Synthesis of deoxyadenosine-5′-(35S)thiomonophosphate [dAMP(35S)] of high specific activity - A key intermediate for the synthesis of Sp-deoxyadenosine-5′-(α-35S)thiotriphosphate [Sp-dATP (α-35S)]

Article abstract of DOI:10.1002/jlcr.531

Unprotected deoxyadenosine 1 was treated with an excess of phosphorus acid and stoichiometric proportions of N, N′-di-p- tolylcarbodiimide in anhydrous pyridine to give deoxyadenosine-5′-monophosphite 2. The latter wax activated with trimethylsilyl chloride followed by sulphurisation with elemental ³⁵S (specific activity > 1000 Ci/mmol) in toluene solution to give deoxyadenosine5′-(³⁵S)-thiomonophosphate [dAMP(³⁵S)] 3. Enzymatic conversion of deoxyadenosine-5′-(³⁵S)-thiomonophosphate to Sp-deoxyadenosine-5′-(α-³⁵S)-thiotriphosphate [Sp-dATP (α-³⁵S)] 5 was carried out following a standard reaction protocol. Copyright

Full text of DOI:10.1002/jlcr.531

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2002; 45: 19–28.
DOI: 10.1002/jlcr.531
Research Article
0
35
Synthesis of deoxyadenosine-5 -( S)-
3
5
thiomonophosphate [dAMP( S)] of high
specific activity – a key intermediate for the
0
35
synthesis of Sp-deoxyadenosine-5 -(a- S)-
3
thiotriphosphate [Sp-dATP (a- S)]
5
K. M. Mathew*, S. Ravi , C. V. Sontakke and H. Chanderz
LC/ILCJP, Board of Radiation and Isotope Technology, BARC Vashi Complex,
Navi Mumbai 400 705, India
y
Summary
Unprotected deoxyadenosine 1 was treated with an excess of phosphorus acid
0
and stoichiometric proportions of N, N -di-p-tolylcarbodiimide in anhydrous
0
pyridine to give deoxyadenosine-5 -monophosphite 2. The latterwas activated
5
3
with trimethylsilyl chloride followed by sulphurisation with elemental
S
(
specific activity>1000 Ci/mmol) in toluene solution to give deoxyadenosine-
0
35 35
-( S)-thiomonophosphate [dAMP( S)] 3. Enzymatic conversion of deox-
5
0
35
0
35
yadenosine-5 -( S)-thiomonophosphate to Sp-deoxyadenosine-5 -(a- S)-thio-
triphosphate [Sp-dATP (a- S)] 5 was carried out following a standard reaction
protocol. Copyright # 2002 John Wiley & Sons, Ltd.
3
5
0
Key Words: deoxyadenosine; deoxyadenosine-5 -monophosphite; sulphurisa-
0
35
tion deoxyadenosine-5 -( S)-thiomonophosphate [dAMP( S)]; deoxynucleo-
35
0
0
35
tide-5 -thiomonophosphate (dNMPS); Sp-deoxyadenosine-5 -(a- S)-thiotri-
3
5
0
phosphate [Sp-dATP (a- S)]; deoxynucleotide-5 -thiotriphosphate (dNTPS)
*Correspondence to: K.M. Mathew, LC/ILCJP, Board of Radiation and Isotope Technology,
BARC Vashi Complex, Navi Mumbai 400 705, India. E-mail: mathew@apsara.barc.ernet.in
y
E-mail: ravisesh@apsara.barc.ernet.in
E-mail: harishch@magnum.barc.ernet.in
z
Copyright # 2002 John Wiley & Sons, Ltd.
Received 25 June 2001
Revised 13 September 2001
Accepted 3 October 2001

