Functionalized tricyclic cytosine analogues provide nucleoside fluorophores with improved photophysical properties and a range of solvent sensitivities

Article abstract of DOI:10.1002/chem.201303410

Tricyclic cytosines (tC and tC^O frameworks) have emerged as a unique class of fluorescent nucleobase analogues that minimally perturb the structure of B-form DNA and that are not quenched in duplex nucleic acids. Systematic derivatization of th

Full text of DOI:10.1002/chem.201303410

DOI: 10.1002/chem.201303410
Full Paper
&
Fluorescent Probes
Functionalized Tricyclic Cytosine Analogues Provide Nucleoside
Fluorophores with Improved Photophysical Properties and
a Range of Solvent Sensitivities
Brittney J. Rodgers,[b] Nada A. Elsharif,[b] Nisha Vashisht,[b] Macy M. Mingus,[b]
Mark A. Mulvahill,[b] Gudrun Stengel,[c] Robert D. Kuchta,[c] and Byron W. Purse*[a, b]
Abstract: Tricyclic cytosines (tC and tCO frameworks) have
emerged as a unique class of fluorescent nucleobase ana-
logues that minimally perturb the structure of B-form DNA
and that are not quenched in duplex nucleic acids. System-
atic derivatization of these frameworks is a likely approach
to improve on and diversify photophysical properties, but
has not so far been examined. Synthetic methods were re-
fined to improve on tolerance for electron-donating and
electron-withdrawing groups, resulting in a series of eight
new, fluorescent cytidine analogues. Photophysical studies
show that substitution of the framework results in a pattern
of effects largely consistent across tC and tCO and provides
nucleoside fluorophores that are brighter than either parent.
Moreover, a range of solvent sensitivities is observed, offer-
ing promise that this family of probes can be extended to
new applications that require reporting on the local environ-
ment.
Introduction
ed, however, by the large degree of structural perturbation,
which can prevent normal interactions with proteins of inter-
est. Moreover, the flexibility of the tether can lead to fluoro-
phore intercalation and can limit the precision of distance
measurements by FRET.[32,33] Another significant, unmet need is
for a way to label nucleic acids for fluorescence microscopy
with sufficiently small structural perturbation so that, for exam-
ple, uptake by competent bacteria can be tracked microscopi-
cally.
Fluorescent nucleoside analogues are used extensively as mo-
lecular probes for biophysical studies and in biotechnology.[1–6]
Especially for the former, there is a great need for new, im-
proved probes with a range of fluorescence properties and
minimal structural perturbation. The vast majority of available
probes either dangle freely from the major groove of the
duplex (e.g., Cy5-dC)[2,7] or are fluorescent nucleobase ana-
logues.[8–28] Most of the latter are either incompatible with
Watson–Crick hydrogen bonding (e.g., pyrene deoxyriboside)[1]
or are almost completely quenched when stacked against
neighboring bases (e.g., the popular, commercially available
(deoxy)ribosides of 2-aminopurine and pyrrolocytosine).[29,30]
The approach of using flexible tethers (as in the Cy5-dC exam-
ple) is appealing because of the predictable fluorescence prop-
erties and the possibility to maintain a useful degree of poly-
merase compatibility.[31] Applications of this approach are limit-
In the past decade, a very promising new class of analogues
has emerged, based on a tricyclic cytidine (tC) scaffold
(Figure 1).[34–42] Although originally developed as stabilizers for
double-stranded nucleic acids (substitution of one tC for C in
duplex oligonucleotides raises the Tm by an average of 38C),[34]
tC and tCO were later found to be unique in that they retain
their fluorescence (Fem ꢀ0.2) in duplex nucleic acids, varying
little with the identity of neighboring bases.[37] The photophy-
sics of these compounds, as free nucleosides and in single
[a] Prof. B. W. Purse
Department of Chemistry and Biochemistry
San Diego State University, 5500 Campanile Dr.
