Facile small scale synthesis of nucleoside 5′-phosphate mixtures

Article abstract of DOI:10.1080/15257770903451546

We present a facile method to phosphorylate small amounts of nucleosides (0.05 mol) into mixtures of their 5′-mono-, di-, and triphosphates in a one-pot reaction. The nucleosides were first converted into their dichlorophosphates using a large excess (15-18 equivalents) of phosphorous oxychloride in trimethylphosphate. The large excess resulted in good dichlorophosphate yields (46-76%) for the four nucleosides tested. Upon the addition of tributylammonium-phosphate with additional tributylamine (20 equivalents both), the dichlorophosphate was converted into a mixture containing equal amounts of the mono-, di-, and triphosphate. The presented method was successfully applied to synthesize mixtures of stable isotope labeled nucleotides, which can be used as internal standards in quantitative mass spectrometric assays.

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Facile Small Scale Synthesis of
Nucleoside 5′-Phosphate Mixtures
Robert S. Jansen a , Hilde Rosing a , Jan HM Schellens b c & Jos H.
Beijnen a c
a Department of Pharmacy & Pharmacology, Slotervaart Hospital/
The Netherlands Cancer Institute, Amsterdam, The Netherlands
b Division of Medical Oncology, The Netherlands Cancer Institute,
Amsterdam, The Netherlands
c Science Faculty, Department of Pharmaceutical Sciences, Utrecht
University, Utrecht, The Netherlands
Available online: 21 Dec 2009
To cite this article: Robert S. Jansen, Hilde Rosing, Jan HM Schellens & Jos H. Beijnen (2009): Facile
Small Scale Synthesis of Nucleoside 5′-Phosphate Mixtures, Nucleosides, Nucleotides and Nucleic
Acids, 29:1, 14-26
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Nucleosides, Nucleotides and Nucleic Acids, 29:14–26, 2010
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903451546
FACILE SMALL SCALE SYNTHESIS OF NUCLEOSIDE 5ꢀ-PHOSPHATE
MIXTURES
Robert S. Jansen,1 Hilde Rosing,1 Jan HM Schellens,2,3 and Jos H. Beijnen1,3
1Department of Pharmacy & Pharmacology, Slotervaart Hospital/The Netherlands Cancer
Institute, Amsterdam, The Netherlands
2Division of Medical Oncology, The Netherlands Cancer Institute, Amsterdam,
The Netherlands
3Science Faculty, Department of Pharmaceutical Sciences, Utrecht University, Utrecht,
The Netherlands
We present a facile method to phosphorylate small amounts of nucleosides (0.05 µmol) into
2
mixtures of their 5ꢁ-mono-, di-, and triphosphates in a one-pot reaction. The nucleosides were first
converted into their dichlorophosphates using a large excess (15–18 equivalents) of phosphorous
oxychloride in trimethylphosphate. The large excess resulted in good dichlorophosphate yields
(46–76%) for the four nucleosides tested. Upon the addition of tributylammonium-phosphate
with additional tributylamine (20 equivalents both), the dichlorophosphate was converted into a
mixture containing equal amounts of the mono-, di-, and triphosphate. The presented method was
successfully applied to synthesize mixtures of stable isotope labeled nucleotides, which can be used as
internal standards in quantitative mass spectrometric assays.
Keywords 5ꢁ-Triphosphates; nucleoside analogs; bioanalysis and mass spectrometry
INTRODUCTION
Nucleoside analogs are used in anti-cancer, anti-(retro)viral, and im-
munosuppressive therapies. The analogs are phosphorylated to nucleoside
mono-, di-, and triphosphates intracellularly. The triphosphate form inhibits
human and viral polymerases and reverse transcriptases and is incorporated
into nucleic acids. Although the mono- and diphosphates are not the main
active metabolites, they can possess pharmacological activity as well.[1,2]
Monitoring of the intracellular concentration of these metabolites is
pivotal in understanding the pharmacology and toxicology of nucleoside
Received 11 May 2009; accepted 23 October 2009.
Address correspondence to Robert S. Jansen, Department of Pharmacy & Pharmacology, Sloter-
vaart Hospital/The Netherlands Cancer Institute, Louwesweg 61066, EC Amsterdam, The Netherlands.
E-mail: Robert.jansen@slz.nl
14

