CDP-Ethanolamine and CDP-Choline: One-pot synthesis and 31P NMR study

Article abstract of DOI:10.1016/j.tetlet.2014.07.076

Herein we report a one-pot multi-step synthesis of the cofactors CDP-Ethanolamine and CDP-Choline starting from cytidine 5′-monophosphate and using commercially available and/or easily prepared reagents. While studying the ³¹P NMR spectrum of CDP-Ethanolamine, an unexpected characteristic for a pyrophosphate diester was observed as it showed a singlet or two doublets depending upon the pH. Therefore, further NMR studies were undertaken to investigate the pH dependence of the peak splitting pattern and measure the acid dissociation constants of the compounds.

Full text of DOI:10.1016/j.tetlet.2014.07.076

Tetrahedron Letters 55 (2014) 5306–5310
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
CDP-Ethanolamine and CDP-Choline: one-pot synthesis and 31P NMR
study
Salma Ghezal a, Maggie S. Thomasson b, Isabelle Lefebvre-Tournier a, Christian Périgaud a,
Megan A. Macnaughtan b, Béatrice Roy a,
⇑
a UMR 5247 CNRS-UM1-UM2, Institut des Biomolécules Max Mousseron, Nucleosides and Phosphorylated Effectors Team, Université Montpellier 2, cc1705, Place E. Bataillon,
34095 Montpellier, France
b Louisiana State University, Department of Chemistry, Baton Rouge, LA 70803, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we report a one-pot multi-step synthesis of the cofactors CDP-Ethanolamine and CDP-Choline
starting from cytidine 50-monophosphate and using commercially available and/or easily prepared
reagents. While studying the 31P NMR spectrum of CDP-Ethanolamine, an unexpected characteristic
for a pyrophosphate diester was observed as it showed a singlet or two doublets depending upon the
pH. Therefore, further NMR studies were undertaken to investigate the pH dependence of the peak split-
ting pattern and measure the acid dissociation constants of the compounds.
Received 14 June 2014
Revised 16 July 2014
Accepted 21 July 2014
Available online 30 July 2014
Keywords:
CDP-Ethanolamine
CDP-Choline
Ó 2014 Elsevier Ltd. All rights reserved.
Pyrophosphate diesters
Phosphorylation
31P NMR
Nucleolipids are organic hydrid compounds that are made up of
a nucleobase, or a nucleos(t)ide or an oligonucleotide, and a lipo-
philic moiety. Nucleolipids possessing both nucleic acid type rec-
ognition potentiality and lipophilic chains are emerging as
valuable building blocks for generating supramolecular systems.
Indeed, these amphiphiles possess a diversity of functional groups
capable of cooperative noncovalent interactions combined with
specific base recognition.1 As part of our ongoing study on
phospholipid metabolomics in Plasmodium falciparum,2 we have
been interested in the synthesis of CDP-Ethanolamine (CDP-Etn)
and CDP-Choline (CDP-Cho), two nucleolipid building blocks used
as essential standards in LC/MS/MS analytical studies. So far, only
a few synthetic methods have been described in the literature for
these two compounds. The reports for CDP-Etn (the last one
appeared in 1972) indicate a long reaction time together with
low yields.3–8 Microbial and enzymatic productions of CDP-Etn
have also been reported but require access to the biological mate-
rials together with specific technology.9–12
the synthesis of their precursors, CDP-Etn and CDP-Cho.3 Coupling
of cytidine 50-monophosphate (CMP) with either O-phosphoryl-
choline or O-phosphoryl-ethanolamine in anhydrous pyridine or
DMF was performed in the presence of a coupling agent leading
to the desired compounds in very low yields, especially for CDP-
Etn.3,14–16 Then, these compounds have also been obtained either
by reacting CDP with ethyleneimine in water (yielding CDP-Etn
in 31%) followed by methylation with methyl iodide to give CDP-
Cho,4,5 or by condensation of activated CMP such as cytidine 50-
phosphoromorpholidate with phosphate esters.6–8,17 Our initial
attempts to synthesize CDP-Etn according to literature data failed
to give the desired compound even in moderate yield. We also
tried, unsuccessfully, a direct coupling of CMP-morpholidate with
O-phosphorylethanolamine in pyridine, under conditions
described for the synthesis of ADP-ethanolamine.18 Consequently,
we decided to set up a more practical reaction protocol for the syn-
thesis of CDP-Etn and CDP-Cho. As regards the synthesis of nucle-
oside 50-diphosphates, three multi-step procedures are widely
used and involve (i) the displacement of a 50-O-tosyl group with
tris(tetra-n-butylammonium) pyrophosphate;19 (ii) the use of
cycloSal-nucleotides as intermediates;20 (iii) the nucleophilic
attack of tri-n-butylammonium phosphate on activated nucleoside
50-phosphorimidazolates.21 Taking into account this latter
approach, we report an efficient one-pot synthesis of the target
derivatives starting from CMP (Scheme 1). In addition, the physico-
chemical analysis of these compounds revealed similar apparent
Eugene P. Kennedy has made an essential contribution to the
study of phospholipid metabolism.13 Thus, the eponymous path-
way depicts de novo pathways of phosphatidylcholine (PC) and
phosphatidylethanolamine (PE). He was also the first to report
⇑
Corresponding author. Tel.: +33 4 67 14 38 79; fax: +33 4 67 04 20 29.