San Diego, CA 92182-1030 (USA)
E-mail: bpurse@mail.sdsu.edu
[b] B. J. Rodgers, N. A. Elsharif, N. Vashisht, M. M. Mingus, M. A. Mulvahill,
Prof. B. W. Purse
Department of Chemistry and Biochemistry, University of Denver
2199 S. University Blvd., Denver, CO 80208 (USA)
[c] Dr. G. Stengel, Prof. R. D. Kuchta
Department of Chemistry and Biochemistry, University of Colorado
596 UCB, Boulder, CO 80303 (USA)
Figure 1. Structure of tricyclic 2’-deoxycytidine analogues tC and tCO base-
pairing with G. Modifications to the canonical structure of 2’-deoxycytidine
are shown in gray.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201303410.
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stranded (ss) and double stranded (ds) DNA are well character-
ized.[42] We have shown that both compounds are remarkably
good substrates for A- and B-family DNA polymerases and T7
RNA polymerase, even to the point of being inserted faster
than dCTP during DNA synthesis both by human Pol a and
Klenow fragment, albeit with approximately 10% misincorpo-
ration of dtC(O)TP across from A (fidelity of RNA synthesis with
T7 RNA polymerase is greater).[43,44] More recently, Wilhelmsson
et al. have developed a nitro-tCO analogue that is a non-emis-
sive FRET acceptor and shown that it can be used with tCO as
a donor for internucleobase FRET.[41] The G clamp molecules
are also members of the tC family.[45,46]
Results and Discussion
Synthesis of fluorescent nucleoside analogues
Synthetic methods for the parent compounds 1a (tC) and 2a
(tCO) are established, but the substituent effects of the EDGs
and EWGs necessitated reoptimization to improve yields and
functional group tolerance. In our hands, the original route to
tCO worked poorly or not at all when applied to the synthesis
of compounds 1b–g, but our modified route provided ade-
quate yields.[34] Moreover, our reoptimization of the route to tC
substantially improves the overall yield (from 10–15% to 43%
for the conversion of 10a to 2a), facilitating the synthesis of
2b,c.[47]
The original synthesis of tCO (1a) entails the activation of
the 4-OH tautomer of 3’,5’-di-O-acetyl-5-bromo-2’-deoxyuridine
as a mesitylsulfonyl ester, formation of a secondary amine by
SNAr of this mesylate, removal of the acetate protective
groups, and ring closure by SNAr of the aryl bromide, promot-
ed by KF in ethanol (Figure 3).[34] Two problems limited the ap-
plication of this route to derivative synthesis. First, chloro and
methoxy groups meta to N of 2-aminophenols attenuate the
nucleophilicity, which has a detrimental effect on secondary
amine formation. Second, chloro and methoxy groups meta to
O attenuate the SNAr reactivity with the aryl bromide, inhibit-
ing ring closure.
Although the three nucleobases of the tC family have
unique properties, they are also significantly limited in that the
emissive compounds (tC and tCO) have nearly overlapping
spectra and they are around 50-fold less bright than fluores-
cein. This brightness is inadequate for use in most single-mole-
cule fluorescence experiments or for labeling nucleic acids for
fluorescence microscopy. Nitro-tCO does not emit. Because it is
difficult to predict, a priori, the photophysical properties of
a molecule based on structure, we set out to design, synthe-
size, and characterize a new set of tC(O) analogues incorporat-
ing electron-donating and -withdrawing groups at varied posi-
tions and with further extensions to conjugation (Figure 2).
Figure 2. Tri- and tetracyclic cytidine analogues synthesized for this study.
