Pyrimidine-fused heterocyclic frameworks based on an N4-arylcytosine scaffold: Synthesis, characterization, and PNA oligomerization of the fluorescent cytosine analogue 5,6-benzopC

Article abstract of DOI:10.1021/jo402873e

A synthesis of an intrinsically fluorescent cytosine analogue 5,6-benzopC has been developed utilizing the reductive Ni-mediated cyclization of an N4-aryl,N4-(Boc)cytosine intermediate as a key step. 5,6-BenzopC was found to possess interesting fluorescence properties (φ = 0.79, EtOH; Stokes shift 113 nm). Peptide nucleic acid (PNA) oligomerization of the 5,6-benzopC monomer was carried out, followed by hybridization studies with complementary deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which showed the modification to be well tolerated in the sequence contexts examined. Initial attempts to synthesize the heterocyclic skeleton present in 5,6-benzopC resulted in the discovery of routes to the pyrimido[1,6-a]benzimidazole, pyrimido[1,6-a]quinazoline, and pyrimido[1,6-a]benzo[b]6-bora-1,3-diazine heterocyclic frameworks.

Full text of DOI:10.1021/jo402873e

Article
pubs.acs.org/joc
Pyrimidine-Fused Heterocyclic Frameworks Based on an N4-
Arylcytosine Scaffold: Synthesis, Characterization, and PNA
Oligomerization of the Fluorescent Cytosine Analogue 5,6-BenzopC
Mojmír Suchy and Robert H. E. Hudson*
́
Department of Chemistry, The University of Western Ontario, London, ON N6A 5B7, Canada
S
* Supporting Information
ABSTRACT: A synthesis of an intrinsically fluorescent cytosine analogue 5,6-
benzopC has been developed utilizing the reductive Ni-mediated cyclization of an
N4-aryl,N4-(Boc)cytosine intermediate as a key step. 5,6-BenzopC was found to
possess interesting fluorescence properties (Φ = 0.79, EtOH; Stoke’s shift 113
nm). Peptide nucleic acid (PNA) oligomerization of the 5,6-benzopC monomer
was carried out, followed by hybridization studies with complementary
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which showed the
modification to be well tolerated in the sequence contexts examined. Initial
attempts to synthesize the heterocyclic skeleton present in 5,6-benzopC resulted in
the discovery of routes to the pyrimido[1,6-a]benzimidazole, pyrimido[1,6-
a]quinazoline, and pyrimido[1,6-a]benzo[b]6-bora-1,3-diazine heterocyclic frame-
works.
would provide access to the pyrimido[5,4-b]indole framework
INTRODUCTION
■
present in tert-butyl 5,6-benzopC acetate (2b). We have
successfully synthesized 5,6-benzopC acetic acid (2a) and
conjugated the corresponding nucleobase analogue with a Boc-
chemistry compatible PNA backbone. The fluorescence
properties associated with this analogue6 have been evaluated.
PNA oligomerization was carried out, followed by hybridization
studies with corresponding DNA and RNA complements.
Unfortunately, a complete quenching of the fluorescence was
observed during these studies even upon binding to a
complementary DNA strand, and possible reasons for this
unexpected behavior are discussed below.
6-Phenylpyrrolocytosine (6-PhpC, 1, Figure 1) is an intrinsi-
cally blue fluorescent nucleobase analogue recently developed
in our laboratory for use in peptide nucleic acids.1 The key step
in the synthesis of 6-PhpC is a one-pot domino Sonogashira
cross-coupling/5-endo-dig annulation reaction between the
corresponding 5-iodocytosine analogues and phenylacetylene.2
The good emissivity of the 6-PhpC chromophore (Φ 0.61,
EtOH)2b along with the fact that 6-PhpC scaffold obeys
Watson−Crick base-pairing rules with guanine (Figure 1) make
this nucleobase analogue an excellent probe for biological
applications.
In addition to the preparation, characterization, and
oligomerization of 5,6-benzopC-containing monomer 2a, we
have also found that tert-butyl 1-oxo-4-bromopyrimido[1,6-
a]benzimidazol-2-ylacetate (4, Figure 1) and tert-butyl 1,10-
dioxo-4-bromopyrimido[1,6-a]quinazolin-2-ylacetate (5, Figure
1) can be obtained in good yields from tert-butyl N4-aryl-5-
bromocytosin-1-ylacetates 3b, 3d, and 3f (Figure 1) by simple
variation of the reaction conditions. As discussed later, there are
several methodologies available for the preparation of the
heterocyclic skeleton present in compound 4, while the highly
oxidized heterocyclic framework of 5 has not been described in
the literature. These unexpected observations have been made
when the substrates 3a−f have been subjected to Cu- and Pd-
catalyzed intramolecular cross-coupling reactions with the
intention to prepare 5,6-benzopC-containing PNA monomer
2a.
Introduction of 6-PhpC into PNA (peptide nucleic acid)
sequences is well tolerated, resulting in increased melting
temperature and reasonable discrimination against binding to
mismatched sequences.2b,c A PNA oligomer containing a
modified 6-PhpC subunit has been shown to inhibit mutant
huntingtin protein (a hallmark of Huntington’s disease) in an
allele-selective manner.3 In a more recent study, 6-PhpC-
modified siRNA was found to have gene silencing potency and
immunostimulatory properties mimicking those of natural short
interfering RNA (siRNA).4 The intrinsic fluorescence of 6-
PhpC has also been exploited to monitor cellular uptake and
distribution of PNA3,5 and RNA4 in the aforementioned
studies.
In order to gain a better understanding of fluorescence and
hybridization properties of 6-PhpC, we chose to prepare a
fused-ring analogue (5,6-benzopC acetic acid, 2a, Figure 1)
suitable for incorporation into PNA. We envisioned that a
transition-metal-mediated cyclization of N4-aryl-5-bromocyto-
sine analogues (3, Figure 1), synthesized here for the first time,
Received: December 27, 2013
Published: March 25, 2014
© 2014 American Chemical Society
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Figure 1. Structures of 6-PhpC (1) and 5,6-benzopC (2) showing a Watson−Crick base pairing with guanine. Structures of N4-arylcytosines 3 and
heterocycles 4−6 described in this study.
An unprecedented heterocyclic skeleton present in tert-butyl
1-oxo-10-hydroxy-4-bromopyrimido[1,6-a]benzo[b]6-bora-1,3-
diazin-2-yl-acetate (6, Figure 1) has been obtained in a similar
fashion by reacting tert-butyl 4-(1,2,4-triazol-1-yl)-5-bromocy-
tosin-1-ylacetate (8, Scheme 1) with 2-aminobenzeneboronic
1,2,4-triazole/POCl3, provided the triazolyl-modified nucleo-
base 8 in 42% overall yield (Scheme 1).8 Compound 8 was
purified by flash column chromatography (FCC) and
1
crystallization. The H NMR spectrum of 8 was found to be
in agreement with that reported by Pedroso et al.,8 and the13C
NMR spectrum of 8 was completely consistent with the
structure.
Scheme 1. Synthesis of 4-Arylaminocytosine Analogues 3a−f
An aromatic nucleophilic substitution of 8 with aniline was
attempted following the procedures reported by Pedroso et al.8
We have found that rigorous exclusion of water was
undesirable, and the addition of a small amount of water
facilitates the nucleophilic substitution of 8 with aniline
(Scheme 1). The reaction was performed at room temperature
(rt) in dioxane containing 10% water, in the absence of any
additional base, and the product cytosine analogue 3a
conveniently precipitated from the reaction mixture and
required no further purification (74% yield, Scheme 1).
A similar methodology was employed to prepare N4-
arylcytosine analogues 3b, 3d, and 3f. These reactions involved
less nucleophilic anilines (2-bromoaniline, 2-chloroaniline, and
anthranilic acid) and were therefore performed at 100 °C using
10% water in dioxane as a solvent without additional base.
Upon cooling to rt, compounds 3b (68% yield) and 3d (50%
yield) crystallized from the reaction mixture, whereas 3f formed
a gel and was subsequently dried and recrystallized from
MeOH/water (1:1) in 86% yield (Scheme 1). Finally,
cyclization substrates 3c and 3e were prepared in ca. 60%
yield by treatment of 3b and 3d with Boc2O in the presence of
DMAP (Scheme 1).
Preparation of 5,6-BenzopC Acetic Acid (2a) and Its
Conjugation with a Boc-OEt PNA Backbone. Several
transition-metal-based protocols were considered for the
cyclization of cytosine analogues 3 to tert-butyl 5,6-benzopC
acetate (2b). Pd-mediated approaches9 (substrates 3b and 3d)
did not prove to be successful and led only to decomposition.
An interesting discovery was made when elemental copper was
used to promote the cyclization of 3b and 3d. This approach
resulted yielded pyrimido[1,6-a]benzimidazol-2-ylacetate (4,
Figure 1), as discussed below. Unfortunately, the desired tert-
butyl 5,6-benzopC acetate (2b) could not be prepared by Cu-
mediated cyclization. We have also investigated an intra-
molecular decarboxylative coupling10 of substrate 3f; however,
this approach only led to intractable mixtures devoid of the
desired product.
acid pinacol ester. As described herein, our overall findings
imply that N4-arylcytosine scaffold is a versatile source of the
diversity in the chemistry of nitrogen-containing heterocycles.
RESULTS AND DISCUSSION
■
Preparation of tert-Butyl N4-Aryl-5-bromocytosin-1-yl
Acetates as Substrates for Cyclization. The substrates for
cyclization reactions, tert-butyl N4-aryl-5-bromocytosin-1-yla-
cetates 3a, 3b, 3d, and 3f, were prepared in four steps from
uracil (7). Bromination of uracil (7),7 followed by alkylation
with tert-butyl bromoacetate and subsequent reaction with
Since we observed the unwanted cyclization involving N3 of
cytosine, a reasonable explanation was that the large groups
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present at C5 of cytosine (Br) and in the ortho position on the
N-arylamine moiety (Br, Cl, COOH) forced substrates 3b, 3d,
and 3f to adopt an anti conformation, thus disfavoring the
formation of the desired pyrimido[5,4-b]indole heterocycle 2b.
We envisioned that use of the bulky tert-butyloxycarbonyl
(Boc) protecting group on the exocyclic amine may favorably
bias the conformation for the desired cyclization and
simultaneously mask the reactivity of the N3 of the cytosine
ring (Scheme 2).
Scheme 3. Synthesis of a PNA Monomer 11b
Scheme 2. Presumed Conformations and Cyclization
Patterns of Substrates 3b and 3c
With the substrate 3c in hand, we turned our attention to a
Ni-mediated cyclization of 3c (NiCl2/PPh3/Zn0).11 This
reaction was performed by in situ preparation of the nickel
catalyst (indicated by the solution turning deep red)11b and
then slowly adding a solution of the cyclization substrate 3c. As
the reaction progressed, the red color slowly disappeared. After
extractive workup, the desired tert-butyl 5,6-benzopC acetate
(2b) was isolated by FCC (blue fluorescent spot on the TLC
plate). Interestingly, the protecting Boc group was not retained
during the cyclization reaction, and attempts to isolate the
intermediate containing the Boc group were not successful. The
reaction was initially executed using the proportions of reagents
as described11b (0.2 equiv of NiCl2, 3.8 equiv of PPh3, and 15
equiv of Zn); however, the yields of the isolated product 2b
were not higher than 10−15%. The yield of 2b was improved
(to 36%) when an equimolar amount of NiCl2 was used
(Scheme 3). A further increase of the amount of NiCl2 did not
provide 2b in higher yield.