E-mail address: beatrice.roy@univ-montp2.fr (B. Roy).
http://dx.doi.org/10.1016/j.tetlet.2014.07.076
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

S. Ghezal et al. / Tetrahedron Letters 55 (2014) 5306–5310
5307
NH2
NH2
N
N
O
O
P
N
O
a, b
N
O
N
P
O
HO
O
N
O
O
O-
OH
O
O
OH OH
O
1
c, g
c, d
NH2
NH2
N
N
O
P
O
O
P
O
P
BocHN
(H3C)3N
N
O
N
O
O
O
P
O
O
O
O
O
O
O-
O-
O-
O-Na+
O
e
O
HO
OH
5
O
2
conversion rate: 80%
isolated yield: 74%
NH2
N
NH2
N
N
O
O
P
O
P
O
+H3N
BocHN
f
N
O
O
P
O
O
P
O
O
O
O
O
O
O-Na+
O-
O-
O-
HO
OH
OH
OH
3
4
conversion rate: 90%
isolated yield: 87%
Scheme 1. Synthesis of CDP-Etn and CDP-Cho. Reagents and conditions: (a) tributylamine, DMF, 50 °C, 10 min; (b) CDI, rt, 30 min; (c) MeOH, 30 min; (d) N-Boc-2-
aminoethylphosphate, ZnCl2, DMF, rt, 24 h; (e) TEAB 1 M pH 9, rt, 3 h; (f) TFA/DCM (1/1, v/v), rt, 1 h then purification by chromatography; (g) choline phosphate salt, ZnCl2,
DMF, rt, 24 h then purification by chromatography.
acid dissociation constants (pKa), but different 31P NMR splitting
patterns that depend on pH.
tion by semi-preparative HPLC was used to isolate CDP-Etn. Alter-
natively, CDP-Etn and CDP-Cho could be purified by anion
exchange chromatography using a gradient of triethylammonium
hydrogen carbonate (TEAB) followed by another reversed-phase
chromatography. Finally, exchange of the triethylammonium
counter-ions by sodium led to compounds 4 and 5 as sodium salts
in 87% and 74% yield, respectively. Thus, these derivatives were
obtained in better yields than those reported previously for their
synthesis. The final products were fully characterized by 1H, 13C,
and 31P NMR spectroscopic analyses and high resolution mass
spectrometry.26
In our NMR experiments, the sample concentration was kept
low enough to avoid intermolecular base stacking and to enable
the detection of clear and sharp NMR signals.27 We noticed that
protons belonging to the nucleobase of CDP-Etn, when purified
by HPLC under acidic conditions, display downfield shifts com-
Our synthetic strategy began with the activation of CMP in the
presence of 1,10-carbonyldiimidazole (CDI). Interestingly, we found
that heating commercial cytidylic acid, the acid form of CMP, with
tributylamine in DMF at 50 °C for 10 min before addition of CDI,
allowed the rapid formation of the corresponding nucleoside
20,30-carbonate 50-phosphorimidazolate 1 (Scheme 1). Without this
preheating step, no activation occurred. After 30 min of activation,
the intermediate
1
was characterized by 31P NMR (d
31P = ꢀ10.2 ppm) and mass spectrometry, then the excess of reac-
tant was quenched with methanol. The progress of the reaction
was monitored by HPLC coupled with a Photo Diode Array detector
since it is a lower material-consuming technique than 31P NMR.22
To circumvent the dual reactivity on both edges of O-phosphory-
lethanolamine as well as to increase its solubility in DMF, we pro-
tected the primary amino group of O-phosphorylethanolamine
with the acid-labile Boc group.23 Then, addition of N-Boc-2-amino-
ethylphosphate and zinc chloride as the catalyst24 to the reaction
mixture led to a complete conversion of phosphorimidazolate 1
into intermediate 2 after 24 h. In situ deprotection of the 20,30-car-
bonate group under basic conditions was quantitative after 3 h.
