A facile two-step synthesis of 8-arylated guanosine mono- and triphosphates (8-aryl GXPs)

Article abstract of DOI:10.1039/b614477b

We report a simple and high-yielding two-step procedure for the preparation of 8-arylated guanosine mono- and triphosphates (8-aryl GXPs). The key step of our synthesis is the Suzuki-Miyaura coupling of unprotected 8-bromo GMP and 8-bromo GTP with various

Full text of DOI:10.1039/b614477b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
A facile two-step synthesis of 8-arylated guanosine mono- and triphosphates
(8-aryl GXPs)
Alice Collier and Gerd Wagner*
Received 4th October 2006, Accepted 3rd November 2006
First published as an Advance Article on the web 13th November 2006
DOI: 10.1039/b614477b
We report a simple and high-yielding two-step procedure for the preparation of 8-arylated guanosine
mono- and triphosphates (8-aryl GXPs). The key step of our synthesis is the Suzuki–Miyaura coupling
of unprotected 8-bromo GMP and 8-bromo GTP with various arylboronic acids in aqueous solution.
The 8-bromoguanosine 5ꢀ-phosphates required as cross-coupling substrates were prepared from
8-bromoguanosine via an optimised Yoshikawa procedure.
Introduction
The 5ꢀ-mono- and triphosphates of the naturally occurring purine
nucleosides adenosine and guanosine (AXPs and GXPs) serve
numerous biological roles, from building blocks for nucleic acids to
enzyme cosubstrates. Non-natural analogues of AXPs and GXPs
are therefore sought after as chemical tools for the investigation of
important biological processes. Base-modified purine nucleotides
have been used as inhibitors of therapeutically relevant proteins,1
as purine receptor antagonists,2 and as probes for RNA structure
and function.3 Shokat and coworkers have employed N6-modified
ATP analogues for the engineering of orthogonal protein kinase–
nucleoside triphosphate pairs.4
In order to explore further the usefulness of non-natural purine
Fig. 1 General synthetic strategy towards the target 8-arylated guanosine
mono- and triphosphates (8-aryl GXPs).
nucleotides as biological tools, robust synthetic methods for the
rapid generation of structurally diverse analogues of AXPs and
GXPs are required. While the direct structural modification of
unprotected nucleotides represents the most elegant and efficient
strategy towards that goal, this approach is complicated by three
factors:
(a) The presence of multiple functional groups, including the
free phosphate group(s), limits the choice of chemistry.
(b) The phosphate ester, pyrophosphate, and glycosidic bonds
all represent sites for potential hydrolytic breakdown.
(c) The water-solubility of unprotected nucleotides necessitates
the use of aqueous reaction media.
Consequently, limited precedence exists for the direct structural
manipulation of unprotected nucleotides in aqueous solution.
Important examples that have been reported to date concern the
Sonogashira and Suzuki–Miyaura cross-coupling of unprotected
pyrimidine5 as well as purine6 nucleotides, as Pd-catalysed cross-
coupling chemistry is well suited to meet the synthetic challenges
outlined above.
identified reaction conditions for the Pd-catalysed cross-coupling
of unprotected halopurine nucleosides in aqueous media.7,8 More
recently, Hocek and coworkers have described the direct cross-
coupling of unprotected 8-bromo AMP and 8-bromo ATP with
boronophenylalanine in aqueous acetonitrile, using a catalytic
system composed of Pd(OAc)2 and TPPTS (triphenylphosphine
trisulfonic acid).6b However, no examples have been reported to
date for the Suzuki–Miyaura cross-coupling of 8-halo GMP and
8-halo GTP.
It is well known that guanosine analogues undergo Pd-catalysed
cross-coupling reactions much less readily than the corresponding
adenosine derivatives.7b,9 It has been noted that there is indeed
a noticeable lack of studies concerning the Pd-catalysed cross-
coupling of 8-bromoguanosine, presumably due to this low
reactivity.10 The reduced reactivity of 8-bromoguanosine in the
Suzuki–Miyaura reaction has been attributed to the coordination
of the transition metal catalyst to the guanine base under basic
conditions (Fig. 2),9 and guanosine has been shown to inhibit the
cross-coupling of both halopurine nucleosides and simple aryl
bromides.9 Because of these complications, the cross-coupling
of guanosine and GXP derivatives is nontrivial. In addition to
the low cross-coupling reactivity of guanosine derivatives, past
attempts at the cross-coupling of guanine nucleotides may also
have been hampered by difficulties in the preparation of the 8-
bromoguanosine phosphates required as substrates for the cross-
coupling reaction, resulting from their limited stability.11
As part of an ongoing investigation into the biological activity
of non-natural nucleotides, we are interested in GXP analogues
bearing additional aryl substituents at position 8 of the guanine
base (Fig. 1). The desired substituents can be installed via
the Suzuki–Miyaura reaction. Previously, we and others have
Centre for Carbohydrate Chemistry, School of Chemical Sciences and
Pharmacy, University of East Anglia, Norwich, UK NR4 7TJ. E-mail:
g.wagner@uea.ac.uk; Fax: +44(0)1603 592003; Tel: +44 (0)1603 593889
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Table 1 Optimisation of phosphorylation conditionsa
Nucleosidec Nucleotide
Entry Conditionsb
(substrate)
(product)
Yield (%)
0
46
34
1
2
3
4
5
6
7
8
H2O
H2O
H2O
H2O–pyridine
H2O–pyridine
H2O–pyridine
8-BrG
8-BrA
8-BrI
8-BrG
8-BrA
8-BrI
n/a
8-Chloro AMP
8-Chloro IMP
8-Bromo GMP (1) 23d
8-Bromo AMP
8-Bromo IMP
14
19
75d
90
Fig. 2 Possible coordination of guanosine derivatives to Pd under basic
conditions (after ref. 9).
