Unusual C7- versus Normal 5′-O-Dimethoxytritylation of 6-Arylpyrrolocytidine Analogs

Article abstract of DOI:10.1021/acs.joc.6b01584

Fluorescent deoxynucleosides possessing the modified bases 6-(2-benzo[b]furyl)- and 6-(2-furyl)pyrrolocytosine (BFpC and FpC) have been synthesized along with the quencher nucleosides possessing 6-{4-[(4-dimethylamino)azo]phenyl}pyrrolocytosine (DABCYLpC) and 6-(p-nitrophenyl)pyrrolocytosine (p-NO₂-PhpC) nucleobase analogs. Standard treatment of BFpC, FpC, DABCYLpC, and p-NO₂-PhpC with dimethoxytrityl chloride (DMT-Cl) led to the unusual substitution on the C7 of the pyrrolocytosine skeleton. The desired 5′-O-DMT-protected nucleoside analogs were synthesized from suitably protected 5′-O-DMT cytidines. Subsequent phosphitylation smoothly afforded BFpC-, FpC-, DABCYLpC-, and p-NO₂-PhpC-derived monomers suitable for standard oligonucleotide synthesis.

Full text of DOI:10.1021/acs.joc.6b01584

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Article
Unusual C7- versus Normal 5’-O-
Dimethoxytritylation of 6-Arylpyrrolocytidine Analogs
Mojmir Suchy, Christie Ettles, James A. Wisner, Augusto Matarazzo, and Robert H. E. Hudson
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01584 • Publication Date (Web): 16 Aug 2016
Downloaded from http://pubs.acs.org on August 18, 2016
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The Journal of Organic Chemistry
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Unusual C7- versus Normal ’-O-Dimethoxytritylation of 6-
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Arylpyrrolocytidine Analogs
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Mojmír Suchý, Christie Ettles, James A. Wisner, Augusto Matarazzo and Robert H. E. Hudson1,2*
1Department of Chemistry and 2The Centre for Advanced Materials and Biomaterials Research, The University of Western
Ontario, London ON, N6A 5B7 CANADA
ABSTRACT: Fluorescent deoxynucleosides possessing the modified bases 6ꢀ(2ꢀbenzo[b]furyl)ꢀ and 6ꢀ(2ꢀfuryl)ꢀpyrrolocytosine
(BFpC and FpC) have been synthesized along with the quencher nucleosides possessing 6ꢀ{4ꢀ[(4ꢀ
dimethylamino)azo]phenylpyrrolocytosine (DABCYLpC) and 6ꢀ(pꢀnitrophenyl)pyrrolocytosine (pꢀNO2ꢀPhpC) nucleobase analogs.
Standard treatment of BFpC, FpC, DABCYLpC and pꢀNO2ꢀPhpC with dimethoxytrityl chloride (DMTꢀCl) led to the unusual subꢀ
stitution on the C7 of the pyrrolocytosine skeleton. The desired 5’ꢀOꢀDMT protected nucleoside analogs were synthesized from
suitably protected 5’ꢀOꢀDMT cytidines. Subsequent phosphitylation smoothly afforded BFpCꢀ, FpCꢀ, DABCYLpCꢀ and pꢀNO2ꢀ
PhpCꢀderived monomers suitable for standard oligonucleotide synthesis.
INTRODUCTION
Within the framework of our continuing interest in the deꢀ
velopment of intrinsically fluorescent nucleobases1 we have
prepared and studied 6ꢀphenylpyrrolocytidine (PhpC, 1, Figure
1). PhpC obeys the WatsonꢀCrick base pairing rules with
guanine (Figure 1), is generally stabilizing to hybridization
when incorporated into DNA, RNA or PNA and possesses
useful probe properties.2 With the aim to tune the fluoresꢀ
cence properties associated with PhpC, we have, among other
structural modifications, also prepared 2ꢀbenzo[b]furylꢀ
(BFpC,3 3, Figure 1) and 2ꢀfurylꢀsubstituted (FpC, 4, Figure 1)
analogs for enhanced fluorescence properties along with the
nucleobase
possessing
a
4ꢀ[(4ꢀ
dimethylaminophenyl)azo]phenyl (DABCYLpC, 5, Figure 1)4
and 6ꢀ(pꢀnitrophenyl) (pꢀNO2ꢀPhpC, 2, Figure 1)2a moieties to
serve as fluorescence quenchers.5
Our intention to use the nucleoside analogs 2-5 for automatꢀ
ed DNA oligomerization required further synthetic transforꢀ
mations. The “gold standard” in automated DNA synthesis
requires the 5’ꢀOH group to be protected with the acid labile
dimethoxytrityl (DMT) group.6 The introduction of the DMT
group into nucleosides has been well documented;7 the reagent
used for this purpose, dimethoxytrityl chloride (DMTꢀCl), is
usually reacted with the unprotected nucleosides under basic,
aprotic conditions (pyridine as solvent) for nucleobases that
lack reactive amino groups.
Figure 1. Chemical structures of PhpC (1), pꢀNO2ꢀPhpC (2),
BFpC (3), FpC (4) and DABCYLpC (5).
As described below, direct treatment of nucleoside analogs
2-5 with DMTꢀchloride resulted in the formation of unusual
C7ꢀDMTꢀsubstituted arylpyrrolocytosine analogs. Detailed
spectral characterization (1 and 2D NMR) of C7ꢀDMTꢀ
substituted arylpyrrolocytosine analogs was carried out and
the results were compared to those acquired for genuine 5’ꢀOꢀ
DMTꢀsubstituted analogs. The structural assignment of C7ꢀ
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Page 2 of 12
DMTꢀsubstituted arylpyrrolocytosine analogs was also supꢀ
(3) can be found elsewhere.3 Characterization of the fluoresꢀ
cence properties associated with FpC (4) will be discussed
below.
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ported by quantum chemical calculations performed on the
aglycone possessing N1ꢀisopropyl substitution as a model for
the deoxyribose.
Treatment of nucleoside analogs 2-5 with DMT-Cl
The desired 5’ꢀOꢀDMTꢀsubstituted phosphoramidites deꢀ
rived from 2-5 suitable for automated DNA oligomerization
Nucleoside analogs 2-5 were treated with DMTꢀCl (Scheme
1) in pyridine at room temperature in the presence of Et3N.
The reaction was found to be unusually sluggish (requiring 24ꢀ
72 h), compared to the same reaction with natural nucleosides,
and in all four instances resulted in incomplete conversion of
starting nucleosides. The reaction of 2-5 with DMTꢀCl proꢀ
duced the DMTꢀprotected products 2a-5a in moderate (38ꢀ
52%, Scheme 1) yield after flash column chromatography
(FCC). Although 5’ꢀOꢀDMT protected nucleobases are
known to be very sensitive to acidic environments, the isolated
products 2a-5a failed to display this property. When analyzed
by thin layer chromatography (TLC), the spots associated with
2a-5a did not turn orange upon staining with 1 M HCl (exꢀ
pected due to DMT cation formation). Moreover, compounds
2a-5a remained stable, i.e. no DMT removal as judged by
TLC and high resolution mass spectrometry (HRꢀMS) analyꢀ
sis, even after prolonged exposure to strong acid (neat TFA,
12 h).
have been prepared by an alternate synthetic route.
The
phosphoramidite derived from FpC (4), Figure 1, was successꢀ
fully incorporated into DNA oligomers by means of automated
solid phase DNA synthesis. Hybridization studies with FpCꢀ
modified DNA oligomers revealed moderate duplex stabilizaꢀ
tion upon hybridization to the complementary strand as well as
fluorescence responsiveness to the perfectly matched compleꢀ
ment wherein diminution fluorescence signal was observed
(G:pC base pair, Figure 1).
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RESULTS AND DISCUSSION
Synthesis of nucleoside analogs 2-5
Nucleoside analogs 2ꢀ5 possessing substituted pyrrolocytoside
nucleobases have been obtained in one pot fashion via a reacꢀ
tion cascade involving a Sonogashira crossꢀcoupling with an
appropriate alkyne and subsequent 5ꢀendoꢀdig cyclization.2a
The synthesis of BFpC (3, Scheme 1) from protected cytosine
6a and 2ꢀethynylbenzo[b]furan (7, Scheme 1) has been deꢀ
scribed elsewhere.3
When PhpC (1)3 was treated with the DMTꢀCl under similar
conditions for a prolonged period of time (up to 10 days was
required), two products were obtained in low yields (1a, 25%;
1b, 19%, Scheme 2), their structures were assigned based on
the analogy with the NMR spectra acquired for 2a-5a, disꢀ
cussed below.
Scheme 1. Preparation of C7-DMT substituted nucleobase
modified nucleosides 2a-5a
Scheme 2. Reaction of PhpC (1) with DMT-Cl
Spectroscopic characterization of 2a-5a
Detailed spectroscopic characterization of 2a-5a was carried
out and the spectra were compared to those associated with
nucleoside analogs 2b-5b, prepared as described later. HRꢀ
MS spectra of 2a-5a were consistent with the presence of the
1
DMT group; this was further supported by the H NMR and
13C NMR spectra associated with 2a-5a. As an illustrative
1
example, the NMR spectra for the benzo[b]furyl derivatives
1
3a,b are shown in Figure 2. For comparison, selected H
NMR chemical shifts associated with compounds 2a-5a are
listed in Table 1.
pꢀNO2ꢀPhpC (2, Scheme 1), FpC (4, Scheme 1) and
DABCYLpC (5, Scheme 1) have been synthesized in the same
manner by reacting suitably protected cytosines 6a or 6b3 with
commercially available pꢀNO2ꢀphenylacetylene (10), 2ꢀ
ethynylfuran (8, prepared in two steps from fural using the
modified literature protocol)8 or DABCYLꢀmodified alkyne 9
(Scheme 1) developed recently by our laboratory.9 Subseꢀ
quent deprotection (Scheme 1) led to preparation of nucleoꢀ
base analogs 2-5 (Scheme 1) in reasonable overall yield (40ꢀ
52%), details in the Experimental Section. A thorough characꢀ
terization of the fluorescence properties associated with BFpC
Initially, upon isolation of 3a from column chromatography,
and expecting the normal 5’ꢀOꢀdimethoxytrityl ether, we were
pleased with the correspondence of the calculated formula
mass and observed HRꢀMS. However, we were perplexed by
1
aspects of the NMR spectrum. For example, the presence of
two D2O exchangeable signals in the spectrum for 3a at δ 5.26
ppm (d, J = 4.0 Hz, 1H) and 4.71 ppm (t, J = 5.5 Hz, 1H) imꢀ
plied that neither 5’ꢀ nor 3’ꢀ OH present in 3 [δ 5.30 ppm (d, J
= 5.5 Hz, 1H) and 5.15 ppm (t, J = 5.5 Hz, 1H), DMSOꢀD6]3
were converted to an ether (Figure 3, panels A and B and
Table 1). Another D2O exchangeable signal (N5ꢀH) associated
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with 3a was present at δ 11.74 ppm (s, 1H), discounting possiꢀ NMR spectra associated with 3a (or any of 2a-5a) our initial
suspicion was that the DMT group was attached to C95b which
was counterintuitive to the presumed sites of the heterocycle
susceptible to electrophilic substitution. Clearly, further strucꢀ
tural analysis was needed to resolve the structural assignment.
