Synthesis of 3′(2′)-O-lysophosphatidylnucleosides - A further application of a chemoenzymatic strategy

Article abstract of DOI:10.1002/1099-0690(200211)2002:21<3622::AID-EJOC3622>3.0.CO;2-4

Mono[(2R)-2,3-dihydropropyl] esters of the four 3′-nucleotides of DNA, prepared from protected nucleoside phosphoramidites and [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methanol, were regioselectively acylated at the C-1 hydroxyl of the glycerol moiety by a lipase-catalyzed transacylation with activated palmitic acid ester in organic solvent, giving the relevant 3′-O-lysophosphatidyl-2′-deoxynucleosides. The synthesis was also adapted for the preparation of 3′-O-lysophosphatidyl derivatives of 5′-deoxy-5-fluorouridine and 5′deoxy-5′-(methylthio)adenosine, with the 2′-O-isomer of the latter compound also being prepared. The enhanced ability of lysophosphatidyl compounds to interact with lipid monolayers was also tested in comparison with that of the relevant free nucleosides. Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002.

Full text of DOI:10.1002/1099-0690(200211)2002:21<3622::AID-EJOC3622>3.0.CO;2-4

FULL PAPER
Synthesis of 3Ј(2Ј)-O-Lysophosphatidylnucleosides ؊ a Further Application of
a Chemoenzymatic Strategy
Rosa Chillemi,[a] Danilo Aleo,[b] Giuseppe Granata,[a] and Sebastiano Sciuto*[a]
Keywords: Antitumor agents / Enzyme catalysis / Lipoconjugates / Nucleosides / Transesterification
Mono[(2R)-2,3-dihydropropyl] esters of the four 3Ј-nucleo-
tides of DNA, prepared from protected nucleoside phos-
phosphatidyl derivatives of 5Ј-deoxy-5-fluorouridine and 5Ј-
deoxy-5Ј-(methylthio)adenosine, with the 2Ј-O-isomer of the
phoramidites and [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]me- latter compound also being prepared. The enhanced ability
thanol, were regioselectively acylated at the C-1 hydroxyl of
the glycerol moiety by a lipase-catalyzed transacylation with
activated palmitic acid ester in organic solvent, giving the
relevant 3Ј-O-lysophosphatidyl-2Ј-deoxynucleosides. The
of lysophosphatidyl compounds to interact with lipid mono-
layers was also tested in comparison with that of the relevant
free nucleosides.
( Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany,
synthesis was also adapted for the preparation of 3Ј-O-lyso- 2002)
Introduction
useful to prepare the lysophosphatidyl conjugates of these
compounds. The covalent attachment of lipophilic moieties
to such nucleoside analogues is particularly useful, since
low-density lipoproteins (LDL) have been claimed as at-
tractive carriers for selective and efficient intracellular deliv-
ery of antineoplastic drugs to tumor cells. Indeed, this
therapeutic approach has already been explored for some
conventional lipophilic antitumor drugs,[2,3] and the avail-
ability of lysophosphatidyl conjugates of antitumor nucle-
oside analogues could then be of great utility for further
pharmacological studies in this field.
However, not all nucleoside compounds known to pos-
sess antiproliferative properties bear a 5Ј-hydroxyl group at
their sugar (or sugar-like) moiety [e.g., 5Ј-deoxy-5-fluorour-
idine (DFUR) or 5Ј-deoxy-5Ј-(methylthio)adenosine
(MTA)], thus denying the possibility of obtaining the relev-
ant 5Ј-O-lysophosphatidyl conjugates. In these cases, the
covalent attachment of the lysophosphatidyl moiety at the
3Ј-(or 2Ј-)hydroxy group of the sugar residue may be an
alternative and reasonably simple way.
The constant quest to produce biologically active com-
pounds with ever better capabilities to interact with cell
membranes has recently encouraged us to develop a general
synthetic preparation of 5Ј-O-lysophosphatidyl conjugates
of nucleosides, some of these being nucleoside analogues
of pharmacological interest.[1] The basic idea behind this
approach is that lipophilic moieties conjugated to nucleos-
ides may significantly enhance the cellular uptake of these
polar molecules, as a consequence of the affinity of the lipo-
philic moieties for membrane structures.
In a two-step chemoenzymatic strategy, mono[(2R)-2,3-
dihydroxypropyl] esters of 5Ј-nucleotides (5Ј-GPNs) were
first prepared from a phosphoramidite of [(4S)-2,2-di-
methyl-1,3-dioxolan-4-yl]methanol and the appropriately
protected nucleosides. Selective acylation at 1-OH of the
glycerol moiety of the 5Ј-GPNs was then achieved by a lip-
ase-catalyzed (Lipozyme) transacylation with activated
fatty acid esters in organic solvents.
We therefore also now wish to report a procedure for the
preparation of 3Ј(2Ј)-O-lysophosphatidyl conjugates of 2Ј-
deoxyribo- or ribonucleosides, by which the synthesis of ly-
sophosphatidyl derivatives of DFUR and MTA has also
been achieved.
By this procedure we succeeded in synthesizing 5Ј-O-lyso-
phosphatidyl derivatives of some deoxyribo- and ribonu-
cleosides that normally occur in nucleic acids, along with
lysophosphatidyl conjugates of two nucleoside analogues of
wide use in current antiviral therapy: AZT and acyclovir.
As other nucleosides showing antineoplastic activity are
of great interest in medicinal chemistry, we also thought it
Results and Discussion
[a]
`
Dipartimento di Scienze Chimiche, Universita di Catania,
viale A. Doria 6, 95125, Catania, Italy
Fax: (internat.) ϩ 39-095/580-138
E-mail: ssciuto@dipchi.unict.it
Fidia Oftal S.p.A.
Corso Italia 141, 95127, Catania, Italy
Fax: (internat.) ϩ 39-095/722-3856
E-mail: daniloaleo@tiscalinet.it
To achieve the preparation of 3Ј-O-lysophosphatidylnu-
cleosides we planned to follow the same two-step chemoen-
zymatic strategy as previously used for the preparation of
5Ј-O-lysophosphatidylnucleosides.[1] Therefore, mono[(2R)-
2,3-dihydroxypropyl] esters of 3Ј-nucleotides (3Ј-GPNs) had
[b]
3622
 2002 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim 1434Ϫ193X/02/1121Ϫ3622 $ 20.00ϩ.50/0 Eur. J. Org. Chem. 2002, 3622Ϫ3631

Synthesis of 3Ј(2Ј)-O-Lysophosphatidylnucleosides
FULL PAPER
to be prepared first, possibly with modification of the
chemical step. These would subsequently be acylated by
Lipozyme in organic solvent. All this was of course based
on the assumption that 3Ј-GPNs could also be recognized
by Lipozyme as substrates.
Synthesis of 3Ј-O-Lysophosphatidyl-2Ј-deoxynucleosides
The synthetic plan was first applied to the preparation of
3Ј-O-lysophosphatidyl derivatives of the four 2Ј-deoxynu-
cleosides that normally occur in DNA.
As shown in Scheme 1, protected mono[(2R)-2,3-dihy-
droxypropyl] esters of 3Ј-(2Ј-deoxy)nucleotides (3Ј-GPdNs)
were prepared from [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]-
methanol and the relevant relevant suitably protected 2Ј-
deoxynucleoside phosphoramidites IϪIV. For this purpose,
nucleoside phosphoramidites currently used in DNA solid-
phase synthesis were employed. Then, after oxidation by
iodine and removal of all protective groups by consecutive
treatment with ammonia and weak acid, free 3Ј-GPdNs
(1؊4) were obtained. Finally, the enzymatic acylation of
each 3Ј-GPdN [as a tetrabutylammonium (TBA) salt] was
achieved by use of Rhizomucor miehei lipase (Lipozyme) in
tert-butyl alcohol and in the presence of trifluoroethyl pal-
mitate (TFEP). At the end of the synthetic run, HPLC ana-
lyses of the crude reaction mixtures in each case revealed
the presence of only one acylation product (5؊8), which
was isolated and characterized on the basis of its spectro-
Scheme 1
able to acylate the primary hydroxyl of the glycerol moiety
selectively. In 3Ј-GPdNs there are two primary hydroxyls
Ϫ both, in principle, susceptible to enzymatic acylation Ϫ
present in the molecule, in addition to a secondary hy-
droxyl. Actually, though, the acylation once again occurred
regioselectively at the primary hydroxyl of the glycerol moi-
ety and not at the free 5Ј-OH of the sugar ring. A prefer-
ence of R. miehei lipase for recognition of that side of 3Ј-
GPdNs molecules which is structurally related to natural
glycerides is evident.
Table 1 reports the conversion values obtained after 48 h
enzymatic acylation of the four 3Ј-GPdNs, in comparison
with those previously found, under the same experimental
conditions, for the 5Ј- counterparts. From these data it ap-
pears that, whereas both the two pyrimidine 3Ј-GPdNs and
their 5Ј-isomers undergo enzymatic acylation essentially to
the same extent, the degrees of conversion of purine 5Ј-
1
scopic features (FAB-MS, NMR). The H and 13C NMR
spectra of compounds 1؊8 are reported in the Exp. Sect.
The complete assignments of individual resonances were
performed with the help of 1HϪ1H COSY, APT, and
HSQC experiments, while 1D decoupling experiments,
1
when necessary, allowed the HϪ1H coupling constants to
be measured.
In the 13C NMR spectra of compounds 5؊8, the reson-
ances of the C-1 and C-2 carbon atoms of the 3-phos-
phoryl-sn-glycerol moiety were shifted downfield (∆δ ϭ
1.2 ppm for pyrimidine and 1.7 ppm for purine nucleotides)
and upfield (∆δ ϭ Ϫ3.65 Ϯ 0.5 ppm), respectively, from the
corresponding resonances of the relevant 3Ј-GPdNs. From
the α- and β-effects of the acylation, these results clearly
indicate that each 3Ј-GPdN had undergone regioselective
acylation at the primary hydroxyl group of its glycerol moi-
ety. Accordingly, in the 1H NMR spectra of these com-
pounds, the C-1 methylene protons of the 3-phosphoryl-sn-
glycerol moiety were shifted downfield (∆δ ϭ 0.46 Ϯ
0.1 ppm) in comparison with the spectra of the pertinent
3Ј-GPdNs. In conclusion, the corresponding 3Ј-O-lysophos-
phatidyl derivative was formed, whichever the starting 2Ј-
deoxynucleoside had been. Confirmatory evidence for the
assigned structures was also provided by HRFAB-MS(Ϫ)
spectra obtained for each compound (see Exp. Sect.).
It has been reported that various lipases (including Lipo-
zyme) in organic solvents in the presence of substrates bear-
ing different kinds of alcoholic functions preferentially acyl-
ate the primary ones.[4,5] Thus, in the case of 5Ј-GPdNs,[1]
bearing only one primary and two secondary hydroxyls, it
Table 1. Lipase-catalyzed palmitoylation of 3Ј- and 5Ј-GPdNs
Substrate
Conversion[a] [%]
3Ј-GPdA
5Ј-GPdA
3Ј-GPdG
5Ј-GPdG
3Ј-GPdC
5Ј-GPdC
3Ј-GPdT
5Ј-GPdT
63
40
32
25
40
37
58
55
[a]
After 48 h incubation, according to the general procedure re-
had not been surprising to find that the enzyme had been ported under Exp. Sect. for the synthesis of compounds 5؊8.