Synthesis of Nucleoside 5ꢁ-Phosphate Mixtures
15
analogs.[3] In the past decade, liquid chromatography coupled to tandem
mass spectrometry (LC-MS/MS) has become the method of choice to ana-
lyze nucleotide analogs in cells.[4] For optimal performance of LC-MS/MS
assays, an internal standard should be used to correct for interferences
caused by other sample constituents. Stable isotope labeled analytes are the
ideal internal standards because they have identical chemical properties, but
their mass difference can be distinguished in the mass spectrometer.[5]
The availability of stable isotope labeled nucleotide analogs is, how-
ever, limited. Many of their nonphosphorylated, stable isotope labeled
nucleosides, on the other hand, are commercially available. We, therefore,
explored the possibility to phosphorylate small quantities of nucleosides to
a mixture of their mono-, di-, and triphosphate to serve as internal standards
for LC-MS/MS analysis.
Many methods have been developed to synthesize nucleoside triphos-
phates, as reviewed by Burgess and Cook.[6] Most of these methods
first convert the nucleoside into an activated monophosphate, such as a
dichlorophosphate or phosphoramidate. A commonly used phosphytilating
agent is phosphorous oxychloride (POCl3). When POCl3 is used in a
trialkylphosphate solution it results in selective phosphorylation of the 5ꢁ-
hydroxyl group of the nucleoside.[7,8]
Addition of tributylammonium(TBA)-pyrophosphate to the thus
formed nucleoside dichlorophosphate results in the formation of the
nucleoside 5ꢁ-triphosphate.[9] Reaction with water, on the other hand,
deactivates the dichlorophosphate intermediate resulting in the formation
of a nucleoside 5ꢁ-monophosphate.
In attempt to synthesize the 5ꢁ-diphosphate, Mishra and Broom added
TBA-phosphate to the nucleoside dichlorophosphate.[10] To their surprise,
the 5ꢁ-triphosphate was formed instead of the 5ꢁ-diphosphate. Similarly,
Hoffmann et al. added crystalline phosphoric acid in combination with
different organic bases.[11] Depending on the organic base added, they
obtained mixtures of 5ꢁ-mono-, di-, and triphosphates in different ratios.
In this article, we describe the optimization of nucleoside phospho-
rylation with POCl3, and the pitfalls that we encountered. Moreover, the
subsequent conversion of the nucleoside dichlorophosphate into mixtures
of mono-, di-, and triphosphates is presented (Scheme 1). We applied these
reactions to obtain stable isotope labeled nucleotides. These mixtures were
successfully applied in quantitative LC-MS/MS analyses.
MATERIALS AND METHODS
Chemicals
2-Chloro-2ꢁ-deoxyadenosine (cladribine, 2CdA, 1a) was obtained from
Sequoia Research Products (Pangbourne, UK), 2ꢁ, 2ꢁ -difluoro 2ꢁ-

