Hydrolytic reactions of thymidine 5′-O-phenyl-N- alkylphosplioramidates, models of nucleoside 5′-monophosphate prodrugs

Article abstract of DOI:10.1002/chem.200700623

To obtain detailed data on the kinetics of hydrolytic reactions of triester-like nucleoside 5′-O-aryl-N-alkylphosphoramidates, potential prodrugs of antiviral nucleoside monophosphates, the hydrolysis of diastereomeric (R_p/S_p) thymidine 5′-{O-phenyl-N- [(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate) (3), a phosphoramidate derived from the methyl ester of L-alanine, has been followed by reversed-phase HPLC over the range from H₀ = 0 to pH 8 at 90°C. According to the time-dependent product distributions, the hydrolysis of 3 proceeds at pH <4 by two parallel routes, namely by nucleophilic displacement of the alaninyl ester moiety by a water molecule and by hydrolysis of the carboxylic ester linkage that allows intramolecular attack of the carboxy group on the phosphorus atom, thereby resulting in the departure of either thymidine or phenol without marked accumulation of any intermediates. Both routes represent about half of the overall disappearance of 3. The departure of phenol eventually leads to the formation of thymidine 5′-phosphate. At pH > 5, the predominant reaction is hydrolysis of the carboxylic ester linkage followed by intramolecular displacement of a phenoxide ion by the carboxylate ion and hydrolysis of the resulting cyclic mixed anhydride into an acyclic diester-like thymidine 5′-phosphoramidate. The latter product accumulated quantitatively without any indication of further decomposition. Hydroxide-ioncatalyzed P-OPh bond cleavage of the starting material 3 occurred as a side reaction. Comparative measurements with thymidine 5′-{N-[(1S)-2- oxo-2-methoxy-1-methylethyl]phosphoramidate( (4) revealed that, under acidic conditions, this diester-like compound is hydrolyzed by P-N bond cleavage three orders of magnitude more rapidly than the triester-like 3. At pH > 5, the stability order is reversed, with 3 being hydrolyzed six times as rapidly as 4. Mechanisms of the partial reactions are discussed.

Full text of DOI:10.1002/chem.200700623

FULL PAPER
DOI: 10.1002/chem.200700623
Hydrolytic Reactions of Thymidine 5’-O-Phenyl-N-Alkylphosphoramidates,
Models of Nucleoside 5’-Monophosphate Prodrugs
Mikko Ora,* Jarno Ojanperä, and Harri Lçnnberg[a]
Abstract: To obtain detailed data on
the kinetics of hydrolytic reactions of
triester-like nucleoside 5’-O-aryl-N-al-
kylphosphoramidates, potential pro-
drugs of antiviral nucleoside mono-
phosphates, the hydrolysis of diastereo-
meric (RP/SP) thymidine 5’-{O-phenyl-
N-[(1S)-2-oxo-2-methoxy-1-methyl-
molecular attack of the carboxy group
on the phosphorus atom, thereby re-
sulting in the departure of either thy-
midine or phenol without marked accu-
mulation of any intermediates.Both
routes represent about half of the over-
all disappearance of 3.The departure
of phenol eventually leads to the for-
mation of thymidine 5’-phosphate.At
pH>5, the predominant reaction is hy-
drolysis of the carboxylic ester linkage
followed by intramolecular displace-
ment of a phenoxide ion by the carbox-
ylate ion and hydrolysis of the resulting
cyclic mixed anhydride into an acyclic
diester-like thymidine 5’-phosphorami-
date.The latter product accumulated
quantitatively without any indication of
further decomposition.Hydroxide-ion-
À
catalyzed P OPh bond cleavage of the
starting material 3 occurred as a side
reaction.Comparative measurements
with thymidine 5’-{N-[(1S)-2-oxo-2-me-
thoxy-1-methylethyl]phosphoramidate}
(4) revealed that, under acidic condi-
tions, this diester-like compound is hy-
ethyl]phosphoramidate} (3),
a phos-
phoramidate derived from the methyl
ester of l-alanine, has been followed
by reversed-phase HPLC over the
range from H0 =0 to pH 8 at 908C.Ac-
cording to the time-dependent product
distributions, the hydrolysis of 3 pro-
ceeds at pH<4 by two parallel routes,
namely by nucleophilic displacement of
the alaninyl ester moiety by a water
molecule and by hydrolysis of the car-
boxylic ester linkage that allows intra-
À
drolyzed by P N bond cleavage three
orders of magnitude more rapidly than
the triester-like 3.At pH >5, the stabil-
ity order is reversed, with 3 being hy-
drolyzed six times as rapidly as 4.
Mechanisms of the partial reactions are
discussed.