The Boc protecting group of compound 3 was then removed with
a TFA/DCM (1/1, v/v) mixture to give CDP-Etn 4. We then applied
the synthetic sequence for CDP-Cho. Treatment of the 20,30-carbon-
ate 50-phosphorimidazolate derivative 1 with choline phosphate25
gave compound 5. The conversion rates (determined by HPLC) for
CDP-Etn 4 and CDP-Cho 5 were 90% and 80%, respectively. Purifica-
pared to those of the sodium salt of CDP-Etn (
Dd = 0.15 ppm for
H5, d = 0.23 ppm for H6). This shift has been attributed to the
D
protonation of cytosine at low pH. Literature data report two spin
AX-systems for CDP-Etn (dP = ꢀ7.95 ppm; dPb = ꢀ8.10 ppm, 2
J
=
a
ab
20.9 Hz) and CDP-Cho (dP = ꢀ8.13 ppm; dPb = ꢀ8.93 ppm,
a
2J b = 21.1 Hz) at pH 7.25.28 However, Robitaille et al. describe a
a
singlet for CDP-Etn at neutral pH.29 In our case, the 31P NMR spec-
trum of CDP-Etn, recorded in D2O, showed only a singlet at
ꢀ11.16 ppm (Fig. 1a). Upon addition of NaOH 1 N (10% of the final
volume), this singlet was instantaneously split into two doublets
(dP = ꢀ10.56 ppm; dPb = ꢀ11.27 ppm, 2J b = 20.9 Hz) as shown in
a
a
Figure 1b. Thus, compared to the 31P NMR of CDP-Cho, where only

5308
S. Ghezal et al. / Tetrahedron Letters 55 (2014) 5306–5310
Figure 1. 31P NMR (81 MHz) spectra of (a) CDP-Etn in D2O. (b) CDP-Etn upon addition of NaOH.
a broadening of the two doublets was observed at low pH, the 31P
NMR pattern of CDP-Etn was highly dependent on pH.
ters fall into two categories: those characterized by a singlet in the
range of ꢀ10.39 to ꢀ14.50 ppm and those characterized by two
These results led us to carry out a comparative study of 31P NMR
shifts of compounds 2, 3, 4, and 5 as well as 20,30-carbonate CDP-
Ethanolamine (intermediate A). As references, a series of naturally
occurring ubiquitous pyrophosphate diesters were also analyzed
doublets with a 31P–31P scalar coupling value (2J b) in the order
a
of 20 Hz. The first category encompasses symmetrical pyrophos-
phate diesters such as dinucleotides27 but also some unsymmetri-
cal pyrophosphates such as co-factors NADPH and NADH,28–30
altritol nicotinamide adenine dinucleotide lithium salt,31 dinucleo-
tide Gp2C,27 or P2-(7-diethylaminocoumarin-4-yl)methyl cytidine
50-diphosphate.32 It should be pointed out that a singlet around
ꢀ10 ppm in 31P NMR, is also characteristic of aromatic phospho-
ramidates.33 For unsymmetrical pyrophosphate diesters, the sin-
glet indicates that the 31P nuclei are in similar chemical
environments due to conformational symmetry about the pyro-
phosphate diesters and the absence of nearby substituents that
strongly influence chemical shift, such as aromatic rings or alkyne
groups. The second category includes compounds having non
(Table 1). We found that the phosphorus atoms P and Pb are mag-
a
netically equivalent in the fully protonated diphosphate moiety of
CDP-Etn, but not in CDP-Cho. Moreover, the magnetic equivalence
of CDP-Etn is broken upon deprotonation of the primary amino
group at high pH.