Eur. J. Org. Chem. 2002, 3622Ϫ3631
3623

R. Chillemi, D. Aleo, G. Granata, S. Sciuto
FULL PAPER
GPdNs are decidedly lower than those of the relevant 3Ј- These optimized experimental conditions were then fol-
compounds. lowed in the course of the syntheses of lysophosphatidyl
These findings may be accounted for by the supposition derivatives of DFUR and MTA discussed below.
that, in purine 5Ј-GPdNs (and unlike in 3Ј-GPdNs), a con-
formational preference could compel the glycerol moiety to
be positioned close to the purine nucleobase, making it not
entirely available for the catalytic site of the enzyme.
Synthesis of 3Ј-O-Lysophosphatidyl-5Ј-deoxy-5-
fluorouridine
The required intermediate for the synthesis of the title
compound is 3Ј-(5Ј-deoxy-5-fluoro)uridylic acid mono-
[(2R)-2,3-dihydroxypropyl] ester (12), which was prepared
first. Unlike the 2Ј-deoxynucleosides, DFUR bears two
secondary hydroxy groups at the ribose ring, and so prior
protection of the 2Ј-hydroxy was necessary (Scheme 2).
For this, DFUR was first treated with two molar equiva-
lents of tert-butyldimethylsilyl chloride (TBDMSCl) to af-
ford, after purification, near equimolar amounts of 3Ј- and
2Ј-O-(tert-butyldimethylsilyl)-5Ј-deoxy-5-fluorouridine (10,
11), which were not separated. The two products were then
treated with 2-cyanoethyl [(4R)-2,2-dimethyl-1,3-dioxolan-
4-yl]methyl diisopropylamidophosphite (9) and converted
into a mixture of 3Ј-[2Ј-O-(tert-butyl(dimethylsilyl)-5Ј-de-
oxy-5-fluoro]uridylic acid mono[(4R)-2,2-dimethyl-1,3-di-
oxolan-4-yl]methyl ester (V) and the relevant 2Ј-isomer,
after oxidation by iodine and subsequent ammonia hydro-
lysis. Compound V, isolated by chromatography, then gave
the desired 12, after mild acid hydrolysis. Enzymatic acyl-
ation of 12, with TFEP as acyl donor, afforded the 3Ј-O-
lysophosphatidyl derivative of 5Ј-deoxy-5-fluorouridine (13)
in good yield. The spectroscopic features (NMR and MS)
of the isolated compound were reasonably consistent with
the assigned structure (see Exp. Sect.).
Preliminary indirect evidence in support of this hypo-
thesis was found by comparison of NMR spectroscopic
data (D2O) of purine 5Ј-GPdNs with those of their 3Ј-coun-
terparts. In the 1H NMR spectra of the former compounds,
the resonances of H-1a and H-1b of the glycerol moiety ap-
peared as double doublets at δ ϭ 3.37 and 3.47 ppm, re-
spectively, for the adenosine derivative, and at δ ϭ 3.43 and
3.52 ppm for the guanosine one. In contrast, the resonances
1
of these two protons in the H NMR spectra of purine 3Ј-
GPdNs appeared at δ ϭ 3.67 and 3.74 ppm both for the
adenosine and for the guanosine derivatives. The upfield
values found for the H-1a and H-1b resonances of the two
purine 5Ј-GPdNs indicated that some constraint in these
compounds causes the C-1 methylene protons of the gly-
cerol moiety to lie inside the shielding cone of the relevant
purine rings. A ROE correlation between the H-8 of the
base and the H-2 or H-3b (overlapped signals) of the gly-
cerol moiety, observable in ROESY experiments on both
purine 5Ј-GPdNs, seemed to support this hypothesis.
As the enzymatic acylations of GPdNs had been per-
1
formed in tert-butyl alcohol, H NMR experiments on 3Ј-
and 5Ј-GPdAs (as TBA salts) were also carried out in
(CD3)3COD (see Exp. Sect.). In this solvent, unlike in D2O,
the resonances of the relevant protons of the glycerol moiet-
ies of the two compounds had similar chemical shifts, the
only observable difference being that the two H-1 and the
two H-3 protons in the 3Ј-GPdA spectrum were isochron-
ous whereas in the 5Ј-GPdA spectrum they were not. More
Synthesis of 3Ј- and 2Ј-O-Lysophosphatidyl-5Ј-deoxy-5Ј-
(methylthio)adenosine
After N6-benzoyl-5Ј-deoxy-5Ј-(methylthio)adenosine (14)
interestingly, NOE difference spectra of 5Ј-GPdA showed had been prepared from commercially available MTA, com-
diagnostic mutual NOEs between H-8 of the base and H2- pounds 17 and 18 were obtained from 14 by the same syn-
3 of the glycerol moiety, indicating a certain proximity of thetic approach as used for the preparation of 12. In this
these protons in space. As was to be expected from the high case, however, the two compounds were isolated and each
viscosity of the solvent, all the observed NOE enhance- of them was separately subjected to the enzymatic acyl-
ments were negative.[6] These data gave further evidence of ation. In this manner, compounds 19 and 20 were obtained
a conformational preference in which the glycerol moiety (Scheme 3), their molecular structures being confirmed on
lies close to the purine nucleobase in purine 5Ј-GPdNs.
the basis of their spectroscopic characteristics (see Exp.
Before we applied the synthetic procedure to the prepara- Sect.).
tion of 3Ј-O-lysophosphatidyl derivatives of DFUR and
Here, once again, a difference was found between the en-
MTA, we thought it necessary to optimize the experimental zymatic acylation rates of compounds 17 and 18. After an
conditions used for enzymatic acylation of 3Ј-GPdNs, to incubation time of 48 h, in fact, 41% conversion was ob-
enhance the yield of this synthetic step significantly. After served for compound 18, compared to 60% conversion
various trials on some 3Ј-GPdNs as model substrates, it was found for the 3Ј- isomer 17. Also in this case a comparison
1
decided to maintain the amount of GPN substrate and the of H NMR spectroscopic data (D2O) of 18 with those of
concentration of acyl donor unchanged from the previously the 3Ј-counterpart showed that the resonances of H-1a and
used experimental conditions, but at the same time to in- H-1b of the glycerol moiety appeared in the spectrum of
crease the total reaction volume and the amount of enzyme the former as double doublets at δ ϭ 3.39 and 3.45 ppm,
(four- and elevenfold, respectively), in addition to which the respectively (the similarity of these values with those found
temperature setting was lowered to 38 °C. As a result, it for purine 5Ј-GPdNs is striking), while these two protons
1
proved possible to prolong the reaction time up to 5 days in the H NMR spectrum of 17 gave signals at δ ϭ 3.68
and to achieve a 90% final yield when 1 was used as sub- and 3.75 ppm. The upfield-shifted values found for the H-
strate (similar results were obtained with compounds 2؊4). 1 resonances of the glycerol moiety of 18 were indicative of
3624
Eur. J. Org. Chem. 2002, 3622Ϫ3631

Synthesis of 3Ј(2Ј)-O-Lysophosphatidylnucleosides
FULL PAPER
Scheme 2
Scheme 3
Eur. J. Org. Chem. 2002, 3622Ϫ3631
3625

R. Chillemi, D. Aleo, G. Granata, S. Sciuto
FULL PAPER
a prevalent conformation in which these protons lie inside also been shown to display antiproliferative activity on gli-
1
the adenine shielding cone. When the H NMR spectra of oma cell lines.[14]
17 and 18 were recorded (as TBA salts) in (CD3)3COD, all
Lysophosphatidyl derivatives of both these compounds,
the glycerol moiety protons of the latter compound ap- which may be viewed as intermediate metabolites of com-
peared shielded relative to the relevant ones of 17 (see Exp. pounds themselves, have pro-drug features and are poten-
Sect.). Furthermore, NOE difference spectra of 18 obtained tially better able than free drugs to permeate cell mem-
in the same solvent showed diagnostic enhancements to H- branes.
8 when H2-1, H-2, or H2-3 of the glycerol moiety were irra-
Finally, it should be expected that, for a given lipid mem-
diated. Similar NOEs could not be obtained with 17. These brane composition, a specific fatty acid chain in the lyso-
data as a whole indicate that a conformational preference phosphatidyl compounds should show the best lipophilic
exists in 18, but not in 17, in which the glycerol moiety lies interactions with membranes. Accordingly, lysophosphati-
close to the purine nucleobase. It is not unlikely that this dyl derivatives of nucleoside drugs may be prepared with
finding may, as has been proposed for purine 5Ј-GPdNs, be ever higher efficiency in targeting drugs against specific
related to a poorer ability of the lipase to recognize 18 as tumor cells by a opportune choice of the acyl donor in the
a substrate.
enzymatic step of the synthesis,. Further work is in progress
in this area.
Lipoaffinity of 3Ј-O-Lysophosphatidylnucleosides
Bearing in mind the aim of this work, the tendency of
the newly synthesized lysophosphatidyl compounds to per-
meate lipophilic areas of biomembranes was indirectly in-
vestigated by evaluation of their interactions with lipid
monolayers,[7,8] in comparison with those of the relevant
free nucleosides. In this approach, interactions between a
pharmacologically active substance and lipids are investig-
ated by injection of the drug into the subphase beneath a
lipid monolayer at a constant surface area. The penetration
of the drug can be estimated by the surface pressure in-
crease of the lipid film, which can be measured by the
Wilhelmy plate method.[8] Figure 1 gives the results of this
test when applied to compound 13 in comparison with
DFUR; the enhanced ability of 13 to penetrate the phos-
phatidylcholine monolayer over that of the free nucleoside
is clear. Similar results, not shown here for brevity, were
obtained from compound 19 in comparison with MTA.
Experimental Section
General: NMR spectra were recorded on a Varian Unity Inova
spectrometer at 500 MHz (1 H) and 125.7 MHz (13C). The chemical
shifts are given in ppm and referenced to: (a) TMS as internal
standard, for the experiments in C6D6, CDCl3, and CD3OD; (b)
the residual HOD signal (δ ϭ 4.82 ppm), for 1H experiments in
D2O; (c) the signal of appropriately added CD3OD (δ ϭ 49.0 ppm),
for 13C experiments in D2O; and (d) the signal of the residual par-
tially deuterated solvent (δ ϭ 1.30 ppm) for 1H experiments in
(CD3)3COD. ROESY experiments were carried out in D2O at 27
°C, with a mixing time of 300 ms. NOE difference spectra were
obtained in (CD3)3COD at 30 °C. High-resolution fast atom bom-
bardment mass spectra (HRFAB-MS) were recorded with a Fisons
ZAB 2SE spectrometer with glycerol as matrix. Column chromato-
graphy was performed on silica gel (40Ϫ63 µm, Merck), and TLC
was carried out on silica gel 60 F254 precoated glass plates
(0.25 mm, Merck). HPLC was performed on a HewlettϪPackard
1050 chromatograph equipped with a UV detector set at 260 nm,
on LiChrospher-100 ODS (5 µm; 4 ϫ 250 mm) and Ultrasphere
ODS (5 µm; 10 ϫ 250 mm) or Zorbax SB-C18 (5 µm; 9.4 ϫ
250 mm) columns for analytical and semipreparative runs, respect-
ively. Modified nucleosides dissolved in water or methanol were
spectroscopically quantified by attributing them the molar extinc-
tion coefficients reported in the literature for the relevant 3Ј-nucle-
otides, DFUR, and MTA. Commercially purchased solvents and
reagents were all of reagent quality. Triethylamine (TEA), petro-
leum ether, tBuOH, dichloromethane, and diethyl ether were
freshly dried and stored under argon as previously reported.[1]
TFEP was prepared by a reported procedure,[15] and its purity was
checked by its chromatographic and spectroscopic properties. Lip-
ase from Rhizomucor miehei (LipozymeTM, immobilized) was from
Novo-Nordisk, Copenhagen, Denmark. Before use the enzyme was
allowed to stand for 45 min in dried tBuOH. For the monolayer
penetration experiments, egg yolk phosphatidylcholine (Fluka),
dissolved in chloroform (0.2 mg/mL) was spread (30 µL) on the
aqueous subphase, which consisted of 0.10  NaCl and 0.05 
phosphate buffer solution (pH 7.4). Surface pressure measurements
were carried out at 25 °C with a NIMA PS4 surface pressure sensor
(NIMA Technology LTD).
