Enzymatic Incorporation of a Coumarin–Guanine Base Pair

Article abstract of DOI:10.1002/anie.201910059

Previous expansions beyond nature's preferred base-pairing interactions have utilized either nonpolar shape-fitting interactions or classical hydrogen bonding. Reported here is a hybrid of these systems. By replacing a single N?H with C?H at a Watson–Crick interface, the design space for new drug candidates and fluorescent nucleobase analogues is dramatically expanded, as demonstrated here by the new, highly fluorescent deoxycytidine mimic 3-glycosyl-5-fluoro-7-methoxy-coumarin-2′-deoxyribose (dC^C). dGTP is selectively incorporated across from a template dC^C during enzymatic DNA synthesis. Likewise, dC^C is selectively incorporated across from a template guanine when dC^C is provided as the triphosphate dC^CTP. DNA polymerase I (Klenow fragment) exhibited about a 10-fold higher affinity for dC^CTP than dCTP, allowing selective incorporation of dC^C in direct competition experiments. These results demonstrate that a single C?H can replace N?H at a Watson–Crick-type interface with preservation of functional selectivity and enhanced activity.

Full text of DOI:10.1002/anie.201910059

Angewandte
Communications
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International Edition: DOI: 10.1002/anie.201910059
German Edition: DOI: 10.1002/ange.201910059
Nucleosides
Enzymatic Incorporation of a Coumarin-Guanine Base Pair
Aaron Johnson, Ashkan Karimi, and Nathan W. Luedtke*
Abstract: Previous expansions beyond natureꢀs preferred
base-pairing interactions have utilized either nonpolar shape-
fitting interactions or classical hydrogen bonding. Reported
rated across from deoxyadenosine (dA) and vice versa in
primer extension reactions.[9] A subsequent X-ray structure
revealed a nearly coplanar orientation of the F–dA base pair
in duplex DNA,[8d] yet, according to thermal denaturation
experiments, F–dA base pairs provided little or no thermal
stabilization of duplex DNA.[8b] Computational studies con-
cluded that F–dA pairing in the active site of DNA
polymerase was due to shape complementary between F
and dA, and that any hydrogen-bonding interactions were
very weak due to the low polarity of F.[9g]
À
here is a hybrid of these systems. By replacing a single N H
À
with C H at a Watson–Crick interface, the design space for
new drug candidates and fluorescent nucleobase analogues is
dramatically expanded, as demonstrated here by the new,
highly fluorescent deoxycytidine mimic 3-glycosyl-5-fluoro-7-
methoxy-coumarin-2’-deoxyribose (dCC). dGTP is selectively
incorporated across from a template dCC during enzymatic
DNA synthesis. Likewise, dCC is selectively incorporated
across from a template guanine when dCC is provided as the
triphosphate dCCTP. DNA polymerase I (Klenow fragment)
exhibited about a 10-fold higher affinity for dCCTP than dCTP,
allowing selective incorporation of dCC in direct competition
Here we report the trisubstituted coumarin dCC (Fig-
ure 1a), which mimics deoxycytidine (dC) during enzymatic
synthesis of DNA. A multidentate interface containing
À
experiments. These results demonstrate that a single C H can
À
replace N H at a Watson–Crick-type interface with preserva-
tion of functional selectivity and enhanced activity.
P
airing beyond A–T and G–C gives fundamental insights
into molecular recognition while providing tools for biotech-
nology and chemical biology.[1] Benner et al. have produced
an artificially expanded genetic information system (AEGIS)
based on classical, multidentate hydrogen bonding.[2] Tricyclic
nucleobase pair scaffolds developed by Matsuda and co-
workers expanded such logical designs to comprise four
hydrogen bonds.[3] In contrast, Romesberg et al. synthesized
large libraries of nonpolar and non-isosteric base-pairing
candidates and screened them for specific and orthogonal
pairing behavior during DNA synthesis.[4] Optimized “hits”
from such screens have been applied in synthetic biology for
creating bacteria with a six letter genetic alphabet,[4c] aptam-
ers,[5] and expression platforms for producing RNAs and
proteins containing non-natural residues.[4d]
Figure 1. a) Proposed dCC–dG base pair and coumarin numbering.
b) Optimized structure of CC–G in the gas phase according to B3LYP/
6–31+G(d).[10] c) Electrostatic potential maps on van der Waals
surfaces of CC, C, F, and T bases containing methyl groups rather than
deoxyribose (dR). Scale: À50 to +25 kcalmolÀ1; calculated using
B3LYP/6–31+G(d) in water. See Table S1 for dipole moments and
partial atomic charges.