For phosphorus-containing molecules, the 31P NMR chemical
shift is sensitive to the local chemical environment. With pyro-
phosphate diesters, the 31P nuclei are affected by protonation
states of the phosphate groups and by the type and conformational
position of substituent groups. In this regard, pyrophosphate dies-

S. Ghezal et al. / Tetrahedron Letters 55 (2014) 5306–5310
5309
Table 1
methylammonium chloride solution containing 10% D2O to main-
tain a constant ionic strength during the titration and to provide
a lock signal for NMR analysis, respectively. Tetramethylammo-
nium hydroxide was used for titration experiments, thus avoiding
polarizable cation interactions with the pyrophosphates. Figure 2
shows the results of the titration for CDP-Etn and CDP-Cho. The
apparent pKa values determined from fitting the titration curves
gave values of 4.35 0.02 and 4.55 0.02 for CDP-Etn and CDP-
Cho, respectively. These values are similar to those reported for
cytosine (pKa = 4.56) and cytidine (pKa = 4.16).38 The 31P NMR
spectra of each compound did not show large changes in the chem-
ical shift or the peak splitting pattern over the pH range tested;
CDP-Etn displayed a second-order splitting pattern and CDP-Cho
displayed an AX-type doublet of doublets (see Supporting informa-
tion). The phosphorus nuclei of CDP-Etn are nearly magnetically
equivalent when the pH is less than 9.5, which suggests that the
molecule adopts a conformation in solution where both substitu-
ents, particularly deprotonation of the cytosine N3, do not greatly
31P NMR chemical shifts and 31P–31P scalar coupling value of pyrophosphate diesters
Compound
dP
dP
2J
ab
ppm
ppm
Hz
2a
R1: Cytidine 20,30-carbonate
R2: N(Boc)-Ethanolamine
R1: Cytidine
ꢀ10.81
ꢀ10.85
ꢀ11.11
ꢀ11.35
ꢀ11.14
ꢀ11.22
ꢀ11.20
ꢀ11.20
ꢀ11.31
ꢀ11.25
ꢀ11.31
21.0
3a
21.1
n.d.
R2: N(Boc)-Ethanolamine
R1: Cytidine
4c
R2: Ethanolamine
5c
R1: Cytidine
ꢀ12.14
ꢀ11.75
21.4
18.5
n.d.
R2: Choline
Ab
R1: Cytidine 20,30-carbonate
R2: Ethanolamine
influence the magnetic environment of P and Pb. The positive
a
NADHb
NAD+b
NADPHb
NADP+b
R1: Adenosine
charge of the amino group likely plays a role in this conformation
because upon deprotonation (pH >9.5), the symmetry is broken.
CDP-Cho is different from CDP-Etn: its phosphorus nuclei are not
R2: Nicotinamide reduced form
R1: Adenosine
ꢀ11.52
ꢀ11.61
20.0
n.d.
R2: Nicotinamide oxidized form
R1: Adenosine 20-phosphate
R2: Nicotinamide reduced form
R1: Adenosine 20-phosphate
R2: Nicotinamide oxidized form
20.5
a
b
c
81 MHz, D2O.
121 MHz, D2O.
162 MHz, D2O.
identical phosphorus centers. Thus, if the 31P chemical shift differ-
ence in Hz (
) is sufficiently greater than the 2J coupling
constant ( /J > 5), the spectrum will show a characteristic first-
D
m
a
b
D
m
order splitting of a doublet of doublets (two spin AX-systems) with
a coupling constant of 17–22 Hz. The oxidized co-factors NADP+ or
NAD+ and their analogues fall into this category.28–31 Other exam-
ples include NDP-sugars (UDP-Glc, UDP-Gal, ADP-Glc, GDP-Man)34
as well as other miscellaneous unsymmetrical pyrophosphates.35
The difference between the reduced and oxidized cofactors is the
conjugation state of the nicotinamide ring. In the oxidized form,
the aromatic conjugation is intact and the ring current has a large
effect on the chemical shift of nearby nuclei. The relative orienta-
tion of the ring to the phosphorus nuclei will affect the direction
and amplitude of the chemical shift. The reduced co-factor nicotin-
amide ring is not conjugated. In the absence of this affect, the 31P
nuclei are magnetically equivalent. This equivalence can be broken
by other means such as interactions with salts. The reduced co-fac-
tor NADPH exhibits a two spin AX-system when the NMR samples
are registered in a sodium tetraborate buffer,30 probably due to the
formation of nucleotide-borate adducts on the nicotinamide unit.36
Another example of how small structural modifications can induce
NMR differences is the comparison of altritol- and hexitol-nicotin-
amide adenine dinucleotide lithium salts (aNAD+ and hNAD+).