Figure 1. Kinetics of surface pressure increase of phosphatidylcho-
line monolayers after dissolving into the aqueous subphase DFUR
(a) [10.1µ] and compound 13 (b) [0.40 µ] (see also Exp. Sect.
under General)
At present, DFUR is widely used, often in combination
with other drugs, in the chemotherapeutic treatment of
tumors.[9Ϫ12] MTA, a potent inhibitor of protein carboxyl-
methyltransferase, is able to induce apoptosis in leukemia
U937 cells.[13] As an inhibitor of protein tyrosine kinase ac-
General Procedure for the Synthesis of Compounds 1Ϫ4: Solutions
of
[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methanol
(22
µL,
tivity stimulated by basic fibroblast growth factor, MTA has 0.18 mmol) and 2.5% tetrazole (1 mL) in anhydrous CH3CN
3626 Eur. J. Org. Chem. 2002, 3622Ϫ3631

Synthesis of 3Ј(2Ј)-O-Lysophosphatidylnucleosides
FULL PAPER
(3 mL) were alternately added dropwise to a stirred solution of
0.12 mmol of compounds I, or II, III, IV in the same solvent. The
magnetically stirred mixture was left at room temperature for 1 h
and then treated with an excess of iodine solution (0.1 ⁾ in THF/
H2O/pyridine (9:1:0.1). After the reaction mixture had been taken
to dryness in vacuo, the residue was dissolved in 20 mL of CHCl3/
nBuOH (9:1) and the solution was extracted with freshly prepared
5% aqueous sodium metabisulfite to remove the excess of iodine.
The organic layer was washed with H2O and the solvents were
evaporated in vacuo. The residue was taken up in concd. ammonia
(10 mL) and the suspension was stirred at room temperature for
1 h, then at 55 °C for 5 h (this step was omitted for 4). After the
reaction mixture had been taken to dryness in vacuo, the residue
was dissolved in 30% aqueous HOAc (10 mL) and the solution was
allowed to stand at room temperature for 5 h. After evaporation of
the solvent in vacuo, the residue was taken up in H2O and extracted
with CH2Cl2. The aqueous layer was then evaporated in vacuo and
the totally deprotected product was purified by column chromato-
graphy on silica gel (iPrOH/H2O/concd. ammonia, 87:10:3). Pure
compounds (1Ϫ4) were obtained as colourless oils.
J4Ј, 5ЈЈ ϭ 4.4 Hz, 1 H, H-4Ј), 4.99 (dddd, J3Ј,2Ј ϭ 6.1, J3Ј,2ЈЈ ϭ 2.7,
J3Ј,4Ј ϭ 2.4, J3Ј, ϭ 7.0 Hz, 1 H, H-3Ј), 6.36 (dd, J1Ј,2Ј ϭ 8.2,
P
J1Ј,2ЈЈ ϭ 6.1 Hz, 1 H, H-1Ј), 8.04 (s, 1 H, H-8) ppm. 13C NMR
(D2O): δ ϭ 40.7 (d, JCCOP ϭ 2.2 Hz, C-2Ј), 64.5 (C-5Ј), 65.0 (C-1
of glycerol), 69.3 (d, JCOP ϭ 4.7 Hz, C-3 of glycerol), 73.6 (d,
JCCOP ϭ 7.7 Hz, C-2 of glycerol), 78.6 (d, JCOP ϭ 5.4 Hz, C-3Ј),
87.1 (C-1Ј), 89.2 (d, JCCOP ϭ 6.1 Hz, C-4Ј), 119.5 (C-5), 141.1 (C-
8), 154.4 (C-4), 156.6 (C-2), 161.7 (C-6) ppm. HRFAB-MS(Ϫ)
calcd. for C13H19N5O9P [M Ϫ H]Ϫ 420.0920, found 420.0938.
3Ј-(2Ј-Deoxy)cytidilic Acid Mono[(2R)-2,3-dihydroxypropyl] Ester
(3): 32.5 mg (71%). 1H NMR (D2O): δ ϭ 2.43 (ddd, J2Ј,2ЈЈ ϭ Ϫ14.3,
J2Ј,1Ј ϭ 7.3, J2Ј,3Ј ϭ 6.4 Hz, 1 H, H-2Ј), 2.68 (ddd, J2ЈЈ,2Ј ϭ Ϫ14.3,
J2ЈЈ,1Ј ϭ 6.3, J2ЈЈ,3Ј ϭ 3.2 Hz, 1 H, H-2ЈЈ), 3.66 (dd, J1a,1b ϭ Ϫ11.7,
J1a,2 ϭ 6.0 Hz, 1 H, H-1a of glycerol), 3.73 (dd, J1b,1a ϭ Ϫ11.7,
J1b,2 ϭ 4.3 Hz, 1 H, H-1b of glycerol), 3.84 (dd, J5ЈЈ,5Ј ϭ Ϫ12.5,
J5ЈЈ,4Ј ϭ 4.9 Hz, 1 H, H-5ЈЈ), 3.90 (dd, partially obscured by next
signal, J5Ј,5ЈЈ ϭ Ϫ12.5, J5Ј,4Ј ϭ 3.5 Hz, 1 H, H-5Ј), 3.91Ϫ4.01 (par-
tially overlapped multiplets, 3 H, H-2 and H-3 of glycerol), 4.30
(ddd, J4Ј,3Ј ϭ 3.2, J4Ј,5Ј ϭ 3.5, J4Ј,5ЈЈ ϭ 4.9 Hz, 1 H, H-4Ј), 6.17 (d,
J5,6 ϭ 7.6 Hz, 1 H, H-5), 6.35 (dd, J1Ј,2Ј ϭ 7.3, J1Ј,2ЈЈ ϭ 6.3 Hz, 1
H, H-1Ј), 7.98 (d, J6,5 ϭ 7.6 Hz, 1 H, H-6) ppm, the H-3Ј resonance
is obscured by the residual HOD signal. 13C NMR (D2O): δ ϭ 41.1
(d, JCCOP ϭ 3.3 Hz, C-2Ј), 64.0 (C-5Ј), 65.0 (C-1 of glycerol), 69.3
(d, JCOP ϭ 5.7 Hz, C-3 of glycerol), 73.6 (d, JCCOP ϭ 7.6 Hz, C-2
of glycerol), 77.9 (d, JCOP ϭ 4.3 Hz, C-3Ј), 88.8 (d, JCCOP ϭ 5.9 Hz,
C-4Ј), 89.1 (C-1Ј), 98.9 (C-5), 145.0 (C-6), 158.5 (C-2), 167.7 (C-4)
3Ј-(2Ј-Deoxy)adenylic Acid Mono[(2R)-2,3-dihydroxypropyl] Ester
(1): 33.1 mg (68%). 1H NMR (D2O): δ ϭ 2.80 (ddd, J2ЈЈ,2Ј ϭ Ϫ14.2,
J2ЈЈ,1Ј ϭ 6.1, J2ЈЈ,3Ј ϭ 2.7 Hz, 1 H, H-2ЈЈ), 2.92 (ddd, J2Ј,2ЈЈ ϭ Ϫ14.2,
J2Ј,1Ј ϭ 7.9, J2Ј,3Ј ϭ 5.9 Hz, 1 H, H-2Ј), 3.67 (dd, J1a,1b ϭ Ϫ11.8,
J1a,2 ϭ 6.0 Hz, 1 H, H-1a of glycerol), 3.74 (dd, J1b,1a ϭ Ϫ11.8,
J1b,2 ϭ 4.3 Hz, 1 H, H-1b of glycerol), 3.87 (dd, J5ЈЈ,5Ј ϭ Ϫ12.7,
ppm. HRFAB-MS(Ϫ) calcd. for C12H19N3O9P [M
Ϫ
H]Ϫ
J5ЈЈ,4Ј ϭ 4.2 Hz, 1 H, H-5ЈЈ), 3.91 (dd, J5Ј,5ЈЈ ϭ Ϫ12.7, J5Ј,4Ј
ϭ
380.0859, found 380.0865.
