CG base pair recognition within DNA triple helices by modified N-methylpyrrolo-dC nucleosides

Article abstract of DOI:10.1039/c0ob00119h

3-Aminophenyl-modified analogues of the bicyclic nucleoside N-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one were synthesised and incorporated directly into triplex-forming oligonucleotides in order to utilise their extended hydrogen bonding motif for recogn

Full text of DOI:10.1039/c0ob00119h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
CG base pair recognition within DNA triple helices by modified
N-methylpyrrolo-dC nucleosides†
Simon R. Gerrard,a Mastoura M. Edrees,a Imenne Bouamaied,b Keith R. Foxc and Tom Brown*a
Received 13th May 2010, Accepted 19th July 2010
DOI: 10.1039/c0ob00119h
3-Aminophenyl-modified analogues of the bicyclic nucleoside N-methyl-3H-pyrrolo[2,3-
d]pyrimidin-2(7H)-one were synthesised and incorporated directly into triplex-forming
oligonucleotides in order to utilise their extended hydrogen bonding motif for recognition of the CG
base pair. All analogues demonstrated strong binding affinity and very good selectivity for CG from
pH 6.2 to 7.0; a marked improvement on previous modifications.
Introduction
Since their discovery in 1957,1 the biomedical and biotechnological
importance of DNA triple helices has been exploited. These
structures have potential applications in gene therapy (antigene
strategy),2 site-directed DNA cleavage2a,3 and repair,2a,4 or as tools
in molecular biology and biotechnology.2a,5 Binding of a triplex-
forming oligonucleotide (TFO) to a specific region of the genome
may block binding of transcription factors and polymerases and
prevent unwinding of the duplex by helicases. This inhibits gene
expression, prevents DNA replication and interrupts cell division
(chemotherapeutics).
DNA triple helices are formed by binding of a single-stranded
TFO to a target duplex, via specific hydrogen bond interactions
within the major groove. GC and AT base pairs form triplets
with the natural bases C+ (protonated) and T respectively. Using
modified nucleosides in TFOs,6 however, significantly enhances
duplex binding affinity and selectivity.
Applications of TFOs require strong, selective binding to
mixed-sequence DNA. However, triple helix-mediated recognition
of Py.Pu base pairs (CG, TA) in double-stranded DNA is a
significantly greater challenge than for the Pu.Py base pairs (GC,
AT), as fewer hydrogen bonding residues are present within
the major groove of the duplex.7 Thymine is the only natural
base capable of binding to CG, but lacks selectivity, actually
forming a more stable triplet with AT. The unnatural base 4HT,
one of several presented as a replacement for T (Fig. 1),8 is
selective for CG, forming one weak C–H ◊ ◊ ◊ O hydrogen bond
and one conventional N–H ◊ ◊ ◊ N hydrogen bond. Ranasinghe
et al.9 examined the N-methylated base MP, an analogue of the
bicyclic nucleoside, 6-methylpyrrolo-dC (MNHP), which has been
investigated as a fluorescent mimic of C in duplex forming probes,
the fluorescence of which is sensitive to hybridisation with G.10
The core structure maintains the hydrogen bonding motif of 4HT,
Fig. 1 Binding models of T.CG, 4HT.CG, MP. CG and APP.CG triplets. R =
2¢-deoxyribofuranose sugar.
whilst contributing extra base-stacking via an extended conjugated
system. Recognition was enhanced further by extending hydrogen
bonding interactions across the CG base pair. This was achieved by
replacing the 6-methyl group of MP with a 2-aminoethyl (AEP) or 3-
aminopropyl group (APP) for binding to C O6 and N7 of G, whilst
introducing additional charge-stabilisation due to protonation of
the pendant amine group. The 4N-methyl group was vital for
maintaining selectivity, as it prevents this C-analogue from binding
to GC base pairs.
These monomers demonstrated enhanced binding affinity to
CG and improved selectivity compared to T, but were similar to
4HT and MP, suggesting the aminopropyl group of APP does not
contribute greatly to binding. From this work we hypothesised that
reducing linker flexibility whilst allowing some rotational freedom
around the aryl-C6 bond might position the amino group more
precisely for effective hydrogen bonding.
aSchool of Chemistry, University of Southampton, Highfield, Southampton,
SO17 1BJ, UK. E-mail: tb2@soton.ac.uk
bATDBio Ltd, School of Chemistry, University of Southampton, Highfield,
Southampton, SO17 1BJ, UK
cSchool of Biological Sciences, University of Southampton, Bassett Crescent
East, Southampton, SO16 7PX, UK
† Electronic supplementary information (ESI) available: 13C NMR, 1H
NMR (for phosphoramidites) and IR data, melting study protocols,
melting data, example melting curves and derivatives, oligonucleotide
sequences and ESMS data. See DOI: 10.1039/c0ob00119h
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To this end, a number of TFOs containing substituted phenyl-
N-methylpyrrolo-dC nucleotides (XP) have been synthesised, with
amino, acetamido, ureido, and guanidino groups in the 3-position
(Fig. 2). Previously our approach has been to incorporate the
furano-dT monomer in to TFOs and convert post-synthetically to
N-methylpyrrolo-dC.11 We now describe the direct incorporation
of the N-methylpyrrolo-dC phosphoramidites in to TFOs, which
is a higher yielding and simpler strategy.
N-methylimidazole and POCl3.12 After methylamine insertion and
deacetylation, the conversion product 3 was obtained in 90% yield
(Scheme 1).
Scheme 1 Synthesis of 5-iodo-4N-methyl-2¢-deoxycytidine substrate 1. i)
Ac2O (1.7 eq), pyridine, 0 ◦C–rt, 3 h; ii) POCl3 (3.8 eq), NMI (12.8 eq),
pyridine, 0 ◦C–rt, 1 h; iii) 40 wt% CH3NH2–H2O, 0 ◦C–rt, 16 h.
Five modified 6-phenyl-N-methylpyrrolo-dC phosphoramidite
monomers were synthesised from this intermediate;
trifluoroacetamido- (AP, 12), acetamido- (AcP, 8b), ureido-
(UP, 8c), guanidino- (GP, 17) and also 6-phenyl-N-methylpyrrolo-
dC phosphoramidite (PhP, 8a). Monomers 8a–c were synthesised
via Pd-catalysed cross-coupling with the appropriate phenyl
acetylene (Scheme 2), followed by CuI-catalysed cyclisation13
then phosphitylation (Scheme 3). Monomers 12 and 17 required
additional steps as described below (Schemes 4, 5).
Scheme 2 Synthesis of alkynes 5a,b i) AcCl (1.0 eq), Et3N (1.0 eq), Et2O,
0
◦C–rt, 1.5 h, ii) phenyl carbamate (2.0 eq), 90 ◦C, 12 h.
Fig. 2 Putative PhP. CG, AP. CG and UP.CG triplet binding motifs, and X
nucleotide. R = 2¢-deoxyribofuranose, R¢ = H (PhP), NH2 (AP), NHCOCH3
AcP), NHCONH2 (UP), NHC(NH2+)NH2 (GP).
P
(
Results and discussion
The preliminary series of nucleosides, based on
a 3H-
furano[2,3-d]pyrimidin-2(7H)-one core, were constructed as de-
scribed by Ranasinghe et al.9a for the synthesis of AEP and
APP. These monomers were incorporated into oligonucleotides and
post-synthetically converted from furano- to N-methylpyrrolo-
pyrimidines, as previously reported (ring-opening with methy-
lamine, then subjected to acid-catalysed cyclisation with DOWEX-
H+ resin).11
Synthesis of N-methylpyrrolo-dC analogues
Scheme 3 Synthesis of PhP 8a, AcP 8b, and UP 8c phosphoramidites. i)
alkyne 5a–c (3.0 eq), Pd(PPh3)4 (0.1 eq), CuI (0.4 eq), Et3N (5.0 eq),
The alternative synthetic strategy, direct synthesis of N-
methylpyrrolo-dC phosphoramidite monomers followed by in-
corporation into TFOs, obviates the need for tedious post-
synthetic modification. This was adopted due to problems with
the previous method caused by incomplete re-cyclisation of the
N-methylpyrrolopyrimidine core (identified by HPLC), giving rise
to secondary transitions in triplex melting studies. The direct syn-
thetic route involved conversion of 4,4¢-dimethoxytrityl (DMTr)
protected 5-iodo-2¢-deoxyuridine 2 into 5-iodo-4N-methyl-2¢-
deoxycytidine 3 via activation of the 4-carbonyl position, using
˚
DMF, dark, rt, 1–3 h; ii) CuI (1.1 eq), Et3N (10.0 eq), 4 A sieves,
DMF, dark, 125 ◦C, 0.5–2 h; iii) 2-cyanoethoxy-N,N-diisopropylamino
chlorophosphine (1.1 eq), DIPEA (2.5 eq), CH2Cl2 or THF–DMF
(1 : 2 v/v), rt, 1–2 h. R = H (a), NHCOCH3 (b), NHCONH2 (c).
Monomer AP 12 was synthesised by the route in Scheme 4, which
A
involved trifluoroacetylation of P monomer precursor 10 under
forcing conditions, followed by phosphitylation. Monomer GP 17
was synthesised by guanidinylation of 10 using isothiourea 15.
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Scheme 4 Synthesis of AP phosphoramidite 12. i) alkyne 4 (3.0 eq),
Pd(PPh3)4 (0.1 eq), CuI (0.4 eq), Et3N (5.0 eq), DMF, dark, rt, 1.5 h;
ii) CuI (1.1 eq), Et3N (10.0 eq), 4 A sieves, DMF, dark, 125 ◦C, 1.5 h; iii)
˚
CF3CO2Et (20.0 eq), DMAP (1.0 eq), Et3N (22.0 eq), CH2Cl2, 0 ◦C–rt,
35 min then 50 ◦C, overnight; iv) 2-cyanoethoxy-N,N-diisopropylamino
chlorophosphine (1.1 eq), DIPEA (2.5 eq), CH2Cl2, rt, 5 h.
