Emissive RNA alphabet

Article abstract of DOI:10.1021/ja206095a

A fluorescent ribonucleoside alphabet consisting of highly emissive purine (^thA, ^thG) and pyrimidine (^thU, ^thC) analogues, all derived from thieno[3,4-d]pyrimidine as the heterocyclic nucleus, is described. Stru

Full text of DOI:10.1021/ja206095a

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Emissive RNA Alphabet
Dongwon Shin, Renatus W. Sinkeldam, and Yitzhak Tor*
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0358, United States
S Supporting Information
b
ABSTRACT: A fluorescent ribonucleoside alphabet con-
sisting of highly emissive purine (thA, thG) and pyrimidine
(thU, thC) analogues, all derived from thieno[3,4-d]-
pyrimidine as the heterocyclic nucleus, is described.
Structural and biophysical analyses demonstrated that the
emissive analogues are faithful isomorphic nucleoside sur-
rogates. Photophysical analysis established that the nucleo-
sides offer highly desirable qualities, including visible
emission, high quantum yield, and responsiveness to envir-
onmental perturbations, traits entirely lacking in their native
counterparts.
he nonemissive nature of the nucleobases found in con-
temporary nucleic acids has triggered the development of
T
Figure 1. Emissive RNA Alphabet.
emissive nucleoside analogues.1 When carefully designed and
employed, fluorescent nucleosides, nucleotides, and oligonucleo-
tides can play an unparalleled role in exploring fundamental bio-
chemical transformations and facilitate the fabrication of bio-
physical and discovery assays.1,2 A key criterion for the design
and successful implementation of such probes is to minimize
structural and functional perturbation, which is an inevitable
consequence of replacing any native residue with a synthetic
probe. This important constraint has led to the development of
emissive nucleoside analogues that display strong structural resem-
blance to their native counterparts, a characteristic we commonly
describe as isomorphicity.1,3
Scheme 1. Syntheses of thG and thCa
Although several emissive nucleoside analogues have been
reported over the past several years,1 a complete alphabet of iso-
morphic fluorescent ribonucleosides derived from a single hetero-
cyclic core has not been described.4 Here we disclose the design
and synthesis as well as the structural, photophysical, and pre-
liminary biophysical characteristics of a complete ribonucleoside
alphabet consisting of highly emissive purine (thA, thG) and
pyrimidine (thU, thC) analogues, all derived from thieno[3,4-d]-
pyrimidine as the heterocyclic nucleus (Figure 1). This parent
heterocycle can be viewed as a precursor to 5,6-modified emissive
pyrimidines and, at the same time, as a purine mimic with
thiophene substituted for the imidazole moiety.5 As schemati-
cally illustrated in Figure 1, glycosylation with D-ribose at N1
of the heterocycle is expected to yield expanded and emissive
pyrimidine nucleoside analogues, while C-glycosylation at the
thiophene’s C2 position is anticipated to provide a viable route to
fluorescent purine C-nucleoside analogues. Appropriate elabora-
tion of the H-bonding faces provides emissive analogues of U,
C, A, and G (Figure 1).6
a Reagents and conditions: (a) (i) Chloroformamidine hydrochloride,
DMSO2, 125 °C, 77%; (ii) dimethylformamide dimethyl acetal, DMF,
98%. (b) β-D-Ribofuranose 1-acetate 2,4,5-tribenzoate, SnCl4, MeNO2,
65 °C, 64%. (c) NH3/MeOH, 65 °C, 84%. (d) (i) Dimethylformamide
dimethyl acetal, DMF, 77%; (ii) DMTrCl, Py, 70%; (iii) TOMCl,
tBu2SnCl2, iPr2NEt, DCE, 13%. (iv) 2-Cyanoethyl N,N-diisopropyl-
chlorophosphoramidite, iPr2NEt, DCM, 0 °CꢀRT, 67%. (e) 2,4,6-
Triisopropylbenzenesulfonyl chloride, TEA, DMAP, CH2Cl2. (f) NH3/
MeOH, 70 °C, 37% over two steps.9,13
The preparation of two analogues is briefly discussed.7,8 The
synthesis of thGstarted with methyl 4-aminothiophene-3-carboxylate
hydrochloride (Scheme 1a). Treatment with chloroformamidine
Received: June 30, 2011
Published: August 25, 2011
r
2011 American Chemical Society
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Table 1. Structurala and Photophysicalb Data for Thieno[3,4-d]pyridmidine Nucleoside Analogues
rmsd (Å)a
sugar pucker
C30-endo
ribose
base
solvent
λabs (ε)
λem (Φ)
Φε
τ
Stokes shift
polarity sensitivityc
68.9
thA
thC
thG
thU
0.0521
0.157
water
341 (7.44)
345 (7.83)
320 (4.53)
326 (4.21)
321 (4.15)
333 (4.53)
304 (3.16)
304 (3.50)
420 (0.21)
411 (0.14)
429 (0.41)
422 (0.01)
453 (0.46)
424 (0.50)
409 (0.41)
378 (0.04)
1562
1096
1857
42
3.9
3.2
5950
5080
8300
7550
9580
