biaryl Pyrimidine
Nucleosides
Guillaume Mata and Nathan W. Luedtke*
Department of Chemistry, University of Zu€rich, Winterthurerstrasse 190,
CH-8057 Zu€rich, Switzerland
luedtke@oci.uzh.ch
Received April 4, 2013
ABSTRACT
Fluorescent pyrimidine analogs containing a fused biphenyl unit were prepared in high yields using stereoselective N-glycosylation and
SuzukiꢀMiyaura cross-coupling reactions. The resulting “pushꢀpull” fluorophores exhibit highly solvatochromic emissions from twisted
intramolecular charge-transfer (TICT) states.
Fluorescence spectroscopy is one of the most powerful
tools in chemical biology.1 Fluorescence-based labeling of
nucleic acids is widely used for genomic sequencing and for
the visualization of DNA replication in vivo.2 The small
size and predictable location of fluorescent nucleobase
analogs3 make them ideally suited for monitoring bio-
chemical transformations,4 conformational changes,5 and
base pairing interactions.6 The environmental sensitivity
of these probes allows for reporting of microenviron-
ment parameters such as polarity,7 viscosity,8 and pH.9
C-Nucleosides containing arene groups such as pyrene
and biphenyl can exhibit high quantum efficiencies of
emission (Figure 1A). When incorporated into DNA, they
can be used as scaffolds for the construction of multi-
chromophore arrays.10 The incorporation of biphenyl
C-nucleosides into duplex DNA has been shown to result
in a zipper-like interstrand stacking recognition motif.11
However, the WatsonꢀCrick hydrogen bonding face in
such derivatives is absent, making them poor nucleobase
mimics. Given the remarkable photophysical properties of
substituted biphenyls,12 and the need for emissive nucleo-
base mimics,3 we designed nucleoside analogs 1aꢀ1h
(Figure 1B). These new analogs contain a biphenyl unit
fused to thymidine in a way that should be compatible with
(1) (a) Lakowicz, J. R. Principles of Fluorescence Spectroscopy;
Springer: New York, 2006. (b) Lavis, L. D.; Raines, R. T. ACS Chem. Biol.
2008, 3, 142. (c) Sletten, E. M.; Bertozzi, C. R. Acc. Chem. Res. 2011, 44,
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(2) (a) Bao, G.; Rhee, W. J.; Tsourkas, A. Annu. Rev. Biomed. Eng.
2009, 11, 25. (b) Neef, A. B.; Luedtke, N. W. Proc. Natl. Acad. Sci. U.S.A.
2011, 108, 20404.
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132, 11896.
(5) Luedtke, N. W.; Dumas, A. J. Am. Chem. Soc. 2010, 132, 18004.
(6) (a) Xie, Y.; Maxson, T.; Tor, Y. Org. Biomol. Chem. 2010, 8, 5053.
(b) Michel, J.; Gueguen, G.; Vercauteren, J.; Moreau, S. Tetrahedron
1997, 53, 8457. (c) Xie, Y.; Dix, A. V.; Tor, Y. J. Am. Chem. Soc. 2009,
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(8) Sinkeldam, R. W.; Wheat, A. J.; Boyaci, H.; Tor, Y. Chem-
PhysChem 2011, 12, 567.
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R. A. J. Am. Chem. Soc. 2007, 129, 1894. (b) Riedl, J.; Pohl, R.; Rulisek,
L.; Hocek, M. J. Org. Chem. 2012, 77, 1026.
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(b) Malinovskii, V. L.; Wenger, D.; Haner, R. Chem. Soc. Rev. 2010,
39, 410.
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1655. (b) Zahn, A.; Leumann, C. J. Chem.;Eur. J. 2008, 14, 1087.
(12) For general reviews about twisted intramolecular charge trans-
fer (TICT) states, see: (a) Bhattacharyya, K.; Chowdhury, M. Chem.