Keywords: hydrolysis
·
kinetics
·
nucleotides
prodrugs
· phosphoramidates ·
Introduction
prodrug has shown higher antiviral potency on cell lines
than the parent nucleoside analogues.[4] It has been suggest-
ed that the increased potency results from enhanced cellular
uptake of the prodrug and subsequent intracellular release
of the monophosphate.[9] Accordingly, the rate-limiting con-
version of the nucleoside analogue into its monophosphate
by thymidine kinase is avoided.The a-amino acid moiety
appears to be essential, since phosphoramidates derived
from simple alkylamines or b-amino acids do not show anti-
viral activity.[10] The release of the monophosphate probably
proceeds by intermediate formation of l-alaninyl phosphor-
amidate 2.[11] Either release of phenol by chemical hydroly-
sis is followed by enzymatic cleavage of the alaninyl ester
linkage, or these two processes take place in reverse
order.[4,9,12] Unfortunately, the kinetics of the chemical hy-
drolysis of nucleoside O-aryl-N-alkylphosphoramidates have
not previously been studied in such a detailed manner that
mechanistic conclusions were possible.The present work is
aimed at filling this gap.The hydrolysis of diastereomeric
The delivery of antiviral nucleoside analogues as appropri-
ately protected monophosphate prodrugs has attracted in-
creasing interest after approval of the bis(isopropoxycarbo-
nyloxymethyl) esters of two acyclic nucleoside phospho-
nates, namely 9-[2-(phosphonomethoxy)ethyl]adenine and
(R)-9-[2-(phosphonomethoxy)propyl]adenine, for clinical
use.[1] Phosphoramidate pronucleotides constitute one class
of such prodrug candidates.[2,3] In particular, aryl esters of
nucleoside 5’-phosphoramidates derived from a-amino acid
esters, such as 1, have received attention.[4–8] This kind of
[a] Dr.M.Ora, J.Ojanperä, Prof.Dr.H.Lçnnberg
Department of Chemistry
University of Turku
20014 Turku (Finland)
Fax : (+358)2-333-6700
E-mail: mikora@utu.fi
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to assign the absolute configu-
ration of the slower and faster
migrating diastereomers as RP
or SP.Thymidine 5 ’-(O-phenyl-
N-isopropylphosphoramidate)
(5) was prepared similarly
from 6 and diisopropylamine
and was used in kinetic meas-
urements as a mixture of the
RP and SP diastereomers.
Thymidine
5’-{N-[(1S)-2-
oxo-2-methoxy-1-methylethyl]-
phosphoramidate} (4) was pre-
pared by the methodology de-
scribed by Zhao and co-work-
ers.[13] Accordingly, thymidine
was first converted into the flu-
oren-9-ylmethyl ester of thymi-
(RP/SP) thymidine 5’-{O-phenyl-N-[(1S)-2-oxo-2-methoxy-1-
methylethyl]phosphoramidate} (3) has been studied over a
wide pH range (from H0 =0 to pH 8) at 908C by analyzing
the composition of aliquots withdrawn from the reaction
mixture at appropriate time intervals by reversed-phase
(RP) HPLC.Comparative
dine 5’-(H-phosphonate) (7) by H-phosphonylation with flu-
oren-9-ylmethyl phenyl H-phosphonate and the product was
subjected to oxidative amination with l-alanine methyl ester
(Scheme 2).Finally, the fluoren-9-ylmethyl group was re-
moved by treatment with piperidine in CH2Cl2.
measurements have been car-
ried out with thymidine 5’-{N-
[(1S)-2-oxo-2-methoxy-1-meth-
ylethyl]phosphoramidate} (4)
and thymidine 5’-(O-phenyl-N-
isopropylphosphoramidate)
(5).
Results
Preparation of thymidine 5’-
Scheme 2.Preparation of thymidine 5 ’-{N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate} (4).Fm:
fluoren-9-ylmethyl.
phosphoramidates: Thymidine
5’-{O-phenyl-N-[(1S)-2-oxo-2-
methoxy-1-methylethyl]phos-
phoramidate} (3) was obtained
as a mixture of RP and SP diastereomers by oxidative amina-
tion of thymidine 5’-(H-phosphonate) phenyl ester (6) with
l-alanine methyl ester (Scheme 1), a method previously
used for the synthesis of nucleoside phosphoramidate die-
sters[13] and for the synthesis of oligoribonucleotide phos-
phoramidates on a solid support.[14] The diastereomers were
separated by reversed-phase HPLC.No attempt was made
Product distribution: The cleavage of the RP and SP diaste-
reomers of thymidine 5’-{O-phenyl-N-[(1S)-2-oxo-2-me-
thoxy-1-methylethyl]phosphoramidate} (3) was followed
over a wide pH range (from H0 =0 to pH 8) at 908C by ana-
lyzing the composition of aliquots withdrawn from the reac-
tion mixture at appropriate time intervals by RP HPLC.
The products were characterized either by spiking with au-
thentic samples or by mass
spectrometric analysis (HPLC/
ESI-MS).
The time-dependent product
distributions obtained with the
faster migrating diastereomer
of 3 under acidic (pH 2) and
slightly basic (pH 8) conditions
are shown in Figures 1 and 2,
respectively.At pH 2, three
Scheme 1.Preparation of thymidine 5 ’-{O-phenyl-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate}
(3).Thy: thymine, Py: pyridine.
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Hydrolysis of nucleoside 5’-O-aryl-N-alkylphosphoramidates
FULL PAPER
Figure 2.Time-dependent product distribution for the hydrolysis of the
faster-eluting diastereomer of thymidine 5’-{O-phenyl-N-[(1S)-2-oxo-2-
methoxy-1-methylethyl]phosphoramidate} (3) in a glycine buffer at pH 8
Figure 1.Time-dependent product distribution for the hydrolysis of the
faster-eluting diastereomer of thymidine 5’-{O-phenyl-N-[(1S)-2-oxo-2-
methoxy-1-methylethyl]phosphoramidate} (3) in 0.01 molLÀ1 aqueous hy-
drogen chloride at 908C (I=0.1 molLÀ1 with NaCl).Compound 3: &,
and 908C ([HA]/[AÀ]=0.02/0.01 molLÀ1
;
I=0.1 molLÀ1 with NaCl).