Fig. 3 Monomers incorporated into oligonucleotides.
Guanidinylating reagent 15 was synthesised by a much improved
synthetic route, in 89% yield compared to the literature yield of
35%.14 Guanidinylation followed by phosphitylation afforded the
desired phosphoramidite monomer (Scheme 5). Careful handling
minimised degradation of these acid- and base-sensitive protected
guanidinyl-nucleosides.
Table 1 UV triplex melting experiment: XP against YZ at pH 6.2, 6.6 and
7.0 (10 mM sodium phosphate, 200 mM NaCl, 1 mM Na2EDTA)a,b
Entry VXW YZ
pH
T
PhP
AP
AcP
UP
GP
1
2
3
4
5
6
7
8
TXT
CG 6.2 26.7 32.2 31.5 28.5 30.9 29.0
GC
AT
TA
CG 6.2
—
—
—
—
—
22.7 22.5 21.4 21.2 20.4
22.9 22.6 21.0 22.2 21.8
18.6 n.d.
21.8 22.3 19.3
TXP
PXT
PXP
TXT
TXP
PXT
PXP
TXT
34.2
34.9
—
—
—
—
—
—
—
—
30.2 36.9 37.0 31.6 34.3 32.1
CG 6.6 20.1 24.9 23.6 21.6 23.6 21.9
n.d. 26.2 24.5 22.7 24.7 22.3
9
10
11
12
23.6 26.8 27.0 23.8 25.6 23.2
22.9 28.9 28.4 24.2 26.4 23.7
CG 7.0
—
18.6 18.3 n.d.
n.d.
n.d.
a Av◦erage Tm values for triplex melt given in ◦C. Heating/cooling at
0.5 C min-1. n.d. = not determined (Tm < 17 ◦C). b M = 5-MedC;P = 5-
(3-aminoprop-1-ynyl)-dU (pdU), V, W = T or P, X = T, PhP, AP, AcP, UP or
GP; purine duplex strand ( Y = C, G, A, T); pyrimidine duplex strand (Z =
G, C, T, A).
Scheme 5 Synthesis of GP phosphoramidite 17. i) carbonate 14 (2.0 eq),
NaHCO3 (3.0 eq), degassed CH2Cl2–H2O (2 : 1 v/v), rt, 8 h; ii) carbonate
14 (1.0 eq), CH2Cl2, rt, 14.5 h; iii) isothiourea 15 (2.4 eq), pyridine (4.8 eq),
˚
4 A sieves, CH2Cl2, reflux, 2 d; iv) 2-cyanoethoxy-N,N-diisopropylamino
chlorophosphine (1.8 eq), DIPEA (2.0 eq), CH2Cl2, rt, 2 h. Compound
15 was synthesised by modification to literature route.14 Compound 14
synthesised according to literature method.15
analysis was conducted using modified TFOs (see ESI, Section B.3
and Table C1), targeting a single CG inversion in a homopurine
tract, in a modified version of Leumann’s triplex mo◦tif.8 UV
melting was performed with a temperature gradient of 0.5 C min-1
at pH 6.2–7.0 (Table 1).
The order of stability of analogues of the triplet in Fig. 2,
based on hydrogen bonding, base-stacking capability and charge,
is predicted to be GP > UP > AcP > AP > PhP > MP > T (MP examined
in previous study11). It was surprising therefore, that the simplest
analogue, PhP, should match or outperform all other monomers
against CG in all TFOs, under all buffer conditions (Table 1,
Five monomers, PhP, AP, AcP, UP and GP (Fig. 3), and the
natural nucleoside T, were thus incorporated into TFOs for
UV and fluorescence melting studies, using standard solid-phase
DNA synthesis, and standard deprotection conditions (conc. aq
ammonia, rt or 55 ◦C, 4–24 h).
Melting studies
Preliminary DNA melting data11 indicated high binding affinities
and good selectivity for CG. In the present study, UV melting
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Fig. 4 UV melting curves (a/c) and derivatives (b/d). a/b: XP (TFO 1-6, TXT, see ESI, Table C1) against CG at pH 6.2 (10 mM sodium phosphate,
200 mM NaCl, 1 mM Na2EDTA). See Table 1, entry 1. c/d: AP (TFO 3, TXT, see ESI, Table C1) against YZ at pH 6.2 (10 mM sodium phosphate,
200 mM NaCl, 1 mM Na2EDTA). See Table 1, entries 1–4.
Table 2 Fluorescence triplex melting experiment: XP against YZ at
pH 6.2, 6.6 and 7.0, or 7.0 + 2 mM spermine* (10 mM sodium phosphate,
200 mM NaCl, 1 mM Na2EDTA)a,b
entries 1,7–12, see Fig. 4a,b). The acetamide, AcP, demonstrated
lowest binding affinity to CG (still higher than T) in all sequences
and buffer conditions, also contradicting our expectations (entries
1,7–11).
Selectivity for CG was high for all monomers at pH 6.2 (Table 1,
◦
Fig. 4c,d). AcP was least selective for CG, (DTm +6.7 C vs. TA,
entry 4), and PhP demonstrated greatest selectivity (+9.3 ◦C vs. AT,
entry 3), similar to AP (+8.9 ◦C vs. AT). PhP would be expected to
exhibit lowest selectivity due to lack of potential hydrogen bonding
interactions with G, necessary for good discrimination between
base pairs.
Sequence-dependence of binding was also assessed by varying
the nucleotides on either side of monomer XP in the TFO. Thymine
and 5-(3-aminoprop-1-ynyl)-2¢-deoxyuridine (pdU) were used
for recognition of the neighbouring AT base pairs, the latter
of which significantly increases triplex stability due to intro-
duction of charge-stabilising interactions and additional base-
stacking.7a
Entry VXW YZ
pH
T
PhP
AP
AcP
UP
GP
1
2
3
4
TXT CG 6.2 44.3 44.0 44.0
43.9
38.8
35.0
41.9
50.3
51.0
55.1
46.9
45.0
50.4
36.2
n.d.
n.d.
33.9
43.8
43.8
46.5
30.9
39.9
36.0
44.3 47.2
39.9 36.9
35.1 37.0
40.7 39.7
52.0 53.0
50.8 51.3
57.8 56.0
46.0 45.0
43.8 47.0
49.6 51.0
36.9 40.3
GC
AT
TA
—
—
—
32.0 36.7
34.6 33.9
37.6 36.7
5
6
7
TXP CG 6.2 47.5 50.8 51.0
PXT 48.4 49.6 49.1
PXP CG 6.2 51.1 55.7 56.8
8
9
GC
AT
—
—
—
—
—
—
—
—
—
—
—
—
—
42.1 45.8
44.0 43.3
48.9 47.2
37.0 37.5
31.0 n.d.
n.d. n.d.
31.0 31.5
44.0 43.2
43.0 42.2
48.1 48.5
n.d. 32.0
39.9 40.6
36.1 36.0
10
11
12
13
14
15
16
17
18
19
20
TA
TXT CG 6.6
GC
AT
TA
n.d.
n.d.
n.d.
30.9
TFOs containing central TFO trinucleotides, PXT and TXP,
are expected to dissociate at a similar temperature (Tm), as only
the sequence order is changed and differences in melting kinetics
should be minimal. PXT-oligonucleotides, however, formed more
stable triplexes than TXP-containing TFOs (pH 6.6, entries 9,10,
33.5 33.0
44.2 46.2
43.0 43.7
48.2 47.2
31.6 32.7
39.9 38.5
37.4 39.0
TXP CG 6.6
PXT
PXP
TXT CG 7.0
PXP
TXT CG 7.0*
◦
A
see ESI, Fig. B1), by up to +2.5 C (X = P). This difference be-
tween sequences is probably due to specific electrostatic, hydrogen
bonding or steric interactions between neighbouring pdU and XP
nucleotides, which are difficult to predict.
a Average Tm values for triplex melt given in ◦C. n.d. = not determined
(Tm < 30 ◦C). b M = 5-MedC, P = 5-(3-aminoprop-1-ynyl)-dU (pdU), V,W =
T or P, X = T, PhP, AP, AcP, UP or GP, Q = DABCYL, F = FAM, H =
hexa(ethylene glycol), HEG, hairpin duplex YZ = CG, GC, AT, TA.
Fluorescence-based triplex melting analysis16 was also con-
ducted, using 5¢-DABCYL-labelled, modified TFOs (see ESI,
Section B.4 and Table C2), targeting a single CG inver-
sion in a homopurine tract of a 5¢-FAM-labelled hairpin du-
plex at pH 6.2–7.0 and at pH 7.0 with 2 mM spermine
(Table 2).
These results correlate more closely with predictions based on
the triplet binding model in Fig. 2. Under all buffer conditions,
for TXT sequences (entries 1,11,18,20, see ESI, Fig. B2), GP
demonstrated the highest Tm values by up to +2.9 ◦C (at pH 6.2),
while AcP performed poorest throughout, except at pH 7.0 where
PhP was poorest (<30 ◦C, entry 18). The neighbouring nucleotides
to XP were varied as previously described for the UV melting
study. It was noted that neighbouring protonated pdU nucleotides
contributed less stabilisation to the GP triplex than the rest,
possibly due to significant electrostatic destabilisation from the
guanidinium moiety, which was also observed in the UV melting.
Relative destabilisation from two neighbouring pdU nucleotides
grows with increasing pH (entries 7,17,19), such that the relative
binding affinity of GP for CG, drops from moderate to lowest.
The binding affinity of UP for CG is also affected to a lesser
extent.
Similar effects were observed for TFOs with PXT or TXP
central trinucleotides. In contrast to UV melting data, PXT-
containing TFOs exhibited a lower binding affinity to the CG
duplex than the corresponding TXP-containing TFOs (entries
5,6,15,16). This is except for the acetamide (AcP), which exhibited
the reverse at pH 6.2 (entries 5,6) and no difference at pH 6.6 (en-
tries 18,19). GP displayed the largest difference between sequences
(-2.8/-2.5 ◦C at pH 6.2/6.6, entries 5,6,15,16, see ESI, Fig. B5).