aNAD+ shows
a
singlet (ꢀ10.68 ppm) in 31P NMR whereas
hNAD+ exhibits two doublets (P = ꢀ10.7 ppm, Pb = ꢀ10.3 ppm,
a
2J b = 20.5 Hz) under the same conditions.31 These compounds dif-
a
fer only by a hydroxyl group, which likely influences the phospho-
rus chemical shifts by forming a salt bridge with the Pb-phosphate
or by influencing the orientation of the nicotinamide ring.
Based on the literature and our observations, the 31P nuclei of
CDP-Etn and CDP-Cho experience different chemical environments
under the same conditions. To determine the effect of pH on the 31
P
Figure 2. Titration curves for (a) CDP-Etn and (b) CDP-Cho in 0.1 M tetramethyl-
ammonium chloride solutions titrated with tetramethylammonium hydroxide. The
pH readings are shown as black circles and the line is the best fit to equations with
(a) three pKa values and (b) two pKa values. The fit statistics of chi-squared (Chisq)
and the correlation coefficient (R) are shown on each plot.
chemical shift and the role of the aliphatic amine and/or cytosine
N3 deprotonation, if any, samples of CDP-Etn and CDP-Cho were
titrated and analyzed with 31P NMR from pH 3–9 to observe the
31P splitting pattern.37 The samples were prepared in 0.1 M tetra-

5310
S. Ghezal et al. / Tetrahedron Letters 55 (2014) 5306–5310
and allowed to cool to room temperature. Then, CDI (250 mg, 1.55 mmol) was
added and the suspension turned to a clear solution after a few minutes. The
anhydrous reaction mixture was stirred for 30 min at room temperature and
magnetically equivalent regardless of pH. Even though the choline
is positively charged like the amino group of CDP-Etn, the unsym-
metrical conformation is favored, likely due to the bulky methyl
groups.
treated with anhydrous methanol (100
excess. After 30 min, anhydrous zinc chloride (59 mg, 0.43 mmol) was added
followed by solution of N-(t-butoxycarbonyl)-2-aminoethylphosphate
lL, 2.48 mmol) to hydrolyze the CDI in
a
To conclude, we have developed a straightforward and efficient
method for the synthesis of CDP conjugates. HPLC coupled with UV
detection was found to be a valuable tool for the follow-up of these
reactions combined as a one-pot procedure. 31P NMR analysis of
CDP-Etn, CDP-Cho, and related compounds revealed a singularity
for CDP-Etn as it exhibits a singlet in D2O. Moreover, to our knowl-
edge, CDP-Etn is the only example of a pyrophosphate diester
showing pH-mediated coalescence of 31P NMR signals. We showed
that this feature is related to the ionization state of the primary
amino group of CDP-Etn.
sodium salt (0.77 M solution in anhydrous DMF, 2 mL, 1.55 mmol). The
reaction mixture was stirred for 24 h at room temperature and then an
aqueous triethylammonium bicarbonate buffer solution (1 M, pH 9, 10 mL)
was added and the resulting mixture was stirred for 2 h at room temperature.