Proton sponge 8-BrG
1
1
Proton spongee 8-BrG
Herein we report a simple two-step procedure for the prepa-
ration of novel 8-arylated GMP and GTP analogues (Fig. 1),
addressing the twin issues of problematic precursor synthesis
and low cross-coupling reactivity of 8-bromo GXPs. We have
optimised the preparation of the 8-bromo GXPs required as
cross-coupling substrates, and we have developed suitable reaction
conditions for the Suzuki–Miyaura reaction of these unprotected
8-bromo GXPs in water. Our synthetic protocol is operationally
simple, does not require any special precautions, and uses only
commercially available reagents. The synthetic method reported
herein provides fast and reliable access to a novel family of 8-
substituted GXP analogues with potentially interesting biological
activity.
a For structures, see Scheme 1. b POCl3: 4 equiv., H2O: 0.5 equiv., pyridine:
4 equiv. c 8-BrG: 8-bromoguanosine; 8-BrA: 8-bromoadenosine; 8-BrI:
8-bromoinosine. d Contaminated with 2ꢀ,3ꢀ-cyclophosphate. e Solvent: ace-
tonitrile instead of triethylphosphate.
procedure for the phosphorylation of 8-bromoguanosine in order
to avoid both halogen exchange and depurination.
We reasoned that both side reactions might be promoted by
hydrogen chloride formed in situ under the aqueous Yoshikawa
conditions, and that use of a base might therefore be beneficial.
While attempts with other bases (e.g. lutidine, proton sponge)
were unsuccessful, addition of pyridine to the reaction prevented
both halogen exchange and, in the case of 8-bromoguanosine,
depurination. However, the desired 8-bromopurine nucleoside 5ꢀ-
monophosphates were isolated in only low yields under these
conditions (Table 1, entries 4–6). In the case of 1 we attributed
the low yield mainly to the formation of a bisphosphorylated side
product, which was identified as the cyclic bisphosphate O2ꢀ ,O3ꢀ -
hydroxyphosphoryl 8-bromoguanosine 5ꢀ-monophosphate (dP
7.43, 24.45 ppm). This observation is in agreement with earlier
reports that the phosphorylation of unprotected nucleosides with
POCl3 is 5ꢀ-selective only in aqueous–acidic, but not in aqueous–
basic medium.12b,14 Finally, the use of proton sponge under
anhydrous conditions15 gave 1 in 75% yield, albeit still slightly
contaminated with traces of the cyclic bisphosphate as indicated
by 1H and 31P NMR (Table 1, entry 7).
Results and discussion
As substrates for the Suzuki–Miyaura reaction of GXPs, 8-
bromo GMP (1) and 8-bromo GTP (2) were required. A
commonly used method for the preparation of non-natural
nucleoside 5ꢀ-monophosphates is Yoshikawa’s procedure for the
selective phosphorylation of unprotected nucleosides in position
5ꢀ (Scheme 1).12 While this approach circumnavigates the need for
lengthy protection–deprotection sequences, it had previously been
reported to be unsuccessful in the case of 8-bromoguanosine.13
In our hands, treatment of 8-bromoguanosine under the original
Yoshikawa conditions (excess POCl3, H2O, trialkylphosphate) led
to cleavage of the glycosidic bond and to a partial exchange of the
8-bromo substituent with chlorine. A mixture of 8-bromo- and 8-
chloroguanine was isolated as the sole product from this reaction
(Table 1, entry 1).
Besides the absence of base, two other experimental parameters
have previously been regarded as critical for the 5ꢀ-selectivity of
the Yoshikawa phosphorylation of unprotected nucleosides: low
reaction temperature and use of trialkylphosphates as solvents.12
We found that while maintaining the reaction temperature at 2–
4
◦C is indeed essential in order to suppress formation of the
bisphosphate side product, use of triethylphosphate is not. We
discovered that on the contrary, changing the solvent to acetoni-
trile completely restored the selectivity of the phosphorylation
reaction for the 5ꢀ position. 8-Bromoguanosine is not initially
soluble in acetonitrile, but a clear solution forms within minutes
upon addition of POCl3 to the reaction at 2 ◦C. After 2 hours,
TLC indicated complete phosphorylation, and 1 was isolated
in 90% yield following purification by ion-pair chromatography
(Table 1, entry 8). While initial experiments were carried out on
a 50 mg scale, the reaction was subsequently scaled up to 500 mg
without difficulty. The use of 0.2M TEAB (pH 7.2) during the
aqueous work-up proved to be critical, as replacement of the buffer
with H2O led to depurination. The protocol was also successfully
adapted for the synthesis of 8-bromo GTP (2) using a modification
of Ludwig’s procedure.16 Instead of the aqueous work-up, the
intermediate dichlorophosphate species resulting from treatment
Scheme 1 Optimisation of phosphorylation conditions.