With the exception of AcP, this Tm difference did not appear to
relate to pH.
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Table 3 Fluorescence/UV triplex melting comparison experiment: X
against CG at pH 6.2 (10 mM sodium phosphate, 200 mM NaCl, 1 mM
Na2EDTA) in fluorescence melting motif (Table 2)a
Conclusion
Five CG recognition monomers were synthesised and incorpo-
rated into TFOs to recognise a CG inversion in a target duplex.
Most demonstrated a high binding affinity/selectivity for CG, and
would be suitable for use in synthesis of oligotherapeutics (gene
therapy, chemotherapy) and other applications. TFO sequence,
however, is the key factor governing the relative differences in
triplex stability and selectivity between recognition monomers.
This is of great importance in design of oligonucleotides for
targeting mixed sequences, as sequence-effects are hard to predict.
These modified nucleotides offer a significant improvement on
current CG recognition monomers.
Method
Base pair Buffer PhP
AP
AcP
UP
GP
Fluorescence CG
UV
6.2
44.0b 44.0b 43.9b 44.3b 47.2b
45.1 45.2 44.6 45.7 48.6
DTm
+1.1 +1.2 +0.7 +1.4 +1.4
a Average Tm values for triplex melt given in ◦C. Heating at 0.5 ◦C min-1.
X = PhP, AP, AcP, UP or GP. b Values from entry 1 of Table 2 for comparison.
The selectivity for CG at pH 6.2 (entries 1–4,7–10) and
pH 6.6 (entries 11–14) varied to a greater extent than in the
UV melting study. For the TXT sequence, GP is most selective
(+7.3–7.5 ◦C vs. TA, see ESI, Fig. B4), and AP and PhP also
showed very good selectivity at pH 6.2 (+7.3/+6.4 ◦C respec-
tively). The urea and acetamide, however, were less selective for
CG.
Selectivity results for the PXP sequence, however, differed
significantly, such that AP is now most selective (+9.6 ◦C),
followed by UP (+8.2 ◦C) and PhP (+6.8 ◦C). AcP and GP are
now of low to moderate selectivity. The relative destabilising
electrostatic influence on GP of the neighbouring protonated pdU
nucleotides has an effect on selectivity, in addition to binding
affinity.
Experimental
General
Reagents for chemical synthesis were purchased from Sigma-
Aldrich, Fluka, Acros Organics, Fisher Scientific or Alfa Aesar
and used without further purification. Pyridine, dichloromethane
(CH2Cl2), triethylamine (Et3N) and N,N-diisopropylethylamine
(DIPEA) were distilled over calcium hydride; phosphorus oxy-
chloride over sodium wire; tetrahydrofuran over sodium wire and
benzophenone; and diethyl ether by passing through a column of
dry alumina then degassing at low temperature, or by degassing
˚
with argon over activated 4 A molecular sieves shortly before
Differences in the DNA sequences were assumed to be
responsible for the relative differences in triplex stability and
selectivity between UV and fluorescence melting studies. In order
to exclude the possibility that these differences were caused by
the method of analysis, UV triplex melting experiments were
conducted on the fluorescence melting motif (TXT sequence,
pH 6.2, Table 3). These data prove that the method of analysis does
not affect the relative recognition properties of the monomers.
All UV melting Tms were on average 1.2 ◦C higher than those
observed by fluorescence melting.
In general, there appears to be an inverse correlation between
steric bulk and CG-recognition capability. The unmodified phenyl
monomer PhP, despite lacking hydrogen bonding functionality for
binding to G, formed the strongest and most selective triplexes
in all UV-melting conditions, closely followed by the aniline
AP. The guanidinium monomer GP, appeared most sequence
dependent, exhibiting excellent binding affinity and selectivity
in most fluorescence melting conditions, yet moderate to poor
binding affinity and moderate to good selectivity in the UV melting
triplex sequence. The acetamide performed poorest of all, both in
terms of binding affinity and selectivity.
Choosing between recognition monomers depends strongly on
TFO sequence. In sequences with few pdU modifications, AP
and PhP monomers are best. In pdU-rich sequences, where the
modification is abutted by T, GP is best at all pHs. However,
with neighbouring pdU nucleotides, the relative stability of the GP
triplex drops, such that UP (pH 6.2) then AP (pH 6.6/7.0) produce
the most stable triplexes. For these reasons, from these studies,
the optimum monomer for CG recognition is the aniline, AP. This
monomer has the highest overall selectivity and binding affinity,
little pH dependence, and its recognition properties appear least
sequence-specific.
use. Phosphitylating reagents, DNA phosphoramidite monomers,
other reagents and solid supports were purchased from Link
Technologies Ltd. or Applied Biosystems Ltd. Deuterated NMR
solvents were purchased from Apollo Scientific Ltd. All reactions
requiring absence of oxygen were carried out under an atmosphere
of argon, and glassware was dried overnight at 120 ◦C before
use. Column chromatography was carried out under air or argon
˚
pressure using Fisher Scientific DAVISIL 60 A (35–70 micron)
silica gel, and reactions were monitored by TLC, using Merck
Kieselgel 60 F254 or Machenary-Nagel Alugram Sil G/UV254 silica
gel plates (0.22 mm thickness, aluminium backed). Compounds
were visualised by irradiation at 254/365 nm, or by staining
with p-anisaldehyde, potassium permanganate, ninhydrin, H2SO4–
ethanol or ceric sulfate, followed by heating. Proton NMR
spectra were recorded using either a Bruker AC300 or Bruker
DPX400 spectrometer. Fluorine and phosphorus NMR spectra
were recorded using a Bruker AC300 spectrometer. NMR spectra
were recorded in deuterated chloroform or dimethylsulfoxide.
Chemical shifts are given in ppm relative to tetramethylsilane, and
spectra are calibrated to the appropriate residual solvent peak.17
J values are accurate to within 0.5 Hz. Assignment was aided
by H–H/H–C correlation experiments. Compound numbering
is shown in Fig. 5. Low-resolution mass spectra were recorded
using electrospray ionisation (ES) on a Fisons VG platform
instrument or on a Waters ZMD quadrupole mass spectrometer,
in HPLC grade acetonitrile or methanol. High-resolution mass
spectra were recorded in HPLC grade acetonitrile or methanol
using electrospray ionisation on a Bruker APEX III FT-ICR mass
spectrometer. Melting points were recorded on a Gallenkamp
Electrothermal melting point apparatus and quoted uncalibrated.
Elemental (CHN) Thermal Combustion Analysis was carried out
by MEDAC Ltd., Egham, Surrey, UK.
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Compound numbering
0
◦C, distilled phosphorous oxychloride (3.00 mL, 32.2 mmol,
3.8 eq) was added dropwise over 20 min. The yellow suspension
was stirred for 20 min then allowed to warm to room temperature
◦
over a further 30 min. The mixture was cooled to 0 C, 40 wt%
MeNH2–water solution (40.5 mL, 468 mmol MeNH2, 55.9 eq
MeNH2) was added, and the cloudy solution was stirred at room
temperature for 18 h. The mixture was concentrated under high
vacuum, dissolved in CH2Cl2 (300 mL) and washed with water
(3 ¥ 75 mL). The combined aqueous fraction was re-extracted
(CH2Cl2, 40 mL), the combined organic layer was washed
with sat. aq KCl (120 mL), dried (Na2SO4) and concentrated
to give a pale yellow foam. Following careful purification by
column chromatography (CH2Cl2–MeOH–Et3N, 98 : 2 : 0.5), and
precipitation with Et2O, the 4N-methyl-dC nucleoside 3 was
afforded, as a white powder (4.92 g, 7.35 mmol, 88%). Rf 0.40
(CH2Cl2–MeOH–Et3N, 90 : 10 : 0.3), 0.36 (CH2Cl2–acetone–Et3N,
80 : 20 : 0.3), 0.57 (acetone–MeOH–Et3N, 90 : 10 : 0.3); Mp 141–
144 ◦C (ethyl acetate–diethyl ether); 1H NMR (400 MHz, DMSO-
d6) d (ppm) 7.93 (1H, s, H6), 7.40 (2H, d, J = 7.5 Hz, H13), 7.32
(2H, t, J = 7.3 Hz, H14), 7.29 (4H, d, J = 9.0 Hz, H9), 7.22 (1H, tt,
J = 2.0, 7.3 Hz, H15), 7.06 (1H, q, J = 4.5 Hz, NHCH3), 6.89 (4H,
d, J = 9.0 Hz, H10), 6.12 (1H, dd, J = 6.2, 7.4 Hz, H1¢), 5.25 (1H, d,
J = 4.3 Hz, 3¢-OH), 4.20 (1H, dt, J = 3.3, 6.4 Hz, H3¢), 3.91 (1H,
dd, J = 3.3, 6.5 Hz, H4¢), 3.74 (6H, s, OCH3), 3.20 (1H, dd, J = 3.3,
10.8 Hz, H5¢), 3.17 (1H, dd, J = 4.5, 10.8 Hz, H5¢), 2.80 (3H, d, J =
4.5 Hz, NHCH3), 2.21 (1H, ddd, J = 3.0, 6.0, 13.3 Hz, H2¢), 2.09
(1H, td, J = 6.8, 13.6 Hz, H2¢); MS (ES+) m/z: 692 ([M + Na]+, 12),
670 ([M + H]+, 3), 303 (DMTr+, 100); (ES-) m/z: 668 ([M - H]-,
100); HRMS (ES+): calcd. for C31H32N3O6I, [M + K]+ 708.0967,
found 708.0962, [M + Na]+ 692.1228, found 692.1219, [M + H]+
670.1409, found 670.1414; Anal. calcd. for C31H32N3O6I (669.51)
requires C, 55.61%; H, 4.82%; N, 6.27%. Found C, 54.80%; H,
4.75%; N, 6.05%.
