Unexpected side product formed during LDA-induced phosphonylation of uridine

Article abstract of DOI:10.1246/cl.170005

The LDA-induced coupling of 2′,3′,5′-O-protected uridine with diethyl chlorophosphate, during the synthesis of 6-phosphonouridine, is accompanied by the formation of an unexpected side product. LDA adds slowly to the C4 position of the 2′,3′,5′-O-protected uridine after the initial reaction with diethyl chlorophosphate. The presence of the phosphorochloridate facilitates the side reaction. This observation accounts for the previously reported low yield when conducting the coupling reaction for longer durations and suggests a new route for the synthesis of N-alkylated 6-phosphonocytidine analogues.

Full text of DOI:10.1246/cl.170005

Received: January 5, 2017 | Accepted: February 3, 2017 | Web Released: February 10, 2017
CL-170005
Unexpected Side Product Formed during LDA-induced Phosphonylation of Uridine
Palash Bhar1 and Stephen L. Bearne*1,2
1Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada
2Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada
(E-mail: sbearne@dal.ca)
O
N
O
N
O
N
The LDA-induced coupling of 2¤,3¤,5¤-O-protected uridine
with diethyl chlorophosphate, during the synthesis of 6-
phosphonouridine, is accompanied by the formation of an
unexpected side product. LDA adds slowly to the C4 position of
the 2¤,3¤,5¤-O-protected uridine after the initial reaction with
diethyl chlorophosphate. The presence of the phosphorochlor-
idate facilitates the side reaction. This observation accounts for
the previously reported low yield when conducting the coupling
reaction for longer durations and suggests a new route for the
synthesis of N-alkylated 6-phosphonocytidine analogues.
HN
HN
HN
THFO
O
O
O
O
O
HO
HO
1
2
O
99%
98%
OH OH
O
O
O
O
1
2
3
3a
3b
3c
O
N
N
OMe
N
HN
N
N
O
P
O
P
O
P
OEt
OEt
OEt
THFO
THFO
THFO
O
N
O
O
O
Keywords: LDA-adduct
| LDA-induced phosphonylation |
O
O
OEt
OEt
OEt
6-Phosphonouridine
O
O
O
O
O
O
Although there is a dearth of syntheses reported for
pyrimidine derivatives bearing a phosphonate group directly
attached to the pyrimidine ring,1-10 the syntheses of C6-
phosphonylated uracil to yield the analogue of orotic acid1 and
its corresponding nucleoside5 and 5¤-monophosphate11 have
been described. Indeed, 6-phosphonouridine-5¤-monophosphate
was a modest competitive inhibitor of orotidine-5¤-monophos-
phate (OMP) decarboxylase from E. coli.11 The synthesis of this
OMP analogue was based on the original reports of Honjo
et al.,5 who employed lithium N,N-diisopropylamide (LDA) to
deprotonate the C6 position of the protected 2¤,3¤-O-isopropyl-
ideneuridine-5¤-O-(tetrahydro-2-furanyl) derivative of uridine.
Honjo and co-workers noted that after conducting the reaction
for 6 h in the presence of LDA followed by quenching, removal
of the 5¤-O-(tetrahydro-2-furanyl) protecting group, and purifi-
cation by silica gel chromatography, the yield was 51%. Upon
optimization of the phosphonylation reaction, Thirumalairajan
et al.11 found that the reaction was complete within 1 h with 70%
yield; however, when the reaction was left for longer times, e.g.,
as reported by Honjo et al., significant accumulation of a side
product was observed. Herein, we report the identity of this side
product and the mechanism for its formation.
The key step for the synthesis of 6-phosphonouridine is
the installation of a phosphonyl group at the C6 position of
compound 3, requiring the removal of the C6 vinylic proton
using the strong non-nucleophilic base LDA prior to the reaction
with diethyl chlorophosphate.5 Upon treatment of the 2¤,3¤,5¤-O-
protected uridine 3 (Scheme 1)5,11 with fresh LDA (generated
in situ) and diethyl chlorophosphate, we observed (TLC, 5%
acetone in CH2Cl2) the formation of the target product 4 after
stirring for 1 h at ¹78 °C. However, not all of the reactant 3
was consumed during this time period. Stirring the reaction for
an additional 5 h, as per Honjo et al.,5 gave a side product
(Rf = 0.64, similar to reactant) that was more nonpolar than
the desired product 4 (Rf = 0.32). Upon warming the reaction
mixture to 0 °C and stirring for longer durations, the amount of
this side product increased relative to the amount of 4. Honjo
4
7
10
4
76%
4
88%
4 46%
O
N
N
OMe
HN
N
N
O
O
O
OEt
OEt
OEt
HO
HO
HO
O
N
P
O
O
P
O
N
P
O
O
OEt
OEt
OEt
O
O
O
O
O
O
5
8
11
5
83%
5
82%
5
68%
O
N
N
O
HN
N
HN
O
O
O
OEt
OEt
OEt
HO
HO
HO
O
O
N
P
O
P
O
N
P
O
O
OEt
OEt
OEt
OH OH
OH OH
OH OH
6
9
6
Scheme 1. Synthesis of 6-(diethyl phosphonyl)uridine derivatives
modified at C4. (1) 2,2-Dimethoxypropane, PTSA, acetone, reflux,
1 h; (2) DHF, PPTS, CH2Cl2, rt, 1 h; (3a) (i) LDA, ¹78 °C, THF, 1 h,
(ii) (EtO)2P(O)Cl, THF, ¹78 °C, 1 h; (3b) (i) LDA, ¹78 °C, THF, 1 h,
(ii) (EtO)2P(O)Cl, THF, ¹78 °C, 6-20 h; (3c) (i) LDA, ¹78 °C, THF,
1 h, (ii) (EtO)2P(O)Cl, THF, ¹78 °C, 6 h, (iii) Excess MeOH, 0 °C,
10 min; (4) PPTS, EtOH, rt, 1 h; (5) 90% Aq. TFA, rt, 30-45 min.
et al.5 also observed this product and suggested that it was the
unreacted starting material. Consequently, we sought to charac-
terize the side product 8 after THF-deprotection.
The 31P NMR spectrum of 8 revealed that the reaction with
the diethyl chlorophosphate had indeed occurred and that the
chemical shift (7.13 ppm) and splitting pattern were very similar
to that observed for 5 (see Supporting Information), indicating
that the phosphorus was in a chemical environment similar to that
of 5. Both the H and 13C NMR spectra showed that the C6-H
1
Chem. Lett. 2017, 46, 609–611 | doi:10.1246/cl.170005
© 2017 The Chemical Society of Japan | 609