After evaporation of the solvents in vacuo and freeze-drying, the residue was
treated with a solution of TFA/DCM (1/1, v/v, 5 mL). After 1 h stirring at room
temperature, the volatiles were removed under reduced pressure and the
resulting residue was purified by semi-preparative HPLC. The pure fractions
were collected and freeze-dried. HPLC: tR = 9.1 min (kmax 280 nm); 1H NMR
(400 MHz, D2O) d 8.19 (d, 1H, H6, J6-5 = 8.0 Hz), 6.30 (d, 1H, H5, J5-6 = 8.0 Hz,),
0
0
0
0
0
0
0
5.93 (d, 1H, H1 , J1 -2 = 3.1 Hz), 4.38–4.31 (m, 4H, H2 H3 H4 H5 a), 4.24–4.19 (m,
3H, H5 b CH2O), 3.29 (t, 2H, CH2N, J = 4.8 Hz); 13C NMR (101 MHz, D2O) d 159.2
0
(s, C4), 148.5 (s, C2), 144.2 (s, C6), 95.3 (s, C5), 89.7 (s, C10), 83.3 (s, C40), 74.4 (s,
C20), 69.1 (s, C30), 64.5 (s, C50), 62.4 (s, CH2O), 40.0 (s, CH2N); 31P NMR
(162 MHz, D2O) d ꢀ11.11 (s). HRMS (ESI) m/z: [M+H]+ Calcd for C11H21N4O11P2
447.0682. Found 447.0690.
Acknowledgment
Alternative purification procedure: The mixture dissolved in water was
purified by Sephadex DEAE A-25 resin ion exchange column chromatography
Funding for this research was provided by Infectiopôle Sud
(PhD grant to S.G.).
with
a linear gradient 0–0.5 M of ammonium bicarbonate, followed by
chromatography on RP-18. The triethylammonium counter ions were
exchanged to sodium by passing the nucleotide solution through a Dowex
50WX8 column. The product, in its sodium trifluoroacetate form, was isolated
as a white solid after freeze-drying (164 mg, 87% yield). 1H NMR (400 MHz,
D2O) d 7.96 (d, 1H, H6, J6-5 = 7.6 Hz), 6.15 (d, 1H, H5, J5-6 = 7.6 Hz), 6.02 (d, 1H,
Supplementary data
Supplementary data (additional experimental procedures; HPLC
analysis, NMR spectra and MS for all compounds herein) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.tetlet.2014.07.076.
0
0
0
0
0
0
0
0
H
1 , J1 -2 = 4.1 Hz), 4.40–4.28 (m, 4H, H2 H3 H4 H5 a), 4.28–4.20 (m, 3H, H5 b
CH2O), 3.33 (t, 2H, CH2N, J = 5 Hz); 13C NMR d (101 MHz, D2O) d 166.2 (s, C4),
163.0 (q, F3C-CO2ꢀ, JC-F = 35.4 Hz), 157.8 (s, C2), 141.4 (s, C6), 116.4 (q, F3C-
CO2ꢀ, JC-F = 291.6 Hz), 96.6 (s, C5), 89.4 (s, C10), 82.6 (d, C40, JC-P = 7.4 Hz), 74.1
(s, C20), 69.3 (s, C30), 64.8 (s, C50), 62.3 (s, CH2O), 39.9 (s, CH2N); 31P NMR
(121 MHz, D2O)
d
ꢀ11.07 (s); HRMS (ESI) m/z: [MꢀH]ꢀ Calcd for
References and notes
C11H18N4O11NaP2 467.0345; Found 467.0347.
1-(b-
D
-Ribofuranosyl)cytosine-50-diphosphate choline (5). A stirred suspension
of the free acid of CMP (100 mg, 0.31 mmol) in DMF (2 mL) and tributylamine
(150 L, 0.62 mmol) was heated for 10 min at 50 °C and allowed to cool at
room temperature. Then, CDI (250 mg, 1.55 mmol) was added and the
suspension turned to a clear solution after a few minutes. The anhydrous
reaction mixture was stirred for 30 min at room temperature and treated with
1. (a) Rosemeyer, H. Chem. Biodivers. 2005, 2, 977–1062; (b) Taib, N.; Aimé, A.;
Houmadi, S.; Castano, S.; Barthelemy, P.; Laguerre, M.; Bestel, I. Langmuir 2012,
28, 7452–7460; (c) Allain, V.; Bourgaux, C.; Couvreur, P. Nucleic Acids Res. 2012,
40, 1891–1903; (d) Aimé, A.; Betzsinna, N.; Patwa, A.; Pokolenko, A.; Bestel, I.;
Barthelemy, P. Bioconjugate Chem. 2013, 24, 1345–1355; (e) Simeone, L.;
Mangiapia, G.; Irace, C.; Di Pascale, A.; Colonna, A.; Ortona, O.; De Napoli, L.;
Montesarchio, D.; Paduano, L. Mol. BioSyst. 2011, 7, 3075–3086.