Fig. 5 Example compound numbering (6c and 16).
Synthesis
3¢-O-Acetyl-5¢-O-(4,4¢-dimethoxytrityl)-5-iodo-2¢-deoxyuridine
(2). To a stirred solution of 5¢-O-(4,4¢-dimethoxytrityl)-5-iodo-2¢-
18
deoxyuridine‡1 (10.0 g, 15.2 mmol◦) in distilled pyridine (40.0 mL),
under an argon atmosphere, at 0 C was added acetic anhydride
(7.20 mL, 76.3 mmol, 5.0 eq) dropwise over 20 min. The
reaction was stirred at 0 ◦C for 15 min then allowed to warm
to room temperature. After 243 hours, the reaction mixture was
concentrated in vacuo and under high vacuum, co-evaporating
with acetone (2 ¥ 50 mL) and diethyl ether (2 ¥ 50 mL), to give
a white foam/syrup. The syrup was dissolved in ethyl acetate
(300 mL) and washed with water (3 ¥ 75 mL). The aqueous
was re-extracted (ethyl acetate, 40 mL) and combined organic
fractions were washed with sat. aq KCl (120 mL), dried (Na2SO4)
and concentrated in vacuo and under high vacuum to give a
white foam. Following purification by column chromatography
(CH2Cl2–MeOH–Et3N, 100 : 0 : 0.5→98 : 2 : 0.5), and drying un-
der high vacuum, the desired product 2 was afforded as a
white foam (10.3 g, 14.7 mmol, 97%). Rf 0.31 (CH2Cl2–acetone–
Et3N, 90 : 10 : 0.3), 0.48 (CH2Cl2–MeOH–Et3N, 95 : 5 : 0.3), 0.33
3-Acetamidophenyl acetylene (5a). 19 To a stirred solution of 3-
ethynylaniline 4 (3.50 mL, 31.1 mmol) and distilled Et3N (4.37 mL,
31.4 mmol, 1.0 eq) in dry, degassed diethyl ether (15.0 mL), at 0 ◦C,
under an argon atmosphere, was added acetyl chloride (2.23 mL,
31.4 mmol, 1.0 eq) in dry, degassed diethyl ether (25.0 mL),
dropwise over 45 min. The reaction was stirred for a further 15 min
then allowed to warm to room temperature over 30 min. The
mixture was filtered, the white precipitate was washed with diethyl
ether (3 ¥ 35 mL) and combined ether fractions were concentrated
in vacuo to give a cream-coloured solid. The solid was triturated
with water (60 mL) to which diethyl ether (100 mL) was added, and
the layers were separated. The ether layer was washed with water
(50 mL), combined aqueous layers were re-extracted (diethyl ether,
20 mL), and combined ether fractions were concentrated in vacuo
and dried under high vacuum to afford the desired product 5a as a
pale brown oil, which crystallised on standing to an off-white waxy
solid (4.79 g, 30.1 mmol, 97%). Rf 0.40 (CH2Cl2–CH3OH–Et3N,
1
(hexane–ethyl acetate–Et3N, 70 : 30 : 0.3); H NMR (400 MHz,
DMSO-d6) d (ppm) 11.75 (1H, s, NH3), 8.05 (1H, s, H6), 7.39 (2H,
d, J = 8.0 Hz, H13), 7.32 (2H, t, J = 7.3 Hz, H14), 7.28 (2H, d,
J = 9.0 Hz, H9), 7.28 (2H, d, J = 9.0 Hz, H9), 7.23 (1H, tt, J =
1.1, 7.2 Hz, H15), 6.89 (4H, d, J = 8.8 Hz, H10), 6.10 (1H, dd, J =
6.1, 8.2 Hz, H1¢), 5.23 (1H, td, J = 2.4, 6.4 Hz, H3¢), 4.07 (1H,
td, J = 2.9, 4.5 Hz, H4¢), 3.74 (6H, s, OCH3), 3.32 (1H, dd, J =
4.9, 10.4 Hz, H5¢), 3.22 (1H, dd, J = 3.1, 10.4 Hz, H5¢), 2.44 (1H,
td, J = 7.0, 11.5 Hz, H2¢), 2.33 (1H, ddd, J = 2.0, 6.0, 14.3 Hz,
H2¢), 2.03 (3H, s, COCH3); MS (ES+) m/z: 721 ([M + Na]+, 12),
303 (DMTr+, 100); (ES-) m/z: 697 ([M - H]-, 100); HRMS (ES+):
calcd for C32H31N2O8I (M), [M + Et3N + H]+ = 800.2402, found
800.2369; [M + Na]+ = 721.1017, found 721.1007.
◦
95 : 5 : 0.3); Mp 92–94 C (chloroform–hexane), lit.19 94–96 ◦C
1
(CCl4); H NMR (300 MHz, DMSO-d6) d (ppm) 10.00 (1H, s,
NHCOCH3), 7.77 (1H, app. t, J = 1.7 Hz, H2), 7.54 (1H, ddd, J =
1.1, 2.0, 8.1 Hz, H4), 7.30 (1H, t, J = 7.9 Hz, H5), 7.13 (1H, td,
J = 1.3, 7.7 Hz, H6), 4.13 (1H, s, C CH), 2.05 (3H, s, COCH3);
MS (ES+) m/z: 160 ([M + H]+, 100), 182 ([M + Na]+, 11), 201
([M + CH3CN + H]+, 7), 223 ([M + CH3CN + Na]+, 17), 242 ([M
+ AcOH + Na]+, 12); (ES-) m/z: 158 ([M - H]-, 100); HRMS
5¢-O-(4,4¢-Dimethoxytrityl)-5-iodo-4N-methyl-2¢deoxycytidine
(3). To a stirred solution of acetylated nucleoside 2 (5.85 g,
8.38 mmol) and N-methylimidazole (8.64 mL, 108 mmol, 12.9 eq)
in distilled pyridine (130.0 mL), under an argon atmosphere, at
‡ Prepared according to literature procedure.18
5092 | Org. Biomol. Chem., 2010, 8, 5087–5096
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(ES+): calcd. for C10H9NO, [2M + Na]+ 341.1260, found 341.1239,
[M + Na]+ 182.0576, found 182.0565, [M + H]+ 160.0757, found
160.0755.
[M + K]+ 682.2314, found 682.2303; [M + Na]+ 666.2575, found
666.2565; [M + H]+ 644.2755, found 644.2755.
5-(3-Acetamidophenyl)ethynyl-5¢-O-(4,4¢-dimethoxytrityl)-4N-
3-Ureidophenyl acetylene (5b). 20 3-Ethynylaniline 4 (3.00 mL,
26.6 mmol) and dry phenyl carbamate (7.31 g, 53.3 mmol, 2.0 eq)
were heated in a sealed tube, under an argon atmosphere, excluding
moisture, in dim light at 90 ◦C for 12 h. The mixture was
cooled to room temperature, removed using warm methanol–
acetone, and dried under high vacuum. Following purification
by column chromatography (CH2Cl2–acetone, 95 : 5→50:50), and
drying under high vacuum, the desired urea 5b was afforded,
as an off-white, papery solid (2.93 g, 18.3 mmol, 69%). Rf
0.31 (CH2Cl2–CH3OH–Et3N, 90 : 10 : 0.3), 0.04 (CH2Cl2–acetone–
Et3N, 90 : 10 : 0.3); 1H NMR (300 MHz, DMSO-d6) d (ppm) 8.61
(1H, s, NH), 7.64 (1H, t, J = 1.7 Hz, H2), 7.32 (1H, ddd, J = 1.1,
2.0, 8.2 Hz, H4), 7.22 (1H, t, J = 7.9 Hz, H5), 7.00 (1H, td J = 1.3,
7.3 Hz, H6), 5.89 (2H, s, NH2), 4.08 (1H, s, C CH); MS (ES+)
m/z: 343 ([2M + Na]+, 21), 183 ([M + Na]+, 70), 102 ([Et3N +
H]+, 100); (ES-) m/z: 159 ([M - H]-, 100); HRMS (ES+): calcd.
for C9H8N2O, [M + Na]+ 183.0529, found 183.0530, [2M + Na]+
343.1171, found 343.1170.
methyl-2¢-deoxycytidine (6b). Obtained from
3
(1.69 g,
2.53 mmol) and alkyne 5b (7.60 mmol, 3.0 eq), after aqueous
workup with 5% w/v aq Na2EDTA (pH 9) and column chro-
matography (CH2Cl2–acetone–pyridine, 90 : 10 : 1 →30 : 70 : 1),
partially purified as
a yellow glassy foam (1.99 g). Rf
0.16 (CH2Cl2–acetone–Et3N, 40 : 60 : 0.3), 0.04 (CH2Cl2–acetone–
pyridine, 80 : 20 : 0.3), 0.28 (CH2Cl2–acetone–Et3N, 20 : 80 : 0.3),
1
0.32 (CH2Cl2–acetone–pyridine, 20 : 80 : 1); H NMR (400 MHz,
DMSO-d6) d (ppm) 9.97 (1H, s, NHCOCH3), 8.00 (1H, s, H6),
7.79 (1H, s, H2¢¢), 7.46 (1H, ddd, J = 1.0, 2.0, 8.5 Hz, H4¢¢), 7.43
(1H, q, J = 5.0 Hz, NHCH3), 7.40 (2H, dd, J = 1.3, 8.3 Hz, H15),
7.29 (2H, d, J = 9.0 Hz, H11), 7.29–7.25 (1H, m, H5¢¢), 7.28 (2H, d,
J = 8.5 Hz, H11), 7.24 (2H, t, J = 7.8 Hz, H16), 7.15 (1H, tt, J =
1.3, 7.3 Hz, H17), 6.90 (1H, td, J = 1.3, 7.8 Hz, H6¢¢), 6.84 (2H, d,
J = 9.0 Hz, H12), 6.83 (2H, d, J = 9.0 Hz, H12), 6.16 (1H, t, J =
6.8 Hz, H1¢), 5.28 (1H, d, J = 4.5 Hz, 3¢-OH), 4.26 (1H, dt, J =
3.5, 6.7 Hz, H3¢), 3.97 (1H, td, J = 3.0, 5.0 Hz, H4¢), 3.66 (6H, s,
OCH3), 3.23 (1H, dd, J = 5.0, 10.5 Hz, H5¢), 3.15 (1H, dd, J = 2.5,
10.5 Hz, H5¢), 2.86 (3H, d, J = 4.5 Hz, NHCH3), 2.27 (1H, ddd,
J = 3.5, 6.3, 13.6 Hz, H2¢), 2.16 (1H, td, J = 6.7, 13.6 Hz, H2¢),
2.06 (3H, s, COCH3); MS (ES+) m/z: 723 ([M + Na]+, 100), 303
(DMT+, 29); (ES-) m/z: 699 ([M - H]-, 100); HRMS (ES+): calcd.
for C41H40N4O7, [M + H]+ 701.2970, found 701.2974.