16
R. S. Jansen et al.
SCHEME 1 Reagents and conditions: POCl3 (15–18 equivalents), trimethylphosphate (TMP),
tributylammonium(TBA)-phosphate (2:1, mole/mole, 20 equivalents), N,N -dimethylformamide
(DMF), 1 M triethylammonium-bicarbonate (TEA-HCO3).
deoxycytidine (gemcitabine, dFdC, 1b)·HCl and 2ꢁ, 2ꢁ -difluoro 2ꢁ-
deoxyuridine (dFdU, 1c) were kindly provided by Eli Lilly and company
(Indianapolis, IN, USA), whereas 5-aza-2ꢁ-deoxycytidine (decitabine, aza-dC,
1d) was purchased from Sigma (St. Louis, MO, USA). 13C, 15N2-labeled
∗
∗
dFdC (∗dFdC, 1b) and dFdU (∗dFdU, 1c) were obtained from Toronto
Research Chemicals (North York, ON, Canada). The structural formulas of
the starting nucleosides are presented in Figure 1.
Trimethylphosphate and N,N -dimethylformamide (DMF) were from
Aldrich (St. Louis, MO, USA), whereas 1 M triethylammonium bicarbon-
˚
ate pH 8.5 and 3 A molecular sieves were from Fluka (St. Louis, MO,
USA). 1,8-Bis(dimethylamino)naphthalene (proton sponge), phosphoric
acid crystals, and tributylammonium (TBA)-pyrophosphate (1.5 moles TBA
per mole pyrophosphate, (1.5:1, mole/mole)) were bought from Sigma.
Phosphorous oxychloride (POCl3) and tributylamine (TBA) were obtained
from Fluka and Sigma Aldrich, respectively. All solvents used in the synthesis
were purchased as anhydrous and required no further drying.
TBA dihydrogenphosphate, potassium dihydrogen phosphate, 25% am-
monia, and 2-propanol were all from Merck (Darmstadt, Germany) and
methanol from Biosolve Ltd. (Amsterdam, The Netherlands).
FIGURE 1 Structural formulas of the nucleosides described in the experiments. The asterisks indicate
the position of 13C- and 15N-atoms in the stable isotope labelled compounds ∗1b and ∗1c.

Synthesis of Nucleoside 5ꢁ-Phosphate Mixtures
17
TBA-phosphate (1:1. mole/mole) in DMF (250 µM) was prepared by
mixing 600 µL TBA with 245 mg phosphoric acid crystals and adding DMF
to 10 mL. TBA-phosphate (2:1, mole/mole) was prepared in the same
way, but by adding 1200 µL TBA. Water attracted during preparation was
˚
removed by overnight storage over 3 A molecular sieves.
TBA-pyrophosphate (2.4:1, mole/mole) in DMF (250 µM) was prepared
by dissolving 0.163 mmol TBA-pyrophosphate (1.5:1, mole/mole) in 620 µL
DMF and adding 33 µL TBA.
Monitoring of Reactions
The reactions were monitored using ion-pairing high performance
liquid chromatography with ultraviolet detection (HPLC-UV). In-process
samples of 10 µL were hydrolyzed in 90 µL 1 M triethylammonium-
bicarbonate on ice. The nucleoside dichlorophosphate intermediates 2a–d
were thus converted into nucleoside monophosphates 3a–d.
HPLC-UV experiments were executed on an Agilent 1100 series liquid
chromatograph system (Agilent Technologies, Palo Alto, CA, USA) con-
sisting of a binary pump, an in-line degasser, autosampler, and ultraviolet
(UV) detector. Data were acquired using Chromeleon 6.50 software (Dionex
Corp., Sunnyvale, CA, USA). The mobile phase consisted of 10 mM tetra-
butylammonium dihydrogenphosphate with 70 mM potassium dihydrogen
phosphate in methanol-water (16:84, v/v) and was delivered isocratically to a
Synergi hydro-RP column (150 × 2.0 mm ID, 4 µm particles; Phenomenex,
Torrance, CA, USA) with a flow of 0.25 mL/min. Injections of 1 µL were
carried out with the autosampler thermostated at 4◦C. Absorption was
measured at 268 nm (1b–5b and ∗1b–∗5b), 264 nm (1a–5a), 258 nm (1c–5c
and ∗1c–∗5c), and 243 nm (1d–5d).
Alternatively, reactions could be monitored semiquantitatively using
thin layer chromatography. Using a capillary, 1 µL of the hydrolyzed in-
process samples was spotted onto a HPTLC silica gel F254 plate (Merck,
Darmstadt, Germany). The reaction products were separated using 2-
propanol, 25% ammonia and water (55:35:10, v/v/v). Formation of the
nucleoside dichlorophosphate (2) was observed under UV light (254 nm)
as the appearance of a spot with an Rf lower (∼0.7) than that of the starting
nucleoside (Rf ∼ 0.9).
Optimization of the Phosphorylation of Nucleoside
Dichlorophosphates
A quantity of 0.05 mmole dFdC·HCl (1b) was treated with POCl3 as
described in the section “Final Phosphorylation Procedure.” After 150
minutes, 150 µL aliquots of the reaction mixture were transferred to