We were able to prepare sufficient amounts of tert-butyl 5,6-
benzopC acetate (2b) to permit detailed spectroscopy
characterization and characterization of its fluorescence proper-
ties and for the conjugation with a Boc-OEt PNA backbone via
the corresponding carboxylic acid derivative 2a as described
below. The formation of tert-butyl 5,6-benzopC acetate (2b)
was not observed when a chlorine-containing substrate 3e was
exposed to the conditions of Ni-mediated cyclization.
Debromination on the cytosine ring took place instead;
corresponding product 9 was isolated in 48% yield (Scheme
3). We have also attempted the Ni-mediated cyclization of
unprotected substrates 3b and 3d; however, only products of
decomposition were detected as indicated by TLC analysis.
The PNA monomer containing the 5,6-benzopC nucleobase
was then accessed via standard transformations. TFA-mediated
tert-butyl group deprotection provided the 5,6-benzopC
derived acid 2a (Scheme 3), which was fully characterized
(see the Supporting Information), followed by HBTU/HOBt12
coupling to the backbone submonomer 1013 and a final
saponification,14 Scheme 3. Although the synthesis of 11b from
uracil (7) involved nine steps and furnished the final product in
1.1% overall yield, this was sufficient material to subsequently
incorporate into oligomers to characterize their hybridization
and fluorescence properties.
Ni-Mediated Cyclization on the Boc-OEt and Boc-OBn
PNA Backbones. Although the 5,6-benzopC-containing PNA
monomer 11b was successfully prepared, our initial synthetic
route suffered from low yields for the Ni-mediated
cyclization11b to give 2b, the subsequent coupling12 of 2a
with the PNA backbone submonomer 10, and the final
saponification of the monomer ester (11a→11b, Scheme 3).
Since we had already explored optimization of the Ni-mediated
cyclization, we aimed for improving the latter two steps in the
aforementioned triad of reactions.
The inefficient coupling of nucleobase acetic acid derivative
2a to the Boc-OEt PNA backbone was partly ascribed to its
poor solubility. Thus, we explored the possibility of coupling
the cyclization substrate to the Boc-OEt PNA backbone and
then performing the Ni-mediated reactions. Happily, the
coupling of the cytosine 3b derivative with carboxyl protecting
group removed proceeded in a much improved yield leading to
intermediate 12a, Scheme 4. The substrate was prepared for
cyclization by installation of the Boc group and then underwent
the Ni-mediated reaction11b to give the Boc-OEt PNA
backbone conjugated 5,6-benzopC 11a in a yield similar to
that achieved earlier for the N4-arylcytosine (3c).
Having established that the Ni-mediated cyclization was
possible on a protected PNA monomer, we addressed the low
yield of isolated product after saponification of 11a to give 11b.
The hydrolysis proceeded smoothly, but the aqueous extractive
workup was found to be somewhat troublesome and
unavoidable, and prolonged exposure of 11b to the aqueous
acid (pH ∼ 3) resulted in partial removal of the Boc group. To
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Scheme 4. Alternative Routes Converge to the PNA Monomer 11b
circumvent this issue, we turned to the Boc-OBn PNA
backbone,15 wherein the ester functionality can be removed
by catalytic hydrogenation. The Boc-OBn PNA backbone (13)
was prepared according to the literature procedures16 and
conjugation of the cytosine derivative 3b proceeded well to give
intermediate 12c. Installation of the Boc group and Ni-
mediated cyclization11b furnished 11c (Scheme 4). Removal of
the benzyl ester from 11c was attempted by a catalytic
hydrogenation17 under an atmosphere of H2 (10% Pd/C, 2 h).
Even under these relatively mild conditions, in addition to the
desired removal of the ester functionality (compound 11b) an
over-reduction was found to take place (1H NMR and MS
spectrometry) giving ca. 1:1 mixture in quantitative yield of the
desired product 11b and the 5,6-dihydropyrimidine product18
(compound 14, Scheme 4). While it has been reported that the
cytosine ring can be restored by the oxidation with DDQ,19 we
were concerned about the compatibility of the rather harsh
(DDQ, heat) reaction conditions with the benzopC PNA
monomer 11b. As an alternative approach we attempted
catalytic oxygenation20 (balloon filled with O2, 10% Pd/C, 18
h) and were pleased to find that the desired 5,6-benzopC PNA
monomer 11b can be obtained in 84% yield (Scheme 4). To
avoid over-reduction, we investigated the hydrogenation of 11c
using 1,4-cyclohexadiene as a hydrogen source.21 This method-
ology failed to remove the benzyl ester; however, we later
found that simply shortening the reaction time of the catalytic
hydrogenation of 11c to 20 min leads to the clean formation of
the desired PNA monomer 11b (88%, Scheme 4).
possible alternative methods to reductively couple the bis-aryl
bromide cytosine analogue 3b, we attempted a Cu0-mediated
cyclization (Cu0, Δ in DMF, Et3N; Scheme 5). TLC analysis of
Scheme 5. Cu-Mediated Cyclization of 3b and 3d to Form
tert-Butyl 1-Oxo-4-bromopyrimido[1,6-a]benzimidazol-2-yl
Acetate (4)
the reaction mixture indicated consumption of the starting
material, and after standard extractive workup a single,
unknown product was isolated by FCC in 76% yield. Key
spectral evidence for determination of the structure came from
the observation in 13C NMR spectrum of a high-field aromatic
signal (δ 91.3 ppm) consistent with the presence a C−Br bond.
The presence of Br was further supported by HRMS analysis
which displayed the typical isotopic pattern and allowed us to
deduce the molecular formula C16H17BrN3O3. Although the
presence of the bromine in the product was not consistent with
the structure of tert-butyl 5,6-benzopC acetate (2b), this
reaction was investigated in more detail. When substrate 3b was
treated under the same reaction conditions, except without Cu0,
no product was formed. Variation of the base did not have an
influence on the outcome of the reaction. When a chlorinated
cytosine analogue 3d was subjected to the reaction conditions
(Cu wire, Et3N, DMF, heat), the same unknown product was
obtained, albeit in lower yield (13%, Scheme 5). In addition to
these observations, the Boc-protected substrates 3c and 3e
The alternative syntheses of 11b discussed herein represents
an overall improvement as the desired monomer 11b can be
prepared in 3.4% overall yield (via the Boc-OEt PNA
backbone) in nine steps from uracil (7) or in 1.8% overall
yield (via the Boc-OBn PNA backbone) in eleven steps from
uracil (7) compared to the original approach that afforded the
monomer 11b in 1.1% overall yield.
Cu-Mediated Formation of Pyrimido[1,6-a]-
benzimidazole Heterocyclic Framework. While exploring
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failed to undergo the Cu-mediated cyclization under various
conditions, resulting in the recovery of starting materials 3c and
3e. Overall, these studies suggested the endocyclic N3 of
cytosine ring is quite capable of nucleophilic displacement of a
halogen atom present in the 2-haloarene moiety attached to the
exocyclic N4. The unknown product was determined to be tert-
butyl 1-oxo-4-bromopyrimido[1,6-a]benzimidazol-2-ylacetate
(4, Scheme 5).
Scheme 6. Cyclization of 3f to Form tert-Butyl 1,10-Dioxo-4-
bromopyrimido[1,6-a]quinqzolin-2-yl Acetate (5)
It is noteworthy that the heterocyclic skeleton present in 4
has been previously prepared in moderate yield (49%) by
treatment of 3′,5′-di-OAc-4-O-[(2,4,6-triisopropylphenyl)-
sulfonyl]-5-bromo-2′-deoxyuridine with o-phenylenediamine
in refluxing THF.22 Only the H NMR spectrum (80 MHz)
1
of the product was provided in the original report;22 thus, we
were only able to carry out a tentative comparison with the
spectral data acquired for heterocycle 4. The chemical shift of
the signal due to H9 (Scheme 5) was determined to be δ 8.31−
8.49 ppm (m, 1H).22 This value is in a good agreement with
our data [δ 8.39 ppm (d, J = 8 Hz, 1H)]. The high chemical
shift associated with this proton is attributed to the anisotropy
of the nearby carbonyl group. The pyrimido[1,6-a]-
benzimidazole heterocyclic skeleton has been well studied,
especially with respect to its toxicology, and several different
methods are available for its preparation.23 Among these, the
reaction of 2′-deoxycytidine with p-benzoquinone has been
previously used to synthesize monomers suitable for DNA
oligomerization.24 Our methodology represents, to the best of
our knowledge, the first methodology for the preparation of
pyrimido[1,6-a]benzimidazole heterocyclic framework relying
on the Cu-catalyzed reaction as a key step.25
esters represents an excellent methodology for the formation of
a wide variety of biaryls. Due to some of the difficulties
encountered in producing the 5,6-benzopC nucleus (Scheme
1), we decided to investigate the potential of an intramolecular
Suzuki−Miyaura reaction on the substrate prepared by the
reaction of 1,2,4-triazolyl-modified cytosine 8 and the
commercially available 2-aminobenzeneboronic acid pinacol
ester (16), Scheme 7. After 24 h of reaction, TLC analysis
Formation of Pyrimido[1,6-a]quinazoline Heterocyclic
Skeleton from Anthranilic Acid Modified Cytosine
Analogue 3f. The anthranilic acid modified analogue 3f was
synthesized with the intention to investigate the possibilities of
an intramolecular decarboxylative coupling10 to form the
heterocyclic skeleton present in tert-butyl 5,6-benzopC acetate
(2b). This approach proved to be unsuccessful and failed to
produce the desired product despite extensive experimentation.
As done previously for 3b, we attempted to protect the
exocyclic N4 of the cytosine ring in 3f with the Boc group. The
reaction was carried out in warm dioxane as shown in the
Scheme 6. After isolation of the product (heterocycle 5), the
1H NMR spectrum was not consistent with the expected
structure as only one signal indicating the presence of tert-butyl
moiety was observed [δ 1.51 ppm (s, 9H)]. In addition, five
Scheme 7. Reaction of Modified Cytosine 8 with Aniline
Derivative 16 to Form tert-Butyl 1-Oxo-10-hydroxy-4-
bromopyrimido[1,6-a]benzo[b]6-bora-1,3-diazin-2-yl
Acetate (6)
1
aromatic signals observed in the H NMR spectrum were
similar in chemical shifts and splitting patterns to those
observed for tert-butyl 1-oxo-4-bromopyrimido[1,6-a]-
benzimidazolylacetate (4). The HRMS spectrum of 5 revealed
the molecular mass of 406 (M + H)+, consistent with the
molecular formula C17H17BrN3O4, and thus the unknown
product was assigned the structure of tert-butyl 1,10-dioxo-4-
bromopyrimido[1,6-a]quinazolinylacetate (5, Scheme 6). The
formation of this heterocyclic compound is rationalized as
depicted in Scheme 6. Presumably, the mixed anhydride 15 is
formed in situ upon treatment with Boc2O, followed by a
nucleophilic attack of N3 of the cytosine ring. The product is
obtained in good yield (78%), and this represents the first
synthesis of the highly oxidized 1,10-dioxopyrimido[1,6-a]-
quinazoline heterocyclic skeleton.