The bromo–chloro exchange reaction, which is accompanied by
a characteristic 13C NMR downfield shift for the carbon atom in
position 8, was also observed in control experiments carried out
with 8-bromoadenosine (dC-8 127.9 ppm) and 8-bromoinosine (dC-8
126.7 ppm) under the same conditions. However, in these cases
the mononucleotides 8-chloro AMP (dC-8 139.1 ppm) and 8-chloro
IMP (dC-8 138.9 ppm) were isolated with the glycosidic bond intact
(Table 1, entries 2 and 3). 8-Chloro AMP proved to be unreactive
during the Pd-catalysed cross-coupling reactions (data not shown),
suggesting that the more reactive bromo substituent is required for
successful cross-coupling. Therefore, we sought to optimise the
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Table 2 Optimisation of conditions for the preparation of 3 via Suzuki–
be active in this study allowed the isolation of 8-phenylguanosine
from the reaction as a white crystalline powder (Table 2, entries 2–
4). The best results with regard to yield and reaction times were
achieved with catalytic systems combining Pd(NO3)2 or Na2Cl4Pd
with the water-soluble phosphine ligand TPPTS (Table 2, en-
tries 2–3). Use of Na2Cl4Pd in combination with a different water-
soluble ligand recently reported by the Buchwald group18a gave no
advantage and led to longer reaction times (Table 2, entry 4).
Because of the lower cost and higher stability of Na2Cl4Pd
in comparison with Pd(NO3)2, we subsequently adopted the
Na2Cl4Pd–TPPTS system as our standard catalytic system. In
order to explore the scope of its applicability, the cross-coupling
of 8-bromoguanosine with a range of electronically diverse
phenylboronic acids was investigated. Under these conditions, all
cross-coupling reactions afforded the product 8-phenylguanosine
derivatives as white crystalline powders in high yields and with
short reaction times (Table 3, 4–6). Encouraged by these results,
we next applied our optimised cross-coupling conditions to
the Suzuki–Miyaura reaction of the nucleotides 1 and 2. With
Na2Cl4Pd–TPPTS as the catalyst, 1 was successfully reacted with
various arylboronic acids (Table 3, 7–10). All product 8-aryl
GMP analogues were obtained in good to excellent yields, and
most reactions were complete within 2 hours. Only the cross-
coupling reaction of 8-bromo GMP with 4-methoxyphenylboronic
acid could not be driven to completion under these conditions,
even after prolonged reaction time. However, the resulting 8-
methoxyphenyl GMP (10) was still obtained in 71% yield from
this reaction. All cross-coupling reactions could be conveniently
monitored by TLC (2-propanol–H2O–35%NH3 6 : 3 : 1), and the
work-up of the reactions required only a single purification step.
Remarkably, both the starting nucleotide 1 as well as the product
8-aryl GMP analogues 7–10 withstood the basic cross-coupling
Miyaura coupling of 8-bromoguanosine with phenylboronic acida
Entry
Pd source
Ligand
Yield (%)
Time/h
1
2
3
4
5
Pd(OAc)2
Pd(NO3)2
Na2Cl4Pd
Na2Cl4Pd
PdCl2
TPPTS
TPPTS
TPPTS
Buchwald ligand
EDTA
61
80
77
69
0
3
1
0.3
17
24
a For structures, see Scheme 2; R = H, X = H.
of 8-bromoguanosine with POCl3–proton sponge was reacted
in situ with tri-n-butylammonium pyrophosphate in DMF17 to
give 2 in 72% yield.
Before attempting the potentially challenging Suzuki–Miyaura
coupling of unprotected nucleotides 1 and 2, we sought to optimise
conditions for the cross-coupling of 8-bromoguanosine. Recently,
we have reported that a catalytic system composed of Pd(OAc)2
as the Pd source and the water-soluble ligand TPPTS7b is superior
to Pd(PPh3)4 for the Suzuki–Miyaura coupling of unprotected
halopurine nucleosides in neat water (Table 2, entry 1).7d Products
from the reaction of 8-bromoguanosine with various arylboronic
acids under these conditions were generally isolated by simple
precipitation from the aqueous reaction. However, most of the 8-
arylguanosine derivatives prepared in this fashion were obtained
as black powders, whose poor solubility precluded further purifi-
cation by either recrystallisation or column chromatography. The
mass spectra of these samples frequently showed a molecular ion
of [M + H + 106]+ indicative of the presence of the aforementioned
guanosine–Pd complex (Fig. 2), which presumably was responsible
for the black colour.
In order to avoid this Pd contamination we tested various
catalytic systems which have recently been reported to be effective
for cross-coupling reactions in aqueous media.6b,7b,18 The prepa-
ration of 8-phenylguanosine (3) via Suzuki–Miyaura coupling
of 8-bromoguanosine and phenylboronic acid was chosen as a
model reaction to identify optimum conditions (Table 2). Out
of the catalytic systems tested, only those with a phosphine
ligand were found to be active (Table 2, entries 1–4) while the
PdCl2–EDTA system was not (Table 2, entry 5). This result is
in agreement with a recent report that Na2Cl4Pd–EDTA is not
an efficient catalyst for the cross-coupling of 8-bromoadenosine,6b
and emphasises the importance of the ligand for cross-coupling
reactions of nucleosides in aqueous media.