18
R. S. Jansen et al.
1.5 mL tubes on ice with 240 µL DMF containing TBA-pyrophosphate
(2.4:1, mole/mole), TBA-phosphate (1:1, mole/mole), TBA-phosphate
(2:1, mole/mole) or no reagent. The reagents were tested in an excess of
5, 10, 15, and 20 equivalents. In-process samples were taken after 1 and 5
minutes, and the reaction was quenched after 10 minutes by the addition of
880 µL 1 M triethylammonium-bicarbonate.
Final Phosphorylation Procedure
The glassware and, if the melting point allowed it, nucleosides, were
dried overnight at 85◦C. In a 10 mL round flask, 0.05 mmole nucleoside
(1 equivalent) was dissolved in 2.5 mL trimethylphosphate together with
0.1 mmol (2 equivalents) proton sponge. dFdC·HCl (1b) and aza-dC (1d)
required gentle heating over a flame to dissolve. POCl3 (0.8 mmol, 16
equivalents) was added dropwise to the stirred solution on ice in about
10 minutes. After addition of all the POCl3, the reaction was continued at
room temperature until maximal nucleoside dichlorophosphate 2a–d was
formed (2.5–6.5 hours). Then, the reaction mixture was again cooled on
ice, and 4 mL cold 250 µM TBA-phosphate (2:1, mole/mole) in DMF was
added under vigorous stirring. After 10 minutes, the reaction mixture was
quenched by pouring it into 20 mL 1 M triethylammonium-bicarbonate
buffer. Finally, the reaction product was lyophilized overnight to remove the
volatile reactants, resulting in a solid white residue.
Purification
Although not required for their use as internal standards, further pu-
rification could be performed after reconstitution of the crude lyophilized
reaction mixture in water. Volumes of 100 µL product (reconstituted in
500 µL water) were injected onto a Biosep DEAE-PEI column (75 × 7.8
mm ID, 7µm particles; Phenomenex) and separated using an ammonium-
bicarbonate gradient (0–900 mM) at 0.5 mL/min. The UV-absorption of
the eluate was monitored and the fractions containing the products were
lyophilized twice to remove all mobile phase components.
Identification
Identity of the synthesized nucleotides was assessed using weak anion
exchange chromatography (3a and 3d–5d) an∗d porous graphitic carbon
∗
chromatography (3b–5b, 3b–∗5b, 3c–5c, and 3c–∗5c) coupled with tan-
dem mass spectrometric detection (HPLC-MS/MS).[12,13] The identity was
confirmed based on retention time and specific mass transitions.