Base-pairing systems containing a combination of classical
and nonclassical hydrogen-bonding interactions have not
been previously reported. If successful, this approach would
loosen the design restrictions for producing new, functional
nucleosides as drug candidates,[6] as well as highly fluorescent
nucleobase analogues.[7] The potential for a CH group to act
as a nonclassical hydrogen-bond donor in a Watson–Crick
interface was previously evaluated in the context of the
nonpolar thymidine isostere difluorotolyl deoxyribose F.[8]
Kool et al. demonstrated that F could be selectively incorpo-
classical and nonclassical hydrogen bonds in dCC–dG pairs
is supported by density-functional theory (DFT) calculations
(Figure 1b), as well as structure–activity relationships in the
enzymatic incorporation and elongation efficiencies of 10
different purine dNTPs across from dCC. Only dGTP itself
and highly related analogues (7-deaza-dGTP, 6-thio-dGTP
and dITP) were efficient substrates for incorporation,
whereas all dNTPs lacking a hydrogen bond acceptor at C6
were rejected as substrates. Together these results support the
presence of a functional, multidentate pairing interaction
between dCC and dG in the active site of a high-fidelity DNA
polymerase.
[*] Dr. A. Johnson, A. Karimi, Prof. Dr. N. W. Luedtke
Department of Chemistry, University of Zurich
Winterthurerstrasse 190, 8057 Zurich (Switzerland)
E-mail: nathan.luedtke@chem.uzh.ch
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201910059.
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The design of dCC was based on previous studies showing
that substituents at the 4- and 5-positions of pyrimidines are
well accommodated in the major groove of duplex DNA and
are compatible with the enzymatic synthesis of DNA and
RNA.[11] The 3-position of coumarin was used as the attach-
ment point to deoxyribose to provide a Watson–Crick face
mimic of cytosine.[12] DFT calculations confirmed that proper
hydrogen-bonding geometries and distances could be ach-
ieved by multidentate pairing of dCC–dG (Figure 1b). To
increase the polarity of the coumarin scaffold while endowing
favorable fluorescence properties,[13] methoxy was placed at
the 7-position and fluorine at the 5-position. This design was
evaluated using DFT calculations (Figure 1c; see Table S1 in
the Supporting Information). Addition of the 7-methoxy
group increased the dipole moment of the coumarin scaffold
by 1.8 Debye, giving a value similar to that of cytosine itself
(see Table S1). The base stacking propensity of the hetero-
nucleoside dCC as a single diastereomer in 10% yield over
the three steps. A b configuration of the anomeric center was
confirmed by NOESY NMR spectroscopy. Consistent with
other reports of 7-substituted coumarins,[13,15] dCC is an
intensively bright fluorophore with a quantum yield of F =
0.71 and molar extinction coefficient e = 15,200 cmÀ1mÀ1 in
water (see Figure S1). The product of these terms
(10800 cmÀ1mÀ1) makes dCC one of the brightest fluorescent
nucleobase analogues reported to date.[7c] Consistent with the
presence of base-stacking interactions and excited-state
electron transfer, quenching of dCC was observed in both
single-stranded (brightness = 79–1300 cmÀ1mÀ1) and double-
stranded DNA (brightness = 59–301 cmÀ1mÀ1; see Table S2).
To evaluate the coding selectivity of dCC when located in
a template strand, the dCC phosphoramidite 6 (Scheme 1) was
synthesized and incorporated into oligonucleotides using
automated DNA synthesis. The dCC-containing oligonucleo-
tides were purified using HPLC and annealed to comple-
mentary primers to give a primer-template duplex containing
an 11 nucleotide 5’-overhang (Figure 2). Primer extension
cycle,[8c] as well as the partial atomic charge (NBO) of C H at
À
the 8-position (see Table S1) were enhanced by including
a fluorine at the 5-position. The final electrostatic potential
map of dCC revealed a similar pattern along the Watson–
Crick face as compared to dC (Figure 1c), and furthermore
highlighted the large differences in polarity between dCC and
F.