Rev. 1993, 93, 507. (b) Rettig, W. Angew. Chem., Int. Ed. Engl. 1986,
25, 971. For photophysical properties of substituted biphenyls, see:
(c) Maus, M.; Rettig, W.; Jonusauskas, G.; Lapouyade, R.; Rulliere,
C. J. Phys. Chem. A 1998, 102, 7393. (d) Maus, M.; Rettig, W.;
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r
10.1021/ol400930s
XXXX American Chemical Society

Scheme 1. Stereoselective N-Glycosylation of 2-Deoxythioriboside
3 and Synthesis of Key Intermediate 2
Figure 1. (A) Chromophoric C-nucleosides containing pyrene and
biphenyl. (B) Emissive biaryl nucleoside analogs 1aꢀ1h. R =
20-deoxyribose, R1 = electron-withdrawing or -donating group.
Scheme 2. Pd-Catalyzed SuzukiꢀMiyaura Cross-Couplinga
and Silyl Groups Deprotection
Figure 2. Key steps toward the synthesis of 1aꢀ1h.
proper WatsonꢀCrick base pairing.6aꢀc Herein, we de-
scribe the synthesis and evaluation of these new fluorescent
nucleobase analogs and provide a detailed analysis of their
structureꢀphotophysical properties relationships.
The synthesis of the biaryl nucleosides 1aꢀ1h was envi-
sioned from the silylated 6-bromo-quinazoline-2,4-(3H)-
dione nucleoside 2 via a Pd-catalyzed SuzukiꢀMiyaura
cross-coupling (Figure 2). In this approach, 2 provides a
versatile intermediate for the coupling of various boronic
acids and esters. Nucleoside 2 can be obtained from a
stereoselective N-glycosylation of 2-deoxythioriboside 3 with
6-bromo-quinazoline-2,4-(1H,3H)-dione 4. According to
our new method,13 2-deoxythioriboside 3was N-glycosylated
with 4 using the combination of N,O-bis-(trimethylsilyl)-
acetamide (BSA), N-iodosuccinimide (NIS), and trimethyl-
silyl trifluoromethanesulfonate (TMSOTf) to afford 5
in 84% yield (5r/5β = 1.0:1.8, >10 g scale, Scheme 1).
To facilitate cross-coupling reactions, the C03-acetate of 5β
was converted into a C03-silyl ether to give 2 in 95% yield
over two steps.14 Suitable conditions for Pd-catalyzed
SuzukiꢀMiyaura cross-coupling between 2 and phenyl
boronic acid were evaluated by screening various palladium
sources, ligands, bases, and solvents.15 Pd(dppf)Cl2 and
AcOK in dioxane at 90 °C were found to be highly effective
for this transformation. The biphenyl product 6a (“Ph ”) was
a Reagents and conditions: 2 (1.0 equiv), ArꢀB(OR)2 (1.5 equiv),
AcOK (1.5 equiv), Pd(dppf)Cl2 (0.05 equiv), dioxane (0.07 M), 90 °C,
15 h. b ArꢀB(OR)2 (2.0 equiv) and AcOK (2.0 equiv) were used. c The
reaction was performed on a 4.0 g scale. 6aꢀ6h: R2 = TIPS, R3
TBDMS; 1aꢀ1h: R2 = R3 = H.
=
(13) Mata, G.; Luedtke, N. W. J. Org. Chem. 2012, 77, 9006.
(14) The influence of the protecting groups on the sugar moiety in
Pd-catalyzed cross-coupling has already been examined. The bulky
TBDMS group prevents coordination of the Pd catalyst. For a review
about Pd-assisted routes to nucleosides, see: Agrofoglio, L. A.;
Gillaizeau, I.; Saito, Y. Chem. Rev. 2003, 103, 1875.
(15) Test reactions indicated that Pd(dppf)Cl2 is a superior catalyst as
compared to Pd(PPh3)2Cl2, Pd2(dba)3/SPHOS, and Pd(OAc)2/SPHOS.
Furthermore, dioxane was found to be superior to toluene and DMF.
isolated in 91% yield (Scheme 2). Electron-donating
and -withdrawing groups on the phenyl boronic acid
were well tolerated, giving good to excellent yields for
biphenyl derivatives 6bꢀ6d (“MeOPh”, 90%), (“Me2NPh”,
67%, performed on a 4.0 g scale), and (“CNPh”, 91%).