2
0
K. M. MATHEW ET AL.
Introduction
0
35
35
Deoxyadenosine-5 -(a- S)-thiotriphosphate [dATP (a- S)] is an analo-
gue of deoxyadenosine-5 -triphosphate (dATP) in which a non-bridging
0
oxygen atom of a-phosphorus is replaced by a sulphur atom. Such a
substitution leads to an additional centre of chirality into the nucleotide
and thus to the existence of a pair of diastereomers. This facilitates
determination of the stereochemical course of an enzymatic reaction
and its importance can be seen from the large number of review articles
1
–9
dealing with this subject.
towards hydrolytic cleavage relative to the corresponding phosphates.
These phosphorothioates are also stable
9
1
0–16
It has been reported.
that the Sp-diastereomer of dATP(a-S) is
polymerised by DNA polymerase in the presence of a primer template
to produce DNA chain containing phosphorothioate internucleotide
linkages. The kinetic constants forthe polyme ri sation of Sp-dATP ( a-S)
are similar to those for dATP, while the Rp-diastereomer is not a
0
substrate for these enzymes. Incorporation of deoxyadenosine-5 -
3
5
35
32
(
a- S)-thiotriphosphate [dATP (a- S)], instead of (a- P)dATP, as
the radioactive label in dideoxynucleotide sequence reactions has three
apparent advantages. (i) The major one is the very sharp definition of
bands on a sequencing gel autoradiography and thus increased
resolution, which is due to the short path length of b particles emitted
3
5
32
by S. The strong emission of P gives diffuse and broad bands.
(ii) The weak emission of S reduces the radiation dose received during
35
handling of the labelled material, and (iii) the reaction products can be
stored at ꢀ208C for periods of 1 week or more before use in
electrophoresis with minimal damage to the labelled DNA molecules.
32
Whereas decay of an incorporated P atom will break the sugar-
phosphate backbone, this does not usually seem to be the case during
35
decay of an incorporated S atom.
0
Deoxynucleotide-5 -thiomonophosphate (dNMPS) has been prepared
by hydrolysis of the products obtained from the reaction of unprotected
deoxynucleosides with thiophosphoryl chloride in triethyl phosphate.
3
5
0
This procedure is not attractive for the
S-deoxynucleotide-5 -
thiotriphosphate ( S-dNTPS) because of limited availability if high
35
35
specific active ( S)-PSCl and low yield in the thiophosphorylation
3
1
7–20
steps.
The reaction of 2-chloro-4H-1,3,2-benodioxaphosphorin-4-
0
one (salicylphosphorochloridite) with the 5 -hydroxy group of the
nucleoside to give an intermediate, which on subsequent reaction with
pyrophosphate followed by oxidation with sulphur and hydrolysis to
Copyright # 2002 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2002; 45: 19–28

3
5
SYNTHESIS OF [dAMP( S)]
21
give a diastereomeric mixture of dNTPS, has been reported by Ludwig
2
1
22
et al. The procedure reported by Chen et al. involves sulphurisation
of the trimethylsilyl derivative of deoxyadenosine monophosphite with
an excess of pulverised sulphur. This procedure as such is not suitable
for a radioactive preparation, because of (i) potential loss of radioactive
sulphurwhile pulve ri sation, (ii) twice the amount of sulphurhas to be
used in the reaction and (iii) the scale of operation is in millimoles, and
the yield of reaction product may be drastically low when the reaction is
carried out at nanomole levels.
Therefore, we have modified this approach for the radioactive
35
0
35
preparation of deoxyadenosine-5 -( S)-thiomonophosphate [dAMP( S)]
of high specific activity (>1000Ci/mmol) – an intermediate for the
3
3
5
synthesis of Sp-dATP(a- S) 5. The salient features of our synthesis are
3
a) S elemental sulphurwas used in toluene the re by avoiding the loss of
5
(
sulphur due to pulverisation. This also reduces the radiolytic decom-
0
position of radioactive sulphur and (b) deoxyadenosine-5 -monopho-
sphite was used in excess compared to radioactive elemental sulphur to
35
give a high yield (85%) of dAMP( S).
0
Deoxyadenosine-5 -monophosphite 2 was prepared by condensing
0
phosphorus acid with deoxyadenosine 1 in the presence of N,N -di-p-
0
22
tolylcarbodiimide.
phosphite 2 was confirmed by oxidising it to deoxyadenosine-5 -
The formation of deoxyadenosine-5 -mono-
0
monophoshpate 6 using KmnO . The reaction products were analysed
4
by HPLC. Attempts were made to optimise the yield of 3 by
increasing the reaction volume by addition of various solvents such
as pyridine, triethylamine, dimethylformamide, methylene chloride
35
and toluene. The yield of dAMP( S) was 85% when toluene was
used as a solvent whereas in other solvents the yield was in the range
5
–10%.
Deoxyadenosine-5 -phosphite 2 was activated with trimethylsilyl
chloride and excess of activated intermediate was allowed to react
0
3
5
with [ S] elemental sulphur in toluene. The reaction products were
analysed by HPLC and TLC. The formation of dAMPS 4 was
23
0
confirmed by desulphurisation with 1-[3 -(dimethylamino)propyl]-3-
ethylcarboiimide to give dAMP 6 which in turn, on enzymatic reaction
with alkaline phosphatase, afforded deoxyadenosine 1. The reaction
products obtained were analysed by HPLC and TLC. It may be
noted that dAMPS 4 is resistant to alkaline phosphatase and is
24
not degraded under these conditions. The reaction products were
3
analysed by HPLC. The presence of dAMP( S) 3 was also further
5
Copyright # 2002 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2002; 45: 19–28