6890
8860
6690
dioxane
water
C20-endo
C20-endo
C10-exo
0.294
0.0525
0.240
0.045
0.158
0.047
15.2
5.0
27.3
107.2
80.8
dioxane
water
1909
2265
1296
140
14.8
13.0
11.5
1.0
dioxane
water
dioxane
a Rmsd values were calculated by overlaying the X-ray crystal structures of the natural nucleosides (adenosine-ADENOS10, cytidine-CYTIDI10,
guanosine-GUANSH10 and uridine-BEURID10 from the CCDC) with the structures of the synthetic analogues reported here.9 For thC and thU, the
corresponding pyrimidine regions were overlaid. thGdmf was crystallized and used15 b λabs, λem, ε, τ, and Stokes shift are reported in nm, nm,
103 Mꢀ1 cmꢀ1, ns, and cmꢀ1, respectively. All photophysical values reflect the average of two independent measurements, and the standard errors of the
mean values are smaller than 1, 0.26, and 0.5 for Φ, Stokes shift, and τ, respectively. c Sensitivity to polarity, expressed in cmꢀ1/(kcal molꢀ1), is equal to
the slope of the linearization depicted in Figure 4c,d.
Figure 3. Comparison of the X-ray crystal structures of thA and
adenosine (ADENOS10 from the CCDC): (a) overlay of the ribose
rings (rmsd = 0.0521 Å); (b) overlay of the nucleobases (rmsd =
0.157 Å).
conformation partially resembling a C20-endo conformation
[Table 1; also see Tables S5 and S6 and Figures S4 and S6 in the
Supporting Information (SI)]. In contrast, the purine analogues
Figure 2. X-ray crystal structures of thieno[3,4-d]pyrimidine analo-
thG and thA exhibit C20-endo and C30-endo ribose pucker, res-
gues: (a) thA; (b) thC; (c) thGdmf; (d) thU.15
pectively, in the solid state, which are the predominant con-
formations adopted by the natural ribonucleosides (Table 1).9,14
Indeed, overlaying the crystal structure of thA with that of adenosine
hydrochloride in dimethylsulfone at 125 °C afforded 2-amino-
(Figure 3 and Figure S3) shows minimal root-mean-square
deviation (rmsd) of the sugar pucker (0.0521 Å). Similarly,
minimal distortion of the ribose conformation is seen for thG
(rmsd = 0.0525 Å; Figure S5).9,15
thieno[3,4-d]pyrimidin-4(3H)-one, which was protected as the
dimethylformamidine derivative.9 This protecting group facili-
tated the regioselective FriedelꢀCrafts C-glycosylation affording
the β-isomer only, an outcome which is similar to a Vorbr€uggen
glycosylation reaction.10,11 Crystal structure analysis confirmed
the assigned stereochemistry (see below).12 The corresponding
nucleoside thG was obtained by removing all of the protecting
groups in methanolic ammonia. For the preparation of thC, the
uridine analogue thU7 was treated with 2,4,6-triisopropylbenze-
nesulfonyl chloride, activating position O4 for nucleophilic dis-
placement (Scheme 1b). Treatment with saturated methanolic
ammonia afforded thC in 37% yield over two steps.13
Crystal structures of the modified ribonucleosides showed
that they all display an anti orientation at their glycosidic linkages
(Figure 2), similar to the preference seen with their natural
counterparts (Table 1). This is particularly notable with thU and
thC, which possess a fused thiophene ring at the 5,6-position of
the pyrimidine. This modification, however, does impact the
observed sugar pucker, which appears to adopt an intermediate
The fundamental spectroscopic properties of the modified
nucleosides thU, thC, thA, and thG are summarized in Table 1.9
The ground-state absorption spectra in aqueous solutions showed
long-wavelength maxima ranging from 304 nm (for thU) to
341 nm (for thA), all significantly red-shifted from those of the
parent native nucleosides (Figure 4a). Excitation at the long-
wavelength absorption maximum gave rise to visible emission
with maxima ranging from 409 nm (for thU) to 453 nm (for thG).9
Respectable to good quantum yields, ranging from 0.21 to
0.46 for thA and thG, respectively, were seen for all of the nucleo-
sides, resulting in noteworthy brightness values (Table 1). Time-
resolved experiments showed that the least-emissive nucleoside
analogue (thA) displays a relatively short excited-state lifetime
(3.9 ns), while the most emissive one (thC) has the longest
(15.2 ns) (Figure S10). Spectra taken in dioxane typically
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Figure 5. Thermal melts of thG-containing RNA. All samples contained
each oligo at 1.0 μM, 20 mM sodium cacodylate, 100 mM NaCl, and
0.5 mM EDTA (pH 7.0): (a) absorbance vs temperature denaturation
profiles; (b) comparison of Tm values for duplexes of thG (1a) and G
(1b) with their complementary and mismatched bases. Errors based on
two independent Tm measurements were less than 0.6 °C.