2. Vo Duy, S.; Besteiro, S.; Berry, L.; Perigaud, C.; Bressolle, F.; Vial, H. J.; Lefebvre-
Tournier, I. Anal. Chim. Acta 2012, 739, 47–55.
l
anhydrous methanol (100 lL, 2.48 mmol) to hydrolyze the CDI excess. After
30 min, anhydrous zinc chloride (59 mg, 0.43 mmol) was added followed by a
solution of choline phosphate (0.77 M solution in anhydrous DMF, 2 mL,
1.55 mmol). The reaction mixture was stirred for 24 h at room temperature.
After evaporation of the solvents in vacuo, CDP-Cho 5 was purified by anion-
exchange chromatography. The residue dissolved in water was purified by
Sephadex DEAE A-25 resin ion exchange column chromatography with a linear
gradient 0–0.5 M of ammonium bicarbonate, followed by chromatography on
RP-18. The triethylammonium counter ions were exchanged to sodium by
passing the nucleotide solution through a Dowex 50WX8 column. 1-(b-D-
ribofuranosyl)cytosine-50-diphosphate choline (sodium salt) was obtained as a
white solid after freeze-drying (115 mg, 74% yield). HPLC: tR = 8.8 min (kmax
276 nm); 1H NMR (400 MHz, D2O) d 7.86 (d, 1H, H6, J6-5 = 7.6 Hz), 6.04 (d, 1H,
3. Kennedy, E. P. J. Biol. Chem. 1956, 222, 185–191.
4. Sanno, Y.; Tanaka, K. Chem. Pharm. Bull. 1960, 8, 753–754.
5. Sanno, Y.; Tanaka, K. Chem. Pharm. Bull. 1962, 10, 231–237.
6. Letters, R.; Michelson, A. M. Bull. Soc. Chim. Biol. 1963, 45, 1353–1361.
7. Tamura, K.; Morozumi, M.; Yoshino, H.; Noda, Y.; Suzuki, M. DE2038262A,
1972.
8. Bergmeyer, H. U.; Haid, E.; Nelboeck-Hochstetter, M.; Weimann, G.
DE2059429A, 1972.
9. Takeda, I.; Watanabe, S.; Shirota, S. JP50009872B, 1975.
10. Tochikura, T.; Kimura, A.; Kawai, H. JP47019089B4, 1972.
11. Schneider, W. C.; Fiscus, W. G.; Lawler, J. A. B. Anal. Biochem. 1966, 14, 121–134.
12. Chojnacki, T.; Metcalfe, R. F. Nature 1966, 210, 947–948.
13. (a) Borkenhagen, L. F.; Kennedy, E. P. J. Biol. Chem. 1957, 227, 951–962; (b)
Gibellini, F.; Smith, T. K. IUBMB Life 2010, 62, 414–428.
14. Kikugawa, K.; Ichino, M. Chem. Pharm. Bull. 1971, 19, 1011–1016.
15. Kikugawa, K.; Ichino, M.; Kawashima, T. Chem. Pharm. Bull. 1971, 19, 2466–
2471.
0
0
0
H5, J5-6 = 7.6 Hz), 5.92 (d, 1H, H1 , J1 -2 = 4.0 Hz), 4.31 (sl, 2H, CH2-O), 4.28–4.08
(m, 5H, H2 H3 H4 H5 a H5 b), 3.60 (t, 2H, CH2-N, J = 4.2 Hz), 3.14 (s, 9H, CH3); 13
C
0
0
0
0
0
NMR (101 MHz, D2O) d 166.2 (s, C4), 157.7 (s, C2), 141.4 (s, C6), 96.6 (s, C5),
89.3 (s, C10), 82.6 (d, C40, JC-P = 8.9 Hz), 74.1 (s, C20), 69.3 (s, C30), 65.9 (s, CH2N),
64.7 (d, C50, JC-P = 5.1 Hz), 59.9 (s, CH2O), 53.9 (s, CH3); 31P NMR (162 MHz,
D2O) d ꢀ11.35 (d, 2J b = 21.4 Hz), ꢀ12.14 (d, 2J b = 21.4 Hz); HRMS (ESI) m/z:
a
a
[M+H]+ Calcd for C14H26N4O11NaP2 511.0971. Found 511.0973.