Our attempts to grow crystals of 2a-5a suitable for an Xꢀray
analysis were unsuccessful and we turned to 2D NMR specꢀ
troscopy (Figure 3). Nucleosides 3a and 5a were subjected to
further scrutiny while the structures of the remaining nucleoꢀ
sides (2a, 4a) were assigned by analogy.
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2
3
4
5
6
7
8
ble alkylation of the pyrroloꢀnitrogen (see the Supporting Inꢀ
formation). The presence of three D2O exchangeable signals
1
in H NMR spectra of 3a (and similarly for 2a-5a, Table 1)
and the lack of reactivity toward acid treatment was consistent
with CꢀC connectivity between the nucleobase and the DMT
group. Also notable in the spectrum was the absence of the
singlet typically attributable to the proton labelled H9 of the
phenylpyrrolocytosine moiety. This proton characteristically
possesses a chemical shift in the approximate range δ 8.50ꢀ
9
1
8.80 ppm.10 Since there are no such signals present in the H
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0.55
H9
0.50
A
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0
H5’
H3’
C H2C l2
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
Chemical Shift (ppm)
0.15
0.10
0.05
H9
B
D2O
C H2C l2
0
0.35
0.30
0.25
0.20
0.15
0.10
0.05
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
Chemical Shift (ppm)
H7
C
H9
H3’
C H2C l2
0
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
Chemical Shift (ppm)
H7
H9
D
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
D2O
C H2C l2
0
-0.01
-0.02
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
Chemical Shift (ppm)
1
Figure 2. Comparison of the low field region of H NMR spectra associated with 3a, panels A (DMSOꢀD6) and B (DMSOꢀD6 + D2O)
with that associated with 3b, panels C (DMSOꢀD6) and D (DMSOꢀD6 + D2O). Note that the signals at δ 5.26 ppm (d, J = 4.0 Hz, 1H) and
4.71 ppm (t, J = 5.5 Hz, 1H) associated with 3a (panel A) disappear upon the D2O shake (3’ and 5’ – OH groups, panel B). The signal at δ
5.45 ppm (d, J = 5.0 Hz, 1H) associated with 3b (panel C) disappears upon the D2O shake (3’ – OH group, panel D), as highlighted by the
red
boxes
and
arrows.
Resonances
due
to
H9
and
H7
protons
are
indicated
by
red
labels.
Below is shown the assignment for the benzo[b]furyl derivaꢀ
tive 3a while the spectra for 5a is presented in the Supporting
Information. The notion of CꢀC linkage of the DMT group
was supported by the presence of quaternary carbon in the
range δ 57.9ꢀ58.8 ppm (see in the Experimental Section and in
the Supporting Information) in the 13C NMR spectra of 3a
(and all of 2a-5a) as well as by the stability of analogs 2a-5a
under acidic conditions (no DMT group removal). All characꢀ
teristic signals due to the presence of deoxyribose subunit
were present in the H NMR spectrum associated with 2a-5a
(see the Supporting Information) thus ruling out the unlikely
derivatization of the carbohydrate moiety with the DMT group
1
The chemical shift of the anomeric carbon present in 3a (δ
87.2 ppm) and 5a (δ 86.6 ppm) was determined by HSQC
NMR as depicted in Figure 3 (3a, panel A, red arrows) or in
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4.62 (t, 1H)
the Supporting Information (5a). The HSQC NMR spectrum
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3
4
5
6
7
8
of 3a indicated that the proton exhibiting δ 7.45 ppm is correꢀ
lated with a carbon δ 139.8 ppm. Similarly, a proton with δ
7.20 ppm (5a) was found to be correlated with a carbon with δ
138.3 ppm, see the Supporting Information.
5.26 (d, 1H)
4.72 (t, 1H)
6.14 (t, 1H)
6.15 (t, 1H)
6.10 (t, 1H)
6.24 (t, 1H)
6.21 (t, 1H)
6.23 (t, 1H)
6.22 (t, 1H)
6.24 (t, 1H)
7.45 (s, 1H)
3a
4a
5a
1b
2b
3b
4b
5b
5.24 (d, 1H)
4.71 (t, 1H)
7.32 (s, 1H)
7.20 (s, 1H)
5.25 (d, 1H)
4.66 (t, 1H)
5.44 (d, 1H)
5.45 (d, 1H)
5.45 (d, 1H)
5.41 (d, 1H)
5.45 (d, 1H)
5.81 (s, 1H)
8.66 (s, 1H)
9
6.10 (s, 1H)
8.77 (s, 1H)
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5.92 (s, 1H)
8.75 (s, 1H)
5.67 (s, 1H)
8.59 (s, 1H)
5.89 (s, 1H)
8.71 (s, 1H)
aChemical shifts are given in ppm (DMSOꢀD6), only multiplicities
of the peaks are provided, for J constants see the Experimental
Section. bCompounds 1b-5b contain only 3’ꢀOH group. cComꢀ
pounds 1a-5a contain only H9 proton.
The possibility of the attachment of the DMT group to other
sites, such as the pendant aryl group present at C6 (pꢀNO2ꢀPh,
2ꢀfuryl, 2ꢀbenzo[b]furyl and DABCYL) is not consistent with
the observed spectra. For instance, two doublets δ 7.85 (d, J =
1
8.5 Hz, 2H) and 7.18 (d, J = 8.5 Hz, 2H) are present in the H
NMR spectrum of 2a (pꢀsubstituted benzene ring), while four
doublets (two pꢀsubstituted benzene rings) δ 7.78 (d, J = 9.0
Hz, 2H); 7.41 (d, J = 8.0 Hz, 2H); 7.04 (d, J = 8.0 Hz, 2H) and
1
6.83 (d, J = 9.0 Hz, 2H) are present in the H NMR spectrum
of 5a (see in the Experimental Section and in the Supporting
Information) indicating that both, pꢀNO2ꢀPh moiety present in
2a and DABCYL moiety present in 5a are intact. In totality,
these results indicate that the DMT group is attached to the C7
of the pyrrolocytosine moiety. Although competitive electroꢀ
philic addition to the heterocycle with ether formation is
somewhat surprising, a similar observation has been made
previously, wherein the reaction of indole with trityl chloride
in pyridine leads to the formation of Cꢀsubstituted 3ꢀtrityl inꢀ
dole as a sole product in 75% yield.11
Figure 3. HSQC correlations associated with 3a (panel A),
the key correlation is indicated by red arrows. HMBC correlaꢀ
tions associated with 3a (panel B), the key correlations are
indicated by red and blue arrows.
HMBC NMR spectra of 3a and 5a revealed the presence of
the correlation between the anomeric carbon and the proton at
δ 7.45 ppm (s, 1H, 3a, Figure 3, panel B, red arrows) and δ
7.20 ppm (s, 1H, 5a, Supporting Information). We have also
observed the HMBC correlations between the carbon with δ
139.8 ppm (3a) and the anomeric proton δ 6.14 (t, J = 6.5 Hz,
1H, 3a), Figure 3, panel B, blue arrows or the analogous correꢀ
lation for 5a between 13C δ 138.3 ppm and 1H δ 6.10 (t, J = 6.5
Hz, 1H see Supporting Information). Overall, the results of
2D NMR studies indicate that the proton at the C9 was not
substituted. In each of 2a-5a, the pyrroloꢀtype proton (H7) has
been replaced and the H9 experiences significant upfield shift
due the anisotropic shielding due to the DMT group.
Quantum chemical calculations
The unusual electrophilic attack on the nucleobase, which
was competitive with Oꢀdimethoxytritylation as observed for
PhpC (1, Scheme 2), was surprising since it was observed to
occur on the pyrrole ring irrespective of the nature of the 6ꢀ
substituent. The isolated yields of the electrophilic substituꢀ
tion products 2aꢀ5a were somewhat affected by the electron
richness character of the aromatic group, yet even the pꢀ
nitrophenylꢀsubstituted pyrrolcytosine gave the substitution
product over ether formation. In order to gain some insight on
the site of reactivity in the heterocyclic skeletons present in 1-
5 toward electrophilic aromatic substitution, quantum chemiꢀ
cal calculations were performed.
1
Table 1. Selected H NMR chemical shifts associated with
compounds 1a-5a and 1b-5b
The calculations were performed on the aglycone possessing
an isopropyl group attached to N1 rather than deoxyribose to
reduce the computational burden. The structures were geomeꢀ
try optimized at the HartreeꢀFock 6ꢀ311+G** level using their
lowest energy conformer (PM6) as a starting point. Surfaces
were constructed in Spartan '14 as ionization potential maps
Compound
3’ꢀ, 5’ꢀOHa,b
anomeric Ha
6.10 (t, 1H)
H7, H9a,c
5.24 (s, 1H)
4.66 (s, 1H)
5.22 (d, 1H)
7.19 (s, 1H)
1a
6.07 (t, 1H)
7.24 (s, 1H)
2a
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on electron density using the HꢀF output (see the Supporting
Fluorescence associated with FpC (4)
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2
3
4
5
6
7
8
Information). The calculations indicate that C7, the observed
site of substitution, possesses the lowest ionization potential in
almost every case. For example, PhpC (1, modeled with an
N1ꢀisopropyl, Figure 4) supports this hypothesis as well as the
above described structural assignment indicating that reactiviꢀ
ty occurs at C7 (pyrrole ring) of the nucleobase analog.
Characterization of the fluorescence properties associated
with FpC (4, Supporting Information) revealed that this strucꢀ
tural modification leads to a fluorophore (Φ 0.58, EtOH,
Stoke’s shift 73 nm) possessing properties comparable to
PhpC (1, Φ 0.61, EtOH, Stoke’s shift 78 nm) developed in our
laboratory previously.2a,3 Solvatochromaticity associated with
FpC (4, Supporting Information) also compared well with that
associated
with
PhpC
(1).3
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Figure 4. Ionization potential map (eV) superimposed to the 6ꢀ
phenylpyrrolcytosineꢀderived model compound.