2
2
K. M. MATHEW ET AL.
confirmed by comparison with a cold specimen sample of dAMPS 4
20
obtained via the PSCl route and both the products were found
3
to be identical. The required intermediate 3 was purified by column
chromatography on DEAE Cellulose column and was enzymatically
35
20
converted to Sp-dATP(a- S) 5 using a standard reaction protocol, in
0% yield. The reaction products were analysed by TLC. The
presence of compound 5 was also confirmed by incorporating it into a
7
2
5
nucleotide chain by Nick Translation reaction and comparison with a
chain obtained from an authentic sample of 5 obtained from M/s
Amersham Pharmacia Biotech. Both the samples showed identical
features (Scheme 1).
Scheme 1.
Copyright # 2002 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2002; 45: 19–28

3
5
SYNTHESIS OF [dAMP( S)]
23
Experimental
Chemicals and methods
0
Deoxyadenosine, deoxyadenosine-5 -monophosphate, trimethylsilyl
0
chloride, N,N -di-p-tolylcarbodiimide, Tris-HCl, 1,4-dithiothreitol,
phosphorus acid thiophosphoryl chloride were obtained from Aldrich.
Adenylate kinase, phosphoenol pyruvate, pyruvate kinase, DEAE
0
Cellulose, DEAE Sephadex A-25 and 1-[3 (dimethylaminopropyl)]-3-
3
ethylcarbodiimide were obtained from Sigma. ( S)Elemental sulphur
5
3
5
(
specific activity>1000 Ci/mmol) and dATP(a- S) were obtained from
35
M/s Amersham Pharmacia Biotech. ( S)Elemental sulphurwas pu ri fied
by HPLC and used for the reaction. The solvents were freshly dried by
standard procedures just prior to use. Thin layer chromatography was
carried out on Merck silica gel plates containing fluorescent indicator
with a solvent system: 1-propanol/ammonia/water, 6:3:1 (v/v/v).
Nucleotides were visualised with UV light. Phosphorothioate nucleo-
tides were visualised with UV light as well as by spraying with iodine
26
azide-reagent. Radioactive nucleotides were identified by autoradio-
graphy. A Shimadzu HPLC was used with a UV-Visible detector of
LDC Milton Roy make.
0
Deoxyadenosine-5 -monophosphite 2. To anhydrous deoxyadenosine 1
(260 mg, 1 mmol) dried at 1008C/0.5 mm Hg for8 h was added
anhydrous pyridine (10 ml) and phosphorus acid (120 mg, 14 mmol)
0
and the mixture stirred for 10 min at room temperature. N,N -di-p-
tolylcarbodiimide (320 mg, 1.4 mmol) was added and stirring continued
for 3 days at room temperature. Deionised water (50 ml) was added and
again the solution stirred for 30 min. The reaction mixture was filtered
to remove di-p-tolylurea and the filtrate was rotary evaporated to
dryness under reduced pressure at 358C. The residue was taken up in
1
M triethylammonium bicarbonate (pH 7.5) (8 ml) and rotary
evaporated to dryness. Excess of triethylamine was removed by repeated
co-evaporation with deionised water under reduced pressure. The
residue left was then dissolved in deionised water (10 ml) and the
solution chromatographed on a column (1 cm ꢁ 40 cm) of DEAE-
cellulose. The column was eluted with a linear gradient formed from
2
.5 l of deionised waterand 2.0 l of 0.4 M i te rthylammonium
bicarbonate (pH 7.5). The eluate was monitored for absorbance at
59 nm. Fractions were collected and analysed by HPLC. The results
are presented in Table 1. The fractions corresponding to deoxyadeno-
2
Copyright # 2002 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2002; 45: 19–28