Table 3 Suzuki–Miyaura coupling of 8-bromoguanosine, 8-bromo GMP
(1), and 8-bromo GTP (2) with various arylboronic acidsa
Compound
R
X
Yield (%)
Time/h
4
H
H
H
PO3
PO3
PO3
PO3
(PO3
(PO3
(PO3
Cl
84
70
84
63
92
90
71b
85
56
65
0.3
3
0.5
0.25
1.3
1
6
1
1
2
5
CH3
OCH3
H
6
2−
2−
2−
2−
)
7
8
Cl
9
CH3
OCH3
H
Cl
CH3
10
11
12
13
4−
4−
4−
3
3
3
)
)
The role of the Pd source was highlighted by the finding
that replacement of Pd(OAc)2 with a water-soluble Pd source
(Pd(NO3)2 or Na2Cl4Pd) results in a cleaner product. In contrast
to the Pd(OAc)2–TPPTS system, all other catalysts that proved to
a For structures, see Scheme 2; Pd source: Na2Cl4Pd; ligand: TPPTS.
b Reaction does not go to completion.
Scheme 2 Suzuki–Miyaura coupling of 8-bromoguanosine, 8-bromo GMP (1), and 8-bromo GTP (2).
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conditions and the reaction temperature of 80 ◦C without signif-
icant decomposition, despite the known instability of nucleoside
phosphates.11 This promising result prompted us to attempt the
cross-coupling of the potentially even more unstable nucleoside
triphosphate 8-bromo GTP (2) under the same conditions, using
three different boronic acids. These reactions also proceeded
smoothly, with short reaction times and good yields (Table 3, 11–
13). During the reaction, no decomposition of the triphosphates
was detected by TLC, and the 31P NMR spectra of all cross-
coupling products showed the triade of signals characteristic for
triphosphates. An important factor contributing to the aston-
ishing stability of the guanosine triphosphate derivatives under
our cross-coupling conditions certainly is the pronounced activity
of the Na2Cl4Pd–TPPTS catalytic system, which allows short
reaction times at relatively low temperature. In contrast, Hocek
and coworkers found that cross-coupling of 8-bromo AMP and
8-bromo ATP requires a much higher temperature of 125 ◦C
when Pd(OAc)2–TPPTS is used as the catalyst, despite the higher
cross-coupling reactivity of adenosine derivatives compared to
guanosine.6b
30.83 for solutions in D2O). Coupling constants (J) are reported
in Hz. Resonance assignments are based on COSY experiments.
Splitting patterns are abbreviated as follows: br, broad; s, singlet; d,
doublet; t, triplet; m, multiplet. Accurate electrospray ionization
mass spectra (HR ESI-MS) were obtained on a Finnigan MAT
900 XLT mass spectrometer. Preparative chromatography was
performed on a Biologic LP chromatography system equipped
with a peristaltic pump and a 254 nm UV Optics Module using the
following conditions: Method A—stationary phase: LiChroprep
RP-18 resin, equilibrated with 0.05M TEAB buffer (pH 6.0–6.7);
gradient: 0–10% MeCN against 0.05M TEAB over 400 mL; flow
rate: 3 mL min−1. Method B—stationary phase: MacroPrep 25Q
washed with H2O; gradient: 0–100% 1M TEAB buffer (pH 7.1–
7.6) against H2O over 400 mL; flow rate: 5 mL min−1. Product
containing fractions were combined and reduced to dryness. The
residue was co-evaporated with methanol to remove residual
TEAB.
8-Bromo GMP (1). A mixture of 8-bromoguanosine19 (50 mg,
0.14 mmol) and proton sponge (180 mg, 0.84 mmol) was sus-
pended in dry acetonitrile (3 mL). Under an atmosphere of N2,
the suspension was stirred for 30 min at room temperature. The
suspension was cooled to 2 ◦C, and POCl3 (51 lL, 0.56 mmol)
was added dropwise while maintaining the temperature at 2–
Conclusions
We have identified suitable reaction conditions for the Suzuki–
Miyaura coupling of unprotected 8-bromoguanosine, 8-bromo
GMP, and 8-bromo GTP with a range of phenylboronic acids
in water, using only commercially available reagents. A catalytic
system composed of Na2Cl4Pd and TPPTS is suitable to overcome
the generally reduced reactivity of guanine nucleosides and nu-
cleotides in Pd-catalysed reactions. We have also optimised the syn-
thesis of the 8-bromoguanosine 5ꢀ-phosphates required as cross-
coupling substrates. The phosphorylation of 8-bromoguanosine
via a modified Yoshikawa procedure cleanly gave 8-bromo GMP
and 8-bromo GTP when carried out in acetonitrile and with
proton sponge as the base. The novel 8-aryl GMP and GTP
derivatives presented in this study are interesting biological tools,
and investigations into their biological activity are currently
underway. Our synthetic conditions may also be applicable to
the direct structural modification of even more complex and
sensitive biomolecules like sugar-nucleotides and oligonucleotides
in aqueous solution.