The bromo-fluoro-methoxycoumarin 1 was synthesized
according to the steps in Scheme S1. Cross-coupling of 1 with
the glycal 2 by a Heck reaction proceeded in a diastereose-
lective fashion (Scheme 1).[14] The resulting silyl enol ether 3
was deprotected to afford the ketone 4. Finally, diastereose-
lective reduction and purification delivered the new C-
Figure 2. a) PEX reactions containing dGTP, dATP, dCTP, and dTTP at
various time points (min.). b) Reactions containing only dATP, dCTP,
and dTTP. All reactions contained 100 nm template + primer, 2 mm of
each dNTP, and 50 nm DNA polymerase (Klenow fragment). Aliquots
were removed at various time points and quenched with 10m urea
and resolved using 20% DPAGE. FAM=fluorescein, M=molecular
weight markers. For comparison with deoxycytidine see Figure S3.
(PEX) reactions were conducted in the presence of dGTP,
dATP, dCTP, and dTTP (Figure 2a). Alternatively, a mixture
of dATP, dCTP, and dTTP, lacking dGTP was used (Fig-
ure 2b). Full-length products [see Figures S2 and S3;
MALDI-TOF-MS (calc. for
C282H349N91O170P26 = 8534;
observed 8539)] were only observed in reactions containing
all four dNTPs, whereas stalling of primer extension across
from dCC was observed in reactions lacking dGTP (Fig-
ure 2b).
To evaluate the thermal stabilities (Tm) of duplexes
containing dCC, synthetic oligonucleotides containing dCC
were annealed to complementary sequences containing
a variable residue (dA, dT, dG, dC, or an abasic site) across
from dCC (see Table S3). While duplexes containing dCC were
less thermally stable (ÀDTm = 2.5–6.28C) than duplexes
containing only canonical base pairs, they were more stable
than duplexes containing an abasic site (ÀDTm = 6.4–8.08C).
When a purine residue was located across from dCC, losses in
thermal stability (ÀDTm) were in the range of 2.5–3.58C.
Larger losses in thermal stability (ÀDTm = 4.2–6.28C) were
observed when a pyrimidine residue was in the opposite
Scheme 1. Synthesis of the dCC nucleoside and phoshoramidite.
Reagents and conditions: a) palladium(II) acetate, tri(o-tolyl)phos-
phine, triethylamine, THF, 668C, 16 h; b) hydrogen fluoride pyridine,
THF, 08C to 268C, 1 h; c) sodium triacetoxyborohydride, acetic acid,
acetonitrile, 08C to 278C, 2.5 h, 10% over 3 steps; d) 4,4’-dimethoxy-
trityl chloride, pyridine, 268C, 20 h, 33%; e) 2-cyanoethyl N,N-diisopro-
pylchlorophosphoramidite, N,N-diisopropylethylamine, DCM, 08C to
268C, 1 h, 89%. DCM=dichloromethane, DMT=4,4’-dimethoxytrityl,
TBS=tert-butyldimethylsilyl, THF=tetrahydrofuran.
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strand. These results are consistent with dCC behaving like
a pyrimidine residue in terms of its impact on Tm values, since
pyrimidine–pyrimidine mismatches are among the most
destabilizing mismatches in duplex DNA.[16] According to
our Tm data, dCC was unable to discriminate between dA and
dG in the opposite strand. However, the variable contribu-
tions made by enthalpy and entropy can result in Tm values
that do not necessarily reflect energetic trends at lower
temperatures.[17]
Figure 4. PEX reactions with variable dNTPs annotated in Figure 3 and
To characterize the base-pairing selectivity of dCC under
physiological conditions with respect to temperature (378C)
and salt, we conducted primer extension reactions using DNA
polymerase I (Klenow fragment) and 10 different, non-
canonical nucleotide triphosphates (Figure 3). Among these,
conducted as in Figure 2 after only 5 minute reaction times. See
Figure S5 for additional replicates.
Figure 5. PEX reactions of four different duplexes in the presence of
dCCTP. Each reaction contained 20 nm template + primer, 2 nm of
DNA polymerase (Klenow fragment) and 400 nm of dCCTP. Aliquots
were quenched with 10m urea and analyzed using 18% DPAG at
608C.
(Figure 5). Primer elongation was most efficient for X = dG.