2
4
K. M. MATHEW ET AL.
0
Table 1. HPLC analysis of phosphites and 5 phosphate
Compound
Retention time (min)
0
0
Deoxyadenosine-3 ,5 -diphosphite
0
6.1
7.0
9.8
14.7
Deoxyadenosine-5 -monophosphate
0
Deoxyadenosine-5 -monophosphite
Deoxyadenosine
0
sine-5 -monophosphite were pooled. The yield was 65% (0.65 mmol)
0
(
0.19–0.21 M bufferf ar ction yielded the deoxyadenosine-5 -monopho-
0
0
sphite whereas deoxyadenosine-3 , 5 -diphosphite was eluted with
.28–0.3 M buffer).
0
0
The presence of deoxyadenosine-5 -monophosphite 2 was also
0
confirmed by oxidising to deoxyadenosine-5 -monophosphate 6 fol-
lowed by HPLC (Table 1).
0
0
Deoxyadenosine-5 -monophosphate 6. Deoxyadenosine-5 -monopho-
sphate 2 (32 mg) was dissolved in water(5 ml) containing sodium
hydrogen carbonate (10 mg). The solution was cooled in ice and 0.155%
aqueous KMnO (5 ml) was added overthe cou sr e of 45 min with
4
stirring. The reaction mixture was filtered and was analysed by HPLC.
Results are presented in Table 1.
Column: C-18 ODS (Shandon, 4 mm ꢁ 25 cm), mobile phase: 300 mM
Tris-HCl (pH 8.0) containing 20 mM MgCl : methanol::90:10 (v/v),
detectorUV-259 nm, flow ra te 1.0 ml/min.
2
35
35
Purification [ S] elemental sulphur. [ S]Elemental sulphurwas
purified by HPLC using methanol/water 20:1 (v/v) as mobile phase
on a column of C-18 ODS (Shandon, 4 mm ꢁ 25 cm) with a flow rate of
2
7
1
3
ml/min. The column was monitored by a UV wavelength of 264 nm.
5
[
used forthe re action).
S] elemental sulphur(S , eluting at 9.7 min, was collected and was
8
0
35
Deoxyadenosine-5 -( S)thiomonophosphate (dAMP S) 3. Deoxya-
35
0
denosine-5 -monophosphite triethylammonium salt (23.13 mmol) was
repeatedly (four times) rotary evaporated with anhydrous pyridine
(1 ml). Anhydrous pyridine (645 ml) was added to the residue followed
by addition of anhydrous triethylamine (16.8 ml, 117.5 mmol) and
trimethylsilyl chloride (15 ml, 117.5 mmol). The reaction mixture was
stirred at room temperature for 45 min. The reaction mixture of 33 nmol
3
5
(
1 ml) was added to 5 mCi (5 nmol, 175 ng) of S elemental sulphurin
toluene (100 ml) and the whole stirred for 1 h. The reaction mixture was
quenched with deionised water (1 ml). The sample was repeatedly rotary
Copyright # 2002 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2002; 45: 19–28