◦
5 C. After the addition of POCl3 was complete, a clear, slightly
yello◦w solution formed within minutes. The reaction was stirred
at 2 C until TLC indicated completion (2–3 h). Under cooling,
the reaction was added dropwise to ice-cold 0.2M TEAB buffer
(40 mL, pH 7.3). The aqueous solution was stored at 4 ◦C
overnight to allow deprotonation of proton sponge, washed
repeatedly with ethyl acetate (4 × 25 mL) and evaporated to
dryness. The residue was purified by ion-pair chromatography
(method A) to give 1 as a glassy solid in 90% yield (79 mg, 1.7 equiv.
of triethylammonium as determined by NMR). dH (400 MHz,
D2O) 5.71 (d, J = 6.0, 1H, H-1ꢀ), 5.06 (t, J = 5.6/5.2, 1H, H-2ꢀ),
4.39–4.36 (m, 1H, H-3ꢀ), 4.04–4.00 (m, 1H, H-4ꢀ), 3.92–3.87 (m,
1H, Ha-5ꢀ), 3.84–3.78 (m, 1H, Hb-5ꢀ), 2.95 (q, J = 7.2, 9.9H, CH2),
1.03 (t, J = 7.2, 15.7H, CH3). dC (100 MHz, D2O) 157.6, 153.6,
152.8, 124.1, 116.7, 89.9, 84.1 (d, JC,P = 8.4), 70.8, 70.4, 64.0, 46.6,
8.3. dP (162 MHz, D2O) 7.48. HRMS (ES, negative) calcd. for
C10H12O8N579BrP 439.9612 (monoanion), found 439.9619.
8-Bromo GTP (2). A mixture of 8-bromoguanosine19 (400 mg,
1.1 mmol) and proton sponge (1.44 g, 6.6 mmol) was suspended
in dry acetonitrile (20 mL). Under an atmosphere of N2, the
suspension was stirred for 30 min at room temperature. The
suspension was cooled to 2 ◦C, and POCl3 (410 lL, 4.5 mmol)
was added dropwise while maintaining the temperature at 2–
Experimental
General
MeCN was distilled from CaH2 prior to use. Dowex resin was
washed with water before use. All other chemicals and solvents
were of commercial quality and used as received unless stated
otherwise. 8-Bromoguanosine was prepared as described by Sheu
and Foote.19 TLC was performed on precoated aluminium plates
(Silica Gel 60 F254, Merck). Compounds were visualized by
exposure to UV light. NMR spectra were recorded at 298 K on a
Varian VXR 400 S spectrometer at 400 MHz (1H) or on a Bruker
Avance DPX-300 spectrometer at 300 MHz (1H). Prior to the
recording of 31P NMR spectra a drop of triethylamine was added to
each sample to suppress line broadening and enhance resolution.
Chemical shifts (d) are reported in ppm and referenced to residual
solvent resonances (for DMSO-d6) or acetone (1H d 2.05, 13C d
◦
5 C. After the addition of POCl3 was complete, a clear, slightly
yellow solution formed within 10 min. The reaction was stirred
at 2 ◦C until TLC indicated complete consumption of starting
material (2.5 h). Under cooling, a cold mixture of Bu3N (1.1 mL,
4.6 mmol) and 1.5M tri-n-butylammonium pyrophosphate in dry
DMF17 (5 mL, 7.5 mmol) was added in one portion. The reaction
was allowed to stir at 4 ◦C for 15 min, and added dropwise
to ice-cold 0.2M TEAB ◦buffer (120 mL, pH 7.2). The aqueous
solution was stored at 4 C overnight to allow deprotonation of
proton sponge, washed repeatedly with ethyl acetate (4 × 80 mL)
and evaporated to dryness. The residue was purified consecutively
by ion-pair (method A, to remove inorganics) and ion-exchange
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(method B, to remove residual 1) chromatography to give 2 as a
glassy solid in 72% yield (709 mg, 2.9 equiv. of triethylammonium
as determined by NMR). dH (400 MHz, D2O) 5.78 (d, J = 6.2,
1H, H-1ꢀ), 5.18 (t, J = 5.9/6.0, 1H, H-2ꢀ), 4.51–4.48 (m, 1H,
H-3ꢀ), 4.16–4.04 (m, 3H, H-4ꢀ, H-5ꢀ), 3.00 (q, J = 7.3, 17.8H,
CH2), 1.08 (t, J = 7.3, 25.3H, CH3). dC (100 MHz, D2O) 157.2,
153.1, 152.4, 123.4, 116.1, 89.1, 83.1, 70.1, 69.3, 64.5, 45.8, 7.5; dP
(162 MHz, D2O) −2.89 (d, J = 20.4), −7.68 (d, J = 19.3), −19.09
(t, J = 20.2). HRMS (ES, negative) calcd. for C10H14O14N579BrP3
599.8939 (monoanion), found 599.8942.
HRMS (ES, positive) calcd. for C17H20O5N5 374.1459 [M + H]+,
found 374.1463.