Figure 3. Synthesis of tCO analogues. Yields: a) 85%, b) over two steps, 6b
40%, 6c 48%, 6d 41%, 6e 20%, 6 f 61%, 6g 44%, c) 1b 20%, 6c 11 %, 1d
24%, 1e 3%, 1 f 11 %, 1g 53%. The parent tCO 1a was synthesized using
the original procedure.[34]
The results show clear patterns in the substituent effects,
changes to Fem and lem, and reveal a range of solvent sensitiv-
ities. (7-Cl)tCO 1e is now the brightest known member of the
tC(O) family in aqueous solution, and the strong solvent-sensi-
tivity of the methoxy derivatives 1b, 1c, 2b, and 2c is expect-
ed to make them useful as a new set of minimally perturbing
and environmentally responsive fluorescence probes.
An improved synthesis begins with the same 3’,5’-di-O-
acetyl-5-bromo-2’-deoxyuridine as starting material, but acti-
vates the O4 using Appel chemistry (PPh3, CCl4) for the conver-
sion to a 4-Cl (Figure 3).[48] The 4-Cl intermediate was used in
situ for SNAr with substituted 2-aminophenols in the presence
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of DBU, resulting in good yields of the corresponding secon-
dary amines for all derivatives tested. Removal of the acetyl
protective groups is required for ring closure, as previously re-
ported by Matteucci,[34] likely owing to the intermediacy of
a 5’,6-ether, itself resulting from reversible 1,4-addition of the
5’-OH to the uracil moiety. The attenuated reactivity of several
of the targeted derivatives necessitated more forceful condi-
tions for this step and the removal of competing nucleophiles.
Nucleophilic deamination of the secondary amine (either by
solvent or H2O) is likely the main competing side reaction. The
efficacy of ring closure could be improved significantly by
switching from KF to CsF and changing solvents to N-methyl-
pyrrolidinone, but the hygroscopicity of CsF made it difficult to
handle without introducing water into the reaction mixture.
Fortunately, a further change to KF, activated by [18]crown-6,
in anhydrous diglyme at 1208C proved to offer a significant
improvement, resulting in substantially improved yields of all
derivatives in a reaction time of approximately 20 min. The for-
mation of what are likely deaminated side products (as evi-
readily available benzothiazole precursors, a reaction previous-
ly carried out using concentrated hydroxide. We found this
procedure to be unreliable for methoxy starting materials 7b,c
and a change to hydrazine provided significant improvement.
Because of the tendency of the resulting 2-amino-(7/8)-me-
thoxythiophenols to undergo oxidative degradation, we per-
formed a controlled, in situ oxidation to the disulfides 8b,c
using hydrogen peroxide, giving a product that could be puri-
fied chromatographically. A one-pot reduction of this disulfide
using triethylphosphine and nucleophilic substitution with 5-
bromouracil gave thioether compounds 9b,c. The methoxy
group meta to N attenuates its nucleophilicity, but condensa-
tion conditions more forcing than those of the original proce-
dure nonetheless produced tricyclic nucleobases 10b,c. The
original ribosylation procedure uses the sodium salt of the nu-
cleobase (from NaH) in a reaction with 3,5-di-O-(p-toluoyl)-2’-
deoxyribofuranosyl chloride (Hoffer’s chlorosugar)[49] and yields
only 10–15%, but we found that substantially better yields of
the 2’-deoxyriboside could be obtained by instead activating
the nucleobases 10a–c as the TMS ether using BSA, followed
by ribosylation in situ (Vorbrꢁggen’s Silyl–Hilbert–Johnson
method).[50,51] Yields were typically around 80% and consisted
of an approximately 1:1 mixture of anomers. The pure b-anom-
ers were separable in around 40% yield from 10. Removal of
the toluoyl groups using methoxide completed the synthesis
of the fluorescent nucleoside analogues 2. Yields were unfortu-
nately significantly lower for compound 2c because of an ap-
parent high sensitivity to oxidative degradation of 8c and 9c
and preferential formation of the a-anomer during 2’-deoxyri-
bosylation.
1
denced by H NMR spectroscopy) was greatly minimized under
these conditions.