Synthesis of Nucleoside 5ꢁ-Phosphate Mixtures
19
RESULTS
Hydrolyzation Buffer
In our initial experiments with 2CdA, we used 2 equivalents of POCl3,
and 2 mL 0.2 M triethylammonium-bicarbonate buffer (4 equivalents) to hy-
drolyze the nucleoside dichlorophosphate 2a to the nucleoside monophos-
phate 3a. The desired product 2CdA monophosphate (2CdAMP, 3a) was,
however, not observed in the final reaction mixture. We did, on the other
hand, mainly observe a peak that we identified as 2-chloro-adenine, the
base of 2CdA, Identification was performed based on the retention time
on a previously developed HPLV-UV system.[13,14] This base is formed after
cleavage of the acid-labile N-glycosidic bond between the sugar and the base
of 2CdA (1a). Although the final pH was around 8.5 we hypothesized that
HCl, formed in the reaction between POCl3 and water, caused local acidity
resulting in the cleavage of this glycosidic bond. Indeed, 2-chloro-adenine
was no longer found in reaction products that were hydrolyzed with 10
mL 1 M triethylammonium-bicarbonate (200 equivalents). A buffer with a
sufficient capacity, thus, prevented acid hydrolysis of the reaction products.
Drying of Nucleosides
Still, the obtained 2CdAMP (3a) yields (∼4%) were far below the
yields commonly reported for other nucleosides (60–90%).[15] HPLC-UV
analysis of the final reaction product showed that it consisted mainly of
unreacted nucleoside. Any water present in the nucleoside starting material
will react with POCl3 to form HCl and phosphate, thereby inactivating
the phosphytilating agent. We argued that traces of water present in the
starting material reacted with the POCl3 instead of the nucleosides. We,
therefore, 1) dried 2CdA (1a) before use (85◦C, overnight) and 2) added
extra POCl3. Both approaches resulted in a marked increase in nucleoside
dichlorophosphate 2a formation (as shown in Figure 2). Using the dried
2CdA (1a) starting material, 2CdAMP (3a) was already observed after the
addition of 1 POCl3 equivalent. In contrast, similar 2CdAMP (3a) formation
was only observed after the addition of 3 POCl3 equivalents to the non-
dried material. Drying of the nucleoside starting material, thus, improves
the phosphorylation reaction. Considerable yields could, however, still be
obtained with the non-dried material if sufficient amounts of POCl3 were
added.
Excess of Phosphorous Oxychloride
Drying of the nucleosides at elevated temperatures (as described in
the section “Final Phosphorylation Procedure”) was, however, not feasible
for compounds with a low melting point such as dFdU (1c) (51–53◦C)

20
R. S. Jansen et al.
FIGURE 2 Formation of 2CdA dichlorophosphate (2a) and the decrease of 2CdA (1a) using dried (•
and ◦, respectively) and non-dried (ꢀ and ꢁ, respectively) 2CdA (1a) starting material. The arrows
represent the addition of POCl3 equivalents to dried (solid arrow) and non-dried (dashed arrow) 2CdA
(1a).
and nucleosides might still contain water after drying overnight. Moreover,
water can originate from solvents, glassware, and the air. Others have dried
nucleosides in vacuum, used distilled POCl3, and carried out reactions
under nitrogen to perform the reaction under anhydrous conditions.[16,17]
We hypothesized that further increasing the POCl3 excess was another,
easier approach.
Throughout the literature, POCl3 is used in small excesses (1.5–3
equivalents).[10,18–20] Risbood et al., on the other hand, recently used a very
large excess (43 equivalents).[21]
We have increased the excess of POCl3 up to 43 equivalents and found
that increasing the amount of POCl3 resulted in improved nucleoside
dichlorophosphate 2b formation. To decrease the risk of interference from
unreacted POCl3 in the subsequent reaction step, we used 15–18 POCl3
equivalents in further experiments. The reaction with this excess of POCl3
was monitored for all experiments. The results are shown in Figure 3.
The phosphorylation of the same dFdU (1c) starting material was repeated
and was found to be reproducible (49.7 and 57.3% after 4 hours). The
reaction was, on the other hand, dependent on the type of nucleoside
and their origin, as illustrated by the difference between dFdC-dFdU (1b-
1c) and between dFdU-∗dFdU (1c–∗1c) phosphorylation. Still, acceptable
∗
∗
nucleoside dichlorophosphate 2b, 2b, 2c, and 2c yields (46–76%) were
obtained if the reaction time was adjusted for each nucleoside. Monitoring
of the reaction is, however, indispensable to obtain optimal yields.