Compound 3: &, thymidine 5’-{N-[(1S)-2-oxo-2-hydroxy-1-methylethyl]-
~
^
thymidine 5’-phenylphosphate (8): , thymidine (9): , thymidine 5’-
*
,
phosphoramidate} (11):
thymidine 5’-{N-[(1S)-2-oxo-2-methoxy-1-
^
phosphate (10): ; thymine:
.
*
~
.
methylethyl]phosphoramidate} (4):
nucleosidic products, that is, thymidine 5’-phenylphosphate
(8), thymidine (9), and thymidine 5’-phosphate (10), were
formed in parallel.In addition, a small amount ( <2%) of
thymine was released, mainly
dergoes rate-limiting breakdown by nucleophilic attack of
water on the phosphorus atom with concomitant departure
by subsequent decomposition
of the initial products.Only at
pH 4 was the amount of thy-
mine marked, up to 14%.Oth-
erwise, the product distribution
remained constant over the pH
range 0–4.None of the prod-
ucts obtained at pH 0–4 accu-
mulated at pH 8, but the hy-
drolysis instead yielded thymi-
dine
5’-{N-[(1S)-2-oxo-2-hy-
droxy-1-methylethyl]phosphor-
amidate} (11) and thymidine
5’-{N-[(1S)-2-oxo-2-methoxy-1-
methylethyl]phosphoramidate}
(4).
For the reasons discussed in
the following section, the nu-
cleosidic products listed above
are believed to be formed
along the pathways indicated
in Scheme 3.Formation of thy-
midine 5’-phenylphosphate (8)
as the major product of the
acid-catalyzed hydrolysis is ex-
pected.Previous studies with
N,O-dialkyl-O-arylphosphor-
amidates[15] and O,O-dialkyl-N-
arylphosphoramidates[16] have
shown that the substrate
Scheme 3.Pathways for the acid- and base-catalyzed hydrolyses of thymidine 5 ’-{O-phenyl-N-[(1S)-2-oxo-2-
methoxy-1-methylethyl]phosphoramidate} (3).Structures in parentheses ( I1–I3) indicate intermediates that
were not detected.
monocation obtained in
a
rapid pre-equilibrium step un-
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M.Ora et al.
of the amine ligand.Evidently, 3 can follow a similar path-
way.By contrast, an explanation for the formation of thymi-
dine (9) and its 5’-phosphate 10 in parallel with the forma-
tion of 8 is not so self-evident.Measurements in 18O-en-
riched water indicated that no 18O was incorporated into the
À
ent studies into the hydrolysis of 4 indicated that the conver-
sion of 4 into 11 is considerably slower than the disappear-
ance of 3.Accordingly, if 11 were formed via 4, the latter
should markedly accumulate as an intermediate.Since this
is not the case, the formation of 11 is most likely initiated
by hydroxide-ion-catalyzed hydrolysis of the carboxylic ester
linkage.The rate of this reaction at pH 6–8 is expected to be
comparable to the rate of the acid-catalyzed hydrolysis at
pH 1–2.[17] Accordingly, sufficiently rapid formation of inter-
mediate I2 appears possible, although no direct evidence for
its existence could be obtained.Evidently, rapid intramolec-
ular attack of the carboxylate ion on the phosphorus atom
takes place, with concomitant departure of the phenoxide
ion.The cyclic phosphoramidate intermediate obtained ( I3)
then undergoes ring-opening by attack of water on the phos-
phorus atom and departure of the carboxylate ion.The acid-
thymidine.In other words, thymidine was released by P
O
À
bond rupture, not by C O fission.Thymidine 5 ’-(O-phenyl-
N-isopropylphosphoramidate) (5) did not give any thymi-
dine or thymidine 5’-phosphate at pH 1, but it was quantita-
tively hydrolyzed to thymidine 5’-phenylphosphate (8),
twice as fast as 3.These two observations leave intramolecu-
lar nucleophilic participation by the carboxy function as the
most plausible alternative for the formation of thymidine
and its 5’-phosphate.A prerequisite for the nucleophilic
attack by the carboxylic acid group on the phosphorus atom
is obviously hydrolysis of the carboxylic ester linkage.The
second-order rate constants for the acid-catalyzed hydrolysis
of saturated carboxylic esters are of the order of
10À2 LmolÀ1 sÀ1 at 908C[17] and, hence, formation of 9 and 10
through initial hydrolysis of the alaninyl ester linkage (I1)
appears possible.Nucleophilic attack of the carboxylic acid
group on the phosphorus atom then results in displacement
of thymidine (9) or phenol.Departure of phenol gives a
cyclic phosphoramidate intermediate (I3), which may de-
compose to thymidine 5’-phosphate (10) by consecutive
À
catalyzed cleavage of the P N bond is too slow at pH>6 to
compete with the hydrolysis of the mixed anhydride.It
should be noted that a similar intramolecular participation
has previously been reported for displacement of a phenox-
ide ion from O-phenyl-N-(carboxymethyl)phosphorami-
date.[20]
pH rate profiles: The pH rate profiles for the breakdown of
the two diastereomers of thymidine 5’-{O-phenyl-N-[(1S)-2-
oxo-2-methoxy-1-methylethyl]phosphoramidate} (3) and the
O-unsubstituted analogue, thymidine 5’-{N-[(1S)-2-oxo-2-
methoxy-1-methylethyl]phosphoramidate} (4), are depicted
in Figure 3.The hydrolytic stability of the diaseteromeric
forms of 3 are very similar.The pH rate profiles for the indi-
vidual partial reactions of the breakdown of phosphorami-
date 3 are depicted in Figure 4.Numerical values for the
partial rate constants are given in Table 1.