B
Org. Lett., Vol. XX, No. XX, XXXX

Table 1. Photophysical Data of Emissive Nucleosides 1aꢀ1h
a
b
1aꢀ1h
solvent
λabs
λem
Stokesc
εd
Φe
Ph
dioxane
water
270
269
280
276
312
297
293
286
298
295
292
286
292
290
269
267
371
404
390
462
459
460
362
388
391
449
432
375
403
463
381
430
10.1
12.4
10.1
14.6
10.3
11.9
6.5
2.49
0.11
(1a)
0.085
0.132
0.035
0.071
0.010
0.206
0.140
0.094
0.024
0.079
0.001
0.152
0.038
0.086
0.075
MeOPh
(1b)
dioxane
water
2.74
2.01
2.07
2.02
1.92
3.03
1.87
Me2NPh
(1c)
dioxane
water
CNPh
(1d)
dioxane
water
7.4
2Th
dioxane
water
8.0
(1e)
11.6
11.1
8.3
2MePy
(1f)
dioxane
water
2Fu
dioxane
water
9.4
(1g)
12.9
10.9
14.2
Figure 4. (A) Plot between the Stokes shift of 1c and ET30 value.
(B) Plot between the quantum yield of 1c and ET value. (C)
3Fu
dioxane
water
30
(1h)
Photograph of 1c (fixed concentration) in organic solvents of
increasing polarity with UVꢀB irradiation.
a λabs are reported at the maximum absorbance wavelength in nm.
b λem in nm. c Stokes shifts are reported in 103 cmꢀ1 d Extinction co-
.
efficients (ε) are given at λmax in H2O and are reported in 104 Mꢀ1 cmꢀ1
.
e 2-Aminopyridine in 0.1 N H2SO4 (Φ = 0.60) was used as the
fluorescent standard for the relative quantum yields (Φ) of 1aꢀ1h.
(See SI for complete fluorescence data, spectra and plots.)
itself (Φ = 0.18),18 but the wavelengths of excitation and
emission of 1aꢀ1h are red-shifted. For example, the
maximal wavelengths of absorbance and emission of the
biphenyl nucleoside Ph (λabs = 270 nm, λem = 371 nm)
are 23 and 59 nm to the red of biphenyl in dioxane (λabs
=
247 nm, λem = 312 nm). The reason for this red-shifted
emission is related to the ability of the pyrimidine group
to serve as an electron-accepting group upon photoexcita-
tion. DFT calculations indicate charge transfer from
the benzene group of Ph to pyrimidine upon going from
HOMO f LUMO (Figure 3). These results suggest the
presence of a pushꢀpull system where the pyrimidine group
acts as an electron acceptor in the excited state. The magni-
tude of this pushꢀpull effect is augmented by electron-
donating groups in 1b and 1c, but diminished by the
cyano group in 1d. Indeed, the calculated HOMOꢀ
LUMO gaps (Me2NPh < MeOPh < Ph < CNPh, see
Supporting Information (SI)) were inversely correlated
with the Stokes shifts of these compounds in dioxane
(Table 1). Taken together, these results are consistent with
the presence of emissive TICT states that are stabilized by
electron-donating groups on the benzene ring.12
To characterize the environmental sensitivity of 1aꢀ1h,
the maximal wavelengths for absorbance and emission
were measured in different solvents, and the Stokes shifts
were plotted against Reichardt’s solvent polarity parameter
(ET30).19 Consistent with the presence of a twisted biaryl bond
in the ground state of these molecules (see SI for DFT
calculations), the absorption maxima of 1aꢀ1h were relatively
insensitive to solvent polarity. The Stokes shifts of these com-
pounds are highly solvatochromic due to changes in their emis-
sion wavelengths. For example, the Stokes shift of Me2NPh
increased from 10.3 ꢁ 103 cmꢀ1 in dioxane (λem = 459 nm)
to 13.9 ꢁ 103 cmꢀ1 in MeCN (λem = 550 nm) to give an ex-
ceptionally large slope of 373 cmꢀ1/kcal molꢀ1 (Figure 4A).
