Aqueous methods for the preparation of 5′-substituted guanosine derivatives

Article abstract of DOI:10.1039/b908727c

We have developed simple, aqueous strategies, that avoid the use of protecting groups and chromatography, for the preparation of a series of 5′-substituted guanosine derivatives.

Full text of DOI:10.1039/b908727c

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Aqueous methods for the preparation of 50-substituted guanosine
derivativesw
ˇ
Paul Brear, Gemma R. Freeman, Mark C. Shankey, Milena Trmcic
´
and David R. W. Hodgson*
Received (in Cambridge, UK) 5th May 2009, Accepted 19th June 2009
First published as an Advance Article on the web 6th July 2009
DOI: 10.1039/b908727c
We have developed simple, aqueous strategies, that avoid the use
of protecting groups and chromatography, for the preparation of
a series of 50-substituted guanosine derivatives.
Guanosine derivatives with modified 50-functionalities have
Scheme 2 Acid-catalysed phosphate ester hydrolysis.
been used in the preparation of modified RNA molecules via
phosphoramidite-mediated oligonucleotide synthesis1 and
in vitro transcription.2–5 In addition, they are required for
the preparation of nucleic acid analogues with non-phosphate-
based linkages.6 Thus, easy access to this class of molecules
is essential. Guanosine derivatives often present a greater
synthetic challenge than the other nucleosides owing to their
poor solubility properties, which limit reactivity in solution.
Chromatographic purification procedures are also hampered
by poor solubility, thus, methodologies that overcome
solubility problems and afford cleanly converted products
that do not require chromatography offer a significant
advance. We now describe simple, aqueous procedures,
that avoid the use of protecting groups and chromatography,
for the preparation of 50-S-bridging thiophosphate, hydrazine,
hydroxylamine and azide derivatives 2a–d from 50-deoxy-50-
iodoguanosine 17 (Scheme 1). In addition, further aqueous
transformations performed on thiophosphate 2a and azide 2d
permitted the facile preparation of thiol 3 and amino 4b
derivatives, respectively (Schemes 2 and 3).
Scheme 3 Proposed mechanism of reaction of thiophosphate ion
with azide 2d. (i) Na3PSO3, water, 110 1C, 1 h, 70%.
iodoguanosine starting material 1a. To improve the kinetics
of the bimolecular displacement process, a large excess of
thiophosphate nucleophile was used and initial concentrations
of both reagent and substrate were high (0.1 M and 2 M,
respectively). Progress of the displacement process was
monitored by 31P NMR spectroscopy. Excess thiophosphate
was removed simply by selective precipitation with methanol
(60% MeOH final volume), and the crude product was
isolated by evaporation followed by freeze-drying. Finally,
sodium iodide from the displacement process was removed by
washing the lyophilised solid with acetone. Whilst the material
that was isolated was not analytically pure (Table 1, ESIw), the
only detectable contaminants were the thiol 2e and inorganic
phosphate. A previous approach to thiophosphate 2a followed
a four step route with HPLC purification after the final step,6
rather than our simple two step approach that avoids
chromatography.
We recently took advantage of the fact that guanosine
derivatives become highly soluble in aqueous solution when
dissolved in alkali, owing to the removal of the N-1 proton.
We have used this approach to facilitate the synthesis of
thiophosphate derivative 2a through solubilisation of
Initial experiments towards the preparation of hydrazine 2b
focused on the use of alkali for the solubilisation of iodide 1,
however, we found that 50% aqueous hydrazine solution
proved to be a remarkably good solvent for 1 in the absence
of alkali. We used 13C NMR spectroscopy to monitor reaction
progress at the 50-methylene centre, and initially we found that
a solution of iodide 1 was attained, which was subsequently
converted to 2b over the course of 16 h. Again, the use of a
highly concentrated reagent solution enhanced the rate of
reaction significantly. The hydrazine 2b was isolated by simple
precipitation from the reaction mixture with methanol,
followed by recrystallisation from methanol, which also
allowed for simple removal of the excess reagent and the
hydrazinium iodide by-product. Although the isolated yield
was modest, owing to the partial solubility of hydrazine 2b in
Scheme 1 Transformations of 50-deoxy-50-iodoguanosine 1.
Department of Chemistry, Durham University, Science Laboratories,
South Road, Durham, UK DH1 3LE.
E-mail: d.r.w.hodgson@durham.ac.uk
w Electronic supplementary information (ESI) available: Experimental
details, 1H, 13C and (where appropriate) 31P NMR spectra of
compounds 2a–d, 3 and 4b. See DOI: 10.1039/b908727c
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Table 1 Reagents and conditions
Reagent
Temp./1C
Time/h
2.5
Product
Purification method
Purity (%)
99a, 95b
Yield (%)
72
Na3SPO3 and
NaOH
H2NNH2
50
2a
Precipitation of excess reagent with MeOH,
removal of solvent, washed with acetone
Precipitation with methanol followed by
recrystallisation from methanol
Removal of solvent/reagent followed by
recrystallisation from water
Crystallisation from reaction mixture
40
16
72
2b
2c
2d
99b
95b
99b
40
63
42
H2NOH
60
NaN3
Reflux
18
a
b
1
Determined by 31P NMR spectroscopy. Determined by H NMR spectroscopy.
methanol, the approach provided easy access to the hydrazine,
which, as far as we are aware, has not been reported elsewhere.
Preliminary experiments towards hydroxylamine 2c revolved
around the use of basic conditions with hydroxylamine
hydrochloride as the source of hydroxylamine, however,
owing to the relatively low concentrations of hydroxylamine
that are attainable using this approach, reaction rates were
exceedingly low. We therefore moved to the use of 50%
aqueous hydroxylamine solution as the source of nucleophile,
again employing basic conditions in order to solubilise iodide
1. Unfortunately, hydroxylamine is an ambident nucleophile,
with O-nucleophilicity being enhanced under basic
conditions,8 therefore, we omitted base and we found that
the iodide 1 slowly dissolved. 13C NMR spectroscopy,
performed directly on the reaction mixture, was used to give
details of reaction progress at the 50-methylene centre. In the
absence of base, O-alkylation was minimised (o2.5%) and
the N-alkylated product 2c was isolated by removal of the
reagent/solvent mixture on the rotary evaporator followed by
recrystallisation from water. The isolated yield was modest
because hydroxylamine 2c showed appreciable solubility in
water, however, in comparison to an earlier six step route,9
which used several protecting groups, our new route offers a
much more concise alternative.
from guanosine, which avoids chromatographic steps,
represents a much simpler alternative.
The reactions between thio acids and azides have received
much attention in recent times for the formation of amides11
and sulfonamides.12 In analogy with these thiocarboxylic acid
methods we hoped to extend this approach towards the use of
thiophosphoric acids with the intention of preparing the
phosphoramidate 4a, however, there was no observable reaction
at room temperature. We therefore heated the reaction
mixture, and, on cooling, we isolated the amine 4b. Although
the oxidation–reduction properties of the thiophosphate ion
have been reported,13 we believe that this is the first synthetic
usage of the thiophosphate ion as a reducing agent. The
reaction likely proceeds via the original target phosphoramidate
4a, but this hydrolyses rapidly under the reaction conditions.
In conclusion, we have developed a suite of reactions
that allow for the simple, facile, aqueous preparation of
several guanosine derivatives, where protecting groups and
time-consuming chromatography steps are avoided.
This work was supported by Durham University.
Notes and references
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A convenient, organic solvent-based approach for the
preparation of azide 2d has already been reported by Dean,7
however, the use of dry dimethylformamide as the solvent
may be considered undesirable in some situations. Thus, we
explored the use of an aqueous system for the preparation of
the azide. Using relatively high concentrations of the starting
iodide 1 and a large excess of NaN3, we were able to obtain
moderate yields of the azide directly through crystallisation on
cooling of the reaction mixture. The large excess of azide
improves the kinetics of the displacement process, which are
hampered by solvation of the azide anion by water molecules.
The thiol 3 was readily prepared through hydrolysis of
thiophosphate ester 2a under mildly acidic conditions
(Scheme 2). Fortunately, the thiol product 3 displays poor
solubility in water, and precipitates from the reaction mixture.
The solid can be isolated via filtration and washing to afford
material that is contaminated with some disulfide oxidation
product (20%), in a reasonable overall yield. Thiol 3 has
been prepared in the past using a novel protected thiolate
nucleophile,10 which has to be prepared in-house, and a
protected guanosine precursor. Thus, our three step method
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This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 4980–4981 | 4981