Synthesis of Nucleoside 5ꢁ-Phosphate Mixtures
21
FIGURE 3 Formation of nucleoside dichlorophosphates of dFdC (2b) (•), 13C, 15N2-dFdC (∗2b) (◦),
dFdU (2c) (ꢀ),13C, 15N2-dFdU (∗2c) (ꢁ) and aza-dC (2d) (ꢂ) with 15–18 equivalents POCl3 added.
Conversion of Nucleoside Dichlorophosphate to Nucleotide
Mixture
Hoffmann et al. showed that the addition of phosphoric acid to a
nucleoside dichlorophosphate (2) in the presence of an organic base,
resulted in the formation of nucleoside mono-, di, and triphosphates (3–5)
in different ratios.[11] Of the organic bases tested, TBA resulted in the most
balanced nucleotide formation. We, therefore, used TBA as organic base in
our experiments.
We performed the reaction with several equivalents of TBA-phosphate
(1:1, mole/mole and 2:1, mole/mole) and TBA-pyrophosphate (2.4:1,
mole/mole). We added extra TBA because Ludwig et al. reported that this
increased the yield in a similar triphosphate synthesis.[9]
The reaction mixtures were assayed after 1, 5, and 10 minutes. The
reaction time of the second reaction step was, however, of minor importance
as the reaction was already complete after 1 minute (data not shown).
The type of reagent and the excess in which it was used were, on
the other hand, pivotal to obtain equimolar nucleotide mixtures. The
effect of the type of reagent is shown in Figure 4. When no reagent was
added the overall yield was the highest, but the product mainly consisted
of nucleoside monophosphate (3b). The amount of di- and triphosphate
(4b and 5b)was higher when TBA-phosphate (1:1, mole/mole) was added
and increased even further with extra TBA (2:1, mole/mole). TBA most
likely increases the activation for the nucleophilic phosphate attack.[11]
With TBA-pyrophosphate (2.4:1, mole/mole), the main product formed

22
R. S. Jansen et al.
FIGURE 4 Effect of the reagent on the formation of dFdC nucleotides 3b–5b (20 equivalents, 10 minute
reaction).
was the triphosphate 5b, but the monophosphate 3b was only slightly less
abundant.
All reagents showed an increased formation of higher phosphates when
applied in a larger excess. The effect of the excess of TBA-phosphate (2:1,
mole/mole) on the formation of the dFdC nucleotides 3b–5b is depicted in
Figure 5.
FIGURE 5 Effect of the excess of TBA-phosphate (2:1, mole/mole) on the formation of dFdC
nucleotides 3b–5b (10 minute reaction).