3.2 Hz, 1 H, H-5Ј), 3.94Ϫ4.05 (partially overlapped multiplets, 3
H, H-2 and H-3 of glycerol), 4.41 (ddd, J4Ј,3Ј ϭ 2.4, J4Ј,5Ј ϭ 3.2,
J4Ј,5ЈЈ ϭ 4.2 Hz, 1 H, H-4Ј), 5.01 (dddd, J3Ј,2Ј ϭ 5.9, J3Ј,2ЈЈ ϭ 2.7,
3Ј-(2Ј-Deoxy)thymidilic Acid Mono[(2R)-2,3-dihydroxypropyl] Ester
(4): 34.7 mg (73%). 1H NMR (D2O): δ ϭ 1.95 (d, J ϭ 1.0 Hz, 3 H,
CH3), 2.48 (ddd, J2Ј,2ЈЈ ϭ Ϫ14.3, J2Ј,1Ј ϭ 7.7, J2Ј,3Ј ϭ 6.4 Hz, 1 H,
H-2Ј), 2.61 (ddd, J2ЈЈ,2Ј ϭ Ϫ14.3, J2ЈЈ,1Ј ϭ 6.2, J2ЈЈ,3Ј ϭ 3.2 Hz, 1 H,
H-2ЈЈ), 3.66 (dd, J1a,1b ϭ Ϫ11.7, J1a,2 ϭ 5.8 Hz, 1 H, H-1a of gly-
cerol), 3.73 (dd, J1b,1a ϭ Ϫ11.7, J1b,2 ϭ 4.2 Hz, 1 H, H-1b of gly-
cerol), 3.85 (dd, J5ЈЈ,5Ј ϭ Ϫ12.6, J5ЈЈ,4Ј ϭ 4.9 Hz, 1 H, H-5ЈЈ), 3.91
(dd, partially obscured by next downfield signal, J5Ј,5ЈЈ ϭ Ϫ12.6,
J5Ј,4Ј ϭ 3.4 Hz, 1 H, H-5Ј), 3.92Ϫ4.01 (overlapped multiplets, 3 H,
H-2 and H-3 of glycerol), 4.26 (ddd, J4Ј,3Ј ϭ 3.2, J4Ј,5Ј ϭ 3.4,
J4Ј,5ЈЈ ϭ 4.9 Hz, 1 H, H-4Ј), 4.85 (ddt partially obscured by the
J3Ј,4Ј ϭ 2.4, J3Ј,P ϭ 7.1 Hz, 1 H, H-3Ј), 6.51 (dd, J1Ј,2Ј ϭ 7.9, J1Ј,2ЈЈ
ϭ
1
6.1 Hz, 1 H, H-1Ј), 8.26 (s, 1 H, H-2), 8.39 (s, 1 H, H-8) ppm. H
NMR (TBA salt, (CD3)3COD): δ ϭ 1.13 (t, J ϭ 7.3 Hz, 12 H,
NϪCH2CH2CH2CH3), 1.58 (m, 8 H, NϪCH2CH2CH2CH3), 1.79
(m, 8 H, NϪCH2CH2CH2CH3), 2.78 (ddd, J2ЈЈ,2Ј ϭ Ϫ13.3, J2ЈЈ,1Ј
5.9, J2ЈЈ,3Ј ϭ 3.4 Hz, 1 H, H-2ЈЈ), 2.91 (ddd, J2Ј,2ЈЈ ϭ Ϫ13.3, J2Ј,1Ј
ϭ
ϭ
7.1, J2Ј,3Ј
ϭ 5.7 Hz, 1 H, H-2Ј), 3.41 (br. t, 8 H, N-
CH2CH2CH2CH3), 3.71 (d, J1,2 ϭ 5.9 Hz, 2 H, H-1 of glycerol),
3.85 (tt, J2,1 ϭ 5.9, J2,3 ϭ 4.4 Hz, 1 H, H-2 of glycerol), 3.95 (dd
partially overlapping with next signal, J5ЈЈ,5Ј ϭ Ϫ12.6, J5ЈЈ,4Ј
2.9 Hz, 1 H, H-5ЈЈ), 3.97 (dd partially overlapping with previous
signal, J5Ј,5ЈЈ ϭ Ϫ12.6, J5Ј,4Ј ϭ 2.3 Hz, 1 H, H-5Ј), 4.08 (dd, J3,2
ϭ
residual HOD signal, J3Ј,2Ј ϭ 6.4, J3Ј,2ЈЈ ϭ 3.2, J3Ј,4Ј ϭ 3.2, J3Ј,P
ϭ
7.3 Hz, 1 H, H-3Ј), 6.37 (dd, J1Ј,2Ј ϭ 7.7, J1Ј,2ЈЈ ϭ 6.2 Hz, 1 H, H-
1Ј), 7.71 (q, J ϭ 1.0 Hz, 1 H, H-6) ppm. 13C NMR (D2O): δ ϭ
14.4 (CH3), 40.5 (d, JCCOP ϭ 2.6 Hz, C-2Ј), 63.9 (C-5Ј), 64.9 (C-1
of glycerol), 69.3 (d, JCOP ϭ 5.6 Hz, C-3 of glycerol), 73.5 (d,
JCCOP ϭ 7.9 Hz, C-2 of glycerol), 77.8 (d, JCOP ϭ 5.1 Hz, C-3Ј),
87.9 (C-1Ј), 88.5 (d, JCCOP ϭ 6.1 Hz, C-4Ј), 114.4 (C-5), 140.4 (C-
6), 154.6 (C-2), 169.4 (C-4) ppm. HRFAB-MS(Ϫ) calcd. for
C13H20N2O10P [M Ϫ H]Ϫ 395.0855, found 395.0838.
ϭ
4.4, J3,P ϭ 9.9 Hz, 2 H, H-3 of glycerol), 4.35 (dt, J4Ј,3Ј ϭ 2.9,
J4Ј,5Ј ϭ 2.3, J4Ј,5ЈЈ ϭ 2.9 Hz, 1 H, H-4Ј), 5.15 (dddd, J3Ј,2Ј ϭ 5.7,
J3Ј,2ЈЈ ϭ 3.4, J3Ј,4Ј ϭ 2.9, J3Ј,P ϭ 7.3 Hz, 1 H, H-3Ј), 6.60 (dd, J1Ј,2Ј
ϭ
7.1, J1Ј,2ЈЈ ϭ 5.9 Hz, 1 H, H-1Ј), 8.29 (s, 1 H, H-2), 8.63 (s, 1 H, H-
8) ppm. 13C NMR (D2O): δ ϭ 41.2 (d, JCCOP ϭ 2.5 Hz, C-2Ј), 64.5
(C-5Ј), 64.9 (C-1 of glycerol), 69.3 (d, JCOP ϭ 6.0 Hz, C-3 of gly-
cerol), 73.5 (d, JCCOP ϭ 6.9 Hz, C-2 of glycerol), 78.7 (d, JCOP
ϭ
5.4 Hz, C-3Ј), 87.7 (C-1Ј), 89.5 (d, JCCOP ϭ 5.3 Hz, C-4Ј), 121.6 (C-
5), 143.5 (C-8), 151.0 (C-4), 153.9 (C-2), 157.3 (C-6) ppm. HRFAB-
MS(Ϫ) calcd. for C13H19N5O8P [M Ϫ H]Ϫ 404.0971, found
404.0963.
2-Cyanoethyl [(4R)-2,2-Dimethyl-1,3-dioxolan-4-yl]methyl Diisopro-
pylamidophosphite (9): The synthesis and the purification of com-
pound 9 were carried out as previously reported.[1] Its purity was
checked by its chromatographic and spectroscopic properties.
3Ј-(2Ј-Deoxy)guanylic Acid Mono[(2R)-2,3-dihydroxypropyl] Ester
5Ј-(2Ј-Deoxy)adenylic Acid Mono[(2R)-2,3-dihydroxypropyl] Ester:
(2): 31.9 mg (63%). 1H NMR (D2O): δ ϭ 2.73 (ddd, J2ЈЈ,2Ј ϭ Ϫ14.1, The synthesis and the purification of the title compound were car-
J2ЈЈ,1Ј ϭ 6.1, J2ЈЈ,3Ј ϭ 2.7 Hz, 1 H, H-2ЈЈ), 2.90 (ddd, J2Ј,2ЈЈ ϭ Ϫ14.1, ried out as previously reported.[1]
J2Ј,1Ј ϭ 8.2, J2Ј,3Ј ϭ 6.1 Hz, 1 H, H-2Ј), 3.67 (dd, J1a,1b ϭ Ϫ11.7, (CD3)3COD]: 1.11 (t,
NϪCH2CH2CH2CH3), 1.56 (m, 8 H, NϪCH2CH2CH2CH3), 1.76
Ϫ
1H NMR [TBA salt,
δ
ϭ
J
ϭ
7.3 Hz, 12 H,
J1a,2 ϭ 5.7 Hz, 1 H, H-1a of glycerol), 3.74 (dd, J1b,1a ϭ Ϫ11.7,
J1b,2 ϭ 3.9 Hz, 1 H, H-1b of glycerol), 3.85 (dd, J5ЈЈ,5Ј ϭ Ϫ12.4,
(m, 8 H, NϪCH2CH2CH2CH3), 2.62 (ddd, J2ЈЈ,2Ј ϭ Ϫ13.3, J2ЈЈ,1Ј
6.7, J2ЈЈ,3Ј ϭ 3.8 Hz, 1 H, H-2ЈЈ), 2.87 (ddd, J2Ј,2ЈЈ ϭ Ϫ13.3, J2Ј,1Ј
ϭ
J5ЈЈ,4Ј ϭ 4.4 Hz, 1 H, H-5ЈЈ), 3.89 (dd, J5Ј,5ЈЈ ϭ Ϫ12.4, J5Ј,4Ј
ϭ
ϭ
3.6 Hz, 1 H, H-5Ј), 3.92Ϫ4.04 (partially overlapped multiplets, 3 6.7, J2Ј,3Ј
ϭ
6.1 Hz,
1
H, H-2Ј), 3.39 (br. t,
8
H,
H, H-2 and H-3 of glycerol), 4.36 (ddd, J4Ј,3Ј ϭ 2.4, J4Ј,5Ј ϭ 3.6, NϪCH2CH2CH2CH3), 3.70 (dd partially overlapping with next sig-
Eur. J. Org. Chem. 2002, 3622Ϫ3631 3627

R. Chillemi, D. Aleo, G. Granata, S. Sciuto
FULL PAPER
nal, J1a,1b ϭ Ϫ11.5, J1a,2 ϭ 5.7 Hz, 1 H, H-1a of glycerol), 3.73 (dd 3Ј- and 2Ј-[5Ј-Deoxy-5Ј-(methylthio)]adenylic Acid Mono[(2R)-2,3-
partially overlapping with previous signal, J1b,1a ϭ Ϫ11.5, J1b,2 dihydroxypropyl] Ester (17 and 18): In an adaptation of a reported
ϭ
6.3 Hz, 1 H, H-1b of glycerol), 3.85 (dddd, J2,1a ϭ 5.7, J2,1b ϭ 6.3, procedure,[17] MTA (100 mg, 0.34 mmol) was first converted into
J2,3a ϭ 3.8, J2,3b ϭ 4.7 Hz, 1 H, H-2 of glycerol), 4.07 (ddd partially
the corresponding N6-benzoyl derivative (14), which was then puri-
overlapping with next signal, J3a,3b ϭ Ϫ11.4, J3a,2 ϭ 3.8, J3a,P
ϭ
fied by column chromatography on silica gel, eluting with a gradi-
10.3 Hz, 1 H, H-3a of glycerol), 4.10 (ddd partially overlapping ent of CH3OH in CH2Cl2 from 0 to 5% (Rf ϭ 0.13, CH2Cl2/
with previous signal, J3b,3a ϭ Ϫ11.4, J3b,2 ϭ 4.7, J3b,P ϭ 10.0 Hz, CH3OH, 95:5); the pure fractions, evaporated in vacuo, gave com-
1 H, H-3b of glycerol), 4.16Ϫ4.22 (overlapping multiplets, 2 H, H- pound 14 as a colourless oil (109 mg, 80%). Selected NMR spectro-
5Ј and H-5ЈЈ), 4.25 (m, 1 H, H-4Ј), 4.81 (ddd, J3Ј,2Ј ϭ 6.1, J3Ј,2ЈЈ
3.8, J3Ј,4Ј ϭ 3.4 Hz, 1 H, H-3Ј), 6.65 (t, J1Ј,2Ј ϭ J1Ј,2ЈЈ ϭ 6.7 Hz, 1
H, H-1Ј), 8.30 (s, 1 H, H-2), 8.67 (s, 1 H, H-8) ppm.
ϭ
scopic data referring to the benzoyl group: 1H NMR (CD3OD):
δ ϭ 7.56 (dd, Jortho ϭ 8.4, Jortho ϭ 7.5 Hz, 2 H, H-3 and H-5), 7.66
(tt, Jortho ϭ 7.5, Jmeta ϭ 1.2 Hz, 1 H, H-4), 8.09 (dd, Jortho ϭ 8.4,
J
meta ϭ 1.2 Hz, 2 H, H-2 and H-6) ppm. 13C NMR (CD3OD): δ ϭ
3Ј-(5Ј-Deoxy-5-fluoro)uridylic Acid Mono[(2R)-2,3-dihydroxypropyl]
Ester (12): A mixture of the 2Ј- and 3Ј-O-(tert-butyldimethylsilyl)
derivatives of DFUR was first obtained by adaptation of the pro-
cedure reported by Usman et al.[16] DFUR (60 mg, 0.24 mmol) was
dissolved in anhydrous DMF (0.5 mL) under an argon atmosphere,
and imidazole (45 mg, 0.66 mmol) and TBDMSCl in THF (1 ,
0.48 mL) were then added to the magnetically stirred solution.