27. Stern, N.; Major, D. T.; Gottlieb, H. E.; Weizman, D.; Fischer, B. Org. Biomol.
Chem. 2010, 8, 4637–4652.
16. Kurihara, T.; Yamamoto, I.; Hata, T. Bull. Chem. Soc. Japan 1975, 48, 1077–1078.
17. Kumar, R. Y.; Chauhan, Y.; Dhake, V.; Tambe, V.; Wagh, A. WO2013128393A1,
2013.
28. Kuesel, A. C.; Graschew, G.; Hull, W. E.; Lorenz, W.; Thielmann, H. W. NMR
Biomed. 1990, 3, 78–89.
29. Robitaille, P.-M. L.; Robitaille, P.; Gordon Brown, G., Jr.; Brown, G. G. J. Magn.
Reson. 1991, 92, 73–84.
30. Ragg, E.; Scaglioni, L.; Mondelli, R.; Carelli, I.; Casini, A.; Torotorella, S. Biochim.
Biophys. Acta 1991, 1076, 49–60.
31. Goulioukina, N.; Wehbe, J.; Marchand, D.; Busson, R.; Lescrinier, E.; Heindl, D.;
Herdewijn, P. Helv. Chim. Acta 2007, 90, 1266–1278.
32. Schönleber, R. O.; Bendig, J.; Hagen, V.; Giese, B. Bioorg. Med. Chem. 2002, 10,
97–101.
18. Zou, Y.; Yin, J. J. Am. Chem. Soc. 2009, 131, 7548–7549.
19. Davisson, V. J.; Davis, D. R.; Dixit, V. M.; Poulter, C. D. J. Org. Chem. 1987, 52,
1794–1801.
20. Warnecke, S.; Meier, C. J. Org. Chem. 2009, 74, 3024–3030.
21. Calleri, E.; Ceruti, S.; Cristalli, G.; Martini, C.; Temporini, C.; Parravicini, C.;
Volpini, R.; Daniele, S.; Caccialanza, G.; Lecca, D.; Lambertucci, C.; Trincavelli,
M. L.; Marucci, G.; Wainer, I. W.; Ranghino, G.; Fantucci, P.; Abbracchio, M. P.;
Massolini, G. J. Med. Chem. 2010, 53, 3489–3501.
33. Lebedev, A. V. Nucl. Acids Res. 1984, 12, 5547–5566.
34. Tanaka, H.; Yoshimura, Y.; Jørgensen, M. R.; Cuesta-Seijo, J. A.; Hindsgaul, O.
Angew. Chem. 2012, 124, 11699–11702.
35. Lin, T.-C.; Fang, J.-M. Tetrahedron Lett. 2011, 52, 2232–2234.
36. Kim, D. H.; Marbois, B. N.; Faull, K. F.; Eckhert, C. D. J. Mass Spectrom. 2003, 38,
632–640.
37. Jancan, I.; Macnaughtan, M. A. Anal. Chim. Acta 2012, 749, 63–69.
38. Vojtylová, T.; Dospivová, D.; Trisková, O.; Pilarová, I.; Lubal, P.; Farková, M.;
Trnková, L.; Táborsky, P. Chem. Papers 2009, 63, 731–737.
22. Roy, B.; Lefebvre, I.; Puy, J.-Y.; Périgaud, C. Tetrahedron Lett. 2011, 52, 1250–
1252.
23. Koseki, K.; Yamashita, S.; Takahashi, S.; Koyama, T. Bioorg. Med. Chem. Lett.
2007, 17, 946–950.
24. Kore, A. R.; Parmar, G. Synth. Commun. 2006, 36, 3393–3399.
25. Geilen, C. C.; Samson, A.; Wieder, T.; Wild, H.; Reutter, W. J. Label. Compd.
Radiopharm. 1992, 31, 1071–1076.
26. 1-(b-
suspension of the CMP (free acid, 100 mg, 0.31 mmol) in anhydrous DMF
(2 mL) and tributylamine (150 L, 0.62 mmol) was heated for 10 min at 50 °C
D A stirred
-Ribofuranosyl)cytosine-50-diphosphate ethanolamine (4).
l