Synthesis of Nucleoside 5ꢁ-Phosphate Mixtures
23
Based on these results, we concluded that 20 equivalents of TBA-
phosphate (2:1, mole/mole) were optimal to obtain an equimolar mixture
of nucleoside mono- di-, and triphosphates (3–5).
Identification
The synthesized 2CdAMP (3a) showed LC-MS/MS characteristics iden-
tical to 2CdAMP reference material[13] (retention time: 3.75 minutes; mass
transition (positive ionization mode): m/z 366 → 170). Using the same
chromatographic system, the aza-dC nucleotides (3d–5d) were detected
(retention time: aza-dCMP: 3.90 minutes, aza-dCDP: 4.77 minutes, aza-
dCTP: 5.10 minutes; mass transition (positive ionization mode): aza-dCMP:
m/z 309 → 113, aza-dCDP: m/z 389 → 113, aza-dCTP: m/z 469 → 113).
∗
Both the dFdC and dFdU nucleotides (3b–5b, 3b–∗5b, 3c–5c, and
∗3c–∗5c) showed LC-MS/MS characteristics conform to reference com-
[12]
pounds and their structure
(retention time: dFdCMP/∗dFdCMP: 5.73
minutes, dFdCDP/∗dFdCDP: 7.99 minutes, dFdCTP/∗dFdCTP: 9.12 min-
utes, dFdUMP/∗dFdUMP: 6.89 minutes, dFdUDP/∗dFdUDP: 10.22 min-
utes, dFdUTP/∗dFdUTP: 11.67 minutes; mass transitions (positive ioniza-
tion mode): dFdCMP m/z 344 → 246, ∗dFdCMP 347 → 249, dFdCDP m/z
424 → 326, ∗dFdCDP 427 → 329, dFdCTP m/z 504 → 326, ∗dFdCTP 507 →
329, dFdUMP m/z 345 → 247, ∗dFdUMP 348 → 250, dFdUDP m/z 425 →
327, ∗dFdUDP 428 → 330, dFdUTP m/z 505 → 247, ∗dFdUTP 508 → 250).
Application to an LC-MS/MS Assay
The final method was successfully used to synthesize mixtures of ∗dFdC-
and ∗dFdU nucleotides (∗3b–∗5b and ∗3c–∗5c). The reaction mixtures could
be used as internal standards for an LC-MS/MS assay without further
∗
purification. Figure 6 shows a typical chromatogram of dFdC and dFdC
and their mono-, di-, and triphosphate using porous graphitic carbon
chromatography, under previously described conditions.[12] dFdCMP and
its internal standard ∗dFdCMP.
∗
Because the synthesized dFdC internal standards (∗3b–∗5b) elute at
the same time as their unlabeled variants, compounds that interfere with
the signal intensity will affect the signal of the dFdC analytes as much as
∗
that of the dFdC analytes. The ratio of the two signals will, therefore,
remain constant under the variable conditions in biological matrices, such
as number of cells per sample. The impact of the use of an internal standard
is shown in F∗igure 7, with dFdCMP as example. Whereas the signals of
dFdCMP and dFdCMP are lower in samples containing a high number of
cells, the ratio of the two signals remains relatively unaffected. At 15 million
cells, for example, the use of the internal standard improves the accuracy of
the assay result from 68% to 90%.

24
R. S. Jansen et al.
FIGURE 6 HPLC-MS/MS chromatograms of a mixture of dFdC, dFdCMP, dFdCDP, and dFdCTP (1b,
3b–5b) and their stable isotope labeled internal standards (∗1b, ∗3b–∗5b). Conditions were as previously
described,[12] with the following mass transitions: m/z 264 → 112 and 267 → 115 for dFdC and ∗dFdC,
m/z 344 → 246 and 347 → 249 for dFdCMP and ∗dFdCMP, m/z 424 → 326 and 427 → 329 for dFdCDP
and ∗dFdCDP, and m/z 504 → 326 and 507 → 329 for dFdCTP and ∗dFdCTP.
We have experienced several problems in the general phosphorylation
methods described by others. Likewise, Risbood et al. were not able to
synthesize dFdCTP using these methods. They, therefore, first synthesized,
isolated, and purified dFdCMP (3b), and then used a second reaction to
phosphorylate dFdCMP to dFdCTP (5b). We found that an POCl3 excess
of 15–18 equivalents resulted in increased nucleoside phosphorylation, and
that the formed nucleoside dichlorophosphate intermediate could readily
be converted into a well balanced mixture of mono-, di-, and triphosphates.

Synthesis of Nucleoside 5ꢁ-Phosphate Mixtures
25
FIGURE 7 Effect of the number of cells on the MS/MS signal intensity of dFdCMP (3b) (◦, dashed
line), ∗dFdCMP (∗3b) (ꢁ, dashed line), and the dFdCMP/∗dFdCMP ratio (ꢀ, solid line).
CONCLUSION
We identified residual water in nucleoside starting material and cleavage
of the N-glycosylic bond as pitfalls in the reaction of nucleosides with POCl3.
These problems could be avoided by using a large excess of POCl3 and a
hydrolyzation buffer with sufficient capacity. By adding TBA-phosphate (2:1,
mole/mole), the formed nucleoside dichlorophosphate can subsequently
be converted into a well-balanced mixture of the mono-, di-, and triphos-
phate. This mixture can readily be used as internal standard for quantitative
tandem mass spectrometric assays, resulting in improved assay performance.
The described reaction can easily be executed in laboratories with lim-
ited experience in organic chemistry, without extensive drying procedures.
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