The product contained dCC according to MALDI-TOF-MS
(calc. for C189H225F1N52O110P16 = 5,497; observed 5,499). Initial
velocities (Vo) measured under conditions of saturating
dCCTP and saturating DNA template were 3- to 15-fold
higher for X = dG (Vo = 1.5 Æ 0.2 nm minÀ1 per nm enzyme) as
compared to X = dA, dT or dC (Vo = 0.1–0.5 nm minÀ1 per nm
enzyme; see Figure S5a). In the case of X = dG, the apparent
Km for dCCTP was unexpectedly in the range of 40–400 nm
(see Figure S5b), reflecting an approximate 10-fold higher
affinity of the enzyme for dCCTP than dCTP itself (see
Figure S5c). Indeed, in direct competition PEX reactions
containing a 1:1 ratio of both dCCTP and dCTP, only dCC
could be detected in the product (see Figure S6). The
resulting dCC nucleotide at the 5’-end of the primer was
competent for further elongation, according to PEX reactions
conducted using a template with a five-nucleotide 5’-overhang
(X = GTTTT3’). The primer was extended to the expected
full-length product in the presence of both dCCTP and dATP
(Figure 6a), whereas misincorporation of dA into the primer
in the absence of dCCTP resulted in chain termination
(Figure 6b).
Figure 3. Nucleobase structures of dNTPs used in PEX reactions.
7-deaza-dGTP and 6-thio-dGTP (B, C) were chosen because
of their high structural similarity with dGTP. Triphosphates
with perturbed Watson–Crick faces were also examined,
including dITP (D), which is considered to be a “universal”
base.[18] 2-Aminopurine (E) usually pairs with thymine but
can occasionally pair with cytosine.[19] 2,6-Diaminopurine (F)
exhibits selectivity for thymine but also causes “knock on”
effects in duplex DNA.[20] Finally, 8-substituted derivatives
containing a syn glycosidic bond were assessed (H–K). Primer
extension reactions were conducted with one dNTP (A–K) in
the presence of dATP, dCTP, and dTTP. Only dGTP itself and
highly related analogues (7-deaza-dGTP, 6-thio-dGTP and
dITP) were efficient substrates, giving full-length products
(Figure 4). Notably, all four of these dNTPs contain a (thio)-
carbonyl group at the 6-position, an NH at the 1-position, and
an anti glycosidic bond. Together these results suggest a highly
specific, multidentate interaction in dCC–dG base pairing
under physiological conditions, consistent with a nonclassical,
In summary, dCC is the first example of a nucleobase
analogue containing both polar and nonpolar hydrogen
bonding in
a multidentate array. DNA polymerase I
(Klenow fragment) exhibited about a 10-fold higher affinity
for dCCTP than dCTP, allowing the selective incorporation of
dCC across from dG in direct competition experiments with
À
dCTP. These results demonstrated that a single C H can
À
replace N H in a Watson–Crick-type interface with partial
À
=
C H···O C hydrogen bond in the active site of the poly-
merase.
preservation of functional specificity and even some
enhanced activities. In some ways, dCC was less functional
than native dC (ex. kcat, processivity, base pairing fidelity),
and in some ways, dCC was more functional than native dC
(ex. polymerase affinity, fluorescence properties). The altered
functionality of dCC suggests possible applications of this
The base-pairing selectivity of dCC was further evaluated
using dCC triphosphate (dCCTP, for synthesis see Scheme S2).
PEX reactions using four different templates containing
a single nucleotide 5’-overhang “X” = dA, dT, dG, or dC
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dCC–dG base pairing interactions exhibited the combined
properties of standard Watson–Crick base pairing and non-
polar interactions that are present, for example, in difluor-
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in duplex DNA, selective in primer extension reactions, and
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Acknowledgements
We gratefully acknowledge the Swiss National Science
Foundation for generous financial support (grant 165949).
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The authors declare no conflict of interest.
Keywords: DNA · fluorescent probe · hydrogen bonding ·
noncovalent interactions · nucleosides
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Bassolino, C. Nancoz, Z. Thiel, E. Bois, E. Vauthey, P. Rivera-
Fuentes, Chem. Sci. 2018, 9, 387 – 391.
Manuscript received: August 7, 2019
Accepted manuscript online: September 4, 2019
Version of record online: && &&, &&&&
Angew. Chem. Int. Ed. 2019, 58, 1 – 6
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
À
Nucleosides
Replacements: A single C H can replace
À
N H at a Watson–Crick-type interface
A. Johnson, A. Karimi,
N. W. Luedtke*
with preservation of functional selectivity
and enhanced activity. As demonstrated
here, dGTP is selectively incorporated
across from the new, highly fluorescent
deoxycytidine mimic 3-gylcosyl-5-fluoro-
7-methoxy-coumarin-2’-deoxyribose
&&&& — &&&&
Enzymatic Incorporation of a Coumarin-
Guanine Base Pair
(dCC) during enzymatic DNA synthesis.
Likewise, dCCTP is selectively incorpo-
rated across from a guanine template.
6
www.angewandte.org
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2019, 58, 1 – 6
These are not the final page numbers!