À
À
À
cleavage of the P N and P O (or C(O) O) bonds, in this
order.Mass spectrometric analyses verified release of ala-
nine (m/z value for [M+H]+: 91.9) in addition to alanine
methyl ester (m/z value for [M+H]+: 104.1). Thymidine 5’-
phosphate undergoes dephosphorylation to thymidine under
slightly acidic conditions.[18] Evidently, this reaction is of
minor importance, since the concentration ratio of 9 and 10
remains constant with time.In principle, intermediate I1
could also be hydrolyzed to form 8.This intermediate does
not, however, accumulate, which means that intramolecular
attack of the carboxy group to give 9 and I3 is too fast to
allow hydrolysis to 8 by intermolecular attack of water to
compete.In striking contrast to triester-like thymidine 5 ’-
{O-phenyl-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phos-
phoramidate} (3), its diester-like analogue, thymidine 5’-{N-
[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate} (4),
At pH values <4, the breakdown of 3, utilizing routes A–
C in Scheme 3, is first-order with respect to oxonium ion
À
concentration.The reaction by initial P N bond cleavage,
giving thymidine 5’-phenylphosphate (8; route A), is approx-
À
reacts at pH values <4 only by P N bond rupture, as ob-
served previously with other diester-like nucleoside phos-
phoramidates.[19]
À
At pH values >5, hydroxide-ion-catalyzed P OPh bond
cleavage to thymidine 5’-{N-[(1S)-2-oxo-2-methoxy-1-meth-
ylethyl]phosphoramidate} (4) and hydrolysis of the alaninyl
methyl ester linkage, thereby giving thymidine 5’-{N-[(1S)-2-
oxo-2-hydroxy-1-methylethyl]phosphoramidate} (11), take
place.While 4 is hydrolyzed at pH values <4 more rapidly
À
than 3 by the P N bond cleavage, the stability order of
these two compounds is reversed with higher pH values and,
hence, 4 accumulates as a stable product at pH values >6,
although only as a minor product (8%).The major product,
11, having the alaninyl ester linkage hydrolyzed, cannot be
formed from 4, since the concentration ratio of these two
products remains constant with time.Moreover, independ-
Figure 3.pH rate profiles for the decomposition of diastereomeric thymi-
dine 5’-{O-phenyl-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphorami-
*
date}s (3; fast-eluting diastereomer &, slowly eluting diastereomer ) and
thymidine 5’-{N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate}
À1
~
(4; ) at 908C. I=0.1 molL with NaCl.
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Hydrolysis of nucleoside 5’-O-aryl-N-alkylphosphoramidates
FULL PAPER
At pH values >5, the breakdown of 3 becomes base cata-
lyzed.The reaction by hydrolysis of the carboxylic ester
linkage, followed by rapid intramolecular displacement of
the phenoxy group, that is, formation of 11 by route D, pre-
dominates.Intermolecular displacement of the phenoxide
ion without preceding hydrolysis of the carboxy ester
(route E) is one order of magnitude slower.The hydrolysis
of the carboxy ester linkage of triester-like phosphoramidite
3 under these conditions is 6 times as fast as the correspond-
ing reaction of its diester-like analogue 4.
Discussion
À
Mechanisms of acid-catalyzed P N bond cleavage
Figure 4.pH rate profiles for the hydrolysis of thymidine 5 ’-{O-phenyl-N-
[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate} (3) into thymi-
(route A): As indicated above, thymidine 5’-{O-phenyl-N-
[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate} (3)
is hydrolyzed under acidic conditions by two concurrent
routes: nucleophilic displacement of the alaninyl ester
moiety by a water molecule (route A in Scheme 3) and hy-
drolysis of the carboxylic ester linkage that allows intramo-
lecular attack of the carboxy group on the phosphorus atom,
thereby resulting in departure
*
^
dine 5’-phenylphosphate (8; kA, ), thymidine (9; kB, ), thymidine 5’-
^
phosphate (10; kC, ), thymidine 5’-{N-[(1S)-2-oxo-2-hydroxy-1-methyl-
&
ethyl]phosphoramidate} (11; kD, ), and thymidine 5’-{N-[(1S)-2-oxo-2-
~
methoxy-1-methylethyl]phosphoramidate} (4; kE,
) at 908C (I=
0.1 molLÀ1 with NaCl).The subscripts of the rate constants refer to the
*
routes indicated in Scheme 3.The dotted ( ) and dashed (&) lines show
À
the corresponding curves for P N bond cleavage and methyl ester hy-
drolysis of 4, respectively.
of either thymidine or phenol
Table 1.Rate constants, given as ki ”105 sÀ1, for the partial reactions of the hydrolysis of the two diastereo-
meric forms of thymidine 5’-{O-phenyl-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate} (3) at 908C
(I=0.1 molLÀ1 with NaCl): kA (formation of 8 by route A), kB (formation of 9 by route B), kC (formation of
10 by route C), kD (formation of 11 by route D), and kE (formation of 4 by route E).In addition, the corre-
sponding data for the hydrolysis of thymidine 5’-{N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate}
(4) are given: kF (formation of 10 from 4) and kG (formation of 11 from 4).