After the mixture had been kept at room temperature for 2 h, the
reaction was quenched with 5% aqueous NaHCO3 (7 mL) and the
mixture was concentrated in vacuo. The residue was coevaporated
with toluene, taken up in CH2Cl2, and extracted with H2O. The
organic layer was taken to dryness in vacuo and the residue was
purified by column chromatography on silica gel; elution with a
gradient of iPrOH in CH2Cl2/TEA (99:1) from 0 to 9% afforded
a mixture of 3Ј-O-(tert-butyldimethylsilyl)-5Ј-deoxy-5-fluorouridine
(10) and 2Ј-O-(tert-butyldimethylsilyl)-5Ј-deoxy-5-fluorouridine
128.8 (C-2 and C-6), 129.2 (C-3 and C-5), 133.2 (C-4), 134.2 (C-
1), 167.2 (CONH) ppm. Compound 14 (109 mg, 0.27 mmol) was
then treated with TBDMSCl as described for DFUR. Column
chromatography on silica gel, eluting first with a gradient of
CH2Cl2/TEA (99:1) in hexane/TEA (99:1) from 50 to 100% and
then with a gradient of iPrOH in CH2Cl2/TEA (99:1) from 0 to
3%, afforded a mixture of compounds 15 and 16 (109.5 mg, 78%
1
yield altogether). Selected H NMR (CDCl3) data referring to the
tert-butyldimethylsilyl moiety: 15, δ ϭ 0.22 and 0.23 (singlets, 6 H
altogether, CH3), 0.98 [s, 9 H, C(CH3)3] ppm ; 16, δ ϭ 0.01 and
0.02 (singlets, 6 H altogether, CH3), 0.88 [s, 9 H, C(CH3)3] ppm.
A solution of compound 9 (136 mg, 0.41 mmol) in anhydrous
CH3CN (1.5 mL) and a 2.5% tetrazole solution (1.5 mL) in the
same solvent were alternately added dropwise to the mixture of 15
and 16 (109 mg, 0.21 mmol) dissolved in anhydrous CH3CN
(11) (62.3 mg; 72% yield, altogether). Selected 1H NMR (CDCl3) (7 mL). The reaction mixture was then treated according to the
data referring to the tert-butyldimethylsilyl moiety: 10, δ ϭ 0.16
general procedure above reported for compounds 1Ϫ3, except that
and 0.17 (singlets, 6 H altogether, CH3), 0.96 [s, 9 H, C(CH3)3] the acid hydrolysis was carried out for 7 h. After evaporation of
ppm; 11, δ ϭ 0.02 and 0.03 (singlets, 6H altogether, CH3), 0.89 [s,
9 H, C(CH3)3] ppm.
the solvent in vacuo, the residue was dissolved in 50% aqueous
CH3OH and purified by HPLC on a Zorbax SB-C18 semiprepar-
ative column, eluting with a gradient of CH3CN in 0.1  triethyl-
ammonium acetate (pH 7.0) from 5 to 20% in 25 min, at the flow
rate of 3.5 mL minϪ1. Compounds 17 (tR ϭ 10.6 min) and 18 (tR ϭ
13.0 min) were obtained as colourless oils.
A solution of compound 9 (110 mg, 0.33 mmol) in anhydrous
CH3CN (1 mL) and a 2.5% tetrazole solution (1.2 mL) in the same
solvent were alternately added dropwise to the mixture of 10 and
11 (61 mg, 0.17 mmol) dissolved in anhydrous CH3CN (6 mL). The
reaction mixture was then treated according to the general proced-
ure above, as reported for compound 4. After ammonia treatment
Compound 17: 32.2 mg (34% yield from the mixture of 15 and 16).
1H NMR (D2O): δ ϭ 2.17 (s, 3 H, SϪCH3), 3.02 (dd, J5ЈЈ,5Ј
ϭ
at room temperature, the solution was evaporated in vacuo and the Ϫ14.4, J5ЈЈ,4Ј ϭ 6.7 Hz, 1 H, H-5ЈЈ), 3.10 (dd, J5Ј,5ЈЈ ϭ Ϫ14.4,
residue was chromatographed on a silica gel column (iPrOH/concd.
ammonia, 95:5). Fractions containing compound V (Rf ϭ 0.20)
were taken to dryness in vacuo, the residue was dissolved in 30%
aqueous HOAc (15 mL), and the solution was allowed to stand for
J5Ј,4Ј ϭ 4.4 Hz, 1 H, H-5Ј), 3.68 (dd, J1a,1b ϭ Ϫ11.7, J1a,2 ϭ 6.0 Hz,
1 H, H-1a of glycerol), 3.75 (dd, J1b,1a ϭ Ϫ11.7, J1b,2 ϭ 4.4 Hz, 1
H, H-1b of glycerol), 3.97Ϫ4.08 (partially overlapped multiplets, 3
H, H-2 and H-3 of glycerol), 4.61 (ddd, J4Ј,3Ј ϭ 4.0, J4Ј,5Ј ϭ 4.4,
7 h at room temperature, to remove the isopropylidene group and J4Ј,5ЈЈ ϭ 6.7 Hz, 1 H, H-4Ј), 4.85 (ddd, J3Ј,2Ј ϭ 5.3, J3Ј,4Ј ϭ 4.0,
to accomplish the 2Ј desilylation. The solution was then evaporated
in vacuo and the residue was purified by column chromatography
on silica gel (iPrOH/H2O/concd. ammonia, 85:10:5 to afford pure
J3Ј,P ϭ 7.8 Hz, 1 H, H-3Ј), 5.08 (dd, J2Ј,1Ј ϭ 5.9, J2Ј,3Ј ϭ 5.3 Hz, 1
H, H-2Ј), 6.19 (d, J1Ј,2Ј ϭ 5.9 Hz, 1 H, H-1Ј), 8.32 (s, 1 H, H-2),
8.45 (s, 1 H, H-8) ppm. 1H NMR (TBA salt, (CD3)3COD): δ ϭ
1.13 (t, J ϭ 7.3 Hz, 12 H, NϪCH2CH2CH2CH3), 1.58 (m, 8 H,
12 as a colourless oil (23.8 mg, 35% yield from the mixture of 10
1
and 11): Rf ϭ 0.40. H NMR (D2O): δ ϭ 1.51 (d, J5Ј,4Ј ϭ 6.3 Hz, NϪCH2CH2CH2CH3), 1.79 (m, 8 H, NϪCH2CH2CH2CH3), 2.22
3 H, H-5Ј), 3.66 (dd, J1a,1b ϭ Ϫ11.8, J1a,2 ϭ 5.8 Hz, 1 H, H-1a of (s, 3 H, SϪCH3), 3.04 (dd, J5ЈЈ,5Ј ϭ Ϫ14.3, J5ЈЈ,4Ј ϭ 6.1 Hz, 1 H,
glycerol), 3.73 (dd, J1b,1a ϭ Ϫ11.8, J1b,2 ϭ 4.3 Hz, 1 H, H-1b of
glycerol), 3.94Ϫ4.05 (partially overlapped multiplets, 3 H, H-2 and
H-3 of glycerol), 4.37Ϫ4.43 (partially overlapped multiplets, 2 H,
H-3Ј, H-4Ј), 4.52 (t, J2Ј,1Ј ϭ 4.9, J2Ј,3Ј ϭ 4.9 Hz, 1 H, H-2Ј), 5.94
(dd, J1Ј,2Ј ϭ 4.9, J1Ј,F ϭ 1.1 Hz, 1 H, H-1Ј), 7.91 (d, J6,F ϭ 6.3 Hz,
H-5ЈЈ), 3.12 (dd, J5Ј,5ЈЈ ϭ Ϫ14.3, J5Ј,4Ј ϭ 4.5 Hz, 1 H, H-5Ј), 3.43
(br. t, 8 H, N-CH2CH2CH2CH3), 3.70 (dd partially overlapping
with next signal, J1a,1b ϭ Ϫ11.5, J1a,2 ϭ 5.6 Hz, 1 H, H-1a of gly-
cerol), 3.73 (dd partially overlapping with previous signal, J1b,1a
Ϫ11.5, J1b,2 ϭ 6.4 Hz, 1 H, H-1b of glycerol), 3.90 (dddd, J2,1a
ϭ
ϭ
1 H, H-6) ppm. 13C NMR (D2O): δ ϭ 20.7 (C-5Ј), 65.0 (C-1 of 5.6, J2,1b ϭ 6.4, J2,3a ϭ 3.9, J2,3b ϭ 4.9 Hz, 1 H, H-2 of glycerol),
glycerol), 69.4 (d, JCOP ϭ 3.8 Hz, C-3 of glycerol), 73.6 (d, JCCOP ϭ
4.12 (ddd partially overlapping with next signal, J3a,3b ϭ Ϫ11.3,
7.9 Hz, C-2 of glycerol), 75.5 (d, JCCOP ϭ 2.4 Hz, C-2Ј), 80.3 (d, J3a,2 ϭ 3.9, J3a,P ϭ 9.0 Hz, 1 H, H-3a of glycerol), 4.18 (ddd par-
JCOP ϭ 4.5 Hz, C-3Ј), 82.5 (d, JCCOP ϭ 3.9 Hz, C-4Ј), 92.4 (C-1Ј), tially overlapping with previous signal, J3b,3a ϭ Ϫ11.3, J3b,2 ϭ 4.9,
128.4 (d, JCCF ϭ 34.4 Hz, C-6), 143.7 (d, JCF ϭ 233.4 Hz, C-5),
153.0 (C-2), 162.3 (d, JCCF ϭ 26.6 Hz, C-4) ppm. HRFAB-MS(Ϫ)
calcd. for C12H17FN2O10P [M Ϫ H]Ϫ 399.0605, found 399.0613.
J3b,P ϭ 8.9 Hz, 1 H, H-3b of glycerol), 4.53 (ddd, J4Ј,3Ј ϭ 4.8,
J4Ј,5Ј ϭ 4.5, J4Ј,5ЈЈ ϭ 6.1 Hz, 1 H, H-4Ј), 4.93 (ddd, J3Ј,2Ј ϭ 5.2,
J3Ј,4Ј ϭ 4.8, J3Ј,P ϭ 6.5 Hz,1 H, H-3Ј), 5.01 (dd, J2Ј,1Ј ϭ 4.9, J2Ј,3Ј
ϭ
3628
Eur. J. Org. Chem. 2002, 3622Ϫ3631

Synthesis of 3Ј(2Ј)-O-Lysophosphatidylnucleosides
5.2 Hz, 1 H, H-2Ј), 6.24 (d, J1Ј,2Ј ϭ 4.9 Hz, 1 H, H-1Ј), 8.33 (s, 1  triethylammonium acetate (pH 7) from 0 to 70% in 40 min and
FULL PAPER
H, H-2), 8.49 (s, 1 H, H-8) ppm. 13C NMR (D2O): δ ϭ 18.2 a flow rate of 2.5 mL minϪ1
.