Once the DMT group installs at the C7 additional derivitizaꢀ
tion of the 5’ꢀOH is not observed, and vice versa, mostlikely
due to steric hindrance. This reactivity of nucleobase modified
nucleosides analogs is rather novel; to the best of our
knowledge it has only been observed once previously for a
tricyclic analog of acyclovir with the stronger electrophile
trityl chloride.12
Synthesis of phosphoramidites 2c-5c
To obtain monomers 2c-5c suitable for automated DNA oliꢀ
gomerization, a different synthetic strategy was utilized. N4ꢀ
benzoylꢀ5ꢀiodoꢀdeoxycytosine (6c), prepared by iodination of
deoxycytosine13 followed by global benzoylation and subseꢀ
quent 3’ꢀ, 5’ꢀOꢀdebenzoylation,14 was reacted with DMTꢀCl
(Scheme 3). The reaction proceeded without difficulty and 5’ꢀ
OꢀDMTꢀN4ꢀbenzoylꢀ5ꢀiodoꢀdeoxycytosine (6d) was obtained
in 68% yield after FCC. The spectra associated with 6d were
fully consistent with its structure. A key step in the syntheꢀ
sis,15 a reaction cascade involving a Sonogashira crossꢀ
coupling between 6d and alkynes 7-10, followed by subseꢀ
quent 5ꢀendoꢀdig cyclization was found to proceed smoothly,
furnishing the 5’ꢀOꢀDMT protected pyrrolocytosine analogs
2b-5b in reasonable yields (45ꢀ59%, Scheme 3) after FCC
purification (see the Supporting Information for experimental
details). 1H, 13C NMR and HRꢀMS spectra were fully conꢀ
sistent with the structure of 2b-5b (see Table 1 for selected
chemical shifts and Supporting Information for the full specꢀ
tra).
Treatment of 2b-5b with 2ꢀcyanoethylꢀN,N,N’,N’ꢀ
tetraisopropylphosphorodiamidite (11, Scheme 3) in the presꢀ
ence of tetrazole16 proceeded smoothly affording the phosphoꢀ
ramidites 2c-5c in reasonable yield (53ꢀ64%) after the purifiꢀ
cation. We found the purification of 3c-5c by preparative
TLC (PTLC) more effective as opposed to FCC, at removal of
Hꢀphosphonate impurities (δ ca. 5ꢀ15 ppm) as judged by 31P
NMR spectroscopy. The PTLC purification failed for the pꢀ
NO2ꢀPhpCꢀderived phosphoramidite 2c; however, it was puriꢀ
fied by precipitation from CH2Cl2/hexanes mixture as deꢀ
scribed in the Experimental. A complete spectral characterizaꢀ
tion of phosphoramidites 2c-5c can be found in the Supporting
Information. By utilizing the above described synthetic methꢀ
odology, the phosphoramidites 2c-5c have been synthesized
that are suitable for automated DNA oligomerization.6
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Scheme 3. Preparation of 5’-O-DMT-nucleobase analogs 2b-5b and corresponding phosphoramidites 2c-5c
1
2
3
4
5
6
7
8
9
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11
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GTAGꢀATXꢀACT
GTAGꢀATXꢀACT
GTAGꢀATCꢀXCT
C
FpC
C
35
40
39
< 20, all
MMs
DNA oligomerization of phosphoramidite 4c and hy-
bridization studies
+5.0
+4.0
< 20, all
MMs
FpCꢀderived phosphoramidite 4c was successfully incorpoꢀ
rated into DNA oligomers. The DNA sequences have been
assembled by automated solid phase DNA synthesis.6 Two
sequences have been prepared as follows: GTAGꢀATXꢀACT
(5’ꢀO → 3’ꢀO, Sequence 1, X = FpC); GTAGꢀATCꢀXCT (5’ꢀ
O → 3’ꢀO, Sequence 2, X = FpC). The sequences were puriꢀ
fied by high performance liquid chromatography (HPLC) and
were characterized by MS (see the Supporting Information for
details).
2o (MMA)
22 (MMC)
24 (MMT)
23 (MMA)
28 (MMC)
35 (MMT)
GTAGꢀATCꢀXCT
FpC
43
aMeasurements were carried out as described in the Experiꢀ
mental Section. Each strand was present at 1 ꢁM in a buffer conꢀ
taining 100 mM NaCl, 10 mM, MgCl2, 10 mM Na2HPO4, 0.1
Hybridization studies with Sequences 1 and 2 were perꢀ
formed at 10ꢀ6 M in a phosphate buffer (pH 7) containing 10
mM MgCl2. Replacement of natural C with FpC resulted in
moderate duplex stabilization (Sequence 1, +4.0 °C, Sequence
2, +5.0 °C) as indicated in Table 2 which are similar to the
results obtained for PhpC (1).15
b
mM EDTA, pH 7.0. MM = mismatch.
The fluorescence response associated with DNA duplexes
featuring Sequences 1 and 2 was found to be somewhat sensiꢀ
tive to the presence of mismatch (ca. two fold fluorescence
increase compared to complementary strand), although no
fluorescenceꢀbased mismatch discrimination was observed
with FpCꢀmodified DNA oligomers. The results of these studꢀ
ies are depicted in Figure 5. Although not studied comprehenꢀ
sively herein, there appears to be some sequence context deꢀ
pendence on the fluorescence response. Sequence 2, which
has deoxycytidine nearest neighbors to the fluorescent nucleoꢀ
tide, shows ca. 65% reduction in fluorescence comparing the
duplex to the single strand while sequence 1, with a deoxyꢀ
adenosine neighbor shows ca. 45% reduction in fluorescence.
Table 2. DNA Hybridization studies
Sequencea
X
Tm
MMb DNA
Tm (° C)
ꢀ Tm
(° C)
(5’ → 3’)
(° C)
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matched sequences for mismatched sequences by selective
1
quenching of the fluorescence signal, in the case of the former.
2
3
4
EXPERIMENTAL SECTION
5
General experimental protocols
6
7
8
9
Reagents were commercially available unless otherwise statꢀ
ed and all solvents were reagent grade unless otherwise stated.
Dry solvents (CH2Cl2, dioxane, DMF, Et2O, THF) for chemiꢀ
cal synthesis were obtained by drying on activated Al2O3 colꢀ
umns in a solvent purification system or by drying over 3Å
molecular sieves (Et3N, MeCN, pyridine). Spectroscopic
grade EtOH has been used to perform spectroscopic studies.
Solvents were removed under reduced pressure in a rotary
evaporator and organic extracts were dried over Na2SO4. Reꢀ
action mixtures involving air sensitive reagents [Pd(PPh3)4,
CuI] were degassed by repeated freezeꢀpumpꢀthaw cycles
using dry N2. FCC was carried out using silica gel (SiO2;
mesh size 230 ꢀ 400 Å). Thinꢀlayer chromatography (TLC)
was carried out on an Al backed silica gel plate with comꢀ
pounds visualized by 1 M HCl, phosphomolybdic acid stain,
and UV light. Preparative thin layer chromatography (PTLC)
was carried out on glass backed silica gel plates (20 × 20 cm),
layer thickness 1000 ꢁm; compounds were visualized by UV
light. 1H, 13C and 31P NMR spectra were recorded on a 400
MHz spectrometer. Chemical shifts (δ) are reported in parts
per million, and are referenced as follows: CD2Cl2 (5.32 ppm),
10
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1
DMSOꢀD6 (2.49 ppm) for H NMR and CD2Cl2 (54.0 ppm),
DMSOꢀD6 (39.5 ppm) for 13C NMR (100 MHz). Ultra perꢀ
formance liquid chromatography (UPLC) was performed usꢀ
ing a BEH C18 column (particle size 1.7ꢁm; 1.0 id × 100 mm)
and HRꢀESIꢀMS and diode array UV detectors. Mobile phase:
Method A: 100% H2O – 100% MeCN (both solvents containꢀ
ing 0.1% HCOOH) over 5 min, linear gradient, then 100%
MeCN over 2 minutes, flow rate 0.1 mL/min. Method B:
100% H2O – 100% MeOH over 5 min, linear gradient, then
100% MeOH over 2 minutes, flow rate 0.1 mL/min. HPLC
purification of the DNA oligomers was carried out on a Miꢀ
crosorbꢀMW C18 100 Ǻ column (4.6 id × 250 mm), using 0.05
M ammonium acetate buffer (AAB, pH 6.5). Mobile phase:
Method C (DMTꢀprotected Sequence 1), 0 min, 99% AAB –
1% MeCN to 60% AAB – 40% MeCN over 30 min, linear
gradient, then 60% AAB – 40% MeCN over 2 min; 1 mL/min.
Method D (DMTꢀprotected Sequence 2), 0 min, 99% AAB –
1% MeCN to 46% AAB – 54% MeCN over 27 min, linear
gradient; 1 mL/min. Method E (Sequence 1), 0 min, 99%
AAB – 1% MeCN to 50% AAB – 50% MeCN over 25 min,
linear gradient; 1 mL/min. Method F (Sequence 2), 0 min,
99% AAB – 1% MeCN to 48% AAB – 52% MeCN over 16
min, linear gradient; 1.2 mL/min. Mass spectra (MS) were
obtained on a mass spectrometer using electrospray ionisation
(ESI) and timeꢀofꢀflight (TOF) analyzer. UVꢀVIS spectra
were acquired using an UVꢀVIS spectrophotometer equipped
with temperature controlled cell holder. Steady state fluoresꢀ
cence spectra were acquired using a PTI quantmaster fluoꢀ
rimeter. All fluorescence measurements were performed using
a 1 cm wide fourꢀsided quartz cuvette. UVꢀVIS measurements
were performed using a 1 cm twoꢀsided quartz glass cuvette.
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57
58
59
60
Figure 5. Fluorescence hybridization studies with FpCꢀ
modified DNA sequences 1 (top) and 2 (bottom); MM = misꢀ
match
CONCLUSIONS
In summary, we have described an unusual aromatic electroꢀ
philic substitution of pyrrolocytosine analogs 1-5 with diꢀ
methoxytrityl chloride (DMTꢀCl) leading to the formation of
C7ꢀDMTꢀsubstituted nucleoside analogs 1a-5a. These obserꢀ
vations prompt us to make a cautionary note for other chemists
involved in development of nucleobase analogs, as we believe
that the unusual reactivity between 1-5 and DMTꢀCl observed
by us may complicate the routine DMTꢀprotection of other
electron rich aromatic and heteroaromatic nucleobase analogs.
This reactivity also suggests a route for the preparation of othꢀ
erwise difficult to access C7ꢀsubstituted pyrrolocytosine anaꢀ
logs via for example electrophilic halogenation and further
derivatization.
A simple reordering of the steps in the synthetic scheme
permitted the preparation of the desired 5’ꢀOꢀDMTꢀprotected
nucleoside analogs 2b-5b; subsequently a sufficient amount of
phosphoramidites 2c-5c for automated DNA oligomerization
were obtained.