Figure 3. Molecular orbital plot of the isolated nucleobase of 1a
(see SI for DFT calculation details).
This approach is also compatible with the synthesis of deriv-
atives containing five-membered heteroaromatic groups
6eꢀ6h. It is known that five-membered, 2-heteroaromatic
boronic acids are prone to undergo protodeboronation
under basic conditions,16 but reactions involving 2 and
thiophene, pyrrole, or furan boronic acids furnished the
corresponding biaryls 6eꢀ6h in good isolated yields
(“2Th”, 62%), (“2MePy”, 82%), (“2Fu”, 64%), and
(“3Fu”, 70%). Treating nucleosides 6aꢀ6h with TBAF
in THF afforded the deprotected compounds 1aꢀ1h in
isolated yields ranging from 70% to 92% (Scheme 2),
and in high purity (>98%) according to 1H NMR.17
The photophysical properties of 1aꢀ1h are summarized
in Table 1. The quantum yields of these compounds are
moderate in water (Φ = 0.14ꢀ0.0002) and in dioxane
(Φ = 0.21ꢀ0.07). These values are very similar to biphenyl
(16) (a) Kinzel, T.; Zhang, Y.; Buchwald, S. L. J. Am. Chem. Soc.
2010, 132, 14073. (b) Thakur, A.; Zhang, K.; Louie, J. Chem. Commun.
2012, 48, 203.
(17) In addition to column chromatography, extensive washingof the
products with MeOH and H2O/AcOH was used to eliminate traces of
TBAF salts. See SI for 1H and 13C NMR spectra.
(18) Berlman, I. B. Handbook of Fluorescence Spectra of Aromatic
Molecules; Academic Press: 1971.
(19) (a) Reichardt, C. Chem. Rev. 1994, 94, 2319. (b) Sinkeldam,
R. W.; Tor, Y. Org. Biomol. Chem. 2007, 5, 2523.
Org. Lett., Vol. XX, No. XX, XXXX
C

Figure 5. Sensitivity of the nucleosides 1aꢀ1h toward solvent
polarity in dioxane, water, and their mixtures. The black bars
represent the slope of the Stokes shift and ET30 value. The gray
bars represent the slope of the fluorescence quantum yield and
ET 30 value. a Due to quenching by H2O, slopes were determined
in organic solvents for 1c and 1f.
Figure 6. (A) Plot between the emission wavelength of 1aꢀ1h
and calculated HOMOꢀLUMO energy gaps (see SI for calcula-
tion details). (B) Plot between the emission wavelength of the
nucleosides 1aꢀ1h and Mayr’s nucleophilicity parameter (N)
of the isolated aryl groups (R1) for benzene, anisole, thiophene,
N-methylpyrrole, and furan in CH2Cl2.20
This effect can easily be observed under UV illumination,
where samples of Me2NPh appear bright blue to dark
orange, depending on the solvent polarity (Figure 4C).
The emission intensity of Me2NPh is highly sensitive to
solvent polarity, with a slope of 0.006 mol kcalꢀ1 from Φ =
0.071 in dioxane to Φ = 0.008 in MeCN (Figure 4B).
Me2NPh exhibits a low quantum yield in water (Φ = 0.010)
that is 2.7-fold higher in D2O(Φ= 0.027, Table S3A SI). This
type of kinetic isotope effect suggests the presence of proton-
transfer reactions between the excited fluorophore and bulk
solvent that can facilitate nonemissive decay in water.
Consistent with the presence of emissive TICT states,12
the emissions from 1aꢀ1h exhibit red-shifting and lower
quantum yields with increasing solvent polarity. The
magnitudes of these effects are compared in Figure 5.