was absent in 8 and that a new doublet appeared at 144.58 ppm
in the 13C NMR spectrum with a J value of 191.2 ppm. These
NMR results are consistent with the presence of a diethyl
phosphonate unit at C6. The chemical shift and coupling constant
data from the 1H and 13C NMR spectra confirmed that the C5-H
was present and exhibited long range coupling with the 31P. The
13C NMR spectrum contained three new signals and a DEPT-135
experiment revealed that these arose from CH and CH3. The
1H NMR spectrum also revealed that, apart from the expected
peaks, there were two additional CH and a pair of two CH3
groups present in the molecule. NMR spectral data, chromato-
graphic behavior, and mass spectrometry results all suggested
that the side product was diethyl 4-N,N-(diisopropylamino)-2¤,3¤-
O-isopropylidene-uridine-6-phosphonate (8), formed from the
addition of LDA at the C4 position of the pyrimidine ring
(Scheme 1).
Time-course monitoring of the LDA-induced phosphonyl-
ation reaction using 31P NMR (Figure 1) revealed that the
formation of 4 (5.76 ppm) occurs within the first 10 min of the
addition of diethyl chlorophosphate and is complete after stirring
for 1 h at ¹78 °C. However, no formation of 7 was observed
during this initial time period. Upon stirring the reaction mixture
for longer durations, 31P NMR analysis revealed that the
formation of 7 (6.82 ppm) occurred after 6 h and was complete
by about 20 h. During this time period, the amount of 4
diminished markedly (Figure 1).
was added to a stirred solution of 3 and LDA. Only the
formation of the 6-methyl derivative of 5¤-O-(tetrahydro-2-
furanyl)-2¤,3¤-O-isopropylideneuridine (21) was detected using
both 1H and 13C NMR spectroscopy. These observations suggest
that diethyl chlorophosphate is required for the formation of the
side product.
To assess whether the addition of MeOH would compete
with the addition of LDA, an excess of methanol (in place of
H2O) was added to quench the reaction mixture of diethyl
chlorophosphate in a solution of 3 and LDA at ¹78 °C. TLC
analysis indicated the formation of a compound (Rf = 0.56, 5%
acetone in CH2Cl2) that was more polar than 7 (cf. Rf = 0.64),
but less polar than 4 (cf. Rf = 0.32). 31P NMR analysis of the
quenched reaction mixture following work-up revealed the
presence of a signal at 4.82 ppm (different from both 4 and 7).
Structural characterization of the isolated compound revealed
that while the phosphonate is still present at C6 (JC6-P = 191 Hz
in 13C NMR spectrum), the methoxy group was present at the C4
position of the uridine ring (i.e., 54.97 ppm corresponding
to OCH3 in the 13C NMR spectrum) rather than LDA. This
observation clearly indicates that after addition of the phospho-
nate group at C6, the C4 site is activated by the phosphoro-
chloridate. Furthermore, the more reactive nucleophile (meth-
oxide) readily outcompetes LDA for the reaction at the C4
position.
These observations suggested to us that the diethyl chloro-
phosphate initially reacted with the carbanion at C6 of 3, giving
rise to 4; however, the excess amount of LDA from the medium
slowly reacted at the C4 position of 4 over time to yield 7. This
expectation is consistent with the non-nucleophilic nature of
LDA arising from the steric hindrance of the two isopropyl
groups.
To determine whether the N,N-diisopropylamine adduct
formed via the addition of LDA to 3 in the absence of diethyl
chlorophosphate, aliquots were withdrawn at various times from
the stirred solution of 3 in LDA at ¹78 °C and quenched by the
addition of H2O. At no point was the N,N-diisopropylamine
adduct detected using either 1H and 13C NMR spectroscopy.
Similarly, in place of diethyl chlorophosphate, methyl iodide
H
O
O
O
Li
N
Li
N
HN
N
N
Li
O
N
R
H
O
N
R
O
N
2nd equiv.
R
14
13
3
O
P
O
P
Cl
O
P
OEt
O
O
O
Li
Li
EtO
OEt
OEt
Cl
2
nd equiv.
N
N
N
EtO
(EtO)2P(O)Cl
O
P
O
P
OEt
OEt
OEt
O
N
R
O
N
O
N
R
Li
OEt
OEt
R
15
16
17
H2O
MeOH
MeI
excess
LDA
O
Li
O
Li
O
HN
P
P
H2O
MeOH
OEt
OEt
O
O
O
N
R
CH3
OEt
OEt
N
N
O
P
O
P
OEt
OEt
21
O
N
R
O
N
R
OEt
OEt
19
20
Li
O
N
O
N
OMe
P
OEt
O
OEt
HN
N
N
O
P
O
O
N
OEt
OEt
OEt
O
P
O
N
R
O
N
R
P
O
N
R
P
OEt
O
N
OEt
OEt
OEt
OEt
R
18
7
4
10
THFO
O
R =
O
O
Figure 1. 31P NMR spectra showing the time-course monitoring of
the LDA-induced phosphonylation of 3. The signals at 5.76 and
6.82 ppm correspond to 31P present in 4 and 7, respectively.
Scheme 2. Proposed mechanisms for formation of the water (4),
LDA (7), and methanol (10) adducts during the LDA-promoted
phosphonylation reaction.
610 | Chem. Lett. 2017, 46, 609–611 | doi:10.1246/cl.170005
© 2017 The Chemical Society of Japan