FpC (4) was found to be a relatively bright blue fluorophore
possessing the properties comparable to those observed for the
PhpC (1) described previously.2a,3 FpCꢀderived phosphoꢀ
ramidite 4a was successfully incorporated into DNA oligoꢀ
mers by means of automated solid phase DNA synthesis. The
incorporation of the FpCꢀmodified nucleobase was found to
have moderate stabilizing effect (ca. +5 °C) for an internal site
of substitution upon hybridization to the complementary
strands of DNA and was able to discriminate perfectly
Reaction of PhpC (1) with DMT-Cl
A flask containing PhpCError! Bookmark not defined. (1,
52 mg, 0.16 mmol) and DMTꢀCl (70 mg, 0.21 mmol) was
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flushed with N2, followed by the addition of dry pyridine (1 were washed with brine (3 × 50 mL), were dried and were
1
2
3
4
5
6
7
8
mL) and dry Et3N (160 ꢁL, 1.14 mmol). The reaction mixture
was stirred at RT (room temperature, N2 atmosphere) for 240 h
(ten days). The reaction was quenched by the addition of
MeOH (100 ꢁL) and was coevaporated with toluene (3 × 60
mL). The residue was subjected to PTLC, eluted with
CH2Cl2/MeOH/NH4OH (360:39:1). The bands containing the
products were carved off the plates, extracted with
CH2Cl2/MeOH/NH4OH (380:19:1), the SiO2 was filtered off
and the filtrates were concentrated, two fractions were obꢀ
tained; the more polar, 7ꢀCꢀDMTꢀPhpC (1a, 25 mg, 25%) and
the less polar 5’ꢀOꢀDMTꢀPhpC (1b, 19 mg, 19%).
concentrated. The residues were subjected to FCC on 30 g
SiO2, eluted with Et2O/acetone (2:1) later replaced with
Et2O/acetone (1:1, 6a + 8), 40 g SiO2, eluted with CH2Cl2 later
replaced with CH2Cl2/MeOH (95:5, 6b + 9) or 80 g SiO2, elutꢀ
ed with CH2Cl2/MeOH (95:5, 6b + 10). Evaporation of the
eluates afforded the desired intermediates 2,3ꢀdiꢀO-TBDMSꢀ
p-NO2ꢀPhpC (orange solid, 350 mg, 62%), 2,3ꢀdiꢀOAcꢀFpC
(yellow solid, 257 mg, 60%) and 2,3ꢀdiꢀO-TBDMSꢀ
DABCYLpC (red solid, 423 mg, 59%).
9
2,3ꢀDiꢀO-AcꢀFpC (257 mg, 0.64 mmol) was suspended in
MeOH (8 mL) and the mixture was cooled to 0 ˚C. K2CO3
(221 mg, 1.6 mmol) was added and the mixture was stirred for
1 h at 0 ˚C. The solvent was evaporated and the residue was
subjected to FCC on 25 g SiO2, eluted with EtOAc/MeOH
(95:5) later replaced with EtOAc/MeOH (9:1). Evaporation of
the eluate afforded FpC [4, 176 mg, 87% (52%, based on 6a)].
Yellow solid, UPLC (method A) tR = 3.75 min. 1H NMR
(DMSOꢀD6) δ 11.81 (s, D2O exch., 1H); 8.67 (s, 1H); 7.80 (d,
J = 2.0 Hz, 1H); 6.93 (d, J = 3.5 Hz, 1H); 6.63 (dd, J = 3.0, 1.5
Hz, 1H); 6.44 (s, 1H); 6.24 (t, J = 6.0 Hz, 1H); 5.28 (d, D2O
exch., J = 4.5 Hz, 1H); 5.12 (t, D2O exch., J = 5.0 Hz, 1H);
4.24 (m, 1H); 3.89 (m, 1H); 3.66 (m, 2H); 2.36 (m, 1H); 2.03
(m, 1H). 13C NMR (DMSOꢀD6) δ 159.6, 153.8, 146.0, 143.7,
136.3, 130.7, 112.0, 108.7, 107.8, 95.3, 87.9, 87.0, 69.9, 61.0,
41.4. HRMS (ESI) m/z: found 318.1079 [M + H]+ (calcd.
318.1090 for C15H16N3O5).
10
11
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22
23
24
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60
7ꢀCꢀDMTꢀPhpC (1a), yellow solid, UPLC (method A) tR =
5.06 min. 1H NMR (DMSOꢀD6) δ 11.29 (s, D2O exch., 1H);
7.19 (s, 1H); 7.02 (m, 14H); 6.63 (m, 4H); 6.10 (t, J = 6.5 Hz,
1H); 5.24 (br s, D2O exch., 1H); 4.66 (br s, D2O exch., 1H);
4.45 (m, 1H); 3.92 (m, 1H); 3.69 (m, 1H); 3.68 (s, 6H); 3.03
(m, 1H); 2.21 (m, 1H); 1.45 (m, 1H). 13C NMR (DMSOꢀD6) δ
157.6, 157.2, 157.1, 153.4, 145.5, 138.0, 137.6, 137.3, 137.2,
132.8, 131.4, 131.3, 130.2, 129.1, 127.2, 127.0, 125.9, 116.4,
112.5, 109.8, 87.6, 86.3, 70.7, 61.9, 58.0, 54.9 (2 × C), 40.8.
HRMS (ESI) m/z: found 630.2626 [M + H]+ (calcd. 630.2604
for C38H36N3O6).
5’ꢀOꢀDMTꢀPhpC (1b), yellow solid, UPLC (method A) tR =
5.59 min. 1H NMR (DMSOꢀD6) δ 11.80 (s, D2O exch., 1H);
8.66 (s, 1H); 7.69 (m, 2H); 7.44 (m, 4H); 7.30 (m, 8H); 6.92
(m, 4H); 6.24 (t, J = 6.0 Hz, 1H); 5.81 (s, 1H); 5.44 (d, D2O
exch., J = 5.0 Hz; 1H); 4.45 (m, 1H); 3.98 (m, 1H); 3.72 (s,
3H); 3.71 (s, 3H); 3.41 (m, 1H); 3.32 (m, 1H); 2.43 (m, 1H);
2.20 (m, 1H). 13C NMR (DMSOꢀD6) δ 159.9, 158.2, 158.1,
153.7, 144.3, 139.2, 135.9, 135.6, 135.2, 130.4, 129.8, 129.7,
128.8, 128.3, 128.0, 127.9, 126.8, 113.3, 96.4, 86.6, 86.1,
85.6, 68.9, 62.5, 55.0 (2 × C), 54.8, 41.5. HRMS (ESI) m/z:
found 630.2578 [M + H]+ (calcd. 630.2604 for C38H36N3O6).
Separate solutions of 2,3ꢀdiꢀO-TBDMSꢀp-NO2ꢀPhpC (171
mg, 0.28 mmol) and diꢀOAcꢀDABCYLpC (423 mg, 0.6
mmol) in THF (3 mL, the former intermediate; 7 mL, the later
intermediate) were cooled to 0˚C, followed by a slow addition
of Et3N · HF (160 ꢁL, 1 mmol, the former intermediate; 295
ꢁL, 1.8 mmol, the later intermediate). The cooling baths were
removed and the stirring continued for 18 h at RT. The solꢀ
vents were evaporated and the residues were subjected to FCC
on 25 g SiO2, eluted with CH2Cl2/MeOH/NH4OH (360:39:1)
or 20 g SiO2, eluted with CH2Cl2/MeOH (9:1). Evaporation of
the eluates afforded pꢀNO2ꢀPhpC [2, 82 mg, 77% (48%, based
on 6b)] or DABCYLpC [5, 230 mg, 81% (48%, based on 6b)].
Synthesis of p-NO2-PhpC (2), BFpC (3), FpC (4) and
DABCYLpC (5)
Nucleobase analog BFpC (3) was prepared as described
elsewhere.3 Round bottom flasks containing N4ꢀbenzoylꢀ5ꢀ
iodoꢀ2’,3’ꢀdiꢀOAcꢀdeoxycytidine (6a,3 577 mg, 1.07 mmol)
and 2ꢀethynylfuran (8, 402 mg, 4.37 mmol) or N4ꢀbenzoylꢀ5ꢀ
iodoꢀ2’,3’ꢀdiꢀO-TBDMSꢀdeoxycytidine (6b,3 700 mg, 1.02
mmol) and DABCYLꢀmodified alkyne (9, 381 mg, 1.52
pꢀNO2ꢀPhpC (2), orange solid, UPLC (method A) tR = 4.00
min. 1H NMR (DMSOꢀD6) δ 12.04 (s, D2O exch., 1H); 8.87 (s,
1H); 8.28 (d, J = 9.0 Hz, 2H); 8.08 (d, J = 9.0 Hz, 2H); 7.08 (s,
1H); 6.24 (t, J = 6.0 Hz, 1H); 5.30 (d, D2O exch., J = 4.5 Hz,
1H); 5.18 (t, D2O exch., J = 5.0 Hz, 1H); 4.25 (m, 1H); 3.93
(m, 1H); 3.69 (m, 2H); 2.40 (m, 1H); 2.07 (m, 1H). 13C NMR
(DMSOꢀD6) δ 159.8, 153.7, 146.3, 138.4, 136.9, 136.8, 125.7,
124.1, 108.8, 101.5, 88.1, 87.4, 69.8, 60.9, 41.5. HRMS (ESI)
mmol)
or
N4ꢀbenzoylꢀ5ꢀiodoꢀ2’,3’ꢀdiꢀO-TBDMSꢀ
deoxycytidine (6b,3 643 mg, 0.94 mmol) and pꢀNO2ꢀ
phenylacetylene (10, 207 mg, 1.4 mmol) were charged with N2
and dry DMF (4 mL, 6a + 8; 2.5 mL, 6b + 10) or dry THF (6
mL, 6b + 9) was added. The mixtures were degassed (see
General experimental protocols), followed by the addition of
Pd(PPh3)4 (6a + 8, 123 mg, 0.11 mmol; 6b + 9, 116 mg, 0.1
mmol; 6b + 10, 108 mg, 0.094 mmol) and CuI (6a + 8, 41 mg,
0.21 mmol; 6b + 9, 19 mg, 0.1 mmol; 6b + 10, 36 mg, 0.19
mmol). The mixtures were degassed again, Et3N (6a + 8, 1.49
mL, 10.66 mmol; 6b + 9, 3 mL, 21.52 mmol; 6b + 10, 1.3 mL,
9.38 mmol) was added and the mixtures were stirred (in the
dark, under N2 atmosphere) for 5 h at 50 °C (6a + 8; 6b + 10),
or 48 h at 55 °C (6b + 9). EtOH and Et3N were added (2 mL
each) and the stirring continued for 5 h at 80 °C (6a + 8), 18 h
at 80 °C (6b + 9) or 18 h at 70 °C (6b + 10). The mixtures
were cooled to RT, were diluted with 4% EDTA solution (50
mL) or brine (6b + 10, 100 mL) and were extracted with
EtOAc (2 × 30 + 20 mL). The combined organic extracts
m/z: found 373.1158 [M
C17H17N4O6).
+
H]+ (calcd. 373.1148 for
DABCYLpC (5), red solid, UPLC (method A) tR = 4.67 min. 1H
NMR (DMSOꢀD6) δ 11.89 (s, D2O exch., 1H); 8.76 (s, 1H); 7.99
(d, J = 8.0 Hz, 2H); 7.82 (m, 4H); 6.85 (m, 3H); 6.26 (t, J = 6.0
Hz, 1H); 5.31 (d, D2O exch., J = 4.0 Hz, 1H); 5.19 (t, D2O exch.,
J = 5.0 Hz, 1H); 4.26 (m, 1H); 3.92 (m, 1H); 3.68 (m, 2H); 3.07
(s, 6H); 2.38 (m, 1H); 2.06 (m, 1H). 13C NMR (DMSOꢀD6) δ
160.0, 153.8, 152.6, 151.8, 142.7, 138.6, 136.7, 131.4, 125.8,
124.9, 122.4, 111.6, 109.2, 98.2, 87.9, 87.1, 69.9, 60.9, 45.7, 41.5.