without marked accumulation
of any intermediates (routes B
and C).Both routes represent
about half of the overall disap-
pearance of 3.Consistent with
intermolecular displacement of
the amine ligand with water,
studies in 18O-enriched water
have shown that the methyl
ester of alanine (m/z value for
[M+H]+: 104.1) is released
without 18O-atom incorpora-
tion, while one 18O atom is in-
corporated into the thymidine
3 (fast diastereomer)
3 (slow diastereomer)
4
pH
kA
kB
kC
kD
kE
kA
kB
kC
kD
kE
kF
kG
0.2
1.0
2.0
3.0
4.0
5.6
7.0
8.0
112
54.3
9.1
1.04
0.11
0.02
54.0
12.4
1.34
0.17
0.03
131
60.3
60.3
21.7
2.55
0.20
0.02
2.84
0.03
1.18
0.03
1.48
0.04
1490
270
0.83
12.5
103
0.05
1.59
12.8
0.95
121
0.05
16.4
0.69
0.27
2.83
22.8
5’-phenylphosphate
product
(8).The reaction most likely
imately as fast as the carboxylic ester hydrolysis, which gives
intermediate I1 (routes B and C).The latter intermediate
gives thymidine (9; route B) and intermediate I3, and,
hence, eventually thymidine 5’-phosphate (10; route C), in
an equimolar ratio.The diester-like analogue, 4, is hydro-
lyzed under acidic conditions 600 times more rapidly than 3.
As discussed above, this reaction proceeds entirely by cleav-
proceeds as depicted in Scheme 4.As suggested previous-
ly[15] for N,O-dialkyl-O-arylphosphoramidates, rapid initial
protonation of the starting material probably gives a mixture
of N- and O-protonated monocations, but the reaction pro-
ceeds through the N-protonated species.Upon nucleophilic
attack of water, the positively charged nitrogen atom takes
an apical position and departs by an SN2-like mechanism.It
is worth noting that this is the only reaction detected with
the N-isopropyl analogue of 3, which contains no functional
group on the N-alkyl moiety.The diester-like phosphorami-
date 4, which is hydrolyzed 600 times faster than 3, evidently
utilizes a rather similar mechanism.Protonation of monoa-
nionic 4 is easier than protonation of neutral triester-like 3.
Again, the site of protonation remains obscure,[21] but the N-
protonated tautomer in all likelihood lies on the reaction
pathway.Upon departure of the amine ligand, a dianionic
metaphosphate-like structure is developed.This increases
À
age of the P N bond.In all likelihood, the reactive species
is the zwitterionic minor tautomer of protonated (neutral) 4,
with the nitrogen atom protonated (cationic) and the phos-
phoryl oxygen atom anionic.[19] This ensures a good leaving
group (neutral amine), together with a good remnant (meta-
phosphate-like structure).Hence, the breakdown of 4 is con-
siderably faster than that of the triester-like O-phenyl deriv-
ative 3 and is too fast to allow competition by hydrolysis of
the alaninyl ester linkage and subsequent intramolecular
attack of the carboxy group.
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M.Ora et al.
tained by hydrolysis of the alaninyl ester linkage most likely
serves as an intramolecular nucleophile (routes B and C),
À
which triggers the rupture of P O bonds.It should be noted
that, while intramolecular attacks of oxygen nucleophiles on
protonated (monocationic) phosphotriesters proceed by
À
attack on the phosphorus atom and, hence, by P O bond
cleavage,[25] the corresponding intermolecular reactions take
[26]
À
place at the carbon atom, that is, by C O bond cleavage.
As discussed in the preceding section, ester hydrolysis ap-
pears to be sufficiently fast to allow intramolecular partici-
pation of the carboxy group.Attack of the carbonyl oxygen
atom of the exposed carboxy group on the phosphorus atom
concerted with transfer of the carboxylic acid proton to the
phosphoryl oxygen atom gives a phosphorane intermediate
with either the phenoxy or thymidine ligand in an apical po-
À
À
Scheme 4.Mechanism for the acid-catalyzed hydrolysis of the P N bond
of thymidine 5’-{O-phenyl-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phos-
phoramidate} (3).a: axial position, e: equatorial position.
sition (Scheme 6).Cleavage of the apical P O bond concert-
the electron density at the phosphorus atom and, hence, the
À
cleavage of the P N bond is facilitated (Scheme 5).The fact
that the solvolysis of phosphoramidic acid,[22] O-alkylphos-
phoramidates,[23] and N-alkylphosphoramidates[24] in aque-
ous alcohol favors the alcoholysis product over the hydroly-
sis product, however, suggests that the mechanism is not
purely dissociative and that the entering nucleophile partici-
pates in the transition state.
Scheme 6.Mechanism for the acid-catalyzed hydrolysis of thymidine 5 ’-
{O-phenyl-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate} (3)
into thymidine (9) and its 5’-phosphate 10.
À
Scheme 5.Mechanism for the acid-catalyzed hydrolysis of the P N bond
of thymidine 5’-{N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphorami-
date} (4).
ed with proton transfer from the phosphoryl oxygen atom to
the departing oxygen atom results in release of either
phenol or thymidine (9).Departure of phenol yields a cyclic
triester-like phosphoramidate, which eventually releases thy-
midine 5’-phosphate (10) by consecutive departure of the
carboxy and amino groups of the alanine moiety.It appears
reasonable to assume that the reactions take place in this
order, since the carboxylate ion is a conjugate base of a
stronger acid than the amide ion.Hydrolysis of the mixed
anhydride then gives a diester-like phosphoramidate, known
À
Mechanisms of acid-catalyzed P O bond cleavage (routes B
and C): As mentioned above, the phosphoramidate triester
3 also yields thymidine (9) and its 5’-phosphate 10 under
À
acidic conditions.Both products are formed by P O bond
rupture, since 18O is not incorporated into the released alco-
hols, that is, thymidine or phenol, but is incorporated into
thymidine 5’-phosphate (10).Since no acid-catalyzed P
bond cleavage takes place with thymidine 5’-(O-phenyl-N-
isopropylphosphoramidate) (5), the carboxy function ob-
À
O
8596
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Chem. Eur. J. 2007, 13, 8591 – 8599

Hydrolysis of nucleoside 5’-O-aryl-N-alkylphosphoramidates
FULL PAPER
to be hydrolytically much less stable than its triester ana-
logue.