Formation of Pyrimido[1,6-a]benzo[b]6-bora-1,3-dia-
zine Heterocyclic Framework. Suzuki−Miyaura coupling26
between aryl halides and aryl boronic acids or aryl boronic acid
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indicated consumption of the starting material 8, and
precipitation was induced adding a small amount of water
and setting aside the reaction at 4 °C. The solid material
1
collected by filtration was then analyzed. Neither the H NMR
nor HRMS spectrum of the product was consistent with the
product of the replacement of 1,2,4-triazolyl moiety with ortho-
substituted pinacol borane ester modified aniline. Only two
aliphatic signals were observed in the 1H NMR spectrum
corresponding to the tert-butyl acetate moiety (δ 4.37 ppm, (s,
2H), 1.26 ppm, (s, 9H)); the signals attributable to the pinacol
borane ester were not present. Mass spectrometry indicated the
molecular mass of 405 (M + H)+, along with an unusual isotope
pattern which was attributed to the presence of both boron and
bromine atoms in the molecule which was consistent with the
molecular formula C16H17BBrN3O4. Additionally, a signal with
δ 19.7 ppm was present in the 11B NMR spectrum indicating
the presence of tricoordinate boron atom.27
The structure and formation of the product can be
rationalized as depicted in Scheme 7. Presumably, the
intermediate 17a (not detected) forms upon reacting the
1,2,4-triazolyl intermediate 8 with 2-aminobenzeneboronic acid
pinacol ester (16). A nucleophilic attack of N3 subsequently
takes place to form the intermediate 17b, which further
undergoes the hydrolysis under the conditions of the reaction
with the formation of heterocycle 6 (tert-butyl 1-oxo-10-
hydroxy-4-bromopyrimido[1,6-a]benzo[b]6-bora-1,3-diazin-2-
ylacetate 6, Scheme 7). The outcome of the reaction is
consistent with the pattern of reactivity observed for N4-
arylcytosines as described herein.
As the heterocyclic framework present in 6 is rather unusual
and to the best of our knowledge unknown,27 we confirmed the
structural assignment by single-crystal X-ray analysis. Although
the compound 6 is poorly soluble in a variety of solvents,
crystals suitable for X-ray diffraction were grown by slow
evaporation from a solution in AcOH. As a consequence of
crystallization from AcOH, the solid-state structure of hetero-
cycle 6 shows an acetate coordinated to the boron center. Thus,
the solid-state structure possesses a zwitterionic character
bearing the positive charge on the nitrogen in the position 5
(formerly N4 of cytosine), while the negative charge is located
at the tetrahedral boron atom bearing the acetate (see the
Supporting Information).
Fluorescence of tert-Butyl 5,6-BenzopC Acetate (2b).
The steady-state fluorescence properties of tert-butyl 5,6-
benzopC acetate (2b) have been investigated: the excitation
maximum was found to be red-shifted from the natural
nucleobases (λexcit = 328 nm, EtOH), and the emission
maximum at λemiss = 441 nm possessed a large Stokes shift of
113 nm (Figure 2). The quantum yield was determined (Φ =
0.79, EtOH) as described previously,2b using pyrene as a
standard.28 The large Stokes shift along with an excellent
quantum yield associated with tert-butyl 5,6-benzopC acetate
(2b) are superior among related compounds previously
described in our laboratory.2
The solvatochromicity of the 5,6-benzopC chromophore was
investigated by a examining comixtures of EtOH and H2O. The
fluorescence emission spectra of solutions of tert-butyl 5,6-
benzopC acetate (2b) in EtOH with varying amounts of water
(20, 40, 60, and 80%) were recorded (Figure 2).2b A substantial
quenching of the fluorescence emission was observed with
increasing water along with a noticeable bathochromic shift of
the emission maxima. Severe quenching (>90%) of the
fluorescence was detected in the solution containing 80%
Figure 2. Steady-state fluorescence spectra of 2b in EtOH (top).
Excitation spectrum in black, emission in blue. Solvatochromicity
study of 2b in EtOH/water mixtures (bottom).
water. Under these conditions, no luminophore aggregation
was evident; however, the limited solubility prevented using
pure water. These results demonstrate that the chromophore
present in tert-butyl 5,6-benzopC acetate (2b) is sensitive to its
environment. The dramatic response to increasing polarity of
the solvent comixtures was an encouraging indication that the
5,6-benzopC may behave as a microenvironmentally responsive
nucleobase once incorporated into an oligomer.
Oligomerization of the PNA Monomer 11b and
Hybridization Studies. PNA oligomerization was carried
out manually using the well-established protocols for Boc PNA
chemistry.29 Three oligomers were prepared as follows. The
position of the unnatural nucleobase is indicated as X: GTA
GAT CXC T-Lys (sequence 1A), Lys-GTA GAT CXC T-Lys
(sequence 1B), or GTA GAT XAC T-Lys (sequence 2).
Oligomers were purified by HPLC and were characterized by
HR-ESI-MS. With the PNA oligomers in hand, their hybrid-
ization properties were investigated; the results are summarized
in Table 1. The incorporation of the 5,6-benzopC nucleobase
into sequences 1A and 1B moderately increases the stability of
the corresponding DNA (ΔTm +3 °C, 1A; ΔTm +6 °C, 1B) and
RNA (ΔTm +5 °C, 1A; ΔTm +1.5 °C, 1B) duplexes, while in
sequence 2 it is tolerated in the DNA duplex showing no
stabilization and a mild destabilization (ΔTm −3 °C) toward
complementary RNA. The differential stabilization of PNA:NA
heteroduplexes based on sequence context is not unusual for
PNAs containing modified bases and can be quite profound in
some instances.2b,30 Sequences 1 and 2 differ mainly by the
flanking bases whereas −CXC− is more stabilizing versus
−TXA−. Although it may be expected for 5,6-benzopC to gain
additional stacking interactions over the purine, examination of
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corresponding DNA and RNA duplexes, in the sequences
examined.
Table 1. Hybridization Studies with PNA Sequences 1A, 1B,
and 2
The fluorescence properties of tert-butyl 5,6-benzopC acetate
(2b) have been determined, revealing unprecedented values for
the Stokes shift (113 nm) and quantum yield (Φ 0.79, EtOH)
associated with pC analogs previously described in our
laboratory.2 Moreover, a strong solvatochromicity was also
observed, indicating that the 5,6-benzopC chromophore is
sensitive to its microenvironment. These properties make the
5,6-benzopC chromophore potentially an excellent comple-
ment to pC analogues previously reported.2,32 Unfortunately, a
complete quenching of the fluorescence was observed upon
binding of the PNA oligomers to the DNA and RNA
complements. This can be attributed to the high sensitivity of
the 5,6-benzopC fluorophore to the aqueous environment.32
Further synthetic modifications of the benzopC scaffold are
underway to probe this phenomenon.
We have also found that a Cu-mediated cyclization of tert-
butyl N4-(-Boc)-N4-(2-halophenyl)-5-bromocytosin-1-ylace-
tates provided an access to tert-butyl 1-oxo-4-bromopyrimido-
[1,6-a]benzimidazol-2-ylacetate (4). The reaction was found to
proceed in good yield in the case of bis-brominated cyclization
substrate. In somewhat related cyclization, a treatment of
anthranilic acid modified cytosine acetate with Boc2O afforded
tert-butyl 1,10-dioxo-4-bromopyrimido[1,6-a]quinqzolin-2-yla-
cetate (5) as a first example of the oxidized heterocyclic
skeleton derived from he pyrimido[1,6-a]quinazoline frame-
work. Finally, an unusual boron-containing heterocyclic frame-
work pyrimido[1,6-a]benzo[b]6-bora-1,3-diazine has been
prepared in a similar manner starting from tert-butyl 4-
(1′,2′,4′-triazole)-1′-yl-5-bromocytosin-1-ylacetate (8) and 2-
aminobenzeneboronic acid pinacol ester (16). In all three cases,
the reactivity of the N3 of the cytosine ring played a crucial
role. These unexpected reactions indicate that N4-arylcytosines
represent an excellent source of diversity in heterocyclic
chemistry and might serve as a template for other cyclization
reactions involving various cytosine analogues.
DNA Tm
(°C)
ΔTm
(°C)
RNA Tm
(°C)
ΔTm
(°C)
a,b
sequence (N→C)
X
GTA GAT CXC
C
53.0
56.0
53.0
59.0
53.0
53.0
65.0
70.0
63.0
65.5
56.0
53.0
T-K
GTA GAT CXC
bpC
C
+3.0
+6.0
0.0
+5.0
+1.5
−3.0
T-K
K-GTA GAT CXC
T-K
K-GTA GAT CXC
bpC
C
T-K
GTA GAT XAC
T-K
GTA GAT XAC
bpC
T-K
a
Measurements were carried out as described in the General
Experimental Procedures. Each strand was present at 2 μM in a
buffer of 100 mM NaCl, 10 mM NaH2PO4, 0.1 mM EDTA, pH 7.0.
No cooperative transition was observed for any case of mismatched
nucleoside across from bpC. K = lysine (Lys); bpC = 5,6-benzopC.
b
the reported structure of a PNA:DNA duplex31 suggests the
geometry of the ring fusion rather favors interactions with the
nucleobase on the pseudo 5′ side. One may speculate that this
would result in less stacking (with the pyrimidine) or a clash
with 5-methyl group of thymine thus leading to −TXA− being
a less favored sequence.
The fluorescence properties of sequences 1A, 1B, and 2 upon
binding to the DNA and RNA complementary strands and
corresponding DNA mismatches have been also investigated.
Unfortunately, nearly complete quenching of the fluorescence
has been observed for all of the three sequences (see the
Supporting Information for details) whether in the single-
stranded or duplex state. This behavior can be attributed to the
high sensitivity of the 5,6-benzopC fluorophore to the aqueous
environment,32 and even binding to a complementary DNA
and RNA does not provide a sufficiently dehydrated environ-
ment to prevent the quenching of the fluorescence. This
observation is also consistent with the results of dilution studies
with the PNA oligomers. We have found that the fluorescence
associated with the PNA oligomers containing 5,6-benzopC
fluorophore is to some extent restored upon dissolving the
oligomers in small amount of water and mixing the solutions
with increasing amounts of dioxane or DMSO (see the
Supporting Information). Our findings indicate that 5,6-
benzopC fluorophore might operate in a hydrophobic environ-
ment and may be useful for binding of ligands/proteins to the
oligomer for which a DNA-based scaffold would be preferred
over that of PNA.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All reagents were commer-
cially available, unless otherwise stated. Organic solvents were HPLC
grade and used as such, except for DMF and dioxane (solvent
purification system) and water (18.2 MΩ·cm−1 Millipore water).