HRMS (ESI) m/z: found 475.2079 [M + H]+ (calcd. 475.2094 for
C25H27N6O4).
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The Journal of Organic Chemistry
6.83 (d, J = 9.0 Hz, 2H); 6.63 (m, 4H); 6.10 (t, J = 6.5 Hz, 1H);
Synthesis of C7-DMT-p-NO2-PhpC (2a), C7-DMT-BFpC
1
2
3
4
5
6
7
8
5.25 (d, D2O exch., J = 4.0 Hz, 1H); 4.66 (t, D2O exch., J = 5.5
Hz, 1H); 3.91 (m, 1H); 3.69 (m, 1H); 3.59 (s, 3H); 3.58 (s, 3H);
3.06 (m, 7H); 3.01 (m, 1H); 2.22 (m, 1H); 1.47 (m, 1H). 13C
NMR (DMSOꢀD6) δ 158.0, 157.5, 153.6, 152.7, 151.1, 145.7,
142.8, 137.5, 137.2, 134.2, 131.6, 130.5, 130.1, 127.5, 124.9,
120.8, 117.3, 112.8, 111.8, 110.0, 87.8, 86.6, 70.9, 62.1, 58.2,
55.0 (2 × C), 40.8, 40.1. HRMS (ESI) m/z: found 777.3431 [M +
H]+ (calcd. 777.3401 for C46H45N6O6).
(3a), C7-DMT-FpC (4a) and C7-DMT-DABCYLpC (5a)
Separate flasks containing pꢀNO2ꢀPhpC (2, 75 mg, 0.2
mmol), BFpC (3, 134 mg, 0.37 mmol), FpC (4, 256 mg, 0.81
mmol) and DABCYLpC (5, 199 mg, 0.42 mmol) and DMTꢀCl
(2, 89 mg, 0.26 mmol; 3, 161 mg, 0.47 mmol; 4, 328 mg, 0.97
mmol; 5, 185 mg, 0.5 mmol) were flushed with N2, followed
by the addition of dry pyridine (2, 1.2 mL; 3, 3 mL; 4, 5 mL;
5, 15 mL) and dry Et3N (2, 200 ꢁL, 1.45 mmol; 3, 350 ꢁL,
2.54 mmol; 4, 790 ꢁL, 5.65 mmol; 5, 2.5 mL, 17.94 mmol).
The reaction mixtures were stirred at RT (N2 atmosphere) for
24 h (2, 3 and 4) or 72 h. The reactions were quenched by the
addition of MeOH (200 ꢁL), the mixtures were coevaporated
with toluene (3 × 100 mL). The residues were subjected to
Synthesis of 5’-O-DMT-p-NO2-PhpC (2b), 5’-O-DMT-
BFpC (3b), 5’-O-DMT-FpC (4b) and 5’-O-DMT-
DABCYLpC (5b)
9
10
11
12
13
14
15
16
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18
19
20
21
22
23
24
25
26
27
28
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31
32
33
34
35
36
37
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42
43
44
45
46
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59
60
Separate round bottom flasks containing N4ꢀbenzoylꢀ5ꢀiodoꢀ
5’ꢀOꢀDMTꢀdeoxycytidine (6d, 250 mg, 0.33 mmol) and 2ꢀ
ethynylbenzo[b]furan (7, 70 mg, 0.49 mmol), 2ꢀethynylfuran
(8, 110 mg, 1.2 mmol), DABCYLꢀmodified alkyne (9, 103
mg, 0.41 mmol) and pꢀNO2ꢀphenylacetylene (10, 80 mg, 0.54
mmol) were charged with N2 and dry DMF (2 mL) was added.
The mixtures were degassed (see General experimental protoꢀ
cols), followed by the addition of Pd(PPh3)4 (38 mg, 0.03
mmol) and CuI (13 mg, 0.07 mmol). The mixtures were deꢀ
gassed again, Et3N (460 ꢁL, 3.3 mmol) was added and the
mixtures were stirred (in the dark, under N2 atmosphere) for 4
h at 50 °C. EtOH and Et3N were added (1 mL each) and the
stirring continued for 18 h at 70 °C. The mixtures were cooled
to room temperature (RT), were diluted with brine (50 mL)
and were extracted with EtOAc (3 × 20 mL). Combined orꢀ
ganic extracts were washed with brine (2 × 50 mL), were dried
and were concentrated. The residues were subjected for FCC
on 30 g SiO2, eluted with CH2Cl2/MeOH/NH4OH (380:19:1)
later replaced with CH2Cl2/MeOH/NH4OH (360:39:1, 6d + 7);
30 g SiO2, eluted with CH2Cl2/MeOH/NH4OH (380:19:1, 6d +
FCC as follows: 2a, 40
g
SiO2, eluted with
CH2Cl2/MeOH/NH4OH (360:39:1); 3a, 25 g SiO2, eluted with
acetone/EtOAc/Et3N (55:40:5); 4a, 70 g SiO2; 5a, 25 g SiO2,
both eluted with CH2Cl2/MeOH/Et3N (90:7:3). Evaporation of
the eluates afforded C7ꢀpꢀNO2ꢀPhpC (2a, 52 mg, 38%), C7ꢀ
DMTꢀBFpC (3a, 127 mg, 52%), C7ꢀDMTꢀFpC (4a, 249 mg,
50%) and C7ꢀDMTꢀDABCYLpC (5a, 151 mg, 49%).
C7ꢀpꢀNO2ꢀPhpC (2a), yellow solid, UPLC (method A) tR =
5.07 min. 1H NMR (DMSOꢀD6) δ 11.49 (s, D2O exch., 1H);
7.85 (d, J = 8.5 Hz, 1H); 7.24 (s, 1H); 7.18 (d, J = 8.5 Hz, 1H);
7.14 (m, 5H); 6.95 (m, 4H); 6.63 (m, 4H); 6.07 (t, J = 6.5 Hz,
1H); 5.22 (d, D2O exch., J = 4.0 Hz, 1H); 4.62 (t, D2O exch., J
= 5.5 Hz, 1H); 3.90 (m, 1H); 3.39 (m, 1H); 3.63 (s, 6H); 3.00
(m, 2H); 2.23 (m, 1H); 1.45 (m, 1H). 13C NMR (DMSOꢀD6) δ
157.7, 157.4, 153.3, 145.9, 145.2, 139.9, 138.7, 137.0, 136.9,
135.3, 131.5, 130.6, 130.4, 127.4, 126.1, 122.0, 118.5, 112.6,
109.6, 87.7, 86.5, 70.7, 61.9, 57.9, 54.9 (2 × C), 40.8. HRMS
(ESI) m/z: found 675.2449 [M + H]+ (calcd. 675.2455 for
C38H35N4O8).
8
and 6d
+
10) or 50
g
SiO2, eluted with
CH2Cl2/MeOH/NH4OH (380:19:1) later replaced with
CH2Cl2/MeOH/NH4OH (360:39:1, 6d + 9). Evaporation of
the eluates afforded 5’ꢀOꢀDMTꢀpꢀNO2ꢀPhpC (2b, 106 mg,
48%, based on 6d), 5’ꢀOꢀDMTꢀBFpC (3b, 100 mg, 45%,
based on 6d), 5’ꢀOꢀDMTꢀFpC (4b, 120 mg, 59%, based on
6d) and 5’ꢀOꢀDMTꢀDABCYLpC (5b, 146 mg, 57%, based on
6d).
C7ꢀDMTꢀBFpC (3a), yellow solid, UPLC (method A) tR =
5.20 min. 1H NMR (DMSOꢀD6) δ 11.74 (s, D2O exch., 1H);
7.50 (d, J = 7.5 Hz, 1H); 7.45 (s, 1H); 7.38 (m, 2H); 7.28 (m,
4H); 7.20 (m, 1H); 7.16 (m, 4H); 7.03 (m, 1H); 6.84 (s, 1H);
6.69 (m, 4H); 6.14 (t, J = 6.5 Hz, 1H); 5.26 (d, D2O exch., J =
4.0 Hz, 1H); 4.72 (t, D2O exch., J = 5.5 Hz, 1H); 3.99 (m, 1H);
3.74 (m, 1H); 3.57 (s, 6H); 3.17 (m, 1H); 3.09 (m, 1H); 2.27
(m, 1H); 1.60 (m, 1H). 13C NMR (DMSOꢀD6) δ 158.5, 157.5,
154.4, 153.7, 147.5, 146.4, 139.8, 138.3 (2 × C), 131.1, 129.9,
128.0, 127.9, 126.5, 126.2, 125.1, 123.3, 121.4 (2 × C), 113.3,
111.2, 109.4, 107.9, 88.3, 87.2, 71.4, 62.3, 58.8, 55.3 (2 × C),
41.5. HRMS (ESI) m/z: found 670.2581 [M + H]+ (calcd.
670.2553 for C40H36N3O7).
5’ꢀOꢀDMTꢀpꢀNO2ꢀPhpC (2b), orange solid, UPLC (method
A) tR = 5.62 min. 1H NMR (DMSOꢀD6) δ 12.04 (s, D2O exch.,
1H); 8.77 (s, 1H); 8.29 (d, J = 9.0 Hz, 1H); 7.93 (d, J = 9.0 Hz,
1H); 7.42 (m, 2H); 7.31 (m, 7H); 6.92 (m, 4H); 6.21 (t, J = 4.5
Hz, 1H); 6.10 (s, 1H); 5.45 (d, D2O exch., J = 4.5 Hz, 1H);
4.43 (m, 1H); 4.01 (m, 1H); 3.71 (s, 3H); 3.70 (s, 3H); 3.36
(m, 2H); 2.45 (m, 1H); 2.21 (m, 1H). 13C NMR (DMSOꢀD6) δ
159.9, 158.2 (2 × C), 153.6, 146.4, 144.5, 137.9, 136.7, 136.6,
135.5, 135.2, 129.9, 129.8, 128.0, 127.9, 126.9, 125.5, 124.2,
113.3, 108.6, 87.0, 86.1, 85.8, 68.9, 62.5, 55.0 (2 × C), 54.9,
41.4. HRMS (ESI) m/z: found 675.2468 [M + H]+ (calcd.
675.2455 for C38H35N4O8).