phosphorus atom.This alternative appears, however, less at-
tractive.Previous comparative studies with O-phenyl-N-car-
boxymethylphosphoramidate and O-phenylphosphoramidate
have shown that intramolecular catalysis by the neighboring
One might also speculate that the formation of thymidine
(9) or its 5’-phosphate 10 is initiated by nucleophilic attack
of the 3’-OH group, and not the carboxy group, on the phos-
phorus atom.This pathway is not, however, utilized under
acidic conditions, because no sign of accumulation of the
very stable thymidine 3’,5’-cyclic monophosphate[25c] or its
phenyl ester[25d] could be detected.
À
carboxylate group accelerates the rupture of the P OPh
bond by a factor of approximately 104 at pH 8.[20] Evidently,
the carboxylate function also produces a remarkable rate
2
À
enhancement of P OPh bond cleavage in I .
While the carboxylate group readily attacks on the neutral
triester-like phosphoramidate center, no similar attack on
the monoanionic diester-like phosphoramidate center of 11
takes place, but 11 accumulates as a stable product at pH 7–
8.An intramolecular attack of the carboxylate group would
give a pentacoordinated intermediate (transition state) in
which the attacking oxygen atom and thymidine occupy the
apical positions, while the nitrogen ligand remains equatori-
al, as it is a member of the same five-membered ring as the
attacking oxygen atom.The dianionic intermediate, even if
it exists, is too unstable to pseudo-rotate,[27] but thymidine
should be released “in-line” with nucleophilic attack.The
fact that 11 does not decompose by this pathway is not self-
evident, as the rate constant for a similar intramolecular dis-
placement of a phenoxide ion from O-phenyl-N-carboxyme-
thylphosphoramidate is 1.4”10À4 sÀ1 at 758C.[20] One should,
however, bear in mind that the 5’-oxyanion of thymidine is a
poor leaving group compared to the phenoxide ion.
À
Mechanism of base-catalyzed intramolecular P O bond
cleavage (route D): Hydrolysis of thymidine 5’-{O-phenyl-N-
[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate} (3)
becomes hydroxide-ion-catalyzed at pH values higher than
5.The predominant product is thymidine 5 ’-{N-[(1S)-2-oxo-
2-hydroxy-1-methylethyl]phosphoramidate} (11; route D in
Scheme 3).This product is most probably formed by an ini-
tial attack of a hydroxide ion on the carbonyl carbon atom
and concomitant elimination of a methoxide ion, followed
by attack of the resulting carboxylate group on the phospho-
rus atom.A pentacoordinated intermediate (or transition
state) is obtained and subsequently decomposes through de-
parture of the phenoxide ion, which is a much better leaving
group than thymidine 5’-oxyanion (Scheme 7).A highly un-
stable cyclic mixed anhydride intermediate is obtained.This
intermediate then undergoes fast subsequent hydrolysis by
attack of a hydroxide ion on the phosphorus atom.Since the
endocylic oxygen atom of the mixed anhydride is a more
electronegative atom than the endocyclic nitrogen atom, it
occupies an apical within the five-membered ring upon
À
À
Mechanism of base-catalyzed intermolecular P O bond
cleavage (route E): At pH 6–8, release of the phenoxide ion
from thymidine 5’-{O-phenyl-N-[(1S)-2-oxo-2-methoxy-1-
methylethyl]phosphoramidate} (3) also takes place by direct
intermolecular displacement by a hydroxide ion.On the
basis of 18O-incorporation studies, the diester-like phosphor-
amidate obtained (4) includes one 18O atom.In all likeli-
attack of the hydroxide ion.Accordingly, only the P
O
bond is cleaved.[19g,26] Consistent with the mechanism pro-
posed, the final product (11) obtained in 18O-enriched water
contained two 18O atoms.The accumulation of the first in-
termediate, with the carboxylic ester linkage hydrolyzed (I2
in Scheme 3), remained too low to be reliably quantified by
HPLC with UV detection, but an m/z value ([MÀH]+
472.3) corresponding to the molecular ion containing one
18O atom could be observed by HPLC/ESI-MS.
À
hood, this P OPh bond fission proceeds by a bimolecular
SN2(P)-type mechanism (Scheme 8) by attack of a hydroxide
ion on the phosphorus atom concerted with the departure of
the phenoxide ion, as was also suggested for N-monosubsti-
tuted O-aryl-O-methyl- and O,O-diphenylphosphoramidates
and their their unsubstituted analogues.[28] Consistent with
the suggested mechanism, thymidine 5’-(O-phenyl-N-isopro-
pylphosphoramidate) (5) yields only thymidine 5’-(N-isopro-
pylphosphoramidate).The re-
The results discussed above do not strictly rule out the
possibility that the phenoxide ion is released from inter-
mediate I2 by nucleophilic attack of a hydroxide ion on the
action is one order of magni-
tude slower than with 3, proba-
bly because the isopropyl
group does not exert a similar
electron-withdrawing effect to
that of the 2-methoxy-2-oxo-1-
methylethyl group.