8-(4-Methoxyphenyl)guanosine (6). The target compound was
obtained from 8-bromoguanosine (98.8 mg, 0.273 mmol) and 4-
methoxyphenylboronic acid according to general procedure A
(0.5 h) as a white crystalline powder in 84% yield (88.6 mg). dH
(400 MHz, DMSO-d6) 10.72 (s, 1H, N–H), 7.56 (d, J = 8.5, 2H,
2Ph), 7.06 (d, J = 8.5, 2H, 2Ph), 6.35 (br s, 2H, NH2), 5.59 (d, J =
6.5, 1H, H-1ꢀ), 5.35 (d, J = 6.3, 1H, OH-2ꢀ), 5.07–4.96 (m, 3H, H-2ꢀ,
OH-3ꢀ, OH-5ꢀ), 4.07–4.03 (m, 1H, H-3ꢀ), 3.80 (br s, 4H, H-4ꢀ, CH3),
3.67–3.62 (m, 1H, Ha-5ꢀ), 3.55–3.49 (m, 1H, Hb-5ꢀ). dC (100 MHz,
DMSO-d6) 160.8, 157.2, 153.6, 152.5, 148.2, 131.3, 123.0, 117.6,
114.7, 89.6, 86.4, 71.3, 70.9, 62.8, 56.0. HRMS (ES, positive) calcd.
for C17H19O6N5 412.1228 [M + Na]+, found 412.1228.
General procedure A for the Suzuki–Miyaura coupling of
8-bromoguanosine
8-Bromoguanosine19 (1 equiv.), Na2Cl4Pd (2.5 mol%), TPPTS
(2.5 equiv. to Pd), K2CO3 (2 equiv.), and arylboronic acid
(1.2 equiv.) were placed in a flask and purged with N2. Degassed
water (6 mL◦) was added via a syringe, and the reaction was
stirred at 80 C under N2 for the given time. Upon completion,
the aqueous reaction was diluted with H2O (20 mL) and the
pH adjusted to 7 using 10% HCl. The resulting precipitate was
collected by filtration and dried in vacuo.
General procedure B for the Suzuki–Miyaura coupling of 8-bromo
GMP (2) and 8-bromo GTP (3)
Na2Cl4Pd (2.5 mol%), TPPTS (2.5 equiv. to Pd), K2CO3
(1.5 equiv.), and arylboronic acid (1.2 equiv.) were placed in a flask
and purged with N2. A solution of 8-bromo nucleotide (1 equiv.)
in degassed water (3 mL) was added via a syringe. The pale yellow
◦
8-Phenylguanosine (3). The target compound was obtained
from 8-bromoguanosine (103.1 mg, 0.285 mmol) and phenyl-
boronic acid according to general procedure A (0.3 h) as a white
crystalline powder in 77% yield (78.7 mg). dH (400 MHz, DMSO-
d6) 10.75 (s, 1H, N–H), 7.65–7.63 (m, 2H, 2Ph), 7.53–7.48 (m,
3H, 3Ph), 6.38 (br s, 2H, NH2), 5.61 (d, J = 6.4, 1H, H-1ꢀ), 5.37
(d, J = 6.4, 1H, OH-2ꢀ), 5.05–4.97 (m, 3H, H-2ꢀ, OH-3ꢀ, OH-5ꢀ),
4.07–4.03 (m, 1H, H-3ꢀ), 3.82–3.79 (m, 1H, H-4ꢀ), 3.68–3.62 (m,
1H, Ha-5ꢀ), 3.55–3.49 (m, 1H, Hb-5ꢀ). dC (100 MHz, DMSO-d6)
157.3, 153.8, 152.7, 148.2, 130.7, 130.1, 129.9, 129.3, 117.8, 89.6,
86.5, 71.3, 70.9, 62.8. HRMS (ES, positive) calcd. for C16H18O5N5
360.1302 [M + H]+, found 360.1301.
solution was stirred at 80 C under N2 for the given time. Upon
completion, the reaction turned dark red/brown. The reaction was
cooled to room temperature, filtered and the pH adjusted to 7 using
Dowex 50WX-8 100 resin (H+ form). The resin was filtered off and
washed with water, and the combined filtrate was evaporated to
dryness. The crude product was dissolved in 0.05M TEAB buffer
and purified by ion-pair chromatography (method A).
8-Phenylguanosine monophosphate (7). The target compound
was prepared from 1 (43.4 mg, 0.072 mmol) and phenylboronic
acid according to general procedure B (15 min). After purification
by ion-pair chromatography (method A, fractions 43–53), 7 was
obtained as a glassy solid in 63% yield (25.0 mg, 1.2 equiv. of
triethylammonium as determined by NMR). dH (400 MHz, D2O)
7.38–7.29 (m, 5H, 5Ph), 5.56 (d, J = 6.0, 1H, H-1ꢀ), 5.02 (t, J =
5.9, 1H, H-2ꢀ), 4.28 (dd, J = 3.6/5.7, 1H, H-3ꢀ), 4.02–3.90 (m,
3H, H-4ꢀ + H2-5ꢀ), 2.97 (q, J = 7.3, 7H, CH2), 1.05 (t, J = 7.3,
10H, CH3). dC (100 MHz, D2O) 159.2, 153.8, 153.3, 150.9, 131.1,
129.8, 129.5, 128.3, 116.6, 89.6, 84.1 (d, JC,P = 8.4), 71.0, 70.7, 65.2
(d, JC,P = 4.6), 47.2, 8.8. dP (162 MHz, D2O) 7.43. HRMS (ES,
negative) calcd. for C16H17O8N5P 438.0813 (monoanion), found
438.0820.