C7ꢀDMTꢀFpC (4a), yellow solid, UPLC (method A) tR
=
4.89 min. 1H NMR (DMSOꢀD6) δ 11.53 (s, D2O exch., 1H);
7.32 (s, 1H); 7.31 (m, 2H); 7.25 (m, 5H); 7.16 (m, 2H); 7.08
(m, 1H); 6.74 (m, 4H); 6.32 (m, 2H); 6.15 (t, J = 6.0 Hz, 1H);
5.24 (d, D2O exch., J = 4.0 Hz, 1H); 4.71 (t, D2O exch., J =
5.5 Hz, 1H); 3.98 (m, 1H); 3.72 (m, 1H); 3.68 (s, 6H); 3.17
(m, 1H); 3.09 (m, 1H); 2.24 (m, 1H); 1.58 (m, 1H). 13C NMR
(DMSOꢀD6) δ 158.0, 157.0, 153.3, 146.1, 144.9, 143.4, 138.5,
137.8, 130.6, 129.3, 127.5, 126.7, 125.6, 118.1, 112.9, 111.4,
111.1, 109.2, 87.8, 86.5, 71.0, 61.8, 58.2, 54.9 (2 × C), 41.0.
HRMS (ESI) m/z: found 620.2413 [M + H]+ (calcd. 620.2397
for C36H34N3O7).
5’ꢀOꢀDMTꢀBFpC (3b), yellow solid, UPLC (method A) tR =
5.84 min. 1H NMR (DMSOꢀD6) δ 12.08 (s, D2O exch., 1H);
8.75 (s, 1H); 7.70 (d, J = 7.5 Hz, 1H); 7.61 (d, J = 8.0 Hz, 1H);
7.42 (m, 2H); 7.31 (m, 10H); 6.92 (m, 4H); 6.23 (t, J = 6.5 Hz,
1H); 5.92 (s, 1H); 5.45 (d, D2O exch., J = 5.0 Hz, 1H); 4.44
(m, 1H); 3.99 (m, 1H); 3.72 (s, 3H); 3.71 (s, 3H); 3.36 (m,
2H); 2.45 (m, 1H); 2.22 (m, 1H). 13C NMR (DMSOꢀD6) δ
159.7, 158.2, 154.3, 153.7, 147.9, 144.3, 137.0, 135.6, 135.3,
129.9, 129.8, 129.7, 128.2, 128.0, 127.9, 126.9, 125.2, 123.5,
121.5, 113.3, 110.9, 103.4, 98.2, 86.9, 86.1, 85.7, 68.9, 62.5,
C7ꢀDMTꢀDABCYLpC (5a), red solid, UPLC (method A) tR
=
5.63 min. 1H NMR (DMSOꢀD6) δ 11.39 (s, D2O exch., 1H); 7.78
(d, J = 9.0 Hz, 2H); 7.41 (d, J = 8.0 Hz, 2H); 7.20 (s, 1H); 7.16
(m, 4H); 7.10 (m, 1H); 7.04 (d, J = 8.0 Hz, 2H); 6.98 (m, 4H);
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The Journal of Organic Chemistry
Page 10 of 12
55.0 (2 × C), 41.5. HRMS (ESI) m/z: found 670.2535 [M +
phosphonate contaminant. This material was dissolved in
H]+ (calcd. 670.2553 for C40H36N3O7).
CH2Cl2, hexanes were added until the turbidity in the solution
persisted and the mixture was set aside for 2 h at ꢀ20 °C. The
separated solid was isolated by filtration, was washed with
1
2
3
4
5
6
7
8
5’ꢀOꢀDMTꢀFpC (4b), yellow solid, UPLC (method A) tR =
5.41 min. 1H NMR (DMSOꢀD6) δ 11.80 (s, D2O exch., 1H);
8.59 (s, 1H); 7.77 (m, 1H); 7.40 (m, 2H); 7.27 (m, 7H); 6.89
(m, 5H); 6.60 (m, 1H); 6.22 (t, J = 5.5 Hz, 1H); 5.67 (s, 1H);
5.41 (d, D2O exch., J = 4.5 Hz, 1H); 4.40 (m, 1H); 3.97 (m,
1H); 3.70 (2 × s, 6H); 3.33 (m, 2H); 2.40 (m, 1H); 2.18 (m,
1H). 13C NMR (DMSOꢀD6) δ 159.6, 158.2, 153.8, 146.0,
144.4, 143.8, 135.9, 135.6, 135.3, 130.7, 129.8, 129.7, 128.0,
127.8, 126.9, 113.3, 112.0, 108.7, 107.8, 94.9, 86.7, 86.1 (2 ×
C), 85.7, 69.1, 62.7, 55.0 (2 × C), 41.5. HRMS (ESI) m/z:
found 620.2374 [M + H]+ (calcd. 620.2397 for C36H34N3O7).
hexanes
and
was
dried
to
afford
3’ꢀ(2ꢀ
cyanoethyldiisopropylphosphoramidite)ꢀ5’ꢀOꢀDMTꢀpꢀNO2ꢀ
PhpC (2c, 70 mg, 53%)
3’ꢀ(2ꢀCyanoethyldiisopropylphosphoramidite)ꢀ5’ꢀOꢀDMTꢀ
pꢀNO2ꢀPhpC (2c), orange solid. 1H NMR (CD2Cl2) δ 12.15
(br s, D2O exch., 1H); 9.07 (s, 0.6H); 9.04 (s, 0.4 H); 8.25 (d, J
= 8.5 Hz, 2H); 8.04 (d, J = 9.0 Hz, 2H); 7.51 (m, 2H); 7.36 (m,
7H); 6.88 (m, 4H); 6.43 (m, 1H); 5.74 (s, 1H); 4.86 (m, 1H);
4.24 (m, 1H); 3.76 (s, 3H); 3.74 (s, 3H); 3.62 (m, 6H); 2.83
(m, 1H); 2.63 (m, 1H); 2.53 (m, 2H); 1.19 (m, 9H), 1.11 (d, J
= 6.5 Hz, 3H). 13C NMR (CD2Cl2) δ 161.0, 159.5, 159.4,
154.9, 147.6, 144.9 (2 × C), 139.0 (2 × C), 138.4, 136.9 (2 ×
C), 136.3, 136.2, 135.9 (2 × C), 130.9 (2 × C), 130.8 (2 × C),
129.0 (2 × C), 128.7, 127.7 (2 × C), 126.8, 124.7, 118.3,
118.2, 113.9, 110.3 (2 × C), 101.4, 101.3, 88.3 (2 × C), 87.7 (2
× C), 86.1 (2 × C), 86.0, 85.9, 71.2, 71.0, 62.2, 62.0, 59.1, 58.9
(2 × C), 58.7, 55.8 (3 × C), 44.0, 43.9, 41.9 (2 × C), 41.5 (2 ×
C), 25.0, 24.9 (3 × C), 24.8, 23.3, 23.2, 21.0, 20.9 (2 × C),
20.8. 31P NMR (CD2Cl2) δ 149.3, 148.8. HRMS (ESI) m/z:
found 875.3534 [M + H]+ (calcd. 875.3533 for C47H52N6O9P).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5’ꢀOꢀDMTꢀDABCYLpC (5b), red solid, UPLC (method A) tR =
6.30 min. 1H NMR (DMSOꢀD6) δ 11.89 (s, D2O exch., 1H); 8.71
(s, 1H); 7.82 (m, 6H); 7.43 (m, 2H); 7.31 (m, 7H); 6.93 (m, 4H);
6.85 (d, J = 9.0 Hz, 2H); 6.24 (t, J = 5.0 Hz, 1H); 5.89 (s, 1H);
5.45 (d, D2O exch., J = 4.5 Hz, 1H); 4.46 (m, 1H); 3.99 (m, 1H);
3.72 (s, 3H); 3.71 (s, 3H); 3.37 (m, 2H); 3.07 (s, 6H); 2.44 (m,
1H); 2.22 (m, 1H). 13C NMR (DMSOꢀD6) δ 160.0, 158.2 (2 × C),
153.7, 152.5, 151.9, 144.4, 142.7, 138.5, 136.3, 135.6, 135.2,
131.2, 129.9, 129.8, 128.0, 127.9, 126.9, 122.4, 113.3, 111.5,
109.0, 97.5, 86.7, 86.1, 85.6, 68.9, 62.4, 55.0 (2 × C), 41.5, 39.8.
HRMS (ESI) m/z: found 777.3400 [M + H]+ (calcd. 777.3401 for
C46H45N6O6).
3’ꢀ(2ꢀCyanoethyldiisopropylphosphoramidite)ꢀ5’ꢀOꢀDMTꢀ
BFpC (3c), yellow solid. 1H NMR (CD2Cl2) δ 12.41 (br s,
D2O exch., 1H); 8.89 (s, 0.5H); 8.84 (s, 0.5 H); 8.02 (m, 1H);
7.65 (d, J = 8.0 Hz, 1H); 7.53 (m, 3H); 7.34 (m, 9H); 6.90 (m,
4H); 6.50 (m, 1H); 5.95 (s, 0.5H); 5.94 (s, 0.5H); 4.81 (m,
1H); 4.28 (m, 1H); 3.87 (m, 1H); 3.77 (s, 3H); 3.76 (s, 3H);
3.60 (m, 5H); 2.85 (m, 1H); 2.55 (m, 3H); 1.21 (m, 9H), 1.13
(d, J = 6.5 Hz, 3H). 13C NMR (CD2Cl2) δ 160.9, 160.8, 159.4
(2 × C), 155.6, 155.0 (2 × C), 148.4, 144.9, 144.8, 137.4,
136.3, 136.2, 136.0, 131.9 (2 × C), 130.8 (2 × C), 130.7 (2 ×
C), 129.8, 128.9 (2 × C), 128.6, 127.7 (2 × C), 125.4, 123.6,
122.2, 118.3, 118.2, 113.9, 111.4, 110.4 (2 × C), 105.3, 99.2,
99.1, 88.3, 87.6, 86.2 (2 × C), 86.0 (2 × C), 72.5, 72.4, 71.8,
71.6, 62.7, 62.4, 59.1, 58.9 (2 × C), 58.8, 58.4, 55.8 (2 × C),
55.7, 43.9, 43.8, 42.0 (2 × C), 41.6 (2 × C), 30.3, 25.0, 24.9,
24.8, 23.2 (2 × C), 22.0, 20.9 (2 × C), 20.8. 31P NMR
(CD2Cl2) δ 149.1, 148.7. HRMS (ESI) m/z: found 870.3630
[M + H]+ (calcd. 870.3632 for C49H53N5O8P).