In summary, thymidine 5’-
{O-phenyl-N-[(1S)-2-oxo-2-me-
thoxy-1-methylethyl]phosphor-
amidate} (3) has been shown
to be hydrolyzed under acidic
conditions (pH<4) partly by
displacement of the N-proton-
Scheme 7.Mechanism for the hydroxide-ion-catalyzed hydrolysis of thymidine 5 ’-{O-phenyl-N-[(1S)-2-oxo-2-
methoxy-1-methylethyl]phosphoramidate} (3) into thymidine 5’-{N-[(1S)-2-oxo-2-hydroxy-1-methylethyl]phos-
phoramidate} (11).
Chem. Eur. J. 2007, 13, 8591 – 8599
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8597

M.Ora et al.
H5’, H5’’), 4.11–3.99 (m, 3H; H4’, NH, CH), 3.73 (s, 3H; OCH3), 2.40
(m, 1H; H2’), 2.14 (m, 1H; H2’’), 1.90 (s, 3H; CH3), 1.39 ppm (d, J=
7.0 Hz, 3H; CH3); 13C NMR (125 MHz, [D]CHCl3, 258C, TMS): d=
174.1, 163.8, 150.4, 135.6, 129.9, 125.3, 120.1, 111.3, 85.0, 84.6, 71.0, 66.1,
52.7, 50.2, 39.8, 20.9, 12.5 ppm; 31P NMR (202 MHz, D2O, 25 8C, H3PO4):
d=3.06 ppm; ESI-MS: m/z: 484.6 [M+H]+.
Thymidine 5’-{N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate}
(4): Thymidine was converted into thymidine 5’-(fluoren-9-ylmethyl-H-
phosphonate) (7) as described previously by Zhao and co-workers[13] and
subjected to oxidative amination in a mixture of pyridine and MeCN in
the presence CCl4, EtN3, and alanine methyl ester, as described above for
3.The product was purified by silica-gel chromatography with a mixture
of CH2Cl2 and MeOH as the eluent (90:10).In a final step, the fluorenyl-
methyl group was removed by treatment with piperidine in CH2Cl2.The
product was purified by silica-gel chromatography with a mixture of
CH2Cl2 and MeOH as the eluent (80:20) and by reversed-phase chroma-
tography on a Lobar RP-18 column (37”440 mm, 40–63 mm) by elution
with a mixture of water and MeCN (92:8).Finally, the product was
passed through an Na+-form Dowex 50-W (100–200 mesh) cation-ex-
change column. 1H NMR (500 MHz, D2O, 25 8C, TMS): d=7.65 (s, 1H;
Scheme 8.Mechanism for the hydroxide-ion-catalyzed hydrolysis of thy-
midine 5’-{O-phenyl-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphor-
amidate} (3) into thymidine 5’-{N-[(1S)-2-oxo-2-methoxy-1-methylethyl]-
phosphoramidate} (4) and a phenoxide ion.
ated amino ligand with water and partly by oxonium-ion-
catalyzed methyl ester hydrolysis followed by intramolecular
nucleophilic attack of the resulting carboxy group on the
phosphorus atom.The latter reaction gives thymidine and
its 5’-phosphate as nucleosidic products.The acid-catalyzed
hydrolysis turns into a base-catalyzed hydrolysis above
pH 5.The predominant product of the base-catalyzed hy-
drolysis is thymidine 5’-{N-[(1S)-2-hydroxy-2-oxo-1-methyle-
thyl]phosphoramidate} (11), obtained by hydroxide-ion-cata-
lyzed methyl ester hydrolysis and subsequent intermolecular
attack of a hydroxide ion on the phosphorus atom.Intermo-
lecular displacement of the phenoxide ion with a hydroxide
ion occurs as a side reaction.In the pH range 0–4, the oxo-
H6), 6.23 (dd, J
1H; H4’), 3.93–3.84 (m, 2H; H5’, H5’’), 3.73 (m, 1H; Ha), 2.28–2.24 (s,
2H; H2’, H2’’), 3.66 (s, 3H; OCH3), 1.80 (d, J(H,H)=1.0 Hz, 3H; CH3),
A
AHCTREUNG
1.22 ppm (d,
J
(H,H)=7.0 Hz, 3H; CH3); 31P NMR (202 MHz, D2O,
C
Thymidine 5’-(O-phenyl-N-isopropylphosphoramidate) (5): Compound 5
was obtained as a mixture of RP and SP diastereomers by a similar
method to that described for 3.The diastereomers were not separated
and the compound was used as a diastereomeric mixture. 5: 1H NMR
(500 MHz, CD3CN, 258C, TMS): d=8.56 (s, 2”1H; NH), 7.48–7.18 (m,
À
nium-ion-catalyzed P N bond cleavage makes the diester-
like thymidine 5’-{N-[(1S)-2-oxo-2-methoxy-1-methylethyl]-
phosphoramidate} (4) 2–3 orders of magnitude more reac-
tive than its triester analogue 3.At pH values >6, the stabil-
ity order is reversed, with triester 3 being decomposed six
times as fast as the diester 4.
2”6H; Ph and H6), 6.90 (d,
J
ACHTREUNG
J(H,H)=8.9 Hz, 1H; NH), 6.20, 6.21 (dd, J
AHCTREUNG
H1’), 4.37 (m, 2”1H; H3’), 4.25 (m, 2”2H; H5’, H5’’), 4.04 (m, 2”1H;
H4’), 3.8 (d, 2”1H; 3’-OH), 3.39 (m, 2H, 2”CH), 2.20 (m, 2”1H; H2’),
2.10 (m, 2”1H; H2’’), 1.81 (d, J(H,H)=1.2 Hz, 3H; CH3), 1.80 (d,
A
J(H,H)=1.2 Hz, 3H; CH3), 1.11, 1.09 ppm (d, J=6.5 Hz, 2”6H; CH3);
31P NMR (202 MHz, [D3]CH3CN, 258C, H3PO4): d=4.68, 4.59 ppm; ESI-
MS: m/z: 440.7 [M+H]+.