Organic extracts were dried with Na2SO4 and solvents were removed
under reduced pressure in a rotary evaporator. Flash column
chromatography (FCC) was carried out using silica gel, mesh size
230 − 400 Å. Thin layer chromatography (TLC) was carried out on Al
backed silica gel plates, compounds were visualized by UV light.
Preparative thin layer chromatography (PTLC) was carried out on
glass backed silica gel plates (20 × 20 cm), layer thickness 1000 μm,
compounds were visualized by UV light. Melting points were obtained
on Fisher−Johns apparatus and are uncorrected. Ultra performance
liquid chromatography (UPLC) was performed using a BEH C18
column (particle size 1.7 μm; 1.0 i.d. × 100 mm) and HR-ESI-MS
detector. Mobile phase: Method A: 100% H2O−100% MeCN (both
solvents containing 0.1% HCOOH) over 5 min, linear gradient, flow
rate 0.1 mL/min. HPLC analysis and purification was performed using
a Microsorb-MV C18 300 Å column (4.6 i.d. × 250 mm). The mobile
phase for method B (sequences 1A and 1B) was 0−5 min, 99% H2O−
1% MeCN (both solvents containing 0.1% TFA) then 60% H2O−40%
MeCN over 25 min, 1 mL/min. The mobile phase for method C
(sequence 2) was 0−5 min, 99% H2O−1% MeCN (both solvents
containing 0.1% TFA) then 100% MeCN over 20 min, 1 mL/min.
CONCLUSIONS
■
In conclusion, we have developed a methodology for the
preparation of the pyrimido[5,4-b]indole heterocyclic skel-
eton33 present in a fluorescent nucleobase analogue tert-butyl
5,6-benzopC acetate (2b). A Ni-mediated cyclization11b of tert-
butyl N4-(Boc)-N4-(2-bromophenyl)-5-bromocytosin-1-ylace-
tate (3c) was employed as a key step. The chemistry is
compatible with both Boc-OEt and Boc-OBn PNA backbones
allowing for the conjugation at an earlier stage of the monomer
synthesis followed by the cyclization to form the corresponding
PNA monomers. The PNA oligomers have been successfully
synthesized by means of manual Boc PNA chemistry.29 The
substitution was found to be well tolerated in the
1
NMR spectra were recorded on 400 MHz spectrometer; for H (400
MHz), δ values were referenced as follows CDCl3 (7.26 ppm),
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CD3OD (3.31 ppm), DMSO-d6 (2.49 ppm) and for 13C (100 MHz)
CDCl3 (77.0 ppm), CD3OD (49.2 ppm), DMSO-d6 (39.5 ppm). The
11B NMR spectrum was acquired in dioxane. Mass spectra (MS) were
of anthranilic acid, a gel formed upon cooling the reaction mixture to
rt; the gel was redissolved in MeOH/H2O (1:1, 50 mL), and the
solution was concentrated to dryness. The residue was crystallized
from MeOH/H2O to afford white crystals which were filtered off and
washed as described above.
obtained using an electron spray ionization (ESI) time-of-flight (TOF)
instrument. Fluorescence and UV spectra measurements were carried
out on respective instruments in spectrophotometric grade solvents.
Quantum yield associated with compound 2a was determined in
EtOH as described previously2b using pyrene as a standard.28
Hybridization and UV melting experiments were carried out in a
buffer containing 100 mM NaCl, 10 mM Na2HPO4, and 0.1 mM
EDTA at pH 7.0. Tm experiments were performed at 2 × 10−6 M for
each strand concentration. Samples were heated to 95 °C and were
allowed to cool to room temperature gradually (ca. 3 h). Denaturation
was performed from 20 to 85 °C at a ramp of 0.5 °C/min. ΔTm values
are the difference between the Tm of sequences 1A, 1B, and 2
containing unnatural nucleobase and control sequences containing
cytosine. The Tm values are an average of three measurements and are
rounded to the nearest 0.5 °C. Tm values were estimated for
cooperative transitions by the first derivative method. Temperature-
dependent UV spectra that lacked upper and lower baselines lacked
sigmoidal shape or were indistinguishable from ssPNA “self-melts”
were deemed not to be cooperative transitions. Samples for the
fluorescence measurements associated with the PNA oligomers were
prepared in the same manner as those used for the determination of
Tm values, using the same buffer, the same pH value and the same
concentration.
Preparation of tert-Butyl-4-(1′,2′,4′-triazol-1′-yl)-5-bromocy-
tosin-1-ylacetate (8). 5-Bromouracil was prepared from uracil (7) as
described in ref 7. 5-Bromouracil (1.44 g, 7.56 mmol) was dissolved in
dry DMF (13 mL), followed by the addition of K2CO3 (1.04 g, 7.56
mmol) and tert-butyl bromoacetate (1.12 mL, 7.56 mmol). The
mixture was stirred at room temperature (rt) for 18 h. DMF was
removed by coevaporation with PhCH3 (3 × 200 mL), and the residue
was partitioned between water (80 mL) and EtOAc (80 + 60 + 40
mL). The combined organic extract was dried and concentrated to
leave crude tert-butyl 5-bromouracil-1-ylacetate (1.91 g, 83%) of
sufficient purity for the next step.
1,2,4-Triazole (3.92 g, 56.71 mmol) was dissolved in dry MeCN
(190 mL), and the solution was cooled to 0 °C followed by the
addition of POCl3 (1.17 mL, 12.6 mmol) and Et3N (13.2 mL, 94.52
mmol). The mixture was stirred for 30 min at 0 °C, and then a
solution of tert-butyl 5-bromouracil-1-ylacetate (1.91 g, 6.3 mmol) in
dry MeCN (70 mL) was added in one portion. The cooling bath was
removed, and the mixture was stirred for 18 h at rt. MeCN was
evaporated; the residue was partitioned between water (60 mL) and
EtOAc (60 + 2 × 40 mL). The combined organic extract was dried
and concentrated; the residue was subjected to FCC on 70 g SiO2,
hexanes/acetone (2:1). Colorless needles started to form upon
concentration of the eluate, hexanes were added, and the mixture
was set aside for 2 h at −5 °C. The crystals were filtered off, washed
with hexanes, and dried to leave tert-butyl 4-(1′,2′,4′-triazol-1′-yl)-5-
bromocytosine-1-ylacetate (8, 1.36 g, 51%, based on 5-bromouracil):
colorless needles; mp 118−120 °C; 1H NMR (CDCl3) δ 9.14 (s, 1H),
8.15 (s, 1H), 8.12 (s, 1H), 4.61 (s, 2H), 1.48 (s, 9H); 13C NMR
(CDCl3) δ 165.2, 156.0, 153.9, 153.4, 152.9, 145.1, 86.5, 84.3, 51.7,
27.9; HRMS (ESI) m/z found 356.0366 [M + H]+ (calcd 356.0358 for
C12H15BrN5O3).
Reaction of tert-Butyl 4-(1′,2′,4′-Triazole-1′-yl)-5-bromocy-
tosin-1-ylacetate (8) with Anilines. A solution of tert-butyl 4-
(1′,2′,4′-triazol-1′-yl)-5-bromocytosin-1-ylacetate (8, 712 mg, 2
mmol) in dioxane (7.2 mL) was treated with anilines as follows:
aniline (365 μL, 4 mmol); 2-bromoaniline (447 mg, 2.6 mmol); 2-
chloroaniline (270 μL, 2.6 mmol); and anthranilic acid (357 mg, 2.6
mmol). Water was added, and the mixtures were stirred at for 4 h at rt
(aniline) or for 48 h at 100 °C. A white crystalline precipitate formed
in the mixture containing the aniline; white crystalline precipitates
formed upon cooling to rt in the case of 2-haloanilines. The mixtures
were set aside for 3−4 h at 4 °C, and separated precipitates were
filtered off with suction, were washed with water and cold (−10 °C)
Et2O, and were dried to provide analytically pure products. In the case
tert-Butyl N4-phenyl-5-bromocytosin-1-ylacetate (3a, 563 mg,
1
74%): white crystalline solid; mp 204−206 °C; H NMR (CDCl3) δ
7.72 (m, 2H), 7.55 (s, 1H), 7.35 (m, 2H), 7.30 (br s, D2O exch, 1H),
7.15 (m, 1H), 4.45 (s, 2H), 1.48 (s, 9H); 13C NMR (CDCl3) δ 166.8,
157.6, 154.6, 145.5, 137.0, 129.0, 125.1, 121.6, 88.0, 83.2, 50.9, 28.0;
HRMS (ESI) m/z found 380.0604 [M + H]+ (calcd 380.0610 for
C16H19BrN3O3).
tert-Butyl N4-(2′-bromophenyl)-5-bromocytosin-1-ylacetate (3b,
1
628 mg, 68%): white crystalline solid; mp 194−196 °C; H NMR
(CDCl3) δ 8.76 (d, J = 8 Hz, 1H), 8.01 (br s, D2O exch, 1H), 7.59 (s,
1H), 7.57 (dd, J = 8, 1.5 Hz, 1H), 7.36 (ddd, J = 8, 8, 1.5 Hz, 1H), 7.02
(ddd, J = 8, 8, 1.5 Hz, 1H), 4.48 (s, 2H), 1.49 (s, 9H); 13C NMR
(CDCl3) δ 166.7, 157.6, 154.5, 145.8, 135.2, 132.2, 128.5, 125.7, 123.1,
114.7, 88.6, 83.4, 51.0, 28.0; HRMS (ESI) m/z found 457.9717 [M +
H]+ (calcd 457.9715 for C16H18Br2N3O3).
tert-Butyl N4-(2′-chlorophenyl)-5-bromocytosin-1-ylacetate (3d,
1
415 mg, 50%): white crystalline solid; mp 270−272 °C; H NMR
(CDCl3) δ 8.80 (dd, J = 8, 1 Hz, 1H), 8.03 (br s, D2O exch, 1H), 7.59
(s, 1H), 7.57 (dd, J = 8, 1 Hz, 1H), 7.32 (m, 1H), 7.08 (ddd, J = 8, 8, 1
Hz, 1H), 4.48 (s, 2H), 1.49 (s, 9H); 13C NMR (CDCl3) δ 166.6,
157.6, 154.5, 145.8, 134.1, 128.9, 127.8, 125.2, 123.9, 122.8, 88.5, 83.4,
51.0, 28.0; HRMS (ESI) m/z found 414.0201 [M + H]+ (calcd
414.0220 for C16H18BrClN3O3).
tert-Butyl N4-(2′-oxycarbonylphenyl)-5-bromocytosin-1-ylace-
tate (3f, 729 mg, 86%): white crystalline solid; mp 276−278 °C;
1H NMR (CD3OD) δ 9.14 (d, J = 8.5 Hz, 1H), 8.14 (s, 1H), 8.13 (m,
1H), 7.62 (m, 1H), 7.20 (m, 1H), 4.51 (s, 2H), 1.50 (s, 9H); 13C
NMR (CD3OD) δ 171.4, 168.7, 160.4, 157.6, 148.8, 142.1, 135.1,
132.7, 124.6, 122.9, 118.7, 90.5, 84.1, 52.7, 28.4; HRMS (ESI) m/z
found 424.0505 [M + H]+ (calcd 424.0508 for C17H19BrN3O5).