(SϪCH3), 38.6 (C-5Ј), 65.0 (C-1 of glycerol), 69.5 (d, JCOP
ϭ
3Ј-O-Lysophosphatidyl Derivative of 2Ј-Deoxyadenosine (5): 20.3 mg
(63% yield from 1). H NMR (CD3OD): δ ϭ 0.90 (t, J ϭ 6.9 Hz,
5.6 Hz, C-3 of glycerol), 73.6 (d, JCCOP ϭ 8.0 Hz, C-2 of glycerol),
75.5 (d, JCCOP ϭ 4.8 Hz, C-2Ј), 78.8 (d, JCOP ϭ 5.2 Hz, C-3Ј), 85.7
(d, JCCOP ϭ 3.6 Hz, C-4Ј), 90.1 (C-1Ј), 122.1 (C-5), 143.8 (C-8),
151.9 (C-4), 153.8 (C-2), 157.1 (C-6) ppm. HRFAB-MS(Ϫ) calcd.
for C14H21N5O8PS [M Ϫ H]Ϫ 450.0848, found 450.0851.
1
3 H, CH3 of palmitoyl), 1.26 and 1.27 (br. singlets, 24 H altogether,
from γ- to ξ-CH2 of palmitoyl), 1.59 (tt, Jβ,α ϭ 7.5, Jβ,γ ϭ 7.0 Hz,
2 H, β-CH2 of palmitoyl), 2.33 (t, J ϭ 7.5 Hz, 2 H, α-CH2 of palmi-
toyl), 2.67 (ddd, J2ЈЈ,2Ј ϭ Ϫ13.7, J2ЈЈ,1Ј ϭ 5.8, J2ЈЈ,3Ј ϭ 1.8 Hz, 1 H,
H-2ЈЈ), 2.91 (ddd, J2Ј,2ЈЈ ϭ Ϫ13.7, J2Ј,1Ј ϭ 8.4, J2Ј,3Ј ϭ 5.7 Hz, 1 H,
H-2Ј), 3.83 (dd, J5ЈЈ,5Ј ϭ Ϫ12.5, J5ЈЈ,4Ј ϭ 2.9 Hz, 1 H, H-5ЈЈ), 3.86
(dd, J5Ј,5ЈЈ ϭ Ϫ12.5, J5Ј,4Ј ϭ 2.6 Hz, 1 H, H-5Ј), 3.90Ϫ4.01 (partially
overlapped multiplets, 3 H, H-2 and H-3 of glycerol), 4.12 (dd,
J1a,1b ϭ Ϫ11.4, J1a,2 ϭ 5.7 Hz, 1 H, H-1a of glycerol), 4.19 (dd,
J1b,1a ϭ Ϫ11.4, J1b,2 ϭ 4.5 Hz, 1 H, H-1b of glycerol), 4.33 (ddd,
J4Ј,3Ј ϭ 1.8, J4Ј,5Ј ϭ 2.6, J4Ј,5ЈЈ ϭ 2.9 Hz, 1 H, H-4Ј), 5.03 (ddt,
J3Ј,2Ј ϭ 5.7, J3Ј,2ЈЈ ϭ 1.8, J3Ј,4Ј ϭ 1.8, J3Ј,P ϭ 6.5 Hz, 1 H, H-3Ј),
6.45 (dd, J1Ј,2Ј ϭ 8.4, J1Ј,2ЈЈ ϭ 5.8 Hz, 1 H, H-1Ј), 8.19 (s, 1 H, H-
2), 8.35 (s, 1 H, H-8) ppm. 13C NMR (CD3OD): δ ϭ 14.4 (CH3 of
palmitoyl), 23.7 (ξ-CH2 of palmitoyl), 26.0 (β-CH2 of palmitoyl),
30.2, 30.3, 30.4, 30.5, 30.66, 30.70, 30.72 (from γ- to µ-CH2 of
palmitoyl), 33.0 (ν-CH2 of palmitoyl), 34.9 (α-CH2 of palmitoyl),
40.5 (d, JCCOP ϭ 3.8 Hz, C-2Ј), 63.6 (C-5Ј), 66.2 (C-1 of glycerol),
67.7 (d, JCOP ϭ 5.5 Hz, C-3 of glycerol), 69.9 (d, JCCOP ϭ 7.6 Hz,
C-2 of glycerol), 77.6 (d, JCOP ϭ 4.3 Hz, C-3Ј), 87.3 (C-1Ј), 88.8
(d, JCCOP ϭ 6.1 Hz, C-4Ј), 121.1 (C-5), 141.7 (C-8), 150.0 (C-4),
153.5 (C-2), 157.5 (C-6), 175.4 (COO) ppm. HRFAB-MS(Ϫ) calcd.
for C29H49N5O9P [M Ϫ H]Ϫ 642.3268, found 642.3260.
Compound 18: 38.9 mg (41% yield from the mixture of 15 and 16).
1H NMR (D2O): δ ϭ 2.19 (s, 3 H, SϪCH3), 3.03 (dd, J5ЈЈ,5Ј
ϭ
Ϫ14.3, J5ЈЈ,4Ј ϭ 6.7 Hz, 1 H, H-5ЈЈ), 3.08 (dd, J5Ј,5ЈЈ ϭ Ϫ14.3,
J5Ј,4Ј ϭ 5.3 Hz, 1 H, H-5Ј), 3.39 (dd, J1a,1b ϭ Ϫ11.8, J1a,2 ϭ 6.1 Hz,
1 H, H-1a of glycerol), 3.45 (dd, partially overlapping with next
signal, J1b,1a ϭ Ϫ11.8, J1b,2 ϭ 4.4 Hz, 1 H, H-1b of glycerol), 3.47
(multiplet partially obscured by previous signal, 1 H, H-3a of gly-
cerol), 3.62 (m, 1 H, H-2 of glycerol), 3.70 (m, 1 H, H-3b of gly-
cerol), 4.43 (ddd, J4Ј,3Ј ϭ 3.7, J4Ј,5Ј ϭ 5.3, J4Ј,5ЈЈ ϭ 6.7 Hz, 1 H, H-
4Ј), 4.61 (dd, J3Ј,2Ј ϭ 5.5, J3Ј,4Ј ϭ 3.7 Hz, 1 H, H-3Ј), 5.33 (ddd,
J2Ј,1Ј ϭ 5.9, J2Ј,3Ј ϭ 5.5, J2Ј,P ϭ 8.8 Hz, 1 H, H-2Ј), 6.28 (d, J1Ј,2Ј
ϭ
1
5.9 Hz, 1 H, H-1Ј), 8.36 (s, 1 H, H-2), 8.48 (s, 1 H, H-8) ppm. H
NMR (TBA salt, (CD3)3COD): δ ϭ 1.11 (t, J ϭ 7.3 Hz, 12 H,
NϪCH2CH2CH2CH3), 1.54 (m, 8 H, NϪCH2CH2CH2CH3), 1.75
(m, 8 H, NϪCH2CH2CH2CH3), 2.20 (s, 3 H, SϪCH3), 2.96 (dd,
J5ЈЈ,5Ј ϭ Ϫ14.1, J5ЈЈ,4Ј ϭ 6.3 Hz, 1 H, H-5ЈЈ), 3.06 (dd, J5Ј,5ЈЈ
ϭ
Ϫ14.1, J5Ј,4Ј 4.8 Hz, H, H-5Ј), 3.36 (br. t, H, N-
ϭ
1
8
CH2CH2CH2CH3), 3.65 (d, J1,2 ϭ 5.9 Hz, 2 H, H-1 of glycerol),
3.81 (ddt, J2,1 ϭ 5.9, J2,3a ϭ 5.0, J2,3b ϭ 4.0 Hz, 1 H, H-2 of gly-
cerol), 3.97 (ddd, J3a,3b ϭ Ϫ11.1, J3a,2 ϭ 5.0, J3a,P ϭ 8.8 Hz, 1 H,
H-3a of glycerol), 4.02 (ddd, J3b,3a ϭ Ϫ11.1, J3b,2 ϭ 4.0, J3b,P
8.4 Hz, 1 H, H-3b of glycerol), 4.37 (ddd, J4Ј,3Ј ϭ 5.4, J4Ј,5Ј ϭ 4.8,
J4Ј,5ЈЈ ϭ 6.3 Hz, 1 H, H-4Ј), 4.78 (m partially obscured by the resid-
3Ј-O-Lysophosphatidyl Derivative of 2Ј-Deoxyguanosine (6): 10.6 mg
ϭ
1
(32% yield from 2). H NMR (CD3OD): δ ϭ 0.90 (t, J ϭ 6.9 Hz,
3H, CH3 of palmitoyl), 1.29 (br. s, 24 H, from γ- to ξ-CH2 of
palmitoyl), 1.59 (m, 2 H, β-CH2 of palmitoyl), 2.33 (t, J ϭ 7.5 Hz,
2 H, α-CH2 of palmitoyl), 2.61 (ddd, J2ЈЈ,2Ј ϭ Ϫ13.8, J2ЈЈ,1Ј ϭ 6.1,
J2ЈЈ,3Ј ϭ 2.6 Hz, 1 H, H-2ЈЈ), 2.81 (ddd, J2Ј,2ЈЈ ϭ Ϫ13.8, J2Ј,1Ј ϭ 7.6,
J2Ј,3Ј ϭ 6.1 Hz, 1 H, H-2Ј), 3.80 (dd, J5ЈЈ,5Ј ϭ Ϫ12.3, J5ЈЈ,4Ј ϭ 3.5 Hz,
1 H, H-5ЈЈ), 3.84 (dd, J5Ј,5ЈЈ ϭ Ϫ12.3, J5Ј,4Ј ϭ 3.5 Hz, 1 H, H-5Ј),
3.90Ϫ4.02 (partially overlapped multiplets, 3 H, H-2 and H-3 of
glycerol), 4.12 (dd, J1a,1b ϭ Ϫ11.3, J1a,2 ϭ 6.1 Hz, 1 H, H-1a of
glycerol), 4.19 (dd, J1b,1a ϭ Ϫ11.3, J1b,2 ϭ 4.6 Hz, 1 H, H-1b of
glycerol), 4.24 (dt, J4Ј,3Ј ϭ 2.7, J4Ј,5Ј ϭ J4Ј,5ЈЈ ϭ 3.5 Hz, 1 H, H-4Ј),
5.02 (dddd, J3Ј,2Ј ϭ 6.1, J3Ј,2ЈЈ ϭ 2.6, J3Ј,4Ј ϭ 2.7, J3Ј,P ϭ 6.7 Hz, 1
H, H-3Ј), 6.27 (dd, J1Ј,2Ј ϭ 7.6, J1Ј,2ЈЈ ϭ 6.1 Hz, 1 H, H-1Ј), 7.94 (s,
1 H, H-8) ppm. 13C NMR (CD3OD): δ ϭ 14.4 (CH3 of palmitoyl),
23.7 (ξ-CH2 of palmitoyl), 26.0 (β-CH2 of palmitoyl), 30.23, 30.36,
30.40, 30.55, 30.67, 30.72, 30.74 (from γ- to µ-CH2 of palmitoyl),
33.0 (ν-CH2 of palmitoyl), 34.9 (α-CH2 of palmitoyl), 40.1 (d,
JCCOP ϭ 3.3 Hz, C-2Ј), 63.6 (C-5Ј), 66.2 (C-1 of glycerol), 67.7 (d,
JCOP ϭ 4.3 Hz, C-3 of glycerol), 69.9 (d, JCCOP ϭ 8.2 Hz, C-2 of
glycerol), 77.5 (d, JCOP ϭ 4.5 Hz, C-3Ј), 86.8 (C-1Ј), 88.5 (d,
ual HOD signal, H-3Ј), 5.22 (ddd, J2Ј,1Ј ϭ 4.3, J2Ј,3Ј ϭ 5.3, J2Ј,P
ϭ
6.3 Hz, 1 H, H-2Ј), 6.37 (d, J1Ј,2Ј ϭ 4.3 Hz, 1 H, H-1Ј), 8.30 (s, 1
H, H-2), 8.55 (s, 1 H, H-8) ppm. 13C NMR (D2O): δ ϭ 18.1
(SϪCH3), 38.6 (C-5Ј), 64.7 (C-1 of glycerol), 69.1 (d, JCOP
ϭ
6.0 Hz, C-3 of glycerol), 73.3 (d, JCCOP ϭ 7.6 Hz, C-2 of glycerol),
74.8 (d, JCCOP ϭ 3.6 Hz, C-3Ј), 79.1 (d, JCOP ϭ 5.2 Hz, C-2Ј), 86.4
(C-4Ј), 89.0 (d, JCCOP ϭ 6.0 Hz, C-1Ј), 121.9 (C-5), 143.5 (C-8),
152.0 (C-4), 155.8 (C-2), 158.5 (C-6) ppm. HRFAB-MS(Ϫ) calcd.
for C14H21N5O8PS [M Ϫ H]Ϫ 450.0848, found 450.0834.