Reaction of 5’-O-DMT-p-NO2-PhpC (2b), 5’-O-DMT-
BFpC (3b), 5’-O-DMT-FpC (4b) and 5’-O-DMT-
DABCYLpC
(5b)
with
2-cyanoethyl-N,N,N’,N’-
tetraisopropylphosporodiamidite (11)
Separate round bottom flasks containing 5’ꢀOꢀDMTꢀpꢀNO2ꢀ
PhpC (2b, 102 mg, 0.15 mmol), 5’ꢀOꢀDMTꢀBFpC (3b, 100
mg, 0.15 mmol), 5’ꢀOꢀDMTꢀFpC (4b, 120 mg, 0.19 mmol) or
5’ꢀOꢀDMTꢀDABCYLpC (5b, 143 mg, 0.18 mmol) and teꢀ
trazole (13 mg, 0.18 mmol, to react with 2b and 3b; 16 mg,
0.23 mmol, to react with 4b; 15 mg, 0.22 mmol, to react with
5b) were flushed with N2, followed by the addition of dry
CH2Cl2 (1.5 mL) and MeCN (1 mL). In the case of 2b only
dry MeCN (1.7 mL) was used. A solution of 2ꢀcyanoethylꢀ
N,N,N’,N’ꢀtetraisopropylphosporodiamidite (11, 54 mg, 0.18
mmol, to react with 2b and 35; 70 mg, 0.23 mmol, to react
with 3b; 66 mg, 0.22 mmol, to react with 4b) in dry MeCN
(500 ꢁL) was added and the mixtures were stirred for 2 h (4 h
for 2b) at RT (N2 atmosphere). The mixtures were diluted
with CH2Cl2 (20 mL) and were washed with saturated Naꢀ
HCO3 solution (10 mL). The aqueous phase was extracted
with CH2Cl2 (10 mL), the combined organic extracts were
dried and were concentrated. The residues were subjected to
PTLC (see General experimental protocols for details) eluting
the plates with CH2Cl2/MeOH/NH4OH (380:19:1). The bands
containing the product were carved off the plates, were exꢀ
tracted with CH2Cl2/MeOH/NH4OH (380:19:1), the SiO2 was
filtered off and the filtrates were concentrated to afford 3’ꢀ(2ꢀ
cyanoethyldiisopropylphosphoramidite)ꢀ5’ꢀOꢀDMTꢀBFpC
3’ꢀ(2ꢀCyanoethyldiisopropylphosphoramidite)ꢀ5’ꢀOꢀDMTꢀ
FpC (4c), yellow solid. 1H NMR (CD2Cl2) δ 12.00 (br s, D2O
exch., 1H); 8.76 (s, 0.5H); 8.71 (s, 0.5 H); 7.58 (m, 1H); 7.50
(m, 3H); 7.33 (m, 7H); 6.87 (m, 4H); 6.54 (m, 1H); 6.44 (m,
1H); 5.74 (s, 0.5H); 5.71 (s, 0.5H); 4.75 (m, 1H); 4.26 (m,
1H); 3.85 (m, 1H); 3.77 (2 × s, 3H); 3.76 (2 × s, 3H); 3.59 (m,
5H); 2.82 (m, 1H); 2.63 (t, J = 6.5 Hz, 1H); 2.50 (t, J = 6.5 Hz,
1H); 2.45 (m, 1H); 1.19 (m, 9H), 1.11 (d, J = 6.5 Hz, 3H). 13C
NMR (CD2Cl2) δ 160.7, 159.4 (2 × C), 155.0 (2 × C), 146.8,
144.9 (2 × C), 143.4, 136.3, 136.2 (2 × C), 136.0, 132.4,
132.3, 130.8 (2 × C), 130.7 (2 × C), 128.9, 128.8, 128.6,
127.7, 127.6, 118.3, 118.2, 113.8, 112.6, 110.6, 110.5, 109.2,
96.3, 96.2, 88.1, 87.5, 86.2, 86.1 (2 × C), 86.0, 72.9, 72.7,
72.0, 71.8, 62.9, 62.5, 59.1, 59.0 (2 × C), 58.8, 58.4, 55.8,
55.7, 43.9, 43.8, 42.0 (2 × C), 41.6 (2 × C), 25.0 (2 × C), 24.9
(2 × C), 24.8, 21.0, 20.9 (2 × C), 20.8. 31P NMR (CD2Cl2) δ
149.0, 148.7. HRMS (ESI) m/z: found 820.3485 [M + H]+
(calcd. 820.3475 for C45H51N5O8P).
(3c,
cyanoethyldiisopropylphosphoramidite)ꢀ5’ꢀOꢀDMTꢀFpC (4c,
92 mg, 58%) and 3’ꢀ(2ꢀ
cyanoethyldiisopropylphosphoramidite)ꢀ5’ꢀOꢀDMTꢀ
83
mg,
64%),
3’ꢀ(2ꢀ
DABCYLpC (5c, 115 mg, 64%). In the case of 5’ꢀOꢀDMTꢀpꢀ
NO2ꢀPhpC (2b) was the residue obtained after the extractive
workup subjected to FCC on 40 g SiO2, eluted with
CH2Cl2/MeOH/NH4OH (380:19:1). Evaporation of the eluate
afforded the desired product containing large amount of Hꢀ
3’ꢀ(2ꢀCyanoethyldiisopropylphosphoramidite)ꢀ5’ꢀOꢀDMTꢀ
DABCYLpC (5c), red solid. 1H NMR (CD2Cl2) δ 11.90 (br s,
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The Journal of Organic Chemistry
D2O exch., 1H); 8.93 (s, 0.5H); 8.88 (s, 0.5 H); 7.94 (m, 2H); 7.88
NH4Cl solution (20 mL), the organic layer was separated and the
1
2
3
4
5
6
7
8
(m, 3H); 7.53 (m, 2H); 7.37 (m, 8H); 6.89 (m, 4H); 6.79 (m, 2H);
6.46 (m, 1H); 5.73 (s, 0.5H); 5.71 (s, 0.5H); 4.83 (m, 1H); 4.25
(m, 1H); 3.83 (m, 1H); 3.77 (2 × s, 3H); 3.76 (2 × s, 3H); 3.60 (m,
5H); 3.09 (s, 6H); 2.84 (m, 1H); 2.59 (t, J = 6.0 Hz, 1H); 2.50 (m,
3H); 2.49 (t, J = 6.5 Hz, 1H); 1.19 (m, 9H), 1.11 (d, J = 6.5 Hz,
3H). 13C NMR (CD2Cl2) δ 160.9 (2 × C), 159.4 (2 × C), 154.9 (2
× C), 153.3, 153.2, 145.0, 144.9, 144.2, 140.2 (2 × C), 137.1,
136.3, 136.2, 136.0, 135.9, 131.7, 130.9 (2 × C), 130.8 (2 × C),
129.0, 128.9, 128.7, 127.7, 127.6, 126.7, 125.5,123.2, 118.3,
118.2, 113.9, 112.0, 110.8, 110.7, 98.3 (2 × C), 88.1 (2 × C), 87.6,
86.0 (2 × C), 86.1 (2 × C), 72.2, 72.1, 71.3, 71.2, 62.5, 62.1, 59.2,
59.0, 58.8, 55.8 (2 × C), 43.9, 43.8, 42.0 (2 × C), 41.6 (2 × C),
40.7, 25.0, 24.9 (2 × C), 24.8, 21.0, 20.9, 20.8. 31P NMR
(CD2Cl2) δ 149.2, 148.8. HRMS (ESI) m/z: found 977.4470 [M +
H]+ (calcd. 977.4479 for C55H62N8O7P).
aqueous layer was extracted with Et2O (20 mL). Combined orꢀ
ganic extract was dried and was concentrated (water bath kept at
RT) to yield crude 2ꢀethynylfuran (8, 290 mg, 99%) as pale orꢀ
ange oil used for the subsequent step immediately without further
purification. 1H NMR spectra of both, 2ꢀ(2,2ꢀdibromovinyl)furan
and 2ꢀethynylfuran (8) were in agreement with those previously
reported.
9
Quantum yield (Φ) and molar extinction coefficient (ε)
determination of FpC (4)
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Quantum yield (Φ) associated with FpC (4) was determined
in EtOH using 9,10ꢀdiphenylꢀanthracene (Φ 0.90) as a standꢀ
ard.17 The quantum yield of FpC (4) was determined to be
0.58.
The molar extinction coefficient (ε) associated with FpC (4)
was determined in EtOH by constructing LambertꢀBeer’s plots
(A = ε × c × l),3 wherein A (absorbance), ε (molar extinction
coefficient), c (concentration), l (light path length ~ 1 cm).
Plots of concentration versus absorbance using five different
concentrations were used. The molar extinction coefficient of
FpC (4) was determined to be 3.26 × 104 Mꢀ1cmꢀ1 (260 nm)
and 6.65 × 103 Mꢀ1cmꢀ1 (379 nm, excitation wavelength).
Preparation
of
N4-benzoyl-5-iodo-5’-O-DMT-
deoxycytidine (6d)
A round bottom flask containing N4ꢀbenzoylꢀ5ꢀiodoꢀ2’ꢀ
deoxycytidine (6c,13,14 687 mg, 1.46 mmol) and DMTꢀCl (643
mg, 1.9 mmol) was flushed with N2, followed by the addition
of dry pyridine (8 mL) and dry Et3N (1.47 mL, 10.52 mmol).
The resulting solution was stirred for 18 h at RT (N2 atmosꢀ
phere), the reaction was quenched with MeOH (200 ꢁL), was
diluted with toluene (150 mL) and was concentrated. The
residue was subjected to FCC on 90 g SiO2, eluted with
CH2Cl2/MeOH/NH4OH (380:19:1). Evaporation of the eluate
afforded N4ꢀbenzoylꢀ5ꢀiodoꢀ5’ꢀOꢀDMTꢀdeoxycytidine (6d,
751 mg, 68%).
Yellow solid, UPLC (method A) tR = 6.31 min. 1H NMR
(DMSOꢀD6) δ 12.92 (s, D2O exch., 1H); 8.23 (m, D2O exch., 3H);
7.62 (m, 1H); 7.53 (m, 2H); 7.42 (m, 2H); 7.32 (m, 7H); 7.23 (m,
1H); 6.91 (m, 4H); 6.11 (t, J = 6.0 Hz, 1H); 5.37 (d, D2O exch., J
= 4.5 Hz, 1H); 4.24 (m, 1H); 3.97 (m, 1H); 3.73 (s, 6H); 3.21 (m,
3H); 2.30 (m, 2H). 13C NMR (DMSOꢀD6) δ 178.1, 158.1, 156.6,
147.2, 146.3, 144.7, 136.3, 135.5, 135.4, 132.8, 129.7, 129.5,
128.3, 128.0, 127.7, 126.7, 113.3, 86.3, 86.1, 85.9, 70.4, 69.6,
63.6, 55.0 (2 × C), 40.3. HRMS (ESI) m/z: found 760.1540 [M +
H]+ (calcd. 760.1520 for C37H35IN3O7).
Preparation, purification and characterization of DNA
Sequences 1 and 2
DNA Sequences 1 and 2 were prepared by automated solid
phase DNA synthesis using standard methods as supplied by
the manufacturer. Coupling times for unmodified nucleoside
phosphoramidite reagents was 60 s and 300 s for modified
nucleosides. The individual resins obtained after each DNA
oligomerization were suspended in concentrated NH4OH soluꢀ
tion, agitated at room temperature for 30 min and then incuꢀ
bated at 55 °C in a sealed vial for 12 hours. Afterwards, NH3
was allowed to evaporate, the resin was separated by filtration
by passing through Pasteur pipette with a cotton plug. The
separate solutions containing crude DMTꢀprotected sequences
1 and 2 were frozen and lyophilized. The pellets were disꢀ
solved in water (700 ꢁL) and were subjected to HPLC purifiꢀ
cation as described in General experimental protocols; DMTꢀ
protected Sequence 1 tR = 29.1 min (Method C); DMTꢀ
protected Sequence 2 tR = 24.5 min (Method D).