The biphenyl derivatives containing electron-donating
substituents MeOPh and Me2NPh exhibit highly solvato-
chromic Stokes shifts and quantum yields. In contrast,
CNPh exhibits diminished solvent sensitivity, and a good
quantum yield over the entire range of solvent polarity
tested (Φ = 0.206ꢀ0.140). Together these results are con-
sistent with 00pushꢀpull00 TICT fluorophores.12 The trends
between the compounds containing a five-membered hetero-
cyclic ring 1eꢀ1h are somewhat more complicated. For
example, 2Fu exhibits quantum yields that are highly sensitive
to solvent polarity (4.2 ꢁ 10ꢀ3 mol kcalꢀ1), whereas it isomer,
3Fu, exhibits very little sensitivity (0.3 ꢁ 10ꢀ3 mol kcalꢀ1).
To systematically compare the theoretical and experi-
mental photophysical properties of the biphenyl deriva-
tives 1aꢀ1d with the biaryl derivatives 1eꢀ1h, the
calculated HOMOꢀLUMO gap for each compound
was plotted against its peak emission wavelength in diox-
ane to give an excellent linear correlation (R2 = 0.981,
Figure 6A). These results suggested that the underlying
photophysical processes are similar for 1aꢀ1h, where
electron-donating groups cause smaller HOMOꢀLUMO
gaps and red-shifted emissions as compared to electron-
withdrawing groups. This conclusion is further supported
by plotting the emission wavelength of each nucleoside
versus the Mayr’s nucleophilicity parameter (N) for the
isolated aryl/heteroaryl group “R1”.20 An excellent linear
correlation between these factors is observed (R2 = 0.983,
Figure 6B). This unusual approach reveals a possible
method to extrapolate nucleophilicityparameters of groups
that cannot be experimentally measured. For example, this
plot can be used to predict a nucleophilicity parameter
for the position 3 of furan (N ≈ ꢀ4.12) based upon the
emission wavelength of 3Fu (λem = 388 nm) in CH2Cl2.
In summary, we have synthesized a new family of fluo-
rescent biaryl pyrimidines in high yield and purity. The
nucleosides exhibit highly solvatochromic emissions from
twisted intramolecular charge-transfer (TICT) states. Previous
studies have demonstrated fluorescent emissions resulting
from charge-transfer recombination in pyrene-modified de-
oxyuridine,21 but to the best of our knowledge, compounds
1aꢀhprovide the first examples of nucleobase analogs that are
“classical” TICT fluorophores.12 Me2NPh (1c) exhibits ab-
sorbance well to the red of unmodified DNA nucleobases and
emissions that are exceptionally sensitive to solvent polar-
ity. Me2NPh is therefore a promising candidate for inves-
tigating local DNA conformational changes by reporting
changes in microenvironmental polarity and solvation.5
Acknowledgment. This work was supported by the
Swiss National Science Foundation (#130074) and the
€
University of Zurich.
Supporting Information Available. Figures S1ꢀ12,
Tables S1ꢀS8, experimental procedures, spectroscopic char-
acterizations, fluorescence spectra, molecular orbital plots,
(20) (a) Gotta, M. F.; Mayr, H. J. Org. Chem. 1998, 63, 9769. (b)
Mayr, H.; Bug, T.; Gotta, M. F.; Hering, N.; Irrgang, B.; Janker, B.;
Kempf, B.; Loos, R.; Ofial, A. R.; Remennikov, G.; Schimmel, H. J. Am.
Chem. Soc. 2001, 123, 9500. (c) Ammer, J.; Nolte, C.; Mayr, H. J. Am.
Chem. Soc. 2012, 134, 13902. (d) Mayr, H.; Ofial, A. R. J. Phys. Org.
Chem. 2008, 21, 584. (e) Mayr, H.; Kempf, B.; Ofial, A. R. Acc. Chem.
Res. 2003, 36, 66.
1
and H and 13C NMR spectra. This material is available
free of charge via the Internet at http://pubs.acs.org.
(21) Netzel, T. L.; Zhao, M.; Nafisi, K.; Headrick, J.; Sigman, M. S.;
Eaton, B. E. J. Am. Chem. Soc. 1995, 117, 9119.
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX