Preparation and purification of 5′-amino-5′-deoxyguanosine- 5′-N-phosphoramidate and its initiation properties with T7 RNA polymerase

Article abstract of DOI:10.1039/b717896d

5′-Amino-5′-deoxyguanosine-5′-N-phosphoramidate (GNHP) was synthesised in four steps from guanosine. Its identity was confirmed using spectroscopic and kinetic studies. A chromatographic method for the purification of this unstable compound was developed. GNHP was found to initiate T7 RNAP promoted transcriptions to afford 5′-O₃P-NH-RNA, which hydrolyses readily to yield 5′-H₂N-RNA that can be conjugated to activated esters.

Full text of DOI:10.1039/b717896d

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Preparation and purification of 5ꢀ-amino-5ꢀ-deoxyguanosine-5ꢀ-N-
phosphoramidate and its initiation properties with T7 RNA polymerase†
David Williamson and David R. W. Hodgson*
Received 19th November 2007, Accepted 16th January 2008
First published as an Advance Article on the web 12th February 2008
DOI: 10.1039/b717896d
5ꢀ-Amino-5ꢀ-deoxyguanosine-5ꢀ-N-phosphoramidate (GNHP) was synthesised in four steps from
guanosine. Its identity was confirmed using spectroscopic and kinetic studies. A chromatographic
method for the purification of this unstable compound was developed. GNHP was found to initiate T7
RNAP promoted transcriptions to afford 5ꢀ-O3P-NH-RNA, which hydrolyses readily to yield
5ꢀ-H2N-RNA that can be conjugated to activated esters.
molecule. Given that the use of 5ꢀ-amino-5ꢀ-deoxyguanosine
Indroduction
by Suga et al. was limited by poor solubility, we decided to
RNA conjugates have been used widely in biological research
improve the solubility of this system by the introduction of a
to determine the nature of protein–nucleic acid interactions,1,2
understand RNA folding problems3 and for the in vitro selection
of ribozymes.4 Whilst automated synthesis can produce many
of these conjugates, the technique is limited to the production
of shorter oligonucleotides; thus for some applications, such as
in vitro selection of ribozymes and the study of longer RNA
sequences, one must rely on enzymatic preparation methods.
RNA molecules that are modified at their 5ꢀ-termini have
been produced by enzymatic incorporation of modified initiator
nucleotides, usually based on guanosine.1,5–8 Unfortunately many
of the synthetic routes towards these initiator molecules are
laborious and act as a deterrent to the use of these systems. We
aimed to develop a simple synthetic route towards an initiator
molecule that would broaden the appeal of this approach to a
wider research community.
The use of amine-based initiators has been investigated by Suga9
and Ja¨schke.5 Once incorporated, the amine functional group can
be further modified in a post-transcriptional step using standard
bioconjugation reagents such as reactive esters.10 Suga’s approach,
namely the use of 5ꢀ-amino-5ꢀ-deoxyguanosine in transcriptions,
whilst appealing owing to the ease of synthesis of the nucleoside,
unfortunately afforded low yields of RNA where the RNA
population contains only a small proportion (∼20%) of the amine
functional group. The likely cause of the poor performance is
the poor solubility of 5ꢀ-amino-5ꢀ-deoxyguanosine in aqueous
solution. Ja¨schke and coworkers have prepared several more
elaborate nucleotides that show good initiation characteristics, but
unfortunately the syntheses of these materials are quite lengthy
and thus limited to specialists. We hoped to develop methodology
that provides a simple synthetic route towards a modified initiator
molecule that is then effectively incorporated at the 5ꢀ-termini of
RNA molecules in the transcription process.
phosphoryl group on the 5ꢀ-amino functional group. The resulting
phosphoramidate-based nucleotide, 5ꢀ-amino-5ꢀ-deoxyguanosine-
5ꢀ-N-phosphoramidate (GNHP), should be soluble at pH 8.0
(optimum pH for T7 RNA polymerase activity11), but the phos-
phoramidate functional group is also hydrolytically labile at this
pH with a predicted half-life on the order of several hours.12 Thus
we hoped that the phosphoramidate would be sufficiently stable to
allow incorporation during transcription whilst being sufficiently
labile to allow facile removal of the phosphoryl group in order to
reveal the 5ꢀ-amino group.
We have recently communicated details of the preparation
of GNHP from 5ꢀ-amino-5ꢀ-deoxyguanosine and usage of crude
GNHP in transcription studies.13 In this paper we shall discuss the
development of the aqueous phosphorylation method that was
used to prepare GNHP, the development of a purification strategy
for GNHP, and the use of purified GNHP in transcription in
comparison to the crude material.
Results and discussion
1. Optimisation of the aminophosphorylation procedure
Schattka and Jastorff attempted the synthesis of GNHP us-
ing 2ꢀ,3ꢀ-isopropylidene-protected 5ꢀ-amino-5ꢀ-deoxyguanosine as
their substrate and ester-protected phosphorylating agents.14
Unfortunately they were unable to remove the isopropylidene
protecting group without also removing the phosphoramidate
group. Thus when designing our synthetic route towards GNHP
we limited ourselves to strategies that avoided protecting groups
and where we could use 5ꢀ-amino-5ꢀ-deoxyguanosine as our
starting material, Scheme 1. A recent report from Dean allowed
us to produce 5ꢀ-amino-5ꢀ-deoxyguanosine via a convenient three-
step procedure.15
We attempted aminophosphorylation using phosphorus oxy-
chloride in acetonitrile, as employed by Hampton and co-workers
in their synthesis of the inosine analogue of GNHP,16 and we
found that a solution of 5ꢀ-amino-5ꢀ-deoxyguanosine could not
be obtained. The use of phosphorus oxychloride with trialkyl
phosphate as the solvent17,18 led to a bis-phosphorylation product
With this goal in mind, we set about designing a simple
strategy for the preparation and use of a new modified initiator
Department of Chemistry, University Science Laboratories, South Road,
Durham, DH1 3LE, England. E-mail: d.r.w.hodgson@durham.ac.uk
† Electronic supplementary information (ESI) available: NMR spectra of
GNHP. See DOI: 10.1039/b717896d
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addition to the desired phosphoramidate, the 31P NMR spectra
showed signals corresponding to by-products that we assigned
to inorganic phosphate and 5ꢀ-amino-5ꢀ-deoxyguanosine-2ꢀ- and
3ꢀ-monophosphates. We found that the experiment that did not
contain DMSO cosolvent led to the highest conversion to the
desired product. There are several factors that may have given
rise to this outcome, the most likely being a mixture of increased
nucleophilicity of hydroxide ion towards POCl3 owing to reduced
solvation at higher DMSO concentrations, and the increased
hydrolytic activity of phosphoramidates (even at higher pHs) in
mixed aqueous–organic systems.22 Together these factors would
both lead to increased production of inorganic phosphate ion and
consequent reduction in the conversion of POCl3 to GNHP.
We now turned our attention to the number of equivalents of
sodium hydroxide in the initial reaction mixture using a wholly
aqueous system. A series of experiments was run where the
initial number of equivalents of sodium hydroxide with respect to
substrate was varied. Phosphorus oxychloride was then added to
each aqueous substrate mixture as a solution in THF. The results
from this series of experiments are summarised in Fig. 1.
Scheme 1 Synthetic strategy towards GNHP.
which we tentatively identified as 5ꢀ-amino-5ꢀ-deoxyguanosine-
5ꢀ-N-phosphoramidate-2ꢀ,3ꢀ-cyclic phosphate based on 31P NMR
and ES-MS analyses.
At this point we investigated a convenient water-based proce-
dure from the Drueckhammer laboratory that had been used for
the preparation of analogues of dihydroxyacetone phosphate.19
The procedure used an aqueous alkaline solution of the sub-
strate, where alkali was added at the same time as phosphorus
oxychloride in order to both hydrolyse P–Cl bonds in the
amino phosphorodichloridate intermediate and at the same time
maintain a high pH, at which the phosphoramidate product is
more stable.12 Unfortunately, hydroxide ion also competes against
the amine substrate as a reactant for phosphorus oxychloride,
and thus a balance between maintaining high pH and minimising
hydroxide ion concentration is needed. We were already aware
that 5ꢀ-amino-5ꢀ-deoxyguanosine showed poor solubility in water,
and thus we began our investigations using dimethyl sulfoxide
as a cosolvent. An initial experiment where the 5ꢀ-amino-5ꢀ-
deoxyguanosine substrate was dissolved in DMSO and phospho-
rus oxychloride in THF, and aqueous sodium hydroxide solution
was added simultaneously by pipette, gave a crude reaction mixture
that contained material giving rise to a triplet signal at ca. 8.5 ppm
in the 1H-coupled 31P NMR spectrum, which corresponds to
the expected signal for the desired phosphoramidate.20 With this
preliminary result in hand, we proceeded to systematically study
the effect of the DMSO–water ratio and the effect of the amount
of sodium hydroxide on reaction outcome.
In order to maintain consistency between experiments with
different ratios of DMSO to water, an excess of sodium hydroxide
was added to each experiment at the outset rather than during
the addition of the phosphorus oxychloride solution, so that the
co-addition rate was no longer a variable. The resulting crude
reaction mixtures were then analysed by 31P NMR spectroscopy.
The DMSO content was varied between 0 and 50%, and for
the experiment that did not contain DMSO we found that 5ꢀ-
amino-5ꢀ-deoxyguanosine was actually soluble in aqueous alkali
(rather than pure water). This improved solubility probably results
from deprotonation of the guanine to provide the water-soluble
guanine anion (pKa of N-1 of guanosine is ca. 9.421). The results
from this series of experiments are summarised in Table 1. In
Fig. 1 Optimisation of NaOH. The number of equivalents of NaOH was
varied and the effect on product distribution was assessed by 31P NMR
spectroscopy.
An optimum of five equivalents was found to give the largest
conversion of phosphorus oxychloride to GNHP with the lowest
level of by-products. This number of equivalents likely represents
a balance between the need to ionise the guanine ring in order
to bring the substrate into solution, ionisation of the 2ꢀ,3ꢀ-cis diol
system, reaction of hydroxide ion with POCl3 and hydrolysis of
the phosphoramidate product once it has been formed. With this
crude material in hand, we began kinetic studies on its hydrolysis
in order to confirm that our material shows the expected pH-
dependent hydrolysis properties, and to determine conditions
under which the material may be purified and isolated.
Table 1 Formation of GNHP as a function of solvent composition
DMSO (%)
GNHP (%)a
2. Kinetic evidence for the phosphoramidate
0
10
20
30
40
50
69
61
48
62
55
48
Crude GNHP was subjected to a hydrolysis study over a range of
pHs in order to demonstrate that the product material displayed
similar kinetic properties to known alkyl phosphoramidates.12,16
We used 31P NMR spectroscopy to monitor the disappearance
of the phosphoramidate signal in a series of buffers of varying pHs.
a Determined by 31P NMR spectroscopy on the crude reaction mixture.
◦
A comparison of our data (obtained at 37 C) with Benkovic’s12
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(obtained at 55 ◦C) shows that both profiles are very similar
in form, with the observed rate constants for Benkovic’s study
being higher in value owing to the elevated temperature used in
their experiments, Fig. 2. We chose 37 ◦C as the temperature
for our study because this is the temperature used for T7 RNA
polymerase-catalysed transcription reactions. The half-life for
hydrolysis of GNHP on the pH-independent plateau was 1.8 h, and
at pH 8.0, which is the optimal pH for T7-catalysed transcription,
the half-life was 2.5 h. Thus the phosphoramidate appeared to
be sufficiently stable to allow incorporation into RNA during
transcription (we use 2.5 h incubations for transcription reactions),
but sufficiently labile to allow facile removal of the phosphoryl
group after (and during) the transcription process.
Fig. 3 Capto Q anion exchange chromatography of crude GNHP using
a gradient of 0.1 M (eluent A) to 1 M NaOH (eluent B).
The identities of the components corresponding to the peaks in
the UV trace were deduced on the basis of an authentic standard
in the case of peak A and the basis of 31P NMR spectroscopy in
the cases of peaks B and C. We were unable to identify the nature
of the component that gave rise to peak D. Integration of the
UV trace (assuming that the components giving rise to each peak
have the same extinction coefficient) gives a product distribution
of 18% A, 64% B, 8% C and 8% D. In comparison with earlier 31
P
NMR studies on the crude material, this chromatographic method
shows that the crude GNHP was less pure than we had previously
estimated. Whilst this clearly did not prevent efficient transcription
using the crude material, it makes it all the more important to
study the effect of purity on transcription properties. After elution
from the ion exchange system, the material was lyophilised and
then subjected to gel filtration chromatography on Sephadex G10
media in order to reduce the salt content. Water was used as the
eluent for this process, and thus a small amount of hydrolysis
of the GNHP occurred as the material passed through the
chromatography column (transit time approximately 20 minutes).
The eluted material was immediately frozen and then lyophilised.
We investigated other strategies for the desalting of the eluted
GNHP, such as ethanol precipitation and reduction of the pH to
the isoelectric point (ca. pH 5, see Section 2); however, these proved
less effective than gel filtration. Gel filtration using a mildly basic
Fig. 2 Kinetic data from 31P NMR studies on the hydrolysis of GNHP
at different pHs. Experimental data points (᭹) were fitted to eqn (1) (see
experimental) with the fitting being represented by the solid line. A plot
of the pH hydrolysis behaviour of butyl-N-phosphoramidate (obtained at
55 ◦C) is shown by the dotted line (data from Benkovic and Sampson).12
3. Chromatographic purification of GNHP
Ideally we wished to avoid the use of time-consuming chro-
matographic steps, and we have seen, from the viewpoint of T7-
promoted transcription, that unpurified material gives satisfactory
yields of RNA, with a reasonable proportion of the RNA
containing the 5ꢀ-amino group.13 In order to assess the effect of
GNHP purity on transcription performance, however, we needed
to develop a method for the purification of this hydrolytically labile
material. Hampton and co-workers were able to effect a series of
selective precipitation steps in order to gain the inosine analogue of
GNHP in a form that was homogeneous by paper electrophoresis
and thin layer chromatography. The nature of our synthetic
approach towards GNHP unfortunately leads to the formation
of some by-products and probably some unconverted starting
material (although 31P NMR does not give any information on
this). Owing to the lower hydrolysis rate of GNHP and the
improved solubility of 5ꢀ-amino-5ꢀ-deoxyguanosine at high pH, we
explored the use of strong anion exchange chromatography using a
gradient of increasing concentration of sodium hydroxide solution
as the eluent. This system provides the necessary increase in salt
concentration that allows elution of the desired product whilst
maintaining a high pH. A chromatographic profile of the crude
mixture shows good separation of four major components, Fig. 3.
23
eluent (e.g. 2 mM K2CO3 or 1 mM NaOH), whilst effective, did
leave the eluted material contaminated with salt, which may have
proven detrimental to the transcription process. A small amount
of the freeze-dried material was then repeatedly lyophilised from
D2O before being subjected to 1H NMR spectroscopy.
4. Spectroscopic evidence for the phosphoramidate
Given the number of potential phosphorylation sites that were
available on the unprotected aminonucleoside, we were eager
to confirm that phosphorylation had indeed taken place on
the amino group of 5ꢀ-amino-5ꢀ-deoxyguanosine rather than one
of the guanine functional groups or the ribose hydroxyls. We
have already discussed the use of 31P NMR spectroscopy, the
significance of the triplet signal and its chemical shift, which we
believe correspond to the phosphoramidate group. Kinetic data
on the hydrolysis of the product are also in agreement with similar
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compounds in the literature. 1H NMR spectroscopy gave further
confirmation of our assignment in the form of a complicated
multiplet corresponding to the 5ꢀ-CH2 group, Fig. 4. In addition
to the ABX system that results from coupling to the 4ꢀ-proton
and geminal coupling between the protons of the methylene
group, the signals corresponding to the 5ꢀ-CH2 group of the
phosphoramidate also show coupling to the phosphorus nucleus
band on the purification gel gives a direct measure of the amount
of RNA produced in each experiment, Fig. 5.
3
within the phosphoramidate. 13C NMR spectroscopy reveals J
Fig. 5 Phosphorimage of a purification gel of transcription reactions
containing increasing concentrations of purified GNHP.
coupling between the phosphorus centre and the 4ꢀ-C atom, but
2J coupling to the 5ꢀ-C was not observed.
As the concentration of purified GNHP was increased the
amount of RNA produced increased initially, but then quickly
tailed off as higher concentrations were attained. The gel pieces
containing the RNA transcripts were then excised from the
purification gel and the RNA was eluted passively from the
gel pieces. During this time the remaining phosphoramidate
groups were expected to hydrolyse completely, thus revealing
5ꢀ-amino groups on a proportion of the RNA transcripts. A
biotinylation assay was then used in order to assess the level
of incorporation of 5ꢀ-amino groups in the RNA transcripts
from each transcription experiment. The transcripts from each
experiment were derivatised using 3-sulfo-N-hydroxysuccinimidyl
biotin, and the level of biotinylation (which corresponds to the
level of 5ꢀ-amine label in the RNA transcripts) was assessed using
a streptavidin (SAv)-dependent gel shift assay. The biotinylated
RNA molecules bind to the streptavidin and their mobilities are
reduced relative to those RNA molecules that are not biotinylated
(i.e. those RNAs that were initiated with GTP rather than our
modified initiator molecule). The biotinylation assays revealed that
the level of incorporation of 5ꢀ-amino group increased steadily as
the concentration of GNHP was increased in the corresponding
transcription experiments, Fig. 6.
Fig. 4 The 5ꢀ-CH2 region of the 1H NMR spectrum of purified
GNHP. A complex multiplet occurs at 2.9 ppm that corresponds to
geminal and vicinal coupling to adjacent protons and vicinal coupling
to phosphorus. The sample is contaminated with a small amount of
5ꢀ-amino-5ꢀ-deoxyguanosine owing to hydrolysis of GNHP.
5. Transcription studies
We have already reported our results on the use of crude GNHP
in transcriptions,13 and they are summarised in Fig. 8A.
We have seen from our chromatography studies that the crude
GNHP contained 18% 5ꢀ-amino-5ꢀ-deoxyguanosine and 64%
GNHP i.e. amine and phosphoramidate in a ratio of 1 : 3.6.
The crude material that was used for transcriptions studies was
originally estimated to contain approximately 90% GNHP on
the basis of 31P NMR spectroscopy, which, of course gave no
direct information on the level of 5ꢀ-amino-5ꢀ-deoxyguanosine.
In addition, given the labile nature of the phosphoramidate, the
balance between amine and phosphoramidate is continuously
changing. After chromatographic purification we were able to
obtain a sample of GNHP with a much reduced content of
5ꢀ-amino-5ꢀ-deoxyguanosine, where amine and phosphoramidate
were present in a 1 : 11.1 ratio by 1H NMR spectroscopy, Fig. 4.
The small amount of 5ꢀ-amino-5ꢀ-deoxyguanosine likely resulted
from hydrolysis of GNHP during desalting.
Fig. 6 Phosphorimage of a gel of biotinylated transcription products
from transcription reactions containing increasing concentrations of
purified GNHP.
In order to confirm that the observed gel shifts arose as a result
of the presence of 5ꢀ-amino groups in the RNAs, which in turn
arose from the use of GNHP in transcriptions, a series of control
experiments were also performed. Transcriptions and subsequent
biotinylations were performed where GNHP, biotinylating agent
and/or streptavidin were omitted and the resulting matrix of
experiments was analysed by urea-PAGE, Fig. 7. The experiments
show clearly that the shifted RNA band is seen only when all
components are present, and thus we conclude that the band arose
as a result of the presence of 5ꢀ-amino groups in the RNAs.
The results of the series of transcription and biotinylation
experiments illustrated in Figs. 5 and 6 have been combined to
produce Fig. 8B. This figure shows that total RNA yield tails off
very quickly as the concentration of purified GNHP is increased in
With the purified material in hand, we began to assess the
effect of purification and, more significantly, the effect of amine
vs. phosphoramidate on transcription performance.
Purified GNHP was used under the same transcription condi-
tions as we employed for the crude material. A series of experi-
ments, where each transcription experiment contained a different
concentration of the purified GNHP, was performed. At the end
of the 2.5 h incubation period the RNA from each experiment was
purified by urea-PAGE. The transcription procedure incorporated
a
32P radiolabel thus the level of radioactivity from each RNA
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incorporation of 5ꢀ-amino groups, the results between the two sets
of experiments are similar, with both series reaching a maximum
of ca. 60% of the RNA-containing 5ꢀ-amino groups at the higher
GNHP concentrations. The result from the crude GNHP system
at 7.5 mM is particularly interesting as the overall RNA yield
is high and the level of incorporation of the 5ꢀ-amino group is
also reasonable. We have to remember that the concentration of
7.5 mM was based on the assumption that the material used
in the synthetic procedure was quantitatively converted to the
phosphoramidate, which we have shown not to be the case. The
results of the chromatography study (Section 3) revealed that the
crude material contained the 5ꢀ-amino-5ꢀ-deoxyguanosine and the
GNHP in approximate proportions of 18% and 64%, respectively.
Thus the actual concentrations of 5ꢀ-amino-5ꢀ-deoxyguanosine
and GNHP in this experiment were closer to 1.35 mM and
4.8 mM, respectively. Although the amine is sparingly soluble
it is an effective initiator, and, when combined with GNHP,
both systems contribute towards initiation of transcription with
both systems leading to the production of 5ꢀ-amino-RNA. The
transcription results from our studies with purified GNHP, where
the concentration of any residual 5ꢀ-amino-5ꢀ-deoxyguanosine is
very much lower (<10%), show inhibition of RNA production at
much lower concentrations of initiator.
Fig. 7 Phosphorimage of gel-shift assays on transcription products from
reactions containing (lanes 1–4) or lacking (lanes 5–8) purified GNHP. A
‘+’ symbol signifies the presence of a reagent, whereas a ‘−’ symbol signifies
its absence.
We believe that the anionic phosphoramidate, whilst an effective
inhibitor, may also inhibit the elongation phase of T7 RNA
polymerase. Martin and Coleman demonstrated that the anionic
phosphate groups of GTP and GMP are not required during tran-
scription initiation.24 Indeed, guanosine shows similar initiation
properties to its phosphorylated homologues. The triphosphate
portions of NTPs are, however, bound in the elongation phase of
polymerisation, and so the phosphoryl group of GNHP may also
bind in competition with GTP in the elongation phase. As GNHP
does not possess the same pyrophosphate leaving group as the
NTPs, its binding to the elongation active site is unproductive
and will lead to inhibition and reduced yield. Famulok and
co-workers have observed similar inhibition effects when using
higher concentrations of phosphorylated guanosine analogues as
initiators.7 Although limited by its poor solubility, the amine is
not charged, and therefore we believe that it does not inhibit
the elongation phase in the same way as the phosphoramidate;
thus RNA production with crude GNHP, which contains a
greater proportion of the amine, is significantly greater. This is
a challenging hypothesis to prove as uncharged nucleosides are
likely to be intrinsically limited in their solubility properties, and
thus we are unable to explore the effect of high concentrations
on transcription. We are, however, investigating the transcription
properties of an uncharged guanosine analogue that shows
reasonable solubility properties,25 and we hope to investigate the
properties of cationic initiator nucleosides in the future.
Fig. 8 Transcription results. The overall yield of RNA relative to control
transcriptions that do not contain GNHP are represented by the heights
of the light grey bars. The level of incorporation of the 5ꢀ-amino group into
transcripts is represented by the height of the darker bars within the shaded
bars. Data in charts A and B correspond to results from experiments on
unpurified GNHP and purified GNHP respectively. The concentrations of
unpurified GNHP were based on the assumption that complete conversion
of 5ꢀ-amino-5ꢀ-deoxyguanosine to GNHP occurred during synthesis.
Conclusions
We have devised and optimised a convenient method for the
preparation of a previously unknown nucleoside phosphorami-
date. Our procedure uses a simple aqueous approach to solubilise
5ꢀ-amino-5ꢀ-deoxyguanosine followed by a chemoselective N-
phosphorylation. The use of alkali may represent a general strat-
egy for the solubilisation of recalcitrant guanosine derivatives and
we are currently exploring its use with 5ꢀ-deoxy-5ꢀ-iodoguanosine
the transcription experiments. The analogous results for the crude
GNHP, Fig. 8A, show a more gradual tailing off of RNA yield at
higher concentrations of GNHP, even when correcting for the fact
that the crude GNHP contained only ca. 64% GNHP. In terms of
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and other guanosine systems. The nucleoside phosphoramidate
was then purified by a combination of ion exchange and gel
filtration chromatographies. The identity of the phosphoramidate
was confirmed by kinetic and spectroscopic techniques before
being used in T7 RNA polymerase-catalysed transcriptions.
We found that crude GNHP gave a superior performance in
transcription studies, and we believe that the uncharged nucleoside
5ꢀ-amino-5ꢀ-deoxyguanosine acts as a supplementary initiator. We
are currently exploring the use of another uncharged guanosine
derivative that displays better solubility characteristics than 5ꢀ-
amino-5ꢀ-deoxyguanosine.
solids, which were each dissolved in water (1 mL) and transferred
to NMR tubes. Product distribution was then assessed by 31P
NMR spectroscopy.
Preparation and purification of GNHP
5ꢀ-Amino-5ꢀ-deoxyguanosine (50 mg, 177 lmol) was dissolved in
a mixture of NaOH(aq) (885 lL of a 1 M volumetric standard,
885 lmol (5 eq)) and water (1 mL), and then cooled to 0 ◦C
on an ice bath. Phosphorus oxychloride (33 lL, 177 lmol) in
anhydrous THF (1 mL) was then added dropwise to the solution
over the course of 10 min. The reaction mixture was diluted
with water (3 mL) and loaded on to a Capto Q strong anion
exchange chromatography column (25 mL bed volume) attached
Experimental
¨
to an Akta Prime Plus automated liquid chromatography system.
5ꢀ-Amino-5ꢀ-deoxyguanosine
Materials were eluted from the column using a gradient starting
with eluent A (0.1 M NaOH) and running to 25% eluent B (1 M
NaOH), and the absorbance at 254 nm of the eluted material
was recorded, Fig. 3. Fractions containing material from each
peak were combined and lyophilised. The residues from each
peak were then dissolved in water (0.75 mL) and subjected to
31P NMR spectroscopy. The material from the peak B, Fig. 3,
Prepared in three steps from guanosine using Dean’s procedure.15
5ꢀ-Amino-5ꢀ-deoxyguanosine was recrystallised from water in
order to produce analytically pure material as off-white crystals.
◦
◦
◦
mp = 218–220 C (dec.); lit., 219–220 C15 or 221 C14; (Found
C, 39.8; H, 5.4; N, 27.7. C10H14N6O4·H2O requires C, 40.0; H, 5.3;
1
N, 28.0%); mmax(KBr disc)/cm−1 2622br (OH), 1702 (C O); H
=
¨
was then subjected to gel filtration chromatography (Akta Prime
NMR, dH(500 MHz, DMSO-d6; Me4Si): 2.72 (1 H, ABX system,
JAB 13.5, JBX 5.5, 5ꢀ-CHAHB), 2.77 (1H, ABX system, JAB 13.5,
JAX 4.5, 5ꢀ-CHAHB), 3.77–3.81 (1H, m, 4ꢀ-HX), 4.08 (1H, t, J 4.5,
3ꢀ-H), 4.44 (1H, t, J 6.8, 2ꢀ-H), 5.66 (2H, d, J 6.5, 1ꢀ-H), 6.52 (1H,
br s, NH2), 7.93 (1H, s, 8-H): dC(100.6 MHz, DMSO-d6; Me4Si):
43.5 (5ꢀ-C), 70.6 (3ꢀ-C), 73.11 (2ꢀ-C), 85.5 (4ꢀ-C), 86.2 (1ꢀ-C), 116.7
(5-C), 135.71 (8-C), 151.3 (4-C), 153.6 (2-C), 156.7 (6-C)): m/z
(ES+) 283.1 ([M + H]+).
Plus automated liquid chromatography system, Sephadex G10,
150 mL bed volume, 5 mL min−1 of water) in order to remove salt.
Fractions containing material absorbing at 254 nm were combined
and lyophilised. The material was then repeatedly lyophilized from
1
D2O in order to reduce the large residual water signal in the H
1
NMR spectrum. Integration of the H NMR spectrum showed
that the sample contained 8% 5ꢀ-amino-5ꢀ-deoxyguanosine. Spec-
troscopic data for GNHP: 1H NMR, dH(500 MHz, D2O; Me4Si):
2.86 (1 H, ABX system, JAB 13.5, JAXP 6.5, 5ꢀ–CHAHB), 2.93 (1 H,
ABX system, JAB 13.5, JBXP 5.5, 5ꢀ-CHAHB), 4.04–4.02 (1H, m,
4ꢀ-HX), 4.20 (1H, t, J 4, 3ꢀ-H), 4.61 (1H, t, J 5.5, 2ꢀ-H), 5.66
(2H, d, J 6.5, 1ꢀ-H), 7.74 (1H, s, 8-H): 13C NMR dC(100.6 MHz,
D2O; Me4Si): 44.4 (5ꢀ-C), 71.5 (3ꢀ-C), 73.1 (2ꢀ-C), 85.4 (d, JCP 10.1,
4ꢀ-C), 86.9 (1ꢀ-C), 117.9 (5-C), 136.2 (8-C), 151.8 (4-C), 161.1 (2-
C), 167.8 (6-C): 31P NMR dP(121 MHz, D2O; H3PO4): 10.2 (t,
88%, phosphoramidate), 4.1 (s, 12%, inorganic phosphate), see
ESI for spectra: FT–MS m/z (ES−): found 361.06661 at a FWHH
resolving power of 106; C10H14N6O7P− requires 361.06671.
Preliminary experiment using mixed aqueous–DMSO solvent
system
5ꢀ-Amino-5ꢀ-deoxyguanosine (20 mg, 68.8 lmol) was dissolved in
DMSO (500 lL, 5.82 mmol), and the solution was then diluted
with water (500 lL) and cooled to 0 ◦C. Phosphorus oxychloride
(7.04 lL, 75 lmol) dissolved in dry THF (34.4 lL) was added
dropwise with sufficient sodium hydroxide solution (stock of
109 mg, 2.7 mmol in 1.1 mL water) in order to ensure that the
pH was always above 10 (by indicator paper). When the addition
of the phosphorus oxychloride was complete, the reaction mixture
was diluted with water to a final volume of 750 lL and the mixture
was transferred to an NMR tube. 31P [1H coupled] NMR, dP(121
MHz): 8.52 (t, OP(O)2–NH–CH2), 2.31 (s, inorganic phosphate):
m/z (LC–ES−) 361 ([GNHP2− + H+]).
Kinetic study on the hydrolysis of GNHP
Owing to the great range of reactivity covered from pH 3 to
10.5, kinetic experiments were performed using three different
methods. For experiments between pH 7.2 and 9, GNHP (final
concentration 30 mM) was dissolved in buffer (0.5 M MES pH 7.2,
0.5 M sodium bicarbonate pH 8.0 or 0.5 M sodium borate pH 9.0
where the pHs were measured after dissolution of GNHP) and the
samples were transferred to the NMR machine (equilibrated to
37 ◦C). Spectra were acquired regularly at fixed time points using
the automated software. For experiments at lower pHs (3 and 3.5)
GNHP was dissolved to a final concentration of 30 mM in 0.5 M
sodium formate buffer and the pH was measured (one experiment
at pH 3.0, one at pH 3.5). Aliquots of were withdrawn from the
stock solutions at 5 min intervals and were quenched directly
into sodium hydroxide solution. The quenched mixtures were then
subjected to 31P NMR spectroscopy. At higher pHs (9.8 and 10.5)
Optimisation of DMSO–water ratio
5ꢀ-Amino-5ꢀ-deoxyguanosine (10 mg, 35 lmol) was added to
DMSO (where applicable) and stirred for 5 min, over which time
full dissolution of the solid occurred. Water was added to the
solution, in most cases resulting in the precipitation of a white
solid. The reaction vessel was then cooled to 0 ◦C (in an ice-
bath), before addition of NaOH(aq) (3.75 mmol) resulted in the
dissolution of all the solid to give a clear solution. Additions (10
lL) of phosphorus oxychloride solution (22.8 lL POCl3 in 77.2 lL
dry THF]) were made at 60 s intervals over the course of 10 min.
The solvents were removed under reduced pressure to give white
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GNHP was dissolved to a final concentration of 30 mM in either
0.5 M sodium carbonate buffer or sodium hydroxide solution
and the pH was measured as pH 9.8 or pH 10.5 respectively.
The samples were transferred to a water bath set at 37 ◦C. The
samples were periodically removed for 31P NMR analysis and then
returned to the water bath. All spectra were integrated and the
ratio of the 31P NMR signal corresponding to GNHP relative to
that inorganic phosphate and GNHP was calculated for each time
point. The fraction of GNHP within the mixture was then plotted
as a function of time, and the data were found to fit simple first
order decay curves. The observed pseudo first order rate constants
are plotted in logarithmic form in Fig. 2. The data were found to
fit eqn (1), which represents a simplified version of the expression
used by Benkovic and Sampson.12 Fitted values of the constants
were kH= 26 M−1 s−1, k0 = 6 × 10−3 s−1 and pKa= 8.6.
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k0
kobs = kH × 10−pH
+
(1)
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We thank the Engineering and Physical Sciences Research Council
for a studentship to D.W. and Durham University for additional
financial support for this project. We are grateful to Prof.
W. T. McAllister for supplying an expression vector for His-tagged
T7 RNA polymerase and to Dr M. J. Cann and Dr M. Skipsey
for assistance with the expression of this protein. We are grateful
to Durham University Chemistry Department technical and
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1062 | Org. Biomol. Chem., 2008, 6, 1056–1062
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