General procedure for Pd-catalysed cross-coupling of aromatic al-
kynes 4, 5a–c with 5-iodo-4N-methyl-2¢-deoxycytidine derivative 3
To a solution of 5¢-O-(4,4¢-dimethoxytrityl)-5-iodo-4N-methyl-2¢-
deoxyuridine 3 (0.8–2.5 mmol) in anhydrous DMF (2.5–6.0 mL),
under an argon atmosphere, in absence of light, was added
CuI (0.4 eq), distilled Et3N (5.0 eq) and aromatic alkyne 4,
5a–c (3.0 eq). The mixture was stirred for 15–20 min then
tetrakis(triphenylphosphine) palladium (0) (0.1 eq) was added and
the reaction was stirred at room temperature for 1–3 h. The reac-
tion mixture was concentrated under high vacuum, co-evaporating
with methanol–toluene or acetone–toluene to give an orange–
brown to brown foam. Aqueous workup or filtration of product
from acetone–methanol (1 : 1) and/or column chromatography
afforded the desired product, 6a and 9 (isolated pure), 6b and
6c (isolated with alkyne dimer impurity and used in following
reaction where impurity was removed).
5-(3-Ureidophenyl)ethynyl-5¢-O-(4,4¢-dimethoxytrityl)-4N -
methyl-2¢-deoxycytidine (6c). Obtained from
3
(1.25 g,
1.87 mmol) and alkyne 5c (5.61 mmol, 3.0 eq), after filtration
and column chromatography (CH2Cl2–acetone–Et3N, 20 : 80 : 0.5
→0:100 : 0.5), partially purified as a pale brown foam (1.49 g)
and used without further purification. Rf 0.18 (CH2Cl2–MeOH–
Et3N, 90 : 10 : 0.3), 0.11 (CH2Cl2–acetone–Et3N, 20 : 80 : 0.3), 0.44
(acetone–MeOH–Et3N, 90 : 10 : 0.3).
5-(3-Aminophenyl)ethynyl-5¢-O-(4,4¢-dimethoxytrityl)-4N -
methyl-2¢-deoxycytidine (9). Obtained from 3 (0.97 g, 1.45 mmol)
and alkyne 4 (4.35 mmol, 3.0 eq), after filtration and column
chromatography (CH2Cl2–MeOH–Et3N, 100 : 0 : 0.5→ 97 : 3 : 0.5)
as a pale orange foam (0.91 g, 1.39 mmol, 95%). Rf 0.31
(CH2Cl2–MeOH–Et3N, 90 : 10 : 0.3); 0.31 (CH2Cl2–acetone–Et3N,
5-Phenylethynyl-5¢-O-(4,4¢-dimethoxytrityl)-4N-methyl-2¢-de-
oxycytidine (6a). Obtained from 3 (0.55 g, 0.82 mmol) and
alkyne 5a (2.64 mmol, 3.0 eq), after filtration and column
chromatography (CH2Cl2–MeOH–Et3N, 90 : 10 : 0.5) as a pale
orange foam (0.47 g, 0.74 mmol, 90%). Rf 0.66 (ethyl acetate–
1
20 : 80 : 0.3), 0.03 (CH2Cl2–acetone–Et3N, 80 : 20 : 0.3); H NMR
(400 MHz, DMSO-d6) d (ppm) 7.94 (1H, s, H6), 7.40 (2H, dd, J =
1.1, 8.4 Hz, H15), 7.35 (1H, q, J = 4.8 Hz, NHCH3), 7.30–7.25 (2H,
m, H16), 7.29 (2H, d, J = 8.0 Hz, H11), 7.29 (2H, d, J = 9.0 Hz,
H11), 7.17 (1H, tt, J = 1.0, 7.3 Hz, H17), 6.96 (1H, t, J = 7.8 Hz,
H5¢¢), 6.86 (2H, d, J = 8.5 Hz, H12), 6.83 (2H, d, J = 8.8 Hz, H12),
6.61 (1H, t, J = 1.6 Hz, H2¢¢), 6.57 (1H, ddd, J = 1.1, 2.4, 8.0 Hz,
H4¢¢), 6.46 (1H, ddd, J = 1.1, 2.5, 7.5 Hz, H6¢¢), 6.15 (1H, app. t, J =
6.7 Hz, H1¢), 5.30 (1H, d, J = 4.3 Hz, 3¢-OH), 5.11 (2H, br s, NH2),
4.25 (1H, td, J = 3.1, 9.0 Hz, H3¢), 3.97 (1H, td, J = 2.4, 4.8 Hz,
H4¢), 3.68 (3H, s, OCH3), 3.67 (3H, s, OCH3), 3.23 (1H, dd, J =
5.0, 10.5 Hz, H5¢), 3.14 (1H, dd, J = 2.5, 10.3 Hz, H5¢), 2.86 (3H,
d, J = 4.8 Hz, NHCH3), 2.27 (1H, ddd, J = 3.0, 5.9, 13.4 Hz, H2¢),
2.13 (1H, td, J ¢ 6.8, 13.6 Hz, H2¢); MS (ES+) m/z: 681 ([M + Na]+,
100); HRMS (ES+): calcd. for C39H38N4O6, [M + Na]+ 681.2684,
found 681.2671; [M + H]+ 659.2864, found 659.2847.
1
MeOH–aq NH3, 5 : 1 : 1); H NMR (400 MHz, CDCl3) d (ppm)
8.20 (1H, s, H6), 7.45 (2H, dd, J = 1.2, 8.6 Hz, H15), 7.35 (2H, d,
J = 9.1 Hz, H11), 7.35 (2H, d, J = 8.9 Hz, H11), 7.31 (1H, tt, J = 1.5,
7.3 Hz, H4¢¢), 7.25 (2H, t, J = 7.1 Hz, H3¢¢), 7.25 (2H, t, J = 7.9 Hz,
H16), 7.17–7.12 (2H, m, H17,H2¢¢), 6.79 (2H, d, J = 8.9 Hz, H12),
6.77 (2H, d, J = 8.9 Hz, H12), 6.38 (1H, dd, J = 6.1, 6.8 Hz, H1¢),
5.77 (1H, q, J = 4.8 Hz, NHCH3), 4.58 (1H, td, J = 2.8, 5.5 Hz,
H3¢), 4.19 (1H, dd, J = 3.2, 6.2 Hz, H4¢), 3.70 (3H, s, OCH3), 3.69
(3H, s, OCH3), 3.41 (1H, dd, J = 3.1, 10.6 Hz, H5¢), 3.34 (1H, dd,
J = 3.8, 10.6 Hz, H5¢), 3.11 (3H, d, J = 5.0 Hz, NHCH3), 3.01
(1H, br s, 3¢-OH), 2.76 (1H, ddd, J = 3.1, 5.9, 13.8 Hz, H2¢), 2.28
(1H, J = 6.7, 13.4 Hz, H2¢); MS (ES+) m/z: 745 ([M + Et3N + H]+,
100), 666 ([M + Na]+, 52); HRMS (ES+): calcd. for C39H37N3O6,
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General procedure for Cu-catalysed cyclisation of
5-alkynyl-4N-methyl-2¢-deoxycytidine nucleosides (6a–c, 9)
2(7H)-one (7c). Obtained from 6c (1.48 g of 1.49 g) af-
ter filtration from methanol, column chromatography ¥ 2
(acetone–MeOH–Et3N, 100 : 0 : 0.5→90 : 10 : 0.5), and precipita-
tion (MeOH–acetone–Et2O) as a very pale yellow powder (1.03 g,
1.47 mmol, 79% over two steps). Rf 0.24 (acetone–MeOH–Et3N,
To a solution of nucleoside 6a-c, 9 (0.8–3.4 mmol) and distilled
Et3N (10.0 eq) in anhydrous DMF (2.0–10.0 mL) with activated
˚
4 A molecular sieves, under an argon atmosphere, in absence of
1
90 : 10 : 0.3), 0.49 (CH2Cl2–MeOH–Et3N, 80 : 20 : 0.3); H NMR
light was added CuI (1.1 eq). The reaction mixture was stirred at
125 ◦C for 0.5–2 h, then cooled to rt, and the solution was removed
and dried under high vacuum to give a dark brown foam/syrup.
Aqueous workup with 5% w/v aq Na2EDTA (pH 9) and sat.
aq KCl or filtration of impurities, then column chromatography
afforded the desired product 7a–c, 10.