Reaction of tert-Butyl N4-(2′-Halophenyl)-5-bromocytosin-
1-ylacetates with Boc2O. Separate mixtures containing tert-butyl
N4-(2′-halophenyl)-5-bromocytosin-1-ylacetates (3b or 3d, 1 mmol),
Boc2O (437 mg, 2 mmol), and DMAP (12 mg, 0.1 mmol) in dry
dioxane (6 mL) were stirred at 60 °C for 90 min (3b) or 1 h (3d).
The mixtures were cooled to rt, diluted with brine (50 mL), and
extracted with EtOAc (2 × 30 mL). Combined organic extracts were
dried and concentrated, and the residues were subjected to FCC
purification on 50 g SiO2, hexanes/EtOAc (1:1). Evaporation of
eluates afforded white solid residue, which was crystallized from
CH2Cl2/hexanes solution.
tert-Butyl N4-Boc-N4-(2′-bromophenyl)-5-bromocytosin-1-ylace-
1
tate (3c, 333 mg, 60%): white crystalline solid; mp 186−188 °C; H
NMR (CDCl3) δ 7.76 (s, 1H), 7.60 (m, 1H), 7.33 (m, 2H), 7.18 (m,
1H), 4.49 (s, 2H), 1.50 (s, 9H), 1.48 (s, 9H); 13C NMR (CDCl3) δ
165.6, 164.6, 154.3, 151.2, 149.1, 139.3, 133.4, 130.8, 129.3, 128.0,
123.1, 95.4, 83.8, 83.7, 51.3, 28.0, 27.9; HRMS (ESI) m/z found
580.0041 [M + Na]+ (calcd 580.0059 for C21H25Br2N3O5Na).
tert-Butyl N4-Boc-N4-(2′-chlorophenyl)-5-bromocytosin-1-ylace-
1
tate (3e, 324 mg, 63%): white crystalline solid; mp 176−178 °C; H
NMR (CDCl3) δ 7.78 (s, 1H), 7.42 (m, 1H), 7.32 (m, 1H), 7.27 (m,
2H), 4.50 (s, 2H), 1.50 (s, 9H), 1.48 (s, 9H). 13C NMR (CDCl3) δ
165.6, 164.8, 154.4, 151.2, 149.1, 137.6, 132.7, 130.5, 130.2, 129.1,
127.4, 95.4, 83.8, 83.7, 51.3, 27.9, 27.8; HRMS (ESI) m/z found
514.0740 [M + H]+ (calcd 514.0744 for C21H26BrClN3O5).
Ni-Mediated Cyclization of tert-Butyl N4-Boc-N4-(2′-bromo-
phenyl)-5-bromocytosin-1-ylacetate (3c). PPh3 (258 mg, 0.99
mmol), NiCl2 (34 mg, 0.26 mmol), and Zn dust (254 mg, 3.89 mmol)
were placed into a round-bottom flask, and the flask was then charged
with N2. After the flask was flushed with N2 gas for 10 min, dry DMF
(1.5 mL) was added, and the mixture was stirred at 80 °C for 5−10
min (formation of dark red catalyst was visible).11b A solution of tert-
butyl N4-Boc-N4-(2′-bromophenyl)-5-bromocytosin-1-ylacetate (3b,
145 mg, 0.26 mmol) in dry DMF (1 mL) was added dropwise over a
period of ca. 3 min. The mixture was stirred for 1 h at 80 °C (N2
atmosphere), cooled to room temperature, diluted with 4% EDTA
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solution (40 mL), and extracted with EtOAc (2 × 20 + 10 mL). The
combined organic extract was washed with brine (2 × 50 mL), dried,
and concentrated. The residue was subjected to FCC purification on
30 g of SiO2, CH2Cl2/MeOH (95:5). Evaporation of the eluate
afforded tert-butyl pyrimido[5,4-b]indol-3-ylacetate or tert-butyl 5,6-
J = 7 Hz, 1.5H), 4.07 (s, 1.5H), 3.50 (m, 1.5H), 3.30 (m, 0.5H), 3.25
(q, J = 6.5 Hz, 1.5H), 3.04 (q, J = 6.5 Hz, 0.5H), 1.39 (s, 6.75H), 1.36
(s, 2.25H), 1.27 (t, J = 7 Hz, 0.75H), 1.18 (t, J = 7 Hz, 2.25H); 13C
NMR (DMSO-d6) δ 169.4 (minor), 169.0 (major), 167.9 (minor),
167.7 (major), 162.2 (major + minor), 155.8 (minor), 155.1 (major),
142.8 (major + minor), 139.9 (major + minor), 128.8 (minor), 126.5
(major + minor), 121.3 (major + minor), 120.4 (major + minor),
119.7 (major + minor), 111.2 (major + minor), 103.4 (major +
minor), 78.0 (major), 77.8 (minor), 61.2 (minor), 60.5 (major), 50.7
(minor), 50.4 (major), 49.3 (minor), 48.0 (major), 47.1 (major), 46.9
(minor), 38.4 (major), 37.7 (minor), 28.2 (major + minor), 14.0
(major + minor); HRMS (ESI) m/z found 472.2173 [M + H]+ (calcd
472.2196 for C23H30N5O6).
1
benzopC acetate (2b) as white solid residue (28 mg, 36%): H NMR
(DMSO-d6) δ 11.69 (br s, D2O exch, 1H), 8.87 (s, 1H), 7.77 (d, J = 8
Hz, 1H), 7.33 (dd, J = 8, 7.5 Hz, 1H), 7.28 (d, J = 8 Hz, 1H) 7.18 (dd,
J = 8, 7.5 Hz, 1H), 4.64 (s, 2H), 1.43 (s, 9H); 13C NMR (DMSO-d6) δ
167.4, 162.3, 155.1, 142.4, 139.9, 126.6, 121.3, 120.3, 119.8, 111.3,
103.5, 81.6, 52.4, 27.7; HRMS (ESI) m/z found 300.1342 [M + H]+
(calcd 300.1348 for C16H18N3O3).
Debromination of tert-Butyl N4-Boc-N4-(2′-chlorophenyl)-5-
bromocytosin-1-ylacetate (3e) under the Conditions of Ni-
Mediated Coupling. PPh3 (199 mg, 0.76 mmol), NiCl2 (5 mg, 0.04
mmol), and Zn dust (200 mg, 3 mmol) were placed into a round-
bottom flask, and the Ni catalyst was prepared as described above. A
solution of tert-butyl N4-Boc-N4-(2′-chlorophenyl)-5-bromocytosin-1-
ylacetate (3e, 103 mg, 0.2 mmol) in dry DMF (1 mL) was added
dropwise over a period of ca. 3 min. The mixture was stirred for 1 h at
80 °C (N2 atmosphere), cooled to room temperature, diluted with 4%
EDTA solution (40 mL), and extracted with EtOAc (2 × 20 mL). The
combined organic extract was washed with brine (2 × 40 mL), dried,
and concentrated. The residue was subjected to FCC purification on
30 g of SiO2, hexanes/EtOAc (1:1). Evaporation of the eluate afforded
tert-butyl N4-Boc-N4-(2′-chlorophenyl)cytosin-1-ylacetate (9) as a
colorless solid (42 mg, 48%): 1H NMR (DMSO-d6) δ 8.08 (d, J = 7.5
Hz, 1H), 7.58 (m, 1H), 7.39 (m, 3H); 7.23 (d, J = 7.5 Hz, 1H), 4.48
(s, 2H), 1.40 (s, 9H); 1.34 (s, 9H); 13C NMR (DMSO-d6) δ 166.9,
164.2, 154.5, 151.3, 149.8, 137.2, 131.8, 130.7, 129.4, 129.3, 127.8,
96.5, 83.1, 81.7, 51.1, 27.6, 27.3; HRMS (ESI) m/z found 436.1656
[M + H]+ (calcd 436.1639 for C21H27ClN3O5).
Hydrolysis of 5,6-benzopC PNA Monomer 11a. 5,6-BenzopC
PNA monomer 11a (76 mg, 0.16 mmol) was dissolved in THF (2
mL), followed by the addition of NaOH solution (2 M, 320 μL, 0.64
mmol). The mixture was vigorously stirred for 40 min at rt and cooled
to 0 °C, and the pH was adjusted to ca. 3 (1 M HCl). The mixture was
diluted with water (5 mL) and extracted with EtOAc (5 × 10 mL).
The combined organic extract was dried and concentrated to leave the
Boc-OH 5,6-benzopC PNA monomer 11b (35 mg, 49%) as a slightly
yellow solid: 1H NMR (DMSO-d6) δ 11.66 (br s, D2O exch, 1H), 8.78
(s, 0.75H), 8.77 (s, 0.25H), 7.81 (d, J = 7.5 Hz, 0.25H), 7.78 (d, J =
7.5 Hz, 0.75H), 7.31 (m, 2H), 7.17 (m, 1H), 6.98 (t, D2O exch, J = 5.5
Hz, 0.75H), 6.78 (t, D2O exch, J = 5.5 Hz, 0.25H), 4.96 (s, 1.5H), 4.78
(s, 0.5H), 4.31 (s, 0.5H), 4.01 (s, 1.5H), 3.40 (m, 2H), 3.25 (m, 1.5H),
3.04 (m, 0.5H), 1.39 (s, 6.75H), 1.37 (s, 2.25H); 13C NMR (DMSO-
d6) δ 170.6 (major + minor), 168.0 (major), 167.4 (minor), 162.2
(major), 162.1 (minor), 155.2 (major), 155.1 (minor), 143.1 (minor),
142.9 (major), 139.9 (major), 139.8 (minor), 128.8 (minor), 128.7
(major), 126.5 (major), 126.4 (minor), 121.3 (major + minor), 120.5
(major), 120.4 (minor), 119.9 (minor), 119.8 (major), 111.3 (major),
11.2 (minor), 103.4 (major + minor), 78.0 (major), 77.7 (minor), 50.8
(major + minor), 50.5 (major + minor), 48.1 (major + minor), 47.1
(major + minor), 28.2 (major + minor); HRMS (ESI) m/z found
444.1877 [M + H]+ (calcd 444.1883 for C21H26N5O6).
Conjugation of tert-Butyl N4-(2′-Bromophenyl)-5-bromocy-
tosin-1-ylacetate (3b) with Boc-OEt PNA Backbone Hydro-
chloride (10) and Boc-OBn PNA Backbone (13). TFA (700 μL)
was added to separate suspensions of tert-butyl N4-(2′-bromophenyl)-
5-bromocytosin-1-ylacetate (3b, 358 mg, 0.78 mmol) in CH2Cl2 (2.1
mL). The mixtures were stirred for 18 h at rt, the solvents were
evaporated, excess TFA was removed by coevaporation with CH2Cl2
(3 × 30 mL), and the residues were dried on the vacuum pump for 24
h. In separate flasks the residues were suspended in dry DMF (3 mL),
followed by the addition of DIPEA (680 μL, 3.9 mmol). The resulting
solutions were cooled to 0 °C, and HBTU (296 mg, 0.78 mmol) and
HOBt (32 mg, 0.23 mmol) were added. The mixtures was stirred for 5
min at 0 °C, followed by the addition of Boc-OEt PNA backbone
hydrochloride (10, 220 mg, 0.78 mmol) or Boc-OBn PNA backbone
(13, 241 mg, 0.78 mmol) and DIPEA (in the case of hydrochloride
10) (680 μL, 3.9 mmol). The mixtures were stirred for 24 h at 50 °C,
were cooled to rt, and were diluted with brine (50 mL). The aqueous
phases were extracted with EtOAc (2 × 30 mL), and the combined
organic extracts were washed with brine (2 × 60 mL), dried, and
concentrated. The residue was subjected to FCC on 60 g SiO2,
CH2Cl2/MeOH (95:5) (conjugate 12a) or 45 g SiO2, CH2Cl2/MeOH
(98:2), later replaced with CH2Cl2/MeOH (95:5) (conjugate 12c).