Preparation of alkynes 7-10
2ꢀEthynylbenzo[b]furan (7)3 and DABCYLꢀmodified alkyne 99
have been prepared as described previously, while pꢀNO2ꢀ
phenylacetylene (10) is commercially available. A modified literꢀ
ature procedure8 has been used to prepare 2ꢀethynylfuran (8) as
follows. A flask containing PPh3 (6.56 g, 25 mmol), CBr4 (8.29
g, 25 mmol) and Zn dust (1.64 g, 25 mmol) was flushed with N2.
Dry CH2Cl2 (60 mL) was added and the mixture was stirred for 24
h at RT (N2 atmosphere). The mixture was then cooled to room
temperature, fural (830 ꢁL, 10 mmol) was added, the cooling bath
was removed and the stirring continued for further 18 h at RT (N2
atmosphere). The reaction mixture was filtered using a short
CELITE pad; the filter was washed with CH2Cl2, the filtrate was
concentrated and the residue was subjected to FCC on 50 g SiO2,
eluted with petroleum ether. Evaporation of the eluate afforded 2ꢀ
(2,2ꢀdibromovinyl)furan (2.29 g, 91%) as slightly yellow oil. 2ꢀ
(2,2ꢀDibromovinyl)furan (800 mg, 3.18 mmol) was dissolved in
dry Et2O (20 mL) and the solution was charged with N2, followed
by cooling to ꢀ78 ˚C. A 1.5 M solution of tꢀBuLi in pentane (8
mL, 12.1 mmol) was added slowly and the mixture was stirred for
1 h (N2 atmosphere) while the cooling bath was allowed to graduꢀ
ally warm up. The reaction was the quenched with saturated
The fractions containing pure DMTꢀprotected DNA Seꢀ
quences 1 and 2 were combined, were frozen and lyophilized,
the residues have been dissolved in H2O (150 ꢁL)/AcOH (600
ꢁL) and the solutions were set aside for 30 min at RT, were
diluted with water, were frozen and lyophilized. The residues
were dissolved in water (800 ꢁL each) and were subjected to
HPLC purification as described in General experimental proꢀ
tocols; Sequence 1 tR = 15.4 min (Method E); MS (ESIꢀTOF)
m/z: found 3114.1 [M ꢀ H]+ (calcd 3114.5); Sequence 2 tR =
11.3 min (Method F); MS (ESIꢀTOF) m/z: found 3090.3 [M ꢀ
H]+ (calcd 3090.5).
Hybridization studies with DNA Sequences 1 and 2
The hybridization properties of the modified DNA oligomers
with fully matched, complementary DNA and singleꢀ
mismatched containing sequences was studied by temperature
dependent UV spectroscopy (i.e. melting experiments).
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2. (a) Hudson, R. H. E.; Dambenieks, A. K.; Viirre, R. D. Synlett
Thermal denaturation experiments were carried out in a buffer
containing 100 mM NaCl, 10 mM MgCl2, 10 mM Na2HPO4,
0.1 mM EDTA at pH 7.0. Tm experiments were performed at
10ꢀ6 M strand concentration. Samples were heated to 95 °C
and were allowed to cool to room temperature slowly (ca. 3 h).
Denaturation was performed from 20 °C to 85 °C at a scan rate
of 0.5 °C/min. ꢂTm values are the difference between the Tm
of the sequences 1 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 or lacked sigꢀ
moidal shape were deemed not to be cooperative transitions.
Samples for the fluorescence measurements associated with
the DNA 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.
2004, 2400ꢀ2402. (b) Wojciechowski, F.; Hudson, R. H. E. J. Am.
Chem. Soc. 2008, 130, 12574ꢀ12575. (c) Wojciechowski, F.; Hudson,
R. H. E. Org. Lett. 2009, 11, 4878ꢀ4881. (d) Hu, J.; Dodd, D. W.;
Hudson, R. H. E.; Corey, D. R. Bioorg. Med. Chem. Lett. 2009, 19,
6181ꢀ6184. (e) Wahba, A. S.; Azizi, F.; Deleavey, G. F.; Brown, C.;
Robert F.; Carrier, M.; Kalota, A.; Gewirtz, A. M.; Pelletier, J.; Hudꢀ
son, R. H. E.; Damha, M. J. ACS Chem. Biol. 2011, 6, 912ꢀ919. (f)
Torres, A. G.; Fabani, M. M.; Vigorito, E.; Williams, D.; AlꢀObaidi,
N.; Wojciechowski, F.; Hudson, R. H. E.; Seitz, O.; Gait, M. J. Nu-
cleic Acids Res. 2012, 40, 2152ꢀ2167.
1
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3. Elmehriki, A. A. H.; Suchý, M.; Chicas, K. J.; Wojciechowski,
F.; Hudson, R. H. E. Artif. DNA, 2014, 5, e29174.
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4. (a) Moustafa, M. E.; Hudson, R. H. E. Nucleos. Nucleot. Nucl.
2011, 30, 740ꢀ751. (b) DABCYL has become the classic quencher
since it demonstration in peptide and oligonucleotide applications:
Matayoshi, E. D.; Wang, G. T.; Krafft, G. A.; Erickson, J. Science,
1990, 247, 954ꢀ958. and (c) Tyagi, S.; Kramer, F.R. Nat. Biotechnol.,
1996, 14, 303ꢀ308.
5. (a) Chicas, K. and Hudson, R. H. E. Expanding the Nucleic Acid
Chemists Toolbox: Fluorescent Cytidine Analogs in Fluorescent
Analogs of Biomolecular Building Blocks Design and Applications.
Wilhelmsson, M. and Tor, Y. Eds.; John Wiley & Sons, Hoboken,
New Jersey, USA. 2016, pp. 174ꢀ203. (b) Ettles, C. Progress Toward
Synthesis of Molecular Beacons Incorporating DABCYL Analog
Quenchers, MSc thesis, The University of Western Ontario, Canada,
2013.
6. (a) The DMT group for 5’ꢀOH protection is well established:
Smith, M.; Rammler, D. H.; Goldberg, I.H; Khorana, H. G. J. Am.
Chem. Soc. 1962, 84, 430ꢀ440. (b) Caruthers, M. H. Science 1985,
230, 281ꢀ285.
7. Reese, C. B. Org. Biomol. Chem. 2005, 3, 3851ꢀ3868, and referꢀ
ences cited therein.
AUTHOR INFORMATION
Corresponding Author
* Robert H. E. Hudson; eꢀmail: rhhudson@uwo.ca
Mojmir Suchy current address: Department of Chemistry and
Biomolecular Sciences, University of Ottawa, Ottawa, ON, Canaꢀ
da K1N 6N5
8. Kyi, S.; Wongkattiya, N.; Warden, A. C.; O’Shea, M. S.; Deighꢀ
ton, M.; Macreadie, I.; Graichen, F. H. M. Bioorg. Med. Chem. Lett.
2010, 20, 4555ꢀ4557.
9. Charles, M. Synthesis and Spectroscopic Studies of Substituted
Pyrrolocytidines. MSc thesis, The University of Western Ontario,
Canada, 2012. For the azo coupling between 4ꢀiodoaniline and N, Nꢀ
dimethylaniline to prepare the corresponding iodinated intermediate
see: Siemeling, U.; Bruhn, C.; Meier, M.; Schirrmacher, C. Z.
Naturforsch. 2008, 63B, 1395ꢀ1401.
10. Chemical shifts (DMSOꢀD6) of protons adjacent to C9 and C7
of an authentic sample of 5’ꢀOꢀDMTꢀ4ꢀmethoxyphenylpC available
in our laboratory were found to be at δ 8.58 ppm (s, 1H) and 5.68 ppm
(s, 1H). The sample was prepared as described in Ghorbaniꢀ
Choghamarni, A. Preparation of Some New Reagents and Their Apꢀ
plications in Organic Reactions: Synthesis of Modified Nucleobases
and Their Incorporation into DNA Oligomers and Their Evaluation of
Fluorescence Properties. PhD thesis, Bu Ali Sina University, Iran,
2007.
11. Butskus, P. F.; Raguotene, N. V. Khim. Geterotsikl. Soedin.
1970, 6, 1056ꢀ1057.
12. Zeidler, J.; Golankiewicz, B. Tetrahedron, 1998, 54, 2941ꢀ
2952.
13. Chang, P. K.; Welch, A. D. J. Med. Chem. 1963, 6, 428ꢀ430.
14. Schaller, H.; Weiman, G.; Lerch, B.; Khorana, H. G. J. Am.
Chem. Soc. 1963, 85, 3821ꢀ3827.
15. 5’ꢀOꢀDMTꢀPhpC was prepared by this route, see Hudson, R. H.
E.; GhorbaniꢀChoghamarni, A. Synlett 2007, 870ꢀ873.
Augusto Matarazzo current address: Toronto Medical Discoveries
Tower, MaRS Centre, Toronto, ON, Canada M5G 1L7
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank The Natural Sciences and Engineering Research
Council of Canada (NSERC grant #RGPIN/203210ꢀ2012) for
the financial support of this work. We thank Dr. Mathew Wilꢀ
lans for the assistance with acquiring 2D NMR spectra and
Professor J. Peter Guthrie for fruitful discussion.
ASSOCIATED CONTENT
Supporting Information
Spectral characterization of 2, 4, 5, 1a-5a, 1b-5b, 2c-5c and 6d.
Computed ionization potential maps for models of 1ꢀ5 and atom
coordinate tables and absolute energies from the quantum calculaꢀ
tions. Characterization of the DNA sequences (Sequence 1, Se-
quence 2).
REFERENCES
1. For the recent reviews on fluorescent nucleobase analogs see (a)
Dodd, D. W.; Hudson, R. H. E. Mini Rev. Org. Chem. 2009, 6, 378ꢀ
391. (b) Sinkeldam, R. W.; Greco, N. J.; Tor, Y. Chem. Rev. 2010,
110, 2579ꢀ2619. (c) Wilhelmsson, L. M. Q. Rev. Biophys.
2010, 43, 159ꢀ183.
16. (a) Nielsen, J.; Taagaard, M.; Marugg, J. E.; van Boom, J. H.;
Dahl, O. Nucleic Acids Res.1986, 14, 7391ꢀ7403. (b) Asanuma, H.;
Shirasuka, K.; Takarada, T.; Kashida, H.; Komiyama, M. J. Am.
Chem. Soc. 2003, 125, 2217ꢀ2223.
17. Eaton, D. F. Pure Appl. Chem. 1988, 60, 1107ꢀ1114.
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