(400 MHz, DMSO-d6) d (ppm) 8.71 (1H, s, NHCONH2), 8.63
(1H, s, H4), 7.63 (1H, t, J = 1.8 Hz, H2¢¢), 7.41 (1H, ddd, J =
1.0, 2.0, 7.0 Hz, H4¢¢), 7.41 (2H, d, J = 7.0 Hz, H14), 7.34 (1H, t,
J = 8.0 Hz, H5¢¢), 7.32 (2H, tt, J = 1.5, 7.0 Hz, H15), 7.29 (2H, d,
J = 9.0 Hz, H10), 7.28 (2H, d, J = 8.5 Hz, H10), 7.28 (1H, t, J =
8.0 Hz, H16), 7.02 (1H, td, J = 1.3, 7.5 Hz, H6¢¢), 6.89 (2H, d, J =
9.0 Hz, H11), 6.89 (2H, d, J = 9.0 Hz, H11), 6.26 (1H, dd, J = 5.0,
6.5 Hz, H1¢), 5.92 (2H, s, CONH2), 5.59 (1H, s, H5), 5.40 (1H, d,
J = 4.8 Hz, 3¢-OH), 4.43 (1H, qn, J = 5.5 Hz, H3¢), 4.00 (1H, dd,
J = 4.0, 8.0 Hz, H4¢), 3.70 (3H, s, OCH3), 3.69 (3H, s, OCH3), 3.46
(3H, s, NCH3), 3.40 (1H, dd, J = 4.0, 10.5 Hz, H5¢), 3.29 (1H, dd,
J = 2.5, 10.5 Hz, H5¢), 2.44 (1H, td, J = 6.3, 13.1 Hz, H2¢), 2.18
(1H, ddd, J = 5.0, 6.5, 13.6 Hz, H2¢); MS (ES+) m/z: 1425 ([2M +
Na]+, 8), 724 ([M + Na]+, 100), 702 ([M + H]+, 50); HRMS (ES+):
calcd. for C40H39N5O7, [M + Na]+ 724.2742, found 724.2733; Anal.
calcd. for C40H39N5O7 (701.77) requires C, 68.46%; H, 5.60%; N,
9.97%. Found C, 65.70%; H, 5.82%; N, 9.35%.
3-N-[2¢-Deoxy-5¢-O-(4,4¢-dimethoxytrityl)-b-D-ribofuranosyl]-
6-phenyl-(2,3H)-N -methylpyrrolo[2,3-d]pyrimidine-2(7H)-one
(7a). Obtained from 6a (0.52 g, 0.80 mmol) after aqueous
workup and column chromatography (CH2Cl2–MeOH–Et3N,
90 : 10 : 0.5), as a pale orange foam (0.37 g, 0.57 mmol, 71%). Rf
0.67 (ethyl acetate–MeOH–aq NH3, 5 : 1 : 1), 1H NMR (400 MHz,
DMSO-d6) d (ppm) 8.80 (1H, s, H4), 7.49–7.44 (1H, m, H4¢¢), 7.46–
7.42 (2H, m, H2¢¢), 7.45 (2H, dd, J = 1.8, 8.1 Hz, H14), 7.41 (2H,
dt, J = 1.9, 7.9 Hz, H3¢¢), 7.35 (2H, d, J = 8.9 Hz, H10), 7.34 (2H, d,
J = 9.1 Hz, H10), 7.29 (2H, tt, J = 1.5, 7.4 Hz, H15), 7.23 (1H, tt,
J = 1.3, 7.2 Hz, H16), 6.83 (2H, d, J = 8.9 Hz, H11), 6.82 (2H, d, J =
8.9 Hz, H11), 6.50 (1H, dd, J = 5.2, 6.1 Hz, H1¢), 5.56 (1H, s, H5),
4.70 (1H, dd, J = 5.3, 10.6 Hz, H3¢), 4.21 (1H, td, J = 2.9, 4.9 Hz,
H4¢), 3.76 (3H, s, OCH3), 3.75 (3H, s, OCH3), 3.57 (3H, s, NCH3),
3.56 (1H, dd, J = 2.9, 10.7 Hz, H5¢), 3.51 (1H, dd, J = 3.3, 10.8 Hz,
H5¢), 3.36 (1H, br s, 3¢-OH), 2.86 (1H, td, J = 6.1, 13.7 Hz, H2¢),
2.42 (1H, ddd, J = 5.3, 6.1, 13.7 Hz, H2¢); MS (ES+) m/z: 745 ([M +
Et3N + H]+, 81), 666 ([M + Na]+, 100), 644 ([M + H]+, 12); HRMS
(ES+): calcd. for C39H37N3O6, [M + K]+ 682.2314, found 682.2304;
[M + Na]+ 666.2575, found 666.2567; [M + H]+ 644.2755, found
644.2754.
3-N-[2¢-Deoxy-5¢-O-(4,4¢-dimethoxytrityl)-b-D-ribofuranosyl]-
6-(3-aminophenyl)-(2,3H)-N -methylpyrrolo[2,3-d]-pyrimidine-
2(7H)-one (10). Obtained from 6c (2.21 g, 3.36 mmol) after fil-
tration from CH2Cl2, column chromatography (CH2Cl2–acetone–
Et3N, 50 : 50 : 0.5→0 : 100 : 0.5), as a pale brown foam (0.71 g,
1.07 mmol, 80%). Rf 0.08 (CH2Cl2–MeOH–Et3N, 95 : 5 : 0.3),
1
0.14 (CH2Cl2–acetone–Et3N, 20 : 80 : 0.3); H NMR (400 MHz,
DMSO-d6) d (ppm) 8.63 (1H, s, H4), 7.41 (2H, d, J = 7.3 Hz, H14),
7.32 (2H, t, J = 7.0 Hz, H15), 7.29 (4H, d, J = 8.8 Hz, H10), 7.25
(1H, tt, J = 1.8, 7.3 Hz, H16), 7.13 (1H, t, J = 7.8 Hz, H5¢¢), 6.90
(2H, d, J = 8.8 Hz, H11), 6.89 (2H, d, J = 9.0 Hz, H11), 6.67 (1H, t,
J = 1.8 Hz, H2¢), 6.64 (1H, ddd, J = 0.8, 2.2, 8.0 Hz, H4¢¢), 6.60 (1H,
td, J = 1.3, 8.0 Hz, H6¢¢), 6.26 (1H, dd, J = 5.0, 6.3 Hz, H1¢), 5.51
(1H, s, H5), 5.42 (1H, d, J = 4.8 Hz, 3¢-OH), 5.26 (2H, br s, NH2),
4.44 (1H, td, J = 5.3, 10.5 Hz, H3¢), 4.00 (1H, dd, J = 3.9, 7.9 Hz,
H4¢), 3.71 (3H, s, OCH3), 3.70 (3H, s, OCH3), 3.44 (3H, s, NCH3),
3.39 (1H, dd, J = 3.9, 10.7 Hz, H5¢), 3.30 (1H, d, J = 2.6, 10.9 Hz,
H5¢), 2.44 (1H, td, J = 6.5, 13.1 Hz, H2¢), 2.19 (1H, ddd, J = 5.1, 6.4,
13.4 Hz, H2¢); MS (ES+) m/z: 681 ([M + Na]+, 100); HRMS (ES+):
calcd. for C39H38N4O6, [M + Na]+ 681.2684, found 681.2677; Anal.
calcd. for C39H38N4O6 (658.74) requires C, 71.11%; H, 5.81%; N,
8.50%. Found C, 69.90%; H, 6.11%; N, 8.41%.
3-N-[2¢-Deoxy-5¢-O-(4,4¢-dimethoxytrityl)-b-D-ribofuranosyl]-
6 - (3 - acetamidophenyl) - (2, 3H) - N - methylpyrrolo [2, 3 - d] pyri -
midine-2(7H)-one (7b). Obtained from 6b (1.92 g of 1.97 g) after
filtration from CH2Cl2 then aqueous workup and column chro-
matography (acetone–MeOH–Et3N, 100 : 0 : 0.5→90:10 : 0.5), as
a yellow powder (1.43 g, 2.04 mmol, 84% over two steps). Rf 0.09
(4 : 1, CH2Cl2–acetone–Et3N, 20 : 80 : 0.3), 0.14 (CH2Cl2–acetone–
1
pyridine, 20 : 80 : 1); H NMR (400 MHz, DMSO-d6) d (ppm)
10.08 (NHCOCH3), 8.65 (1H, s, H4), 7.79 (1H, br s, H2¢¢), 7.61
(1H, br d, J = 8.0 Hz, H4¢¢), 7.42 (1H, t, J = 8.0 Hz, H5¢¢), 7.40 (2H,
d, J = 7.0 Hz, H14), 7.32 (2H, t, J = 7.0 Hz, H15), 7.29 (2H, d, J =
8.5 Hz, H10), 7.28 (2H, d, J = 9.0 Hz, H10), 7.25 (1H, t, J = 7.0 Hz,
H16), 7.16 (1H, br d, J = 8.0 Hz, H6¢¢), 6.90 (2H, d, J = 9.0 Hz,
H11), 6.89 (2H, d, J = 9.0 Hz, H11), 6.26 (1H, dd, J = 5.3, 6.3 Hz,
H1¢), 5.62 (1H, s, H5), 5.39 (1H, d, J = 5.0 Hz, 3¢-OH), 4.43 (1H,
td, J = 5.5, 11.0 Hz, H3¢), 4.01 (1H, dd, J = 4.0, 8.0 Hz, H4¢), 3.70
(3H, s, OCH3), 3.69 (3H, s, OCH3), 3.47 (3H, s, NCH3), 3.40 (1H,
dd, J = 4.0, 11.0 Hz, H5¢), 3.30 (1H, dd, J = 3.0, 11.0 Hz, H5¢), 2.45
(1H, td, J = 6.5, 13.6 Hz, H2¢), 2.19 (1H, ddd, J = 5.0, 6.5, 13.6 Hz,
H2¢), 2.08 (3H, s, COCH3); MS (ES+) m/z: 803 ([M + Et3N + H]+,
14), 723 ([M + Na]+, 100); (ES-) m/z: 699 ([M - H]-, 100); HRMS
(ES+): calcd. for C41H40N4O7, [M + H]+ 701.2970, found 701.2965.