The very different reactivity of aryl alcohols and aryl thiols
necessitates a different route to X=S (tC) derivatives (Figure 1
and Figure 4). Matteucci’s and Wilhelmsson’s syntheses of tC
Photophysical measurements
In order to compare the effects of substituents and to deter-
mine how the new analogues compare with tC and tCO, we
carried out photophysical measurements in 1ꢂPBS buffer, sol-
vent mixtures, and 1,4-dioxane (Table 1 and Figure 5). The re-
sults show substituent effects that are consistent across the tC
and tCO derivatives. (7-Cl)tCO (1e) is now the brightest known
member of this family in an aqueous environment, and the sol-
vent effects point to highly desirable properties for biophysical
probe development.
In all cases, substitution of the tC and tCO frameworks had
little effect on labs but a significant influence of lem. The addi-
tion of methoxy groups to either the 7- or 8-position of tCO
red-shifted lem by 27 and 26 nm, respectively [(7-MeO)tCO and
(8-MeO)tCO] in buffer. The position of this substituent had
a stronger effect on lem of the tC framework, with red-shifts of
35 nm for (8-MeO)tC and only 5 nm for (7-MeO)tC. In 1,4-diox-
ane, the pattern of the effects is the same with the overall
magnitude of the influence diminished. Although the influence
of methoxy substitution is consistent for tC and tCO, the differ-
ences are largest at the 8-position, which is perhaps not sur-
prising because this position is in direct resonance conjugation
with the O or S atom.
Figure 4. Synthesis of tC analogues. Yields: a) 8b>98%, 8c 60%, b) 9b
86%, 9c 24%, c) 10b 86%, 10c 27%, d) 2a 43%, 2b 39%, 2c 8% over two
steps.
2a begins with 2-aminothiophenol,[34,47] but derivatives of this
compound have very limited commercial availability and are
highly sensitive to oxidative degradation. 4- and 5-Methoxy-2-
aminothiophenol are accessible through ring opening of more
Substitution of the tCO framework with chloro groups [(7-
Cl)tCO and (8-Cl)tCO, 1e and 1d, respectively] resulted in
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Because the substituents affected lem much more than labs
,
the Stokes shift is strongly correlated to lem (r=0.98). As
a result of its significantly red-shifted lem (8-MeO)tC 2b has the
greatest Stokes shift of the tC family in water: 159 nm. Tetracy-
clic analogue 1g has the smallest Stokes shift of the series of
compounds in buffer: 73 nm.
Using mixtures of 1ꢂPBS buffer and 1,4-dioxane, we mea-
sured the effect of solvent polarity on Stokes shift, calibrated
to the ET(30) scale of Reichardt’s dye (Table 1 and the Support-
ing Information).[18] The results show that substituents impart
significant differences to polarity sensitivity. Substitution gener-
ally decreases the polarity sensitivity with the tCO derivatives,
whereas it increases polarity sensitivity of the tC compounds.
(8-Cl)tCO is now the least solvent-sensitive known member of
the tC family, both in terms of this Stokes shift measurement
and Fem. In contrast, (8-MeO)tC has the most solvent sensitive
photophysical properties.
The introduction of substituents, including extending the
conjugated framework, in general causes a decrease in the ob-
served absorptivity of the compounds. An exception to this
trend is seen with the chloro substituents in 1,4-dioxane. The
decrease is greatest for the methoxy groups when the func-
tionality is in the 7-position, but in buffer the smallest absorp-
tivity is seen when a chloro group is in the 8-position. In con-
trast, the absorptivity of the methoxy analogues is relatively in-
sensitive to solvent polarity.