3
5
SYNTHESIS OF [dAMP( S)]
25
0
35
Table 2. HPLC analysis of deoxyadenosine-5 -( S)thiomonophosphate
Compound
Retention time (min)
0
Deoxyadenosine-5 -thiomonophosphate
5.2
5.2
7.2
9.3
0
35
Deoxyadenosine-5 -( S)thiomonophosphate
0
Deoxyadenosine-5 -monophosphate
0
Deoxyadenosine-5 -monophosphite
Deoxyadenosine
14.2
evaporated with deionised water under reduced pressure to completely
remove the pyridine. The residue was taken up in deionised water (1 ml)
and loaded on a DEAE-Cellulose column (1 cm ꢁ 40 cm). Elution was
carried out with a linear gradient formed of 2 l each of deionised water
and 0.6 M triethylammonium bicarbonate buffer. The flow rate was
kept at 1 ml/min. The column was monitored for both radioactivity and
UV absorbance at 259 nm. Fractions were collected and analysed by
0
HPLC (Table 2). The fraction corresponding to deoxyadenosine-5 -
5
3
35
S)-thiomonophosphate [dAMP( S)] was pooled, giving a yield of
(
8
nosine-5 -( S)-thiomonophosphate [dAMP( S)] 3.
0% (4 mCi, 4 nmol). Bufferf ra ction of 0.34–0.45 M yielded deoxyade-
35
0
35
35
Formation of dAMP( S) was confirmed by preparing a cold sample
of dAMPS 4 with 600 nmol of elemental sulphurusing the above
procedure.
0
Analysis of deoxyadenosine-5 -thiomonophosphate (dAMPS)
dAMPS in the reaction mixture was identified by using TLC (results
presented in Table 4) and also by desulphurisation followed by reaction
with alkaline phosphatase to give deoxyadenosine.
dAMPS 4 (100 nmol) in 0.5 M Tris–HCl buffer (pH=7.5) (0.5 ml) was
0
stirred with 1-[3 (dimethylaminopropyl]-3-ethylcarbodiimide at room
temperature for 1 h. The dAMP formed was analysed by HPLC and
TLC (Tables 2 and 3). To the reaction mixture, three units of alkaline
phosphatase were added and stirred at room temperature. Under these
conditions dAMP was converted to deoxyadenosine. The reaction
mixture was analysed by HPLC and TLC and the results are presented
in Tables 2 and 3.
0
Deoxyadenosine-5 -thiomonophosphate (dAMPS) 4. To anhydrous
deoxyadenosine 1 (2 mmol, 535 mg) dried at 1008C/0.5 mm Hg for8 h
was added anhydrous triethylphosphate (10 ml) and anhydrous pyridine
(4.1 mmol, 330 ml) and the mixtures stirred at 1008C until almost all the
Copyright # 2002 John Wiley & Sons, Ltd.
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6
K. M. MATHEW ET AL.
deoxyadenosine dissolved (ꢂ10 min). Afterthe solution has been cooled
in an ice bath, freshly distilled thiophosphoryl chloride (8 mmol, 800 ml)
was added and the reaction mixture stirred at 08C for1 h. Un re acted
thiophoshoryl chloride was then removed under pressure at 308C. The
residue left was again cooled in an ice bath and a solution of aqueous
pyridine (8 mmol, 660 ml in 500 ml water) was added. The pH was then
raised from 5 to 9 by addition of 1 M NaOH and the solution loaded on
to a column (2.3 ꢁ 41 cm) of DEAE-Sephadex-A25. Elution was carried
out at 48C with a linear gradient of 1.5 l of 0.1 M triethylammonium
bicarbonate buffer (pH 7.5) and 1.5 l of 0.4 M triethylammonium
bicarbonate buffer (pH 7.5). The eluate was monitored for absorbance
at 259 nm. Fractions were collected and analysed by HPLC and TLC
0
(Tables 2 and 3). The fraction corresponding to deoxyadenosine-5 -
thiomonophosphate (dAMPS) were pooled. The product was identical
with that obtained in the previous synthesis.
Column: C-18 ODS (Shandon, 4 mm ꢁ 25 cm), mobile phase: 300 mM
Tris–HCl (pH 8.0) containing 20 mM MgCl : methanol::90:10 (v/v),
2
detectorUV-259 nm, flow ra te 1.0 ml/min.
35
0
35
Sp-Deoxyadenosine-5 -(a- S)thiotriphosphate [Sp-dATP (a- S)] 5.
A mixture of dAMP( S) 3 (3 nmol, 3 mCi), ATP (3 nmol), phosphoenol
35
pyruvate (1 nmol), magnesium chloride (1 nmol), potassium chloride
(60 nmol), Tris–HCl (pH 8.0) (42 nmol), 1,4-dithiothreitol (1 nmol),
adenylate kinase (790 units/ml) and pyuravate kinase (35 units/ml) was
stirred at room temperature. The progress of the reaction was
monitored by TLC. The reaction mixture was chromatographed on a
column (1 cm ꢁ 30 cm) of DEAE-Sephadex A-25 (bicarbonate form)
and was eluted at 48C with a linearg ar dient of 400 ml of 0.2 M
0
Table 3. Thin layer chromatography of deoxyadenosine-5 -thiomonophosphate
Compound
R Value
f
Deoxyadenosine
Deoxyadenosine-5 -thiomonophosphate
Deoxyadenosine-5 -monophosphate
0.60
0.45
0.19
0
0
3
5
Table 4. Thin layer chromatography of dAMP( S) and Sp-dATP[a- S]
35
Compound R value
f
3
dAMP( S)
Sp-dATP[a- S]
5
0.45
0.12
3
5
Copyright # 2002 John Wiley & Sons, Ltd.
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3
5
SYNTHESIS OF [dAMP( S)]
27
triethylammonium bicarbonate buffer (pH 7.5) and 0.75 M triethylam-
monium bicarbonate buffer (pH 7.5). The eluant from the column was
checked by TLC (the results are presented in Table 4). The fractions
35
corresponding to Sp-dATP(a- S) were pooled, giving a yield of 70%
(2.1 mCi).
The product 4 was incorporated into a nucleotide chain by Nick
translation reaction and its performance compared with authentic
3
5
dATP[a- S] obtained from M/s Amersham Pharmacia Biotech. Both
the samples gave identical incorporation.
Acknowledgements
The authors wish to express their thanks to Dr N. Ramamoorthy, Chief
Executive, BRIT, Dr N. Sivaprasad, General Manager, ILCJP, BRIT
and Mr A.U. Garana, Deputy General Manger, LC/ILCJP, BRIT for
their support and encouragement.
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