3-N-[2¢-Deoxy-5¢-O-(4,4¢-dimethoxytrityl)-b-D-ribofuranosyl]-
6-(3-trifluoroacetamidophenyl)-(2,3H)-N -methylpyrrolo[2,3-d]-
pyrimidine-2(7H)-one (11). To a stirred solution of nucleoside
10 (1.50 g, 2.28 mmol) in distilled CH2Cl2 (8.0 mL), under an
argon atmosphere, at room temperature, was added distilled Et3N
˚
(50.1 mmol, 22.0 eq), activated 4 A molecular sieves and DMAP
(0.76 mmol, 0.3 eq). After cooling in ice, ethyl trifluoroacetate
(45.5 mmol, 20.0 eq) was added dropwise over 20 min. The
reaction was stirred at 0 ◦C for a further 10 min, allowed to
warm to room temperature then refluxed under argon for 17 h.
On cooling, the mixture was concentrated to a mobile syrup
then redissolved in MeCN and filtered. The filtrate was dried,
redissolved in CH2Cl2 (150 mL), washed with water (60 mL) and
3-N-[2¢-Deoxy-5¢-O-(4,4¢-dimethoxytrityl)-b-D-ribofuranosyl]-
6 - (3 - ureidophenyl) - (2,3H) - N - methylpyrrolo[2,3 - d]pyrimidine -
5094 | Org. Biomol. Chem., 2010, 8, 5087–5096
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the aqueous was re-extracted with CH2Cl2 (2 ¥ 50 mL). Com-
bined organic fractions were dried (Na2SO4), and concentrated
in vacuo and under high vacuum to give a yellow gum. Following
column chromatography (ethyl acetate–MeOH–Et3N, 95 : 5 : 0.5),
the product 11 was afforded as a pale yellow foam (1.11 g,
1.47 mmol, 65%). Rf 0.31 (CH2Cl2–acetone–Et3N, 30 : 70 : 0.3),
0.08 (CH2Cl2–acetone–Et3N, 80 : 20 : 0.3); 19F NMR (282 MHz,
stirred solution of nucleoside 10 (2.07 g, 3.14 mmol) in distilled
CH2Cl2 (15 mL), under an argon atmosphere, at room temper-
˚
ature, was added activated 4 A molecular sieves. After 15 min,
guanidinylating reagent 15 (7.59 mmol, 2.4 eq) then distilled
pyridine (15.2 mmol, 4.8 eq) were added, and the mixture was
refluxed under argon for 2 d. Argon-degassed CH2Cl2 (200 mL)
was added, the solution was washed with sat. aq KCl (100 mL),
dried (Na2SO4), and concentrated in vacuo and under high vacuum
to give a pale yellow foam. Following purification by column
chromatography (CH2Cl2–acetone, 50 : 50→90:10), on silica gel
pre-equilibrated with pyridine, the desired guanidinylated product
16 was afforded, as a pale yellow foam (1.25 g, 1.40 mmol, 44%).
Rf 0.37 (CH2Cl2–acetone, 20 : 80); 1H NMR (400 MHz, CDCl3) d
(ppm) 11.57 (1H, s, CONH), 9.91 (1H, s, Aryl-NH), 8.52 (1H, s,
H4), 7.48 (1H, t, J = 1.8 Hz, H2¢¢), 7.23 (1H, ddd, J = 1.0, 2.0,
8.0 Hz, H4¢¢), 7.18–7.15 (1H, m, H5¢¢), 7.15 (2H, d, J = 7.0 Hz, H14),
7.04 (4H, d, J = 9.0 Hz, H10), 7.04–6.99 (1H, m, H16), 6.98 (2H, t,
J = 9.0 Hz, H15), 6.95 (1H, br d, J = 8.5 Hz, H6¢¢), 6.54 (2H, d, J =
9.0 Hz, H11), 6.53 (2H, d, J = 9.0 Hz, H11), 6.19 (1H, t, J = 5.5 Hz,
H1¢), 5.29 (1H, s, H5), 4.39 (1H, q, J = 5.5 Hz, H3¢), 4.19 (2H, br
t, J = 5.8 Hz, H24), 4.00 (2H, br t, J = 5.8 Hz, H20), 3.90 (1H, td,
J = 3.0, 5.0 Hz, H4¢), 3.46 (3H, s, OCH3), 3.45 (3H, s, OCH3), 3.34
(3H, s, NCH3), 3.26 (1H, dd, J = 3.0, 11.0 Hz, H5¢), 3.21 (1H, dd,
J = 3.3, 10.8 Hz, H5¢), 2.59–2.52 (1H, m, H2¢), 2.53 (2H, br t, J =
6.0 Hz, H23), 2.41 (2H, br t, J = 5.8 Hz, H19), 2.11 (1H, ddd, J =
5.5, 6.5, 13.6 Hz, H2¢); MS (ES+) m/z: 918 ([M + Na]+, 100), 895
([M + H]+, 10); HRMS (ES+): calcd. for C48H46N8O10, [M + Na]+
917.3229, found 917.3234.
1
DMSO-d6) d (ppm) -73.6 (NHCOCF3); H NMR (400 MHz,
DMSO-d6) d (ppm) 11.36 (1H, s, NHCOCF3), 8.68 (1H, s, H4),
7.83 (1H, br s, H2¢¢), 7.75 (1H, td, J = 0.9, 8.3 Hz, H4¢¢), 7.55 (1H,
t, J = 8.0 Hz, H5¢¢), 7.40 (2H, d, J = 8.0 Hz, H14), 7.37 (1H, d, J =
8.2 Hz, H6¢¢), 7.32 (2H, t, J = 7.3 Hz, H15), 7.29 (2H, d, J = 8.3 Hz,
H10), 7.28 (2H, d, J = 9.0 Hz, H10), 7.24 (1H, t, J = 7.5 Hz, H16),
6.89 (2H, d, J = 8.5 Hz, H11), 6.88 (2H, d, J = 9.0 Hz, H11), 6.25
(1H, app. t, J = 5.7 Hz, H1¢), 5.65 (1H, s, H5), 5.42 (1H, d, J =
4.3 Hz, 3¢-OH), 4.42 (1H, qn, J = 5.0 Hz, H3¢), 4.00 (1H, dd, J =
3.9, 7.4 Hz, H4¢), 3.69 (3H, s, OCH3), 3.69 (3H, s, OCH3), 3.49
(3H, s, NCH3), 3.39 (1H, d, J = 7.3 Hz, H5¢), 3.35 (1H, d, J =
7.3 Hz, H5¢), 2.45 (1H, td, J ¢ 6.5, 13.1 Hz, H2¢), 2.19 (1H, ddd,
J = 5.5, 6.1, 13.2 Hz, H2¢); MS (ES+) m/z: 793 ([M + K]+, 15),
777 ([M + Na]+, 99), 303 (DMT+, 100); HRMS (ES+): calcd. for
C41H37N4O7F3, [M + Na]+ 777.2507, found 777.2509.
N, N¢ - Bis - [ (2 - cyanoethoxy) carbonyl] - S - methylisothiourea
(15).14,21 A mixture of S-methylisothiourea hemisulfate 13 (6.03 g,
43.3 mmol) and NaHCO3 (10.9 g, 130 mmol, 3.0 eq) in
argon-degassed, distilled water (50.0 mL) was stirred, under
an argon atmosphere, at room temperature, for 30 min. To
this was added degassed CH2Cl2 (150.0 mL) followed by (2-
cyanoethyl)-N-succinimidyl carbonate (CEOC-succinimide)§ 14
(18.9 g, 86.6 mmol, 2.0 eq) and the mixture was stirred vigorously,
under an argon atmosphere, at room temperature for 8 h. The
mixture was diluted with CH2Cl2 (100 mL) and water (100 mL),
agitated and separated. The aqueous layer was re-extracted
(CH2Cl2, 3 ¥ 100 mL) and combined CH2Cl2 fractions were dried
(Na2SO4) and concentrated in vacuo and under high vacuum to
give a mixture of mono- and bis-modified isothiourea as a viscous,
cloudy yellow syrup.
The syrup was re-dissolved in distilled CH2Cl2 (65.0 mL), and
further treated with CEOC-succinimide 14 (9.47 g, 43.3 mmol,
1.0 eq), and the mixture was stirred under an argon atmosphere, at
room temperature for 1412 hours. The yellow solution was diluted
with CH2Cl2 (135 mL) then washed with sat. aq KCl (80 mL). The
aqueous fraction was re-extracted (CH2Cl2, 2 ¥ 30 mL) and the
combined organic fractions were dried (Na2SO4) and concentrated
in vacuo to give a cloudy, yellow syrup. Following purification
by column chromatography (CH2Cl2–ethyl acetate, 95 : 5), and
drying under high vacuum, the desired bis-modified product 30
was afforded as a hard, powdery white solid (11.0 g, 38.7 mmol,
89%). Rf 0.46 (CH2Cl2–ethyl acetate, 75 : 25), 0.28 (CH2Cl2–ethyl
acetate, 90 : 10); 1H NMR (400 MHz, CDCl3) d (ppm) 11.77 (1H, s,
NH), 4.40 (2H, t, J = 6.3 Hz, H3), 4.36 (2H, t, J = 6.0 Hz, H3¢),
2.78 (4H, t, J = 6.5 Hz, CH2CN), 2.44 (3H, s, SCH3); MS (ES+)
m/z: 307 ([M + Na]+, 100), 285 ([M + H]+, 12).
General procedure for phosphitylation of 6-aryl-N-methylpyrrolo-
dC nucleosides 7a–c, 11, 16
To a solution of nucleoside 7a–c, 11, 16 (0.25–1.95 mmol)
in distilled CH2Cl2, THF or THF–DMF, strictly under an
argon atmosphere and excluding moisture, distilled DIPEA
(2.0–2.5 eq) was added, followed by chloro-phosphitylating
reagent, 2-cyanoethoxy-N,N-diisopropylaminochlorophosphine
(1.1–1.5 eq) dropwise and the reaction was stirred at room
temperature for 1–5 h. The mixture was diluted with distilled
DCM or argon-degassed ethyl acetate and washed with degassed
sat. aq KCl, dried under argon (Na2SO4) and concentrated
in vacuo. Purification by column chromatography using degassed
solvents, on silica gel pre-equilibrated with Et3N or pyridine,
under argon pressure, afforded the desired air- and acid-sensitive
phosphoramidites 8a–c, 12, 17. Product 8c was further purified by
precipitation (distilled CH2Cl2–degassed hexane).