Based on these observations, we propose the mechanism
shown in Scheme 2 to account for the formation of the LDA
addition product. Initially, phosphonylation at C6 occurs after
deprotonation of the 2¤,3¤,5¤-O-protected uridine by LDA to
yield 16. Subsequently, the reaction likely proceeds via a
phosphorylated intermediate, similar to those formed when
phosphorochloridates are used to activate the O4 positions of
uridine for amination.12-15 Hence, the reaction of 16 with a
second equivalent of diethyl chlorophosphate leads to phospho-
rylation of the C4 oxygen to give intermediate 17. This reactive
phosphate intermediate can slowly react with LDA, despite
its low nucleophilicity,16 to furnish 7, or, when quenched with
methanol or water, yield the 4-methoxy derivative 10 or the
target product 4, respectively. Syntheses of alkylated cytosine
derivatives from uridine are reported in the literature. Typically,
transformations involve activation at C4 position of uridine
either as 4-thiouridine17-20 or via the transformation of the 4-oxo
group to its corresponding enol-form bearing a good leaving.21
In summary, during the LDA-facilitated installation of a
phosphonate group at the C6 position of a uridine ring, the
formation of a 4-N,N-(diisopropylamine) adduct of 6-phospho-
nouridine occurred. This observation highlights the need for
kinetic control when conducting LDA-mediated reactions with
uridine derivatives in the presence of activating reagents such as
phosphorochloridates. Furthermore, our observation suggests a
new route for the synthesis of 4-N-alkylated 6-phosphonocyti-
dine analogues, a relatively unexplored class of nucleoside
analogues. Indeed, 4-N,N-(dialkyl) derivatives of cytidine have
been shown to inhibit ribonuclease22 and nucleoside transport
systems,23,24 as well as reduce the silencing activity in siRNA-
mediated gene silencing, making such modified base useful as
negative microRNA controls.25
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References and Notes
1
M. H. Maguire, R. K. Ralph, G. Shaw, J. Chem. Soc. 1958,
2299.
Chem. Lett. 2017, 46, 609–611 | doi:10.1246/cl.170005
© 2017 The Chemical Society of Japan | 611