Kinetic measurements: The reactions were carried out in sealed tubes im-
mersed in a thermostated water bath (90.0Æ0.18C).The oxonium-ion
concentration of the reaction solutions was adjusted with hydrogen chlo-
ride, sodium hydroxide and formate, acetate, (N-[2-hydroxyethyl]pipera-
zine-N-[2-ethanesulfonic acid]) (HEPES), and glycine buffers.The oxoni-
um-ion concentrations of the buffer solutions were calculated with the
aid of the known pKa values of the buffer acids under the experimental
conditions.[29] Low buffer concentrations were used (30–60 mmolLÀ1).
The initial substrate concentration was approximately 0.1 mmolLÀ1.The
composition of samples withdrawn at appropriate intervals was analyzed
on a Hypersil ODS 5 column (4”250 mm, 5 mm) by using mixtures of
Experimental Section
Thymidine
5’-{O-phenyl-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phos-
phoramidate} (3): Thymidine (9, 0.24 g, 1.00 mmol) in an anhydrous pyri-
dine (2.5 mL) was added dropwise to diphenyl phosphite (288 mL,
1.50 mmol) in anhydrous pyridine (2.5 mL) at À58C.The thymidine 5 ’-
hydrogenphosphonate phenyl ester obtained was not isolated, but the so-
lution was stirred for 20 min at room temperature, after which l-alanine
methyl ester (0.21 g, 1.5 mmol), MeCN (5.0 mL), CCl4 (6.2 mL), and
Et3N (0.5 mL) were added. After being stirred for 75 min, the solution
was evaporated to dryness.The crude product was isolated by a conven-
tional aqueous work up and purified on a silica gel column eluted with a
mixture of CH2Cl2 and MeOH (90:10).The diastereomers were purified
and separated by reversed-phase chromatography on a Lobar RP-18
column (37”440 mm, 40–63 mm) by elution with a mixture of water and
MeCN (80:20, v/v).
MeCN and an acetic acid/sodium acetate buffer (0.045/0.015 molLÀ1
)
containing 0.1 molLÀ1 ammonium chloride as an eluent.Good separation
of the product mixtures of 3 and 4 was obtained by using a 3 min isocrat-
ic elution with the buffer containing 0.5% MeCN, followed by a linear
gradient of the eluent (over 18 min) up to 20.0% MeCN. After this, iso-
cratic elution with the final eluent was continued.Signals were recorded
on a UV detector at a wavelength of 267 nm.The observed retention
1
The faster-eluted diastereomer of 3: H NMR (500 MHz, [D]CHCl3, 258C,
TMS): d=9.35 (s, 1H; NH), 7.45–7.17 (m, 6H; H6, Ph), 6.33 (dd,
times (tR) for the products of
3 with RP HPLC (flow rate was
1 mLminÀ1) were 6.8 (10), 8.6 (thymine), 14.8 (11), 15.0 (9), 20.2 (4), 22.9
(8), and 24.6 min (phenol). The observed retention times for the starting
materials were 36.5 (faster-eluting diastereomer of 3) and 37.2 min (more
slowly eluting diastereomer of 3).The reaction products were identified
by the mass spectra (LCMS).A mixture of MeCN and aqueous ammoni-
um acetate (5 mmolLÀ1) or formic acid (0.1%) was used as the eluent.
The contribution of the buffer catalysis to the observed rate constants
was insignificant even at the higher buffer concentration employed
(0.2 molLÀ1).The product of alkaline hydrolysis of 3, assigned as the
phosphoramidate diester 11, was additionally isolated by RP HPLC (C18
J(H,H)=6.5, 6.5 Hz, 1H; H1’), 4.52 (m, 1H; H3’), 4.40 (dd, J
3.0 Hz, 1H; H5’), 4.35 (dd, J(H,H)=7.0, 3.0 Hz, 1H; H5’’), 4.01–4.11 (m,
3H; H4’, NH, CH), 3.71 (s, 3H; OCH3), 2.34 (m, 1H; H2’), 2.02 (m, 1H;
H2’’), 1.90 (s, 3H; CH3), 1.38 ppm (d, (H,H)=6.5 Hz, 3H; CH3);
A
AHCTREUNG
J
ACHTREUNG
13C NMR (125 MHz, [D]CHCl3, 258C, TMS): d=174.1, 163.9, 150.5,
135.5, 129.8, 125.3, 120.0, 111.2, 84.8, 84.6, 70.6, 65.9, 52.7, 50.4, 39.8, 20.8,
12.5 ppm; 31P NMR (202 MHz, D2O, 25 8C, H3PO4): d=3.38 ppm; ESI-
MS: m/z: 484.6 [MÀH]+.
The more slowlyeluted diastereomer of
[D]CHCl3, 258C, TMS): d=9.10 (s, 1H; NH), 7.37–7.18 (m, 6H; H6, Ph),
3: 1H NMR (500 MHz,
1
6.27 (dd, J
A
column) and characterized by H NMR and 31P NMR spectroscopy.
8598
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Chem. Eur. J. 2007, 13, 8591 – 8599

Hydrolysis of nucleoside 5’-O-aryl-N-alkylphosphoramidates
Diester 11: 1H NMR (500 MHz, D2O, 25