Evaporation of the eluates afforded corresponding bis-brominated
PNA monomers 12a and 12c.
BenzopC Acetic Acid (2a). TFA (300 μL) and TES (100 μL)
were added to a suspension of the tert-butyl pyrimido[5,6-b]indole-3-
ylacetate (2b, 27 mg, 0.09 mmol) in CH2Cl2 (3 mL). The mixture was
stirred for 18 h at rt, the solvent was evaporated, excess TFA was
removed by coevaporation of the residue with CH2Cl2 (3 × 30 mL),
and the residue was used for the conjugation with the Boc-OEt and
Boc-OBn PNA backbones without further purification. Analytical
samples of 2a were obtained by subjecting the residue to the PTLC
using CH2Cl2/MeOH/NH4OH (80:19:1) as an eluent. The band with
Rf 0.05 was carved off the plate, the silica was extracted with MeOH
and filtered off, and the solvent was evaporated to leave pyrimido[5,4-
b]indol-3-ylacetic acid or benzopC acetic acid (2a, 9 mg, 41%) as a
1
slightly yellow solid: H NMR (DMSO-d6) δ 11.57 (br s, D2O exch,
1H), 8.81 (s, 1H), 7.77 (d, J = 7.5 Hz, 1H), 7.29 (m, 2H), 7.14 (dd, J
= 7.5, 6.5 Hz, 1H), 4.50 (s, 2H); 13C NMR (DMSO-d6) δ 161.9,
155.6, 143.0, 139.8, 126.2, 121.1, 120.6, 119.7, 111.1, 103.0, 41.1;
HRMS (ESI) m/z found 244.0714 [M + H]+ (calcd 244.0722 for
C12H10N3O3).
Conjugation of Benzo-PC Acetic Acid (2a) with Boc-OEt PNA
Backbone Hydrochloride (10). Crude benzo-PC acetic acid (2a)
prepared from 0.15 mmol of 2b (by treatment with TFA/TES as
described above) was dissolved in dry DMF (600 μL), diisopropy-
lethylamine (DIPEA, 135 μL, 0.77 mmol) was added, and the mixture
was cooled to 0 °C. HBTU (58 mg, 0.15 mmol) and HOBt (6 mg,
0.05 mmol) were added, and the mixture was stirred for 5 min at 0 °C.
Boc-OEt PNA backbone hydrochloride (10, 43 mg, 0.15 mmol) and
DIPEA (135 μL, 0.77 mmol) were added, the cooling bath was
removed, and the mixture was stirred for 18 h at 50 °C. It was then
cooled to rt and diluted with brine (40 mL). The aqueous phase was
extracted with EtOAc (2 × 20 mL), and the combined organic extract
was washed with brine (2 × 40 mL), dried, and concentrated. The
residue was subjected to FCC on 20 g SiO2, CH2Cl2/MeOH (95:5).
Evaporation of the eluate afforded 5,6-benzopC PNA monomer 11a as
a white solid (23 mg, 32%): 1H NMR (DMSO-d6) δ 11.64 (br s, D2O
exch, 1H), 8.76 (s, 0.75H), 8.75 (s, 0.25H), 7.80 (d, J = 8 Hz, 0.25H),
7.78 (d, J = 8 Hz, 0.75H), 7.35 (m, 2H), 7.17 (m, 1H), 6.99 (t, D2O
exch, J = 5 Hz, 0.75H), 6.78 (t, D2O exch, J = 5 Hz, 0.25H), 4.96 (s,
1.5H), 4.78 (s, 0.5H), 4.41 (s, 0.5H), 4.21 (q, J = 7 Hz, 0.5H), 4.08 (q,
Boc-OEt PNA monomer 12a (429 mg, 87%): slightly yellow foam;
1H NMR (DMSO-d6) δ 8.85 (s, D2O exch, 1H), 8.09 (s, 1H), 7.72 (d,
J = 8 Hz, 1H), 7.65 (m, 1H), 7.44 (dd, J = 8, 8 Hz, 1H), 7.24 (dd, J =
8, 8 Hz, 1H), 6.94 (t, D2O exch, J = 5.5 Hz, 0.75H), 6.75 (t, D2O exch,
J = 5.5 Hz, 0.25H), 4.73 (s, 1.5H), 4.55 (s, 0.5H), 4.31 (s, 0.5H), 4.18
(q, J = 7 Hz, 0.5H), 4.08 (q, J = 7 Hz, 1.5H), 4.04 (s, 1.5H), 3.41 (m,
1.5H), 3.32 (m, 0.5H), 3.18 (q, J = 6 Hz, 1.5H), 3.02 (q, J = 6 Hz,
0.5H), 1.38 (s, 9H); 1.24 (t, J = 7 Hz, 0.75H), 1.18 (t, J = 7 Hz,
2.25H); 13C NMR (DMSO-d6) δ 169.3 (minor), 169.0 (major), 167.7
(minor), 167.4 (major), 159.0 (major + minor), 155.7 (major), 155.5
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(minor), 153.8 (major + minor), 148.2 (minor), 148.1 (major), 136.5
(major + minor), 132.6 (major + minor), 128.6 (major + minor),
128.2 (major), 128.0 (minor), 120.6 (major + minor), 85.5 (major +
minor), 78.0 (major), 77.8 (minor), 61.1 (minor), 60.5 (major), 49.1
(minor), 48.8 (major), 47.9 (major + minor), 47.0 (major + minor),
38.2 (major + minor), 28.2 (major + minor), 14.0 (major + minor);
HRMS (ESI) m/z found 630.0581 [M + H]+ (calcd 630.0563 for
C23H30Br2N5O6).
minor), 129.7 (major + minor), 128.6 (minor), 128.5 (minor) 128.4
(2 × major), 128.2 (minor), 128.1 (major + minor), 127.9 (major),
122.6 (major + minor), 93.7 (major + minor), 83.2 (major + minor),
78.1 (major), 77.8 (minor), 66.4 (minor), 66.0 (major), 49.8 (major +
minor), 48.0 (major + minor), 47.1 (major + minor), 38.2 (major +
minor), 28.2 (major + minor), 27.5 (major + minor); HRMS (ESI)
m/z found 792.1244 [M + H]+ (calcd 792.1222 for C33H40Br2N5O8).
Ni-Mediated Cyclization of Boc-Protected Bis-brominated
PNA Monomer 12b and 12d. PPh3 (230 mg, 0.88 mmol), NiCl2
(30 mg, 0.23 mmol), and Zn dust (227 mg, 3.47 mmol) in dry DMF
(1 mL) were used to form the Ni catalyst as described above. Separate
solutions of Boc-protected bis-brominated PNA monomers 12b (169
mg, 0.23 mmol) and 12d (185 mg, 0.23 mmol) in dry DMF (1 mL)
were added dropwise over a period of ca. 3 min to the separate
solutions of the freshly prepared Ni catalyst (N2 atmosphere). The
mixtures were stirred for 30 min at 80 °C (N2 atmosphere), were
cooled to room temperature, were diluted with 4% EDTA solution (40
mL), and were extracted with EtOAc (20 + 3 × 10 mL). Combined
organic extracts were washed with brine (2 × 50 mL), dried, and
concentrated. The residues was subjected to FCC purification on 25 g
SiO2, CH2Cl2/MeOH (95:5), later replaced with CH2Cl2/MeOH
(9:1). Evaporation of the eluates afforded 5,6-benzopC PNA
monomers 11a and 11c as white solid residue (36 mg, 33%).
Boc-OBn PNA monomer 12c (425 mg, 79%): slightly yellow foam;
1H NMR (DMSO-d6) δ 8.87 (s, D2O exch, 1H), 8.09 (s, 0.75H), 8.05
(s, 0.25H), 7.72 (d, J = 8 Hz, 1H), 7.66 (d, J = 8 Hz, 1H), 7.40 (m,
6H); 7.24 (dd, J = 8, 8 Hz, 1H), 6.97 (t, D2O exch, J = 5.5 Hz, 0.75H),
6.77 (m, D2O exch, 0.25H), 5.21 (s, 0.5 H); 5.13 (s, 1.5 H); 4.76 (s,
1.5H), 4.59 (s, 0.5H), 4.41 (s, 0.5H), 4.13 (s, 1.5H), 3.44 (m, 1.5H),
3.34 (m, 0.5H), 3.19 (m, 1.5H), 3.01 (m, 0.5H), 1.38 (s, 6.75H), 1.37
(s, 2.25 H); 13C NMR (DMSO-d6) δ 172.1 (major + minor), 169.3
(minor), 168.9 (major), 167.8 (minor), 167.5 (major), 164.6 (major +
minor), 159.0 (major + minor), 155.8 (major), 155.6 (minor), 153.9
(major + minor), 148.1 (major + minor), 136.5 (major), 136.1
(minor), 135.8 (major), 135.6 (minor), 132.6 (major + minor), 128.6
(major + minor), 128.5 (2 × major + minor), 128.4 (minor), 128.2
(major + minor), 128.1 (major), 128.0 (2 × minor), 127.9 (major),
120.6 (major + minor), 85.6 (major + minor), 78.1 (major), 77.8
(minor), 66.0 (major), 65.4 (minor), 50.0 (major), 49.1 (minor), 48.8
(major), 48.3 (minor), 48.0 (major), 47.0 (minor), 38.2 (major +
minor), 28.3 (minor), 28.2 (major); HRMS (ESI) m/z found
692.0710 [M + H]+ (calcd 692.0719 for C28H32Br2N5O6).
Boc-OEt 5,6-benzopC PNA monomer 11a (36 mg, 33%): colorless
solid, for the complete spectral characterization see above.