General Procedure for the Enzymatic Synthesis of 3Ј-O-Lysophos-
phatidyl Derivatives of 2Ј-Deoxynucleosides (5؊8): The enzymatic
synthesis and the purification of the title compounds were accomp-
lished according to previous work.[1] Each 3ЈGPdN (50 µmol) was
first converted into the relevant TBA salt by passing it through a
column of Dowex-50 W (TBA form). The aqueous eluate was taken
to dryness in vacuo and the residue was allowed to stand under
reduced pressure over P2O5 overnight. The residue was then dis-
solved in dried tBuOH (20 mL) and the solution was added to-
gether with TFEP (500 µmol) and Lipozyme (40 mg). The suspen-
sion was shaken at 240 rpm for 48 h at 40 °C. Each reaction course
was followed by HPLC monitoring of the rising amount of the
JCCOP ϭ 6.4 Hz, C-4Ј), 139.2 (C-8), 153.3 (C-4), 155.6 (C-2), 160.6
(C-6), 175.4 (COO) ppm, C-5 not detectable. HRFAB-MS(Ϫ)
calcd. for C29H49N5O10P [M Ϫ H]Ϫ 658.3217, found 658.3222.
3Ј-O-Lysophosphotidyl Derivative of 2Ј-Deoxycytidine (7): 12.4 mg
1
relevant 3Ј-O-lysophosphatidyldeoxynucleoside. HPLC analyses, (40% yield from 3). H NMR (CD3OD): δ ϭ 0.90 (t, J ϭ 7.0 Hz,
performed on Lichrospher-100 ODS with a linear gradient of
3 H, CH3 of palmitoyl), 1.28 (br. s, 24 H, from γ- to ξ-CH2 of
CH3CN in 0.1  triethylammonium acetate (pH 7) from 0 to 70% palmitoyl), 1.61 (m, 2 H, β-CH2 of palmitoyl), 2.29 (ddd, J2Ј,2ЈЈ
ϭ
over 30 min at a flow rate of 1.0 mL minϪ1, in each case showed
only one peak other than that of the substrate. The enzyme was
then filtered off and the solvent was evaporated in vacuo. The res-
idue was taken up in a pentane/H2O (1:1) mixture and the aqueous
layer was concentrated in vacuo. The residue containing the acyl-
ated product was then purified by semipreparative HPLC on an
Ultrasphere ODS column, with a linear gradient of CH3CN in 0.1
Ϫ13.8, J2Ј,1Ј ϭ 7.1, J2Ј,3Ј ϭ 6.6 Hz, 1 H, H-2Ј), 2.35 (t, J ϭ 7.5 Hz,
2 H, α-CH2 of palmitoyl), 2.60 (ddd, J2ЈЈ,2Ј ϭ Ϫ13.8, J2ЈЈ,1Ј ϭ 6.0,
J2ЈЈ,3Ј ϭ 3.3 Hz, 1 H, H-2ЈЈ), 3.82 (d, J5Ј,4Ј ϭ 2.9 Hz, 2 H, H-5Ј),
3.87Ϫ3.94 (partially overlapped multiplets, 2 H, H-3 of glycerol),
3.97 (m, 1 H, H-2 of glycerol), 4.11 (dd, J1a,1b ϭ Ϫ11.2, J1a,2
ϭ
5.9 Hz, 1 H, H-1a of glycerol), 4.18 (dd, partially obscured by H-
4Ј signal, J1b,1a ϭ Ϫ11.2, J1b,2 ϭ 4.5 Hz, 1 H, H-1b of glycerol),
Eur. J. Org. Chem. 2002, 3622Ϫ3631
3629

R. Chillemi, D. Aleo, G. Granata, S. Sciuto
FULL PAPER
4.19 (dt, partially overlapping with the previous signal, J4Ј,3Ј ϭ 3.5, JCCOP ϭ 6.8 Hz, C-2 of glycerol), 74.4 (d, JCCOP ϭ 3.0 Hz, C-2Ј),
J4Ј,5Ј ϭ 2.9 Hz, 1 H, H-4Ј), 4.82 (dddd, J3Ј,2Ј ϭ 6.6, J3Ј,2ЈЈ ϭ 3.3, 79.3 (d, JCOP ϭ 4.6 Hz, C-3Ј), 80.2 (d, JCCOP ϭ 5.3 Hz, C-4Ј), 91.0
J3Ј,4Ј ϭ 3.5, J3Ј,P ϭ 7.3 Hz, 1 H, H-3Ј), 6.01 (d, J5,6 ϭ 7.7 Hz, 1 H, (C-1Ј), 126.0 (d, JCCF ϭ 34.0 Hz, C-6), 142.0 (d, JCF ϭ 233.5 Hz,
H-5), 6.26 (dd, J1Ј,2Ј ϭ 7.1, J1Ј,2ЈЈ ϭ 6.0 Hz, 1 H, H-1Ј), 8.23 (d, C-5), 150.9 (C-2), 159.4 (d, JCCF ϭ 25.9 Hz, C-4), 175.4 (COO)
J
6,5 ϭ 7.7 Hz, 1 H, H-6) ppm. 13C NMR (CD3OD): δ ϭ 14.4 (CH3
ppm. HRFAB-MS(Ϫ) calcd. for C28H47FN2O11P [M Ϫ H]Ϫ
of palmitoyl), 23.7 (ξ-CH2 of palmitoyl), 26.0 (β-CH2 of palmitoyl), 637.2902, found 637.2922. Before elemental analysis, a water solu-
30.2, 30.41, 30.45, 30.6, 30.72, 30.75, 30.77 (from γ- to µ-CH2 of tion of chromatographically pure 13 was lyophilized and the re-
palmitoyl), 33.0 (ν-CH2 of palmitoyl), 34.9 (α-CH2 of palmitoyl),
sulting highly hygroscopic powder was allowed to stand overnight
40.9 (d, JCCOP ϭ 3.8 Hz, C-2Ј), 62.4 (C-5Ј), 66.2 (C-1 of glycerol), over P2O5. C28H48FN2O11P·H2O (656.68): calcd. C 51.21, H 7.67,
67.6 (d, JCOP ϭ 5.6 Hz, C-3 of glycerol), 69.9 (d, JCCOP ϭ 7.5 Hz, N 4.27; found C 51.35, H 7.69, N 4.26.
C-2 of glycerol), 76.0 (d, JCOP ϭ 3.4 Hz, C-3Ј), 87.7 (C-1Ј), 88.1
5Ј-O-Lysophosphatidyl Derivative of 5Ј-Deoxy-5Ј-(methylthio)ad-
enosine (19): Compound 17 (TBA salt; 50 µmol) was worked up as
described above for compound 12. After incubation and isolation,
pure compound 19 was obtained by HPLC on Zorbax SB-C18 se-
(d, JCCOP ϭ 6.2 Hz, C-4Ј), 95.4 (C-5), 144.5 (C-6), 153.7 (C-2),
164.6 (C-4), 175.4 (COO) ppm. HRFAB-MS(Ϫ) calcd. for
C28H49N3O10P [M Ϫ H]Ϫ 618.3156, found 618.3163.