3-N-[2¢-Deoxy-5¢-O-(4,4¢-dimethoxytrityl)-b-D-ribofuranosyl]-
6-phenyl-(2,3H)-N -methylpyrrolo[2,3-d]pyrimidine-2(7H)-one-
3¢-O-(2-O-cyanoethyl-N,N-diisopropyl) phosphoramidite (8a).
Obtained from 7a (0.37 g, 0.57 mmol) and chloro-phosphitylating
reagent (0.63 mmol, 1.1 eq), with DIPEA (1.42 mmol, 2.5 eq)
in CH2Cl2, after column chromatography (ethyl acetate–MeCN–
Et3N, 50 : 50 : 0.1), as a distereomeric mixture (ca. 4 : 3), as
a pale yellow foam (0.32 g, 0.38 mmol, 66%). Rf 0.44 (ethyl
acetate–MeCN, 1 : 1); 31P NMR (121 MHz, CDCl3) d (ppm)
150.3, 149.5 (PIII); MS (ES+) m/z: 945 ([M + Et3N + H]+, 74), 866
([M + Na]+, 100), 844 ([M + H]+, 71).
3-N-[2¢-Deoxy-5¢-O-(4,4¢-dimethoxytrityl)-b-D-ribofuranosyl]-
6 - (3 - {N,N¢ - bis - [(2 - cyanoethoxy)carbonyl]guanidinyl}phenyl) -
(2,3H)-N-methylpyrrolo[2,3-d]pyrimidine-2(7H)-one (16). To a
§ Prepared according to literature procedure.15,21b
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Org. Biomol. Chem., 2010, 8, 5087–5096 | 5095
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3-N-[2¢-Deoxy-5¢-O-(4,4¢-dimethoxytrityl)-b-D-ribofuranosyl]-
6 - (3 - acetamidophenyl) - (2,3H) - N - methylpyrrolo[2,3 - d]pyrimi -
dine-2(7H)-one-3¢-O-(2-O-cyanoethyl-N,N-diisopropyl) phospho-
ramidite (8b). Obtained from 7b (1.37 g, 1.95 mmol) and
chloro-phosphitylating reagent (2.15 mmol, 1.1 eq), with DIPEA
(4.91 mmol, 2.5 eq) in THF, after column chromatography
(CH2Cl2–acetone–Et3N, 30 : 70 : 0.5), as a distereomeric mixture
(ca. 1 : 1), as a pale yellow foam (1.51 g, 1.68 mmol, 86%). Rf 0.33,
0.46 (CH2Cl2–acetone–Et3N, 20 : 80 : 0.3); 31P NMR (121 MHz,
DMSO-d6) d (ppm) 149.2, 149.0 (PIII); MS (ES+) m/z: 939 ([M
+ K]+, 5), 923 ([M + Na]+, 100), 901 ([M + H]+, 9); HRMS
(ES+): calcd. for C50H57N6O8P, [M + Et3N + H]+ 1002.5253,
found 1002.5261; [M + Na]+ 923.3868, found 923.3886; [M + H]+
901.4048, found 901.4033.
MS (ES+) m/z: 1197 ([M + Et3N + H]+, 48), 1118 ([M + Na]+, 58),
1096 ([M + H]+, 100).
Acknowledgements
We gratefully acknowledge the BBSRC and The Ministry of
Higher Education (Egypt) for funding, and ATDBio Ltd. for
synthesis and purification of oligonucleotides.
Notes and references
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3-N-[2¢-Deoxy-5¢-O-(4,4¢-dimethoxytrityl)-b-D-ribofuranosyl]-
6 - (3 - ureidophenyl) - (2,3H) - N - methylpyrrolo[2,3 - d]pyrimidine -
2(7H)-one-3¢-O-(2-O-cyanoethyl-N,N-diisopropyl)
phospho-
ramidite (8c). Obtained from 7b (0.92 g, 1.31 mmol) and
chloro-phosphitylating reagent (1.46 mmol, 1.1 eq), with DIPEA
(3.30 mmol, 2.5 eq) in THF–DMF (5 : 1, v/v), after column
chromatography (acetone–MeOH–Et3N, 100 : 0 : 0.5→98:2 : 0.5),
as a distereomeric mixture (ca. 1 : 1), as a pale yellow foam (0.60 g,
0.67 mmol, 51%). Rf 0.30 (acetone–CH2Cl2–Et3N, 90 : 10 : 0.3),
0.24, 0.31 (methanol–acetone–Et3N, 5 : 95 : 0.3), 0.17 (acetone–
Et3N, 100 : 0.3); 31P NMR (121 MHz, DMSO-d6) d (ppm) 149.2,
149.0 (PIII); MS (ES+) m/z: 940 ([M + K]+, 8), 924 ([M + Na]+,
45), 902 ([M + H]+, 78), 303 (DMT+, 100); HRMS (ES+): calcd.
for C49H56N7O8P, [M + Na]+ 924.3820, found 924.3825; [M + H]+
902.4001, found 902.4000.
6 S. Hildbrand, A. Blaser, S. P. Parel and C. J. Leumann, J. Am. Chem.
Soc., 1997, 119, 5499–5511.
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8 I. Pre´vot-Halter and C. J. Leumann, Bioorg. Med. Chem. Lett., 1999,
9, 2657–2660.
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Brown, Chem. Commun., 2005, 2555–2557; (b) D. A. Rusling, V. E. C.
Powers, R. T. Ranasinghe, Y. Wang, S. D. Osborne, T. Brown and K. R.
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3-N-[2¢-Deoxy-5¢-O-(4,4¢-dimethoxytrityl)-b-D-ribofuranosyl]-
6 - (3 - trifluoroacetamidophenyl) - (2, 3H) - N - methylpyrrolo[2,3 -
d]pyrimidine-2(7H)-one-3¢-O-(2-O-cyanoethyl-N,N-diisopropyl)
phosphoramidite (12). Obtained from 11 (0.19 g, 0.25 mmol) and
chloro-phosphitylating reagent (0.29 mmol, 1.2 eq), with DIPEA
(0.63 mmol, 2.5 eq) in CH2Cl2, after column chromatography
(CH2Cl2–acetone–Et3N, 99 : 1 : 0.5→95 : 5 : 0.5), as a distereomeric
mixture (ca. 2 : 1), as a pale yellow foam (0.17, 0.18 mmol,
70%). Rf 0.50, 0.57 (CH2Cl2–acetone–Et3N, 50 : 50 : 0.3), 0.29,
0.34 (CH2Cl2–acetone–Et3N, 80 : 20 : 0.3), 0.18, 0.25 (CH2Cl2–
Et3N, 100 : 0.3); 31P NMR (121 MHz, DMSO-d6) d (ppm) 149.1,
148.9 (PIII); 19F NMR (282 MHz, DMSO-d6) d (ppm) -73.5
(NHCOCF3); MS (ES+) m/z: 977 ([M + Na]+, 100), 955 ([M +
H]+, 13); (ES-) m/z: 953 ([M - H]-, 100); HRMS (ES+): calcd. for
C50H54N6O8F3P, [M + Na]+ 977.3585, found 977.3591; [M + H]+
955.3766, found 955.3783.
11 S. R. Gerrard, N. Srinivasan, K. R. Fox and T. Brown, Nucleosides,
Nucleotides Nucleic Acids, 2007, 26, 1363–1367.
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13 Japan Pat., JP99-255499.
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¨
15 M. Manoharan, T. P. Prakash, I. Barber-Peoc’h, B. Bhat, G. Vasquez,
B. S. Ross and P. D. Cook, J. Org. Chem., 1999, 64, 6468–
6472.
3-N-[2¢-Deoxy-5¢-O-(4,4¢-dimethoxytrityl)-b-D-ribofuranosyl]-
6-(3-{N,N¢-bis-[(2-cyanoethoxy)carbonyl]guanidinyl}phenyl)-
(2,3H)-N -methylpyrrolo[2,3-d]pyrimidin-2(7H)-one-3¢-O-(2-O-
cyanoethyl-N,N-diisopropyl) phosphoramidite (17). Obtained
from 16 (0.50 g, 0.56 mmol) and chloro-phosphitylating reagent
(0.84 mmol, 1.5 eq), with DIPEA (1.15 mmol, 2.0 eq) in CH2Cl2,
after column chromatography (ethyl acetate–acetone–pyridine,
5 : 95 : 1), as a distereomeric mixture (ca. 1 : 1), as a pale yellow
foam (0.29 g, 0.27 mmol, 47%). Rf 0.68, 0.69, (CH2Cl2–acetone,
20 : 80); 31P NMR (121 MHz, CDCl3) d (ppm) 150.3, 149.4 (PIII);
16 R. A. J. Darby, M. Sollogoub, C. McKeen, L. Brown, A. Risitano, N.
Brown, C. Barton, T. Brown and K. R. Fox, Nucleic Acids Res., 2002,
30, 39e.
17 H. E. Gottlieb, V. Kotlyar and A. Nudelman, J. Org. Chem., 1997, 62,
7512–7515.
18 B. Classon and B. Samuelsson, Acta Chem. Scand., Ser. B, 1985, 39b,
501–504.
19 USA Pat., US 4162265.
20 S. J. Vadhia, PhD, University of Southampton, 2007.
21 (a) T. P. Prakash, A. Puschl, E. Lesnik, V. Mohan, V. Tereshko, M. Egli
and M. Manoharan, Org. Lett., 2004, 6, 1971–1974; (b) USA Pat., US
20060160763.
5096 | Org. Biomol. Chem., 2010, 8, 5087–5096
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