Figure 4. (a) Absorption (dashed lines) and emission (solid lines)
spectra of thA (green), thC (purple), thG (orange), and thU (blue) in
water. (b) Absorption (dashed lines) and emission (solid lines) spectra
of thU in water (blue) and dioxane (red) and mixtures thereof (10, 30,
and 70% v/v water in dioxane, black lines). Emission intensities were
corrected to reflect an optical density of 0.1 at λex. (c, d) Stokes shifts vs
ET(30) correlations for (c) purines thA (9) and thG (0) and (d)
pyrimidines thC (b) and thU (O). The orange lines represent linear fits.
Note: the data point for thA in dioxane [ET(30) = 36.0 kcal molꢀ1] was
excluded from the linear fit.9,18
quenching upon incorporation into oligonucleotides, which is a
result of diverse quenching pathways facilitated by neighboring
nucleobases. Of significance therefore is the observation that
oligonucleotide 1a, having the emissive thG “sandwiched”
between two potentially quenching G residues, still displayed
strong visible emission with a relative quantum yield of 0.10
(Figure S13).9,21
The structural, biophysical, and spectroscopic characteristics
of this set of emissive RNA nucleosides illustrate highly desirable
traits, including native WatsonꢀCrick faces, unparalleled struc-
tural isomorphicity with respect to native nucleosides, minimal
perturbation upon incorporation into duplexes, and intense
visible emission. In particular, we note that while numerous emissive
pyrimidine analogues have been reported in recent years,1,22ꢀ25
highly emissive and responsive isomorphic purines are rare, with
the classical 2-aminopurine, introduced in 1969,19a still being the
most prevalently used.26 Expanding the repertoire of isomorphic
purine analogues, as demonstrated here for the thieno[3,4-d]-
pyrimidine-based thA and thG nucleosides, is therefore significant
and is expected to advance the biophysical probing of RNA-
related processes.27
showed slightly bathochromically shifted absorption maxima and
hypsochromically shifted emission maxima (Table 1), suggest-
ing that these chromophores possess charge-transfer character
in their excited states.16 Sensitivity to environmental polarity,
assessed by determining the Stokes shifts and emission inten-
sities in water/dioxane mixtures, showed all of the nucleosides to
be highly responsive, albeit to different extents (Figure 4bꢀd and
Figures S7 and S8).17,18 For example, while the impact of solvent
polarity on the Stokes shifts of thC was modest, its impact on the
emission quantum yield was dramatic (Table 1). In contrast,
polarity substantially impacted the Stokes shifts displayed by thG,
while a minimal effect on the emission quantum yield was seen
(Table 1).
In a preliminary exploration of the impact of such isomorphic
nucleotides (particularly the incorporation of a modified
purine) on the stability of RNA duplexes, the 17-mer oligonu-
cleotide 1a containing thG at a central position was synthesized
using the thG phosphoramidite (Scheme 1a) and compared to
the unmodified strand 1b (Figure 5).9 Hybridization to the
complementary oligonucleotide 2a and all of the mismatched
strands 2bꢀd was followed by thermal denaturation experi-
ments. Replacing a G residue with thG yielded a modified
duplex 1a/2a that was slightly more stable than the native
unmodified one 1b/2a (ΔTm = +1.0 °C) (Figure 5). Impor-
tantly, an entirely analogous stability trend was seen for all of the
mismatched thG-containing duplexes 1a/2bꢀd relative to the
corresponding native hybrids 1b/2bꢀd (Figure 5b). This sug-
gests minimal perturbation of the resulting duplexes, corrobor-
ating the highly isomorphic character of the emissive purine
analogue.
’ ASSOCIATED CONTENT
S
Supporting Information. Synthetic details, X-ray crys-
b
tallographic data (CIF), photophysical data, thermal denatura-
tion measurements, and MALDIꢀTOF MS spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
ytor@ucsd.edu
’ ACKNOWLEDGMENT
We thank the National Institutes of Health for generous
support (GM 069773) and the National Science Foundation
for an instrumentation grant (CHE-0741968).
One drawback of many emissive nucleoside analogues, in-
cluding the classical 2-aminopurine,1,19,20 is their significant
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