3Ј-O-Lysophosphatidyl Derivative of 2Ј-Deoxythymidine (8): 18.4 mg mipreparative column with a linear gradient of CH3CN in 0.1 
1
(58% yield from 4). H NMR (CD3OD): δ ϭ 0.90 (t, J ϭ 7.0 Hz, triethylammonium acetate (pH 7.0) from 0 to 80% in 40 min, at
3 H, CH3 of palmitoyl), 1.28 (br. s, 24 H, from γ- to ξ-CH2 of the flow rate of 3.5 mL minϪ1. 31.2 mg (90% yield from 17). 1H
palmitoyl), 1.61 (tt, Jβ,α ϭ 7.5, Jβ,γ ϭ 7.0 Hz, 2 H, β-CH2 of palmi-
toyl), 1.88 (d, J ϭ 1.1 Hz, 3 H, CH3 of thymine), 2.29 (ddd, J2Ј,2ЈЈ
NMR (CD3OD): δ ϭ 0.90 (t, J ϭ 7.0 Hz, 3 H, CH3 of palmitoyl),
1.27 and 1.29 (br. singlets, 24 H altogether, from γ- to ξ-CH2 of
ϭ
Ϫ13.8, J2Ј,1Ј ϭ 7.9, J2Ј,3Ј ϭ 6.0 Hz, 1 H, H-2Ј), 2.35 (t, J ϭ 7.5 Hz, palmitoyl), 1.60 (m, 2 H, β-CH2 of palmitoyl), 2.11 (s, 3 H,
2 H, α-CH2 of palmitoyl), 2.48 (ddd, J2ЈЈ,2Ј ϭ Ϫ13.8, J2ЈЈ,1Ј ϭ 5.9, SϪCH3), 2.34 (t, J ϭ 7.5 Hz, 2 H, α-CH2 of palmitoyl), 2.95 (dd,
J2ЈЈ,3Ј ϭ 2.2 Hz, 1 H, H-2ЈЈ), 3.81 (d, J5Ј,4Ј ϭ 2.7 Hz, 2 H, H-5Ј), J5ЈЈ,5Ј ϭ Ϫ14.2, J5ЈЈ,4Ј ϭ 6.6 Hz, 1 H, H-5ЈЈ), 3.02 (dd, J5Ј,5ЈЈ
ϭ
3.87Ϫ4.00 (partially overlapped multiplets, 3 H, H-2 and H-3 of
glycerol), 4.11 (dd, J1a,1b ϭ Ϫ11.4 Hz, J1a,2 ϭ 5.7 Hz, 1 H, H-1a
Ϫ14.2, J5Ј,4Ј ϭ 4.5 Hz, 1 H, H-5Ј), 3.97Ϫ4.03 (overlapped mul-
tiplets, 3 H, H-2 and H-3 of glycerol), 4.12 (dd, J1a,1b ϭ Ϫ11.4,
of glycerol), 4.14 (dt, J4Ј,3Ј ϭ 2.9, J4Ј,5Ј ϭ 2.7 Hz, 1 H, H-4Ј), 4.18 J1a,2 ϭ 5.5 Hz, 1 H, H-1a of glycerol), 4.19 (dd, J1b,1a ϭ Ϫ11.4,
(dd, J1b,1a ϭ Ϫ11.4, J1b,2 ϭ 4.3 Hz, 1 H, H-1b of glycerol), 6.30 J1b,2 ϭ 4.4 Hz, 1 H, H-1b of glycerol), 4.45 (ddd, J4Ј,3Ј ϭ 4.3,
(dd, J1Ј,2Ј ϭ 7.9, J1Ј,2ЈЈ ϭ 5.9 Hz, 1 H, H-1Ј), 7.86 (q, J ϭ 1.1 Hz,
1 H, H-6) ppm, the H-3Ј resonance is obscured by the residual
J4Ј,5Ј ϭ 4.5, J4Ј,5ЈЈ ϭ 6.6 Hz, 1 H, H-4Ј), 4.80 (ddd, J3Ј,2Ј ϭ 5.2,
J3Ј,4Ј ϭ 4.3, J3Ј,P ϭ 7.4 Hz, 1 H, H-3Ј), 4.96 (dd, J2Ј,1Ј ϭ 5.4, J2Ј,3Ј
ϭ
HOD signal. 13C NMR (CD3OD): δ ϭ 12.5 (CH3 of thymine), 5.2 Hz, 1 H, H-2Ј), 6.06 (d, J1Ј,2Ј ϭ 5.4 Hz, 1 H, H-1Ј), 8.22 (s, 1
14.5 (CH3 of palmitoyl), 23.8 (ξ-CH2 of palmitoyl), 26.0 (β-CH2 of
palmitoyl), 30.3, 30.5, 30.7, 30.8 (from γ- to µ-CH2 of palmitoyl),
H, H-2), 8.36 (s, 1 H, H-8) ppm. 13C NMR (CD3OD): δ ϭ 14.4
(CH3 of palmitoyl), 16.7 (SϪCH3), 23.7 (ξ-CH2 of palmitoyl), 26.0
33.1 (ν-CH2 of palmitoyl), 34.9 (α-CH2 of palmitoyl), 40.1 (d, (β-CH2 of palmitoyl), 30.22, 30.32, 30.40, 30.46, 30.60, 30.75, 30.77
JCCOP ϭ 3.5 Hz, C-2Ј), 62.8 (C-5Ј), 66.1 (C-1 of glycerol), 67.6 (d, (from γ- to µ-CH2 of palmitoyl), 33.1 (ν-CH2 of palmitoyl), 34.9
JCOP ϭ 5.6 Hz, C-3 of glycerol), 69.8 (d, JCCOP ϭ 8.3 Hz, C-2 of (α-CH2 of palmitoyl), 37.4 (C-5Ј), 66.2 (C-1 of glycerol), 67.9 (d,
glycerol), 76.8 (d, JCOP ϭ 4.9 Hz, C-3Ј), 86.2 (C-1Ј), 87.7 (d, JCOP ϭ 5.6 Hz, C-3 of glycerol), 69.9 (d, JCCOP ϭ 8.0 Hz, C-2 of
CCOP ϭ 5.7 Hz, C-4Ј), 111.6 (C-5), 138.1 (C-6), 151.5 (C-2), 168.5 glycerol), 74.5 (d, JCCOP ϭ 3.6 Hz, C-2Ј), 77.6 (d, JCOP ϭ 5.2 Hz,
J
(C-4), 175. 4 (COO) pp m. HR FA B- MS ( Ϫ ) c a l c d . fo r
C-3Ј), 84.8 (d, JCCOP ϭ 5.2 Hz, C-4Ј), 89.6 (C-1Ј), 120.6 (C-5),
141.4 (C-8), 150.9 (C-4), 153.9 (C-2), 157.3 (C-6), 175.4 (COO)
ppm. HRFAB-MS(Ϫ) calcd. for C30H51N5O9PS [M Ϫ H]Ϫ
688.3145, found 688.3151. For elemental analysis purpose, pure 19
from preparative HPLC was lyophilized and the resulting dried
p owd e r wa s a l l owe d t o s t a n d ov e r n i g h t ove r P 2 O 5 .
C30H52N5O9PS·H2O (707.82): calcd. C 50.91, H 7.69, N 9.89;
found C 51.01, H 7.72, N 9.91.
C29H50N2O11P [M Ϫ H]Ϫ 633.3152, found 633.3164.
3Ј-O-Lysophosphatidyl Derivative of 5Ј-Deoxy-5-fluorouridine (13):
Compound 12 (TBA salt; 50 µmol) was allowed to stand under
reduced pressure over P2O5 overnight, was then dissolved in dried
tBuOH (80 mL), and the solution was added together with TFEP
(2.0 mmol) and Lipozyme (440 mg). The suspension was shaken at
240 rpm for 5 days at 38 °C. The analytical monitoring of the reac-
tion course, and the isolation and the purification of the acylated
product were accomplished as described for compounds 5Ϫ8.
2Ј-O-Lysophosphatidyl Derivative of 5Ј-Deoxy-5Ј-(methylthio)ad-
enosine (20): To obtain the title compound, 18 (TBA salt; 50 µmol)
28.7 mg (90% yield from 12). 1H NMR (CD3OD): δ ϭ 0.90 (t, J ϭ was treated by the same procedure as described above for the relev-
6.9 Hz, 3 H, CH3 of palmitoyl), 1.29 (br. s, 24 H, from γ- to ξ-CH2
of palmitoyl), 1.43 (d, J5Ј,4Ј ϭ 6.3 Hz, 3 H, H-5Ј), 1.61 (tt, Jβ,α
7.5, Jβ,γ ϭ 7.0 Hz, 2 H, β-CH2 of palmitoyl), 2.35 (t, J ϭ 7.5 Hz,
ant 3Ј- isomer (17). 28.9 mg (84% yield from 18). 1H NMR
(CD3OD): δ ϭ 0.90 (t, J ϭ 7.0 Hz, 3 H, CH3 of palmitoyl), 1.28
and 1.29 (br. singlets, 24 H altogether, from γ- to ξ-CH2 of palmi-
ϭ
2 H, α-CH2 of palmitoyl), 3.92Ϫ4.00 (overlapped multiplets, 3 H, toyl), 1.60 (quintet, J ϭ 7.5 Hz, 2 H, β-CH2 of palmitoyl), 2.10 (s,
H-2 and H-3 of glycerol), 4.11 (dd, J1a,1b ϭ Ϫ11.4, J1a,2 ϭ 5.6 Hz, 3 H, SϪCH3), 2.33 (t, J ϭ 7.5 Hz, 2 H, α-CH2 of palmitoyl), 2.87
1 H, H-1a of glycerol), 4.18 (dd, J1b,1a ϭ Ϫ11.4, J1b,2 ϭ 4.2 Hz, 1
H, H-1b of glycerol), 4.23 (dq, J4Ј,3Ј ϭ 5.4, J4Ј,5Ј ϭ 6.3 Hz, 1 H, H-
(dd, J5ЈЈ,5Ј ϭ Ϫ14.1, J5ЈЈ,4Ј ϭ 6.3 Hz, 1 H, H-5ЈЈ), 2.96 (dd, J5Ј,5ЈЈ
Ϫ14.1, J5Ј,4Ј ϭ 5.4 Hz, 1 H, H-5Ј), 3.72 (m, 1 H, H-3a of glycerol),
ϭ
4Ј), 4.27 (ddd, J3Ј,2Ј ϭ 5.1, J3Ј,4Ј ϭ 5.4, J3Ј,P ϭ 6.7 Hz, 1 H, H-3Ј), 3.81Ϫ3.87 (overlapped multiplets, 2 H, H-2 and H-3b of glycerol),
4.33 (dd, J2Ј,1Ј ϭ 4.6, J2Ј,3Ј ϭ 5.1 Hz, 1 H, H-2Ј), 5.85 (dd, J1Ј,2Ј 4.01 (dd, J1a,1b ϭ Ϫ11.5, J1a,2 ϭ 5.8 Hz, 1 H, H-1a of glycerol),
ϭ
4.6, J1Ј,F ϭ 1.0 Hz, 1 H, H-1Ј), 7.76 (d, J6,F ϭ 6.5 Hz, 1 H, H-6) 4.05 (dd, J1b,1a ϭ Ϫ11.5, J1b,2 ϭ 4.4 Hz, 1 H, H-1b of glycerol),
ppm. 13C NMR (CD3OD): δ ϭ 14.4 (CH3 of palmitoyl), 18.9 (C-
5Ј), 23.7 (ξ-CH2 of palmitoyl), 26.0 (β-CH2 of palmitoyl), 30.23,
30.40, 30.44, 30.6, 30.71, 30.74, 30.76 (from γ- to µ-CH2 of palmi-
4.23 (ddd, J4Ј,3Ј ϭ 4.7, J4Ј,5Ј ϭ 5.4, J4Ј,5ЈЈ ϭ 6.3 Hz, 1 H, H-4Ј), 4.60
(dd, J3Ј,2Ј ϭ 5.2, J3Ј,4Ј ϭ 4.7 Hz, 1 H, H-3Ј), 5.28 (ddd, J2Ј,1Ј ϭ 4.9,
J2Ј,3Ј ϭ 5.2, J2Ј,P ϭ 7.5 Hz, 1 H, H-2Ј), 6.20 (d, J1Ј,2Ј ϭ 4.9 Hz, 1
toyl), 33.0 (ν-CH2 of palmitoyl), 34.9 (α-CH2 of palmitoyl), 66.2 H, H-1Ј), 8.22 (s, 1 H, H-2), 8.36 (s, 1 H, H-8) ppm. 13C NMR
(C-1 of glycerol), 67.8 (d, JCOP ϭ 4.3 Hz, C-3 of glycerol), 69.9 (d,
(CD3OD): δ ϭ 14.4 (CH3 of palmitoyl), 16.5 (SϪCH3), 23.7 (ξ-
Eur. J. Org. Chem. 2002, 3622Ϫ3631
3630

Synthesis of 3Ј(2Ј)-O-Lysophosphatidylnucleosides
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of palmitoyl), 34.9 (α-CH2 of palmitoyl), 37.4 (C-5Ј), 66.1 (C-1 of
glycerol), 67.8 (d, JCOP ϭ 5.6 Hz, C-3 of glycerol), 69.7 (d, JCCOP ϭ
7.7 Hz, C-2 of glycerol), 73.7 (d, JCCOP ϭ 3.0 Hz, C-3Ј), 78.1 (d,
JCOP ϭ 5.1 Hz, C-2Ј), 85.5 (C-4Ј), 88.7 (d, JCCOP ϭ 6.7 Hz, C-1Ј),
142.0 (C-8), 151.1 (C-4), 153.5 (C-2), 157.0 (C-6), 175.4 (COO)
ppm, C-5 not detectable. HRFAB-MS(Ϫ) calcd. for C30H51N5O9PS
[M Ϫ H]Ϫ 688.3145, found 688.3155.
[8]
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Acknowledgments
The authors wish to thank Dr. G. Nicolosi (CNR of Valverde,
Italy) for a generous gift of Lipozyme and Dr. P. Fagone (Depart-
ment of Chemistry, University of Southampton, England) for per-
forming the monolayer experiments. This work was supported by
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3631