Boc-OBn 5,6-benzopC PNA monomer 11c (38 mg, 31%):
1
colorless solid; H NMR (DMSO-d6) δ 11.65 (br s, D2O exch, 1H),
8.76 (s, 0.75H), 8.73 (s, 0.25H), 7.80 (d, J = 8 Hz, 0.25H), 7.77 (d, J =
7.5 Hz, 0.75H), 7.37 (m, 7H); 7.17 (m, 1H), 7.01 (t, D2O exch, J = 5.5
Hz, 0.75H), 6.80 (t, D2O exch, J = 5 Hz, 0.25H), 5.24 (s, 0.5H), 5.13
(s, 1.5H), 4.99 (s, 1.5H), 4.82 (s, 0.5H), 4.51 (s, 0.5H), 4.17 (s, 1.5H),
3.53 (t, J = 6.5 Hz, 1.5H), 3.35 (m, 0.5H), 3.26 (q, J = 6 Hz, 1.5H),
3.05 (q, J = 6.5 Hz, 0.5H), 1.39 (s, 6.75H); 1.36 (s, 2.25H); 13C NMR
(DMSO-d6) δ 169.5 (minor), 169.0 (major), 168.1 (minor), 167.8
(major), 162.2 (major + minor), 155.8 (major + minor), 155.1 (major
+ minor), 142.9 (major + minor), 139.9 (major + minor), 135.8
(major), 135.6 (minor), 128.5 (2 × minor), 128.4 (2 × major), 128.2
(minor), 128.1 (major + minor), 127.9 (major), 126.6 (major +
minor), 121.3 (major + minor), 120.4 (major + minor), 119.9
(minor), 119.8 (major), 111.3 (major + minor), 103.5 (major +
minor), 78.1 (major), 77.8 (minor), 66.6 (minor), 66.0 (major), 50.8
(minor), 50.5 (major), 49.3 (minor), 48.1 (major), 47.2 (major +
minor), 38.4 (major + minor), 28.2 (major + minor); HRMS (ESI)
m/z found 534.2330 [M + H]+ (calcd 534.2353 for C28H32N5O6).
Hydrogenolytic Removal of the Bn Group from the
Monomer 11c. 10% Pd/C (50 mg) was added to a solution of the
Boc-OBn 5,6-benzopC PNA monomer 11c (55 mg, 0.1 mmol) in
MeOH (5.5 mL) and THF (1.5 mL). The mixture was vigorously
stirred for 20 min at rt in an atmosphere of H2 (balloon filled with H2),
the catalyst was filtered off using a Celite pad, the filter was washed
with MeOH, and the filtrate was concentrated to leave the Boc-OH
5,6-benzopC PNA monomer 11b (40 mg, 88%) as a slightly yellow
solid. For the complete spectral characterization see above.
When the reaction mixture (using the same amount of starting
material 11c) was stirred for 2 h at rt under in H2 atmosphere (balloon
filled with H2), a 1:1 mixture of the desired Boc-OH 5,6-benzopC
PNA monomer 11b and the product of the cytosine ring
overreduction (compound 14) was obtained in quantitative yield
(44 mg) upon removal of the catalyst by filtration through a Celite
plug. The mixture of 11c and 14 (41 mg, 0.092 mmol) was dissolved
in MeOH (3 mL), water (1 mL), and DMF (100 μL), and 10% Pd/C
(25 mg) was added. The mixture was vigorously stirred for 18 h at rt in
the atmosphere of O2 (balloon filled with O2), the catalyst was filtered
off (Pasteur pipet with a cotton plug), and the filtrate was
concentrated to leave the Boc-OH 5,6-benzopC PNA monomer 11b
(34 mg, 84%) as a slightly yellow solid. For the complete spectral
characterization see above.
Boc-Protection of Bis-brominated PNA Monomers 12a and
12c. Separate solutions of bis-brominated PNA monomers 12a (413
mg, 0.65 mmol) and 12c (451 mg, 0.65 mmol), Boc2O (286 mg, 1.31
mmol), and DMAP (8 mg, 0.07 mmol) in dry dioxane (12a, 3.5 mL;
12c, 2.5 mL) were stirred for 18 h at 50 °C (monomer 12a) or 48 h at
70 °C (monomer 12c). The mixtures were cooled to rt and were
diluted with brine (50 mL). The aqueous phases were extracted with
EtOAc (2 × 30 mL), and combined organic extracts were dried and
concentrated. The residue was subjected to FCC on 60 g SiO2,
CH2Cl2/MeOH (95:5) (conjugate 12b) or 40 g SiO2, CH2Cl2/MeOH
(98:2) (conjugate 12d). Evaporation of the eluates afforded Boc-
protected bis-brominated PNA monomers 12b and 12d.
Boc-protected Boc-OEt PNA monomer 12b (338 mg, 71%):
slightly yellow foam; 1H NMR (DMSO-d6) δ 8.47 (s, 1H), 7.73 (dd, J
= 8, 1.5 Hz, 1H), 7.45 (ddd, J = 8, 8, 1.5 Hz, 1H), 7.31 (m, 2H), 6.95
(t, D2O exch, J = 6 Hz, 0.75H), 6.74 (t, D2O exch, J = 6 Hz, 0.25H),
4.87 (s, 1.5H), 4.69 (s, 0.5H), 4.32 (s, 0.5H), 4.18 (q, J = 7 Hz, 0.5H),
4.08 (q, J = 7 Hz, 1.5H), 4.05 (s, 1.5H), 3.42 (m, 1.5H), 3.32 (m,
0.5H), 3.18 (q, J = 6 Hz, 1.5H), 3.01 (q, J = 6 Hz, 0.5H), 1.45 (s, 9H);
1.37 (s, 9H); 1.24 (t, J = 7 Hz, 0.75H), 1.18 (t, J = 7 Hz, 2.25H); 13C
NMR (DMSO-d6) δ 169.2 (minor), 168.8 (major), 166.8 (minor),
166.4 (major), 163.7 (major + minor), 155.7 (major + minor), 153.7
(major + minor), 152.2 (minor), 152.1 (major), 150.5 (major +
minor), 139.3 (major + minor), 133.2 (major + minor), 130.5 (major
+ minor), 129.7 (major + minor), 128.6 (major + minor), 122.6
(major + minor), 93.6 (major + minor), 83.2 (major + minor), 78.0
(major), 77.8 (minor), 61.2 (minor), 60.5 (major), 50.1 (minor), 49.8
(major), 49.2 (minor), 47.9 (major), 47.02 (major + minor), 38.9
(major), 38.2 (minor), 28.1 (major + minor), 27.5 (major + minor),
14.0 (major + minor); HRMS (ESI) m/z found 730.1107 [M + H]+
(calcd 730.1087 for C28H38Br2N5O8).
Boc-protected Boc-OBn PNA monomer 12d (232 mg, 45%):
slightly yellow foam; 1H NMR (DMSO-d6) δ 8.47 (s, 0.75H), 8.45 (s,
0.25H), 7.73 (d, J = 8 Hz, 1H), 7.38 (m, 8H); 6.97 (t, D2O exch, J =
5.5 Hz, 0.75H), 6.76 (t, D2O exch, J = 5.5 Hz, 0.25H), 5.21 (s, 0.5H),
5.13 (s, 1.5H), 4.90 (s, 1.5H), 4.74 (s, 0.5H), 4.42 (s, 0.5H), 4.14 (s,
1.5H), 3.44 (m, 1.5H), 3.34 (m, 0.5H), 3.20 (m, 1.5H), 3.02 (m,
0.5H), 1.45 (s, 9H); 1.37 (s, 6.75H); 1.36 (s, 2.25H); 13C NMR
(DMSO-d6) δ 169.2 (minor), 168.8 (major), 166.8 (minor), 166.5
(major), 163.8 (major), 163.7 (minor), 162.3 (major + minor), 155.8
(major + minor), 153.7 (major + minor), 152.2 (minor), 152.1
(major), 150.6 (major + minor), 139.4 (major + minor), 135.7
(major), 135.5 (minor), 133.3 (major + minor), 130.5 (major +
Cu-Mediated Cyclization of tert-Butyl N4-(2′-Halophenyl)-5-
bromocytosin-1-ylacetates. Cu wire (46 mg, 0.72 mmol) and Et3N
(140 μL, 1 mmol) were added to a solution of tert-butyl N4-(2′-
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The Journal of Organic Chemistry
Article
bromophenyl)-5-bromocytosin-1-ylacetate (3b, 115 mg, 0.25 mmol)
in DMF (1 mL). The mixture was stirred for 48 h at 70 °C, cooled to
room temperature, diluted with 10% Na2SO3 solution (20 mL), and
extracted with EtOAc (20 + 10 mL). The combined organic extract
was washed with brine (2 × 30 mL), dried, and concentrated. The
residue was subjected to FCC purification on 25 g SiO2, hexanes/
acetone (5:1). Evaporation of the eluate afforded tert-butyl 1-oxo-4-
bromopyrimido[1,6-a]benzimidazol-2-ylacetate (4) as a slightly yellow
solid (72 mg, 76%). Treatment of tert-butyl N4-(2′-chlorophenyl)-5-
bromocytosin-1-ylacetate (3d, 104 mg, 0.25 mmol) was carried out for
48 h at 120 °C, affording 12 mg (13%) of the same product after the
workup procedure described above: 1H NMR (CDCl3) δ 8.39 (d, J = 8
Hz, 1H), 7.91 (d, J = 8 Hz, 1H), 7.51 (ddd, J = 8, 8, 1 Hz, 1H), 7.44
(ddd, J = 8, 8, 1 Hz, 1H), 7.36 (s, 1H), 4.60 (s, 2H), 1.50 (s, 9H); 13C
NMR (CDCl3) δ 166.1, 146.7, 146.3, 143.5, 135.4, 131.0, 126.2, 124.6,
120.0, 115.3, 91.3, 83.9, 50.2, 28.0; HRMS (ESI) m/z found 378.0455
[M + H]+ (calcd 378.0453 for C16H17BrN3O3).
were dissolved in water (1 mL) and were subjected to HPLC
purification as described in the General Experimental Procedures. The
fractions containing the desired PNA sequences were frozen and
lyophilized, and the pure PNA sequences were dissolved in water to
achieve the final concentration of ca. 1 − 1.5 optimum density (OD)
units. Resulting solutions have been used for the hybridization studies
as described in the General Experimental Procedures.
PNA sequence 1A: HPLC (method B) tR 23.0 min; HRMS (ESI)
m/z found 2904.1881 [M
+
H]+ (calcd 2904.1971 for
C119H151N58O32).
PNA sequence 1B: HPLC (nethod B) tR 23.5 min; HRMS (ESI)
m/z found 3032.2772 [M
+
H]+ (calcd 3032.2921 for
C125H163N60O33).
PNA sequence 2: HPLC (method C) tR 19.2 min; HRMS (ESI) m/
z found 2928.2060 [M + H]+ (calcd 2928.2084 for C120H151N60O31).
ASSOCIATED CONTENT
■
Boc2O-Mediated Cyclization of tert-Butyl N4-(2′-Oxycarbo-
nylphenyl)-5-bromocytosin-1-ylacetate (3f). A mixture of tert-
butyl N4-(2′-oxycarbonylphenyl)-5-bromocytosin-1-ylacetate (3f, 212
mg, 0.5 mmol), Boc2O (218 mg, 1 mmol), and DMAP (6 mg, 0.05
mmol) in dry dioxane (3 mL) was stirred for 1 h at 60 °C. The
mixture was cooled to rt, diluted with brine (30 mL), and extracted
with EtOAc (2 × 20 mL). The combined organic extract was dried and
concentrated, and the residue was subjected to FCC purification on 30
g of SiO2, hexanes/acetone (2:1). Concentration of the eluate resulted
in the formation of crystals, hexanes were added, and the mixture was
set aside for 2 h at −10 °C. The crystals were filtered off, washed with
hexanes, and dried to give tert-butyl 1,10-dioxo-4-bromopyrimido[1,6-
a]quinqzolin-2-ylacetate (5) as slightly yellow crystals (158 mg, 78%):
S
* Supporting Information
1H and 13C NMR spectra, UPLC chromatograms, and HR-ESI
mass spectra of compounds 2a,b, 3a−f, 4−6, 8, 9, 11a,b, and
1
12a−d. H NMR spectrum of 1:1 mixture of compounds 11b
and 14. 11B NMR spectrum of compound 6. Detailed X-ray
characterization of compound 6 (CIF). UPLC chromatograms
and HR-ESI mass spectra of sequences 1A, 1B, and 2.