Although tC and tCO are known to maintain fluorescence in
duplex oligonucleotides (tC becomes slightly brighter and tCO
slightly less bright), these analogues show variance in quantum
yield depending on the solvent. tC and tCO were found to
have a threefold and 1.5-fold increase in quantum yield, re-
spectively, when the change was made from buffer to 1,4-diox-
ane. Similar increases in Fem on going to nonhydrogen bond-
ing solvents have been reported previously for tC.[52] The pres-
ence of the chloro substituent, in either position on tCO, atte-
nuated solvent effects on quantum yield. The opposite is true
when the substituents are methoxy groups. Although methoxy
substituents attenuated the solvent sensitivity of e, Fem for the
analogues with methoxy groups is much more sensitive to sol-
vent polarity than that for the corresponding parent com-
pounds, with the greatest difference observed for 8-MeO-tC or
-tCO (1b and 2b). The analogues with methoxy groups have
a relatively high Fem in 1,4-dioxane, but much less so in buffer.
Notably, (8-MeO)tC increases in fluorescence intensity by
almost 30-fold on going from buffer to dioxane. The presence
of a fourth aromatic ring (tetCO) shows solvent sensitivity for
Fem comparable to that of parent tCO. In dioxane, (8-Cl)tCO
shows a greater quantum yield of emission than tCO. In buffer,
tetCO, (7-Cl)tCO, and (8-Cl)tCO, all have greater Fem than the tCO
parent.
Figure 5. Spectroscopic properties of new analogues (Figure 2) and compari-
son with tC and tCO as measured in 1ꢂPBS buffer.
a slight blue-shift of lem by 4–5 nm in aqueous buffer, but in
1,4-dioxane, lem for (7-Cl)tCO was red-shifted by 4 nm and lem
for (8-Cl)tCO was blue-shifted by 25 nm. Fluoro substitution at
the 7-position of tCO [(7-F)tCO] gives a 10 nm red-shift of lem in
dioxane but nearly matches the emission of the parent in
buffer. Tetracyclic compound tetCO 1g has lem blue-shifted by
4 nm in dioxane and 26 nm in buffer with respect to the
parent tCO.
As a result of the combined effects of substitution on ab-
sorptivity and quantum yield, both chloro analogues (7-Cl)tCO
and (8-Cl)tCO are brighter than the parent tCO. (7-Cl)tCO is now
the brightest known member of the tC family in buffer, more
than 40% brighter than tCO. In 1,4-dioxane, the chloro ana-
logues (8-Cl)tCO and (7-Cl)tCO are also the brightest known flu-
orophores of this family. The least bright analogues in each
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growing toolkit of fluorescent
probes for the study of nucleic
acid structure, recognition, and
metabolism.
Table 1. Photophysical properties of analogues 1–2 dissolved in 1ꢂPBS buffer and in 1,4-dioxane.
Compound
Solvent
lmax,abs lmax,em Stokes shift
e
Fem Brightness Polarity
[nm]
[nm]
[nm]
[LmolÀ1 cmÀ1
]
(e·Fem
)
sensitivity[a]
1a tCO
dioxane 357
442
460
449
486
449
487
417
455
446
456
452
462
438
434
472
503
490
538
477
508
85
105
89
125
91
127
51
94
83
99
86
102
76
7400
6800
4800
4000
2700
3000
8500
5600
8700
6700
2700
1800
6700
5300
4300
4700
3800
3800
2200
2000
0.46 3400
0.24 1600
0.38 1800
6
1ꢂPBS 355
1b (8-MeO)tCO dioxane 360
1ꢂPBS 361
dioxane 358
1ꢂPBS 360
dioxane 366
1ꢂPBS 361
dioxane 363
1ꢂPBS 357
dioxane 366
1ꢂPBS 360
dioxane 362
1ꢂPBS 361
dioxane 369
1ꢂPBS 377
dioxane 372
1ꢂPBS 379
dioxane 372
1ꢂPBS 379
5
2
2
4
5
7
3
7
7
Experimental Section
0.05
0.37 1000
0.05 150
200
1c (7-MeO)tCO
1d (8-Cl)tCO
1e (7-Cl)tCO
1 f (7-F)tCO
1g tetCO
Details of the synthesis and charac-
terization of the new compounds,
in addition to modified procedures
for the synthesis of tC 2a and tCO
1a, are provided in the Supporting
Information. Photophysical mea-
surements were performed at least
in triplicate using samples dis-
solved in 1ꢂPBS buffer, 1,4-diox-
ane (spectrophotometric grade), or
mixtures thereof. Absorption spec-
tra were recorded using a Varian
Cary 100 Bio UV–visible spectro-
0.49 4200
0.35 2000
0.46 4000
0.35 2300
0.41 1100
0.17
300
0.43 2900
0.27 1400
0.34 1500
73
2a tC
103
126
118
159
105
129
0.09
400
2b (8-MeO)tC
2c (7-MeO)tC
0.28 1100
0.01
0.27
0.05
40
600
100
photometer
and
fluorescence
spectra were recorded using
a Varian Cary Eclipse fluorescence
spectrophotometer. Fluorescence
[a] Expressed as the slope of Stokes shift plotted against solvent polarity as measured using the Dimroth–
Reichardt ET(30) scale. Units are 101 cmÀ1/[kcalmolÀ1].