8-(4-Chlorophenyl)guanosine (4). The target compound was
obtained from 8-bromoguanosine (100.5 mg, 0.278 mmol) and 4-
chlorophenylboronic acid according to general procedure A (0.5 h)
as a white crystalline powder in 84% yield (91.9 mg). dH (400 MHz,
DMSO-d6) 10.78 (s, 1H, N–H), 7.66 (d, J = 8.5, 2H, 2Ph), 7.58
(d, J = 8.6, 2H, 2Ph), 6.42 (br s, 2H, NH2), 5.58 (d, J = 6.6,
1H, H-1ꢀ), 5.35 (d, J = 6.4, 1H, OH-2ꢀ), 5.05–4.94 (m, 3H, H-2ꢀ,
OH-3ꢀ, OH-5ꢀ), 4.06–4.03 (m, 1H, H-3ꢀ), 3.83–3.80 (m, 1H, H-4ꢀ),
3.67–3.62 (m, 1H, Ha-5ꢀ), 3.55–3.50 (m, 1H, Hb-5ꢀ). dC (100 MHz,
DMSO-d6) 157.2, 153.9, 152.8, 146.9, 135.0, 131.5, 129.6, 129.4,
117.8, 89.5, 86.5, 71.1, 70.9, 62.6. HRMS (ES, positive) calcd. for
C16H17O5N535Cl 394.0913 [M + H]+, found 394.0913.
8-(4-Chlorophenyl)guanosine monophosphate (8). The target
compound was prepared from 1 (50.0 mg, 0.079 mmol) and
4-chlorophenylboronic acid according to general procedure B
(1.3 h). After purification by ion-pair chromatography (method
A, fractions 59–70), 8 was obtained as a glassy solid in 92%
yield (43.9 mg, 1.2 equiv. of triethylammonium as determined
by NMR). dH (400 MHz, D2O) 7.18 (s, 4H, 4Ph), 5.51 (d, J =
5.1, 1H, H-1ꢀ), 4.94 (t, J = 5.5, 1H, H-2ꢀ), 4.44–4.41 (m, 1H, H-
3ꢀ), 4.09–3.94 (m, 3H, H-4ꢀ + H2-5ꢀ), 2.99 (q, J = 7.3, 8H, CH2),
1.08 (t, J = 7.3, 12H, CH3). dC (100 MHz, D2O) 158.8, 153.6,
8-(4-Methylphenyl)guanosine (5). The target compound was
obtained from 8-bromoguanosine (101.5 mg, 0.280 mmol) and 4-
methylphenylboronic acid according to general procedure A (3 h)
as a white crystalline powder in 70% yield (73.1 mg). dH (400 MHz,
DMSO-d6) 10.73 (s, 1H, N–H), 7.52 (d, J = 8.1, 2H, 2Ph), 7.31
(d, J = 8.2, 2H, 2Ph), 6.36 (br s, 2H, NH2), 5.59 (d, J = 6.6,
1H, H-1ꢀ), 5.34 (d, J = 6.4, 1H, OH-2ꢀ), 5.05–4.95 (m, 3H, H-2ꢀ,
OH-3ꢀ, OH-5ꢀ), 4.05–4.03 (m, 1H, H-3ꢀ), 3.80–3.78 (m, 1H, H-
4ꢀ), 3.65–3.62 (m, 1H, Ha-5ꢀ), 3.54–3.51 (m, 1H, Hb-5ꢀ), 2.36 (s,
3H, CH3). dC (100 MHz, DMSO-d6) 157.3, 152.7, 152.6, 140.3,
139.8, 129.8, 129.8, 127.9, 117.7, 89.6, 86.4, 71.3, 70.9, 62.7, 21.6.
153.0, 149.0, 136.4, 130.6, 129.4, 126.4, 116.4, 90.1, 83.7 (d, JC,P
=
8.2), 71.0, 70.8, 65.2 (d, JC,P = 4.3), 47.2, 8.8. dP (162 MHz, D2O)
7.40. HRMS (ES, negative) calcd. for C16H16O8N5ClP 472.0430
(monoanion), found 472.0423.
4530 | Org. Biomol. Chem., 2006, 4, 4526–4532
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8-(4-Methylphenyl)guanosine monophosphate (9). The target
compound was prepared from 1 (49.6 mg, 0.078 mmol) and
4-methylphenylboronic acid according to general procedure B
(1 h). After purification by ion-pair chromatography (method
A, fractions 59–72), 9 was obtained as a glassy solid in 90%
yield (41.5 mg, 1.4 equiv. of triethylammonium as determined by
NMR). dH (400 MHz, D2O) 7.28 (d, J = 7.2, 2H, 2Ph), 7.19 (d, J =
8.0, 2H, 2Ph), 5.72 (d, J = 5.2, 1H, H-1ꢀ), 5.05 (t, J = 5.6/5.2, 1H,
H-2ꢀ), 4.57–4.55 (m, 1H, H-3ꢀ), 4.26–4.11 (m, 3H, H-4ꢀ + H2-5ꢀ),
3.15 (q, J = 7.6/7.2, 8.6H, CH2), 2.34 (s, 3H, CH3–Ph), 1.24 (t,
J = 7.2/7.6 Hz, 12.9H, CH3). dC (100 MHz, D2O) 158.9, 153.4,
(ES, negative) calcd. for C16H18O14N5ClP3 631.9757 (monoanion),
found 631.9763.