Fluorescence studies with sequences 1A, 1B, and 2.This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: robert.hudson@uwo.ca.
1
■
mp 210−212 °C; H NMR (CDCl3) δ 8.31 (dd, J = 8, 1.5 Hz, 1H),
7.78 (m, 2H), 7.49 (m, 1H), 7.38 (s, 1H) 4.42 (s, 2H), 1.51 (s, 9H);
13C NMR (CDCl3) δ 165.9, 159.6, 146.7, 145.7, 144.6, 138.1, 135.4,
127.9, 127.4 (2 × C), 120.6, 96.7, 83.8, 50.7, 28.0; HRMS (ESI) m/z
found 406.0403 [M + H]+ (calcd 406.0402 for C17H17BrN3O4).
Reaction of tert-Butyl-4-(1′,2′,4′-triazol-1′-yl)-5-bromocyto-
sin-1-ylacetate (8) with 2-Aminobenzeneboronic Acid Pinacol
Ester (16). A solution of tert-butyl 4-(1′,2′,4′-triazol-1′-yl)-5-
bromocytosin-1-ylacetate (7, 178 mg, 0.5 mmol) and 2-amino-
benzeneboronic acid pinacol ester (16, 126 mg, 0.58 mmol) in
dioxane (1.8 mL) and water (200 μL) was stirred for 24 h at 95 °C.
The mixture was cooled to room temperature, a small amount of water
(ca. 1−2 mL) was added, and the mixture was set aside for 2 h at 3 °C.
The precipitate which formed was filtered off with suction,
consecutively washed with water and cold (−10 °C) Et2O, and
dried. The mother liquor was concentrated to ca. one-half of its
original volume and set aside for 2 h at 3 °C, and the second crop of
the product was treated as described above to afford tert-butyl 1-oxo-
10-hydroxy-4-bromopyrimido[1,6-a]benzo[b]6-bora-1,3-diazin-2-yla-
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank The Natural Sciences and Engineering Research
Council (NSERC) of Canada for the financial support of this
work. We thank Mr. Jonathan Dube, Dr. Mathew Willans, and
Prof. Paul J. Ragogna for their help with the acquisition of 11B
NMR spectrum of compound 6 and for a fruitful discussion on
this topic. Dr. Paul Boyle is acknowledged for performing the
X-ray crystallographic analysis of compound 6 and subsequent
structure solution.
REFERENCES
■
1
(1) For recent reviews on fluorescent nucleobase analogues, see:
(a) Dodd, D. W.; Hudson, R. H. E. Mini Rev. Org. Chem. 2009, 6,
378−391. (b) Sinkeldam, R. W.; Greco, N. J.; Tor, Y. Chem. Rev. 2010,
110, 2579−2619. (c) Wilhelmsson, L. M. Q. Rev. Biophys. 2010, 43,
159−183.
cetate (6, 166 mg, 66%) as a slightly yellow solid: H NMR (DMSO-
d6) δ 10.46 (s, D2O exch, 1H), 8.48 (m, 1H), 8.26 (m, 1H), 8.02 (m,
1H), 7.45 (dd, J = 8, 8 Hz, 1H), 7.22 (dd, J = 7.5, 7.5 Hz, 1H) 4.37 (s,
2H), 1.26 (s, 9H); 13C NMR (DMSO-d6) δ 166.5, 155.6, 152.1, 147.9,
141.9, 135.1, 130.4 (2 × C), 124.1, 118.9, 85.8, 81.8, 51.0, 27.4; 11B
NMR (dioxane) δ 19.7; HRMS (ESI) m/z found 405.0497 [M + H]+
(calcd 405.0495 for C16H17BBrN3O4).
Synthesis of PNA Sequences 1A, 1B, and 2. PNA synthesis was
performed using the established protocols for the manual Boc PNA
synthesis.29 A Boc-Lys(2-Cl-Cbz)-modified resin (200 mg) was
downloaded with the T monomer to give the loading of 0.063
mmol/g.34 The resulting resin was divided into two parts: the first part
of the resin (110 mg, 6.89 × 10−6 mol) was used to prepare the PNA
sequences 1A and 1B, and the second part of the resin (70 mg, 4.30 ×
10−6 mol) was used to prepare the PNA sequence 2. Incorporation of
the unnatural monomer 11b required repeated (twice) coupling. After
the synthesis was finished, the conjugates were cleaved off from the
resin,29 and crude PNA oligomers were obtained by the precipitation
with cold (−10 °C) Et2O, giving 15 mg of the crude PNA sequence
1A, 14 mg of the crude PNA sequence 1B, and 25 mg of the crude
PNA sequence 2. Approximately 2−3 mg of the crude PNA sequences
(2) (a) Hudson, R. H. E.; Dambenieks, A. K.; Viirre, R. D. Synlett
2004, 2400−2402. (b) Wojciechowski, F.; Hudson, R. H. E. J. Am.
Chem. Soc. 2008, 130, 12574−12575. (c) Wojciechowski, F.; Hudson,
R. H. E. Org. Lett. 2009, 11, 4878−4881.
(3) Hu, J.; Dodd, D. W.; Hudson, R. H. E.; Corey, D. R. Bioorg. Med.
Chem. Lett. 2009, 19, 6181−6184.
(4) 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, 912−919.
(5) Torres, A. G.; Fabani, M. M.; Vigorito, E.; Williams, D.; Al-
Obaidi, N.; Wojciechowski, F.; Hudson, R. H. E.; Seitz, O.; Gait, M. J.
Nucleic Acids Res. 2012, 40, 2152−2167.
(6) Matteucci and von Krosigk previously reported the synthesis of a
DNA 5,6-benzopC monomer, which was termed a carbazole
nucleoside, and demonstrated its oligomerization. They did not
describe the fluorescence associated with this intrinsic fluorophore. We
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The Journal of Organic Chemistry
Article
have chosen to name this molecule as a congener of pyrrolocytosine
(pC) to emphasize the structural relationship between the two.
Matteucci, M. D.; von Krosigk, U. Tetrahedron Lett. 1996, 37, 5057−
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Tetrahedron 2002, 58, 9633−9695. (b) Ackermann, L. Synlett 2007,
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(27) For the recent review on boron-containing heterocycles, see:
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boron into DNA and RNA oligomers, see: (b) Martin, A. R.; Vasseur,
J. J.; Smietana, M. Chem. Soc. Rev. 2013, 42, 5684−5713. Somewhat
related heterocycles are also described in: Groziak, M. P.; Ganguly, A.
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(28) Eaton, D. F. Pure Appl. Chem. 1988, 60, 1107−1114.
(29) Methods in Molecular Biology. In Peptide Nucleic acids: Methods
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(11) For a recent review on Ni-catalyzed biaryl coupling, see:
(a) Yamaguchi, J.; Muto, K.; Itami, K. Eur. J. Org. Chem. 2013, 19−30.
We have adopted the procedure described in: (b) Colon, I.; Kelsey, D.
R. J. Org. Chem. 1986, 51, 2627−2637.
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Buchardt, O. J. Org. Chem. 1994, 59, 5767−5773.
(15) We were also interested in preparing the 5,6-benzopC PNA
monomer derived from the Fmoc-O-t-Bu PNA backbone for
automated PNA synthesis. Although this monomer was prepared in
small amounts (data not shown), the synthetic procedures were
neither reproducible nor scalable. The sensitive Fmoc group appeared
to be incompatible with the reaction conditions used for the Ni-
mediated cyclization. We have also attempted to carry out the Boc-
Fmoc replacement of the monomer 11b as described in: Hamzavi, R.;
Dolle, F.; Tavitian, B.; Dahl, O.; Nielsen, P. E. Bioconjugate Chem.
2003, 14, 941−94−54. However, the isolation by FCC was tedious
and afforded only traces of the desired product.
(30) Ortega, J.-A.; Blas, J. R.; Orozco, M.; Grandas, A.; Pedroso, E.;
Robles, J. Org. Lett. 2007, 9, 4503−4506.
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(32) Elmehriki, A. A. H.; Suchy, M.; Chicas, K. J.; Wojciechowski, F.;
́
Hudson, R. H. E. Artif. DNA 2014, submitted.
(33) The pyrimido[5,4-b]indole heterocyclic skeleton has been
previously synthesized. See, for example, ref 5 or: (a) Monge, A.;
Palop, J. A.; Goni, T.; Martinez-Crespo, F.; Recalde, I.; Fernandez-
̃
Alvarez, E. J. Heterocycl Chem. 1986, 23, 647−649. (b) Showalter, H.
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(34) The loading of the resin was determined as described in: Sarin,
V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Anal. Biochem. 1981,
117, 147−157.
(16) Boc-OBn PNA backbone 13 was prepared by KF/CELITE-
promoted alkylation of mono-Boc-ethylenediamine with benzyl
bromoacetate as described in: (a) Viirre, R. D.; Hudson, R. H. E.
Org. Lett. 2001, 3, 3931−3934. Mono-Boc-ethylenediamine was
prepared as described in: (b) Krapcho, A. P.; Kuell, C. S. Synth.
Commun. 1990, 20, 2559−2564. For the preparation of KF/CELITE,
see: (c) Ando, T.; Yamawaki, J. Chem. Lett. 1979, 45−46.
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(20) We assumed that a restoration of the heteroaromatic ring
system present in 5,6-benzopC PNA monomer 11b might be sufficient
as a driving force for the catalytic oxygenation to take place.
(21) (a) Watkins, B. E.; Kiely, J. S.; Rapoport, H. J. Am. Chem. Soc.
1982, 104, 5702−5708. (b) Johnson, D. C., II; Widlanski, T. S. Org.
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(23) For selected examples, see: (a) Smolii, O. B.; Muzychka, L. V.;
Chernega, A. N.; Drach, B. S. Zh. Obsch. Chim. 2002, 72, 1807−1812.
(b) Bakavoli, M.; Nikpour, M.; Ebrahimi, A. R.; Taghizadeh, A.;
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(24) Chenna, A.; Singer, B. Chem. Res. Toxicol. 1995, 8, 865−874.
(25) Somewhat related methodology based on the intramolecular
Cu-catalyzed reaction to form purine-fused nitrogen heterocycles has
been described recently; see: Qu, G. R.; Liang, L.; Niu, H. Y.; Rao, W.
H.; Guo, H. M.; Fossey, J. S. Org. Lett. 2012, 14, 4494−4497.
(26) For selected reviews on Suzuki−Miyaura coupling and some
other catalytic arylations, see: (a) Kotha, S.; Lahiri, K.; Kashinath, D.
3347
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