spectra were corrected using pub-
lished standard curves for the
commercially available fluoro-
family are those which have the methoxy group in the 8-posi-
tion [(8-MeO)tC and (8-MeO)tCO], but these analogues are ex-
citing prospective molecular probes because of the large in-
crease in brightness on going to a less-polar environment. This
property should be useful for probing molecular recognition of
nucleic acids and may also be useful for reporting on the envi-
ronmental changes during hybridization of oligonucleotides to
form a duplex.
phores 1,1,4,4-tetraphenyl-1,3-butadiene, Coumarin 153, and 4-(di-
cyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran.[53]
Absorption and emission spectra for all compounds 1 and 2 are in-
cluded in the Supporting Information. Quantum yields were deter-
mined using the comparative method of Williams et al. and a solu-
tion of quinine sulfate dissolved in 0.1m H2SO4 as a reference with
an accepted Fem =0.54.[54] All quantum yield measurements were
performed with tC 2a as a second standard to ensure the accuracy
of comparisons. Example plots for quantum yield measurements
are shown in the Supporting Information. Polarity sensitivity was
measured as the slope of a line defined by plotting Stokes shift
against solvent polarity from the Dimroth–Reichardt ET(30) scale.
The accuracy of this determination was in some cases limited by
changes to the vibrational fine structure that occurred when
changing solvents, sometimes manifest in abrupt changes to
lmax,em (all relevant spectra are shown in the Supporting Informa-
tion).
Conclusion
Although the chemical literature reports many examples of nu-
cleobase-derived fluorophores, there is a dearth of data for
substituent effects on the photophysical properties of the
most important of these probes. In this work, we have de-
signed and synthesized a new set of analogues of the tC
family using synthetic procedures reoptimized for improved
yields and access to greater structural diversity. Photophysical
characterization of the analogues in buffer, 1,4-dioxane, and
mixtures thereof reveals that substituents have similar effects
whether attached to tC or tCO. Generally, chloro compounds
gave brighter fluorescence and methoxy compounds gave dra-
matically increased solvent sensitivity. Because the patterns of
effects hold across tC and tCO, future derivatives can be
screened using whichever framework offers easier synthetic
access, and then the most desired substituents can be added
to the other framework, if desired, with predictable effect.
The results of this work encourage a full study of the com-
patibility of the new probes with DNA- and RNA-processing
and polymerizing enzymes and a full photophysical characteri-
zation in single-stranded and duplex oligonucleotides. We an-
ticipate that they will make highly valuable additions to the
Acknowledgements
The authors gratefully acknowledge the support of the NIH for
this research (grant GM093943 to B.W.P. and AI59764 to R.D.K.).
Keywords: DNA · fluorescent probes · nucleosides · RNA ·
structure–activity relationships
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Received: August 29, 2013
Published online on December 5, 2013
Chem. Eur. J. 2014, 20, 2010 – 2015
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