8-(4-Methylphenyl)guanosine triphosphate (13). The target
compound was prepared from 2 (59.2 mg, 0.066 mmol) and
4-methylphenylboronic acid according to general procedure B
(2 h). After purification by ion-pair chromatography (method
A, fractions 41–54), 13 was obtained as a glassy solid in 65%
yield (40.4 mg, 3.3 equiv. of triethylammonium as determined by
NMR). dH (400 MHz, D2O) 7.24 (d, J = 7.9, 2H, 2Ph), 7.14 (d,
J = 8.0, 2H, 2Ph), 5.60 (d, J = 5.7, 1H, H-1ꢀ), 5.10 (t, J = 5.6, 1H,
H-2ꢀ), 4.49–4.47 (m, 1H, H-3ꢀ), 4.23–4.06 (m, 3H, H-4ꢀ + H2-5ꢀ),
2.99 (q, J = 7.3, 20H, CH2), 2.23 (s, 3H, CH3–Ph), 1.08 (t, J = 7.3,
30H, CH3). dC (100 MHz, D2O) 159.0, 153.7, 153.3, 150.8, 141.9,
130.1, 129.6, 125.0, 116.1, 89.8, 83.9 (d, JC,P = 8.7), 71.0, 70.7,
65.9 (d, JC,P = 3.2), 47.2, 21.2, 8.8. dP (162 MHz, D2O) −2.87 (d,
J = 20.7), −7.58 (d, J = 19.7), −19.00 (t, J = 20.1). HRMS (ES,
negative) calcd. for C17H21O14N5P3 612.0303 (monoanion), found
612.0310.
152.0, 150.4, 141.5, 129.9, 129.2, 124.9, 116.3, 90.1, 83.7 (d, JC,P
=
8.6), 71.0, 70.9, 65.2 (d, JC,P = 4.3), 47.2, 21.1, 8.8. dP (162 MHz,
D2O) 7.41. HRMS (ES, negative) calcd. for C17H19O8N5P 452.0980
(monoanion), found 452.0977.
8-(4-Methoxyphenyl)guanosine monophosphate (10). The tar-
get compound was prepared from 1 (49.7 mg, 0.078 mmol) and
4-methoxyphenylboronic acid according to general procedure B
(6 h). After purification by ion-pair chromatography (method
A, fractions 48–60), 10 was obtained as a glassy solid in 71%
yield (32.1 mg, 1.1 equiv. of triethylammonium as determined by
NMR). dH (400 MHz, D2O) 7.20 (d, J = 8.7, 2H, 2Ph), 6.76 (d, J =
8.8, 2H, 2Ph), 5.56 (d, J = 5.4, 1H, H-1ꢀ), 4.96 (t, J = 5.6, 1H, H-
2ꢀ), 4.40–4.37 (m, 1H, H-3ꢀ), 4.09–3.93 (m, 3H, H-4ꢀ + H2-5ꢀ), 3.68
(s, 3H, CH3–O), 3.00 (q, J = 7.4, 6.5H, CH2), 1.08 (t, J = 7.4, 10H,
CH3). dC (100 MHz, D2O) 160.9, 158.9, 153.4, 153.0, 150.2, 131.1,
120.6, 116.2, 114.6, 89.9, 83.7 (d, JC,P = 8.4), 70.9, 70.8, 65.3 (d,
JC,P = 5.1), 56.0, 47.2, 8.8. dP (162 MHz, D2O) 7.45. HRMS (ES,
negative) calcd. for C17H19O9N5P 468.0926 (monoanion), found
468.0930.
Acknowledgements
Financial support by the Nuffield Foundation (NAL/01036/A)
and the Royal Society (Research Grant Scheme) is gratefully
acknowledged. We thank the University of East Anglia for a
studentship, and the EPSRC National Mass Spectrometry Service
Centre, Swansea, for the recording of mass spectra. We also thank
Rob Field for many helpful discussions.
References
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8-Phenylguanosine triphosphate (11). The target compound
was prepared from 2 (60.5 mg, 0.068 mmol) and phenylboronic
acid according to general procedure B (1 h). After purification
by ion-pair chromatography (method A, fractions 33–52), 11 was
obtained as a glassy solid in 85% yield (53.3 mg, 3.1 equiv. of
triethylammonium as determined by NMR). dH (400 MHz, D2O)
7.45–7.37 (m, 5H, 5Ph), 5.61 (d, J = 6.1, 1H, H-1ꢀ), 5.19 (t, J =
5.8, 1H, H-2ꢀ), 4.46–4.44 (m, 1H, H-3ꢀ), 4.18–4.06 (m, 3H, H-4ꢀ +
H2-5ꢀ), 2.99 (q, J = 7.3, 18H, CH2), 1.08 (t, J = 7.3, 28H, CH3).
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compound was prepared from 2 (59.2 mg, 0.066 mmol) and
4-chlorophenylboronic acid according to general procedure B
(1 h). After purification by ion-pair chromatography (method
A, fractions 53–72), 12 was obtained as a glassy solid in 56%
yield (35.8 mg, 3.2 equiv. of triethylammonium as determined by
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1H, H-1ꢀ), 5.18 (t, J = 5.8, 1H, H-2ꢀ), 4.48–4.46 (m, 1H, H-3ꢀ),
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4532 | Org. Biomol. Chem., 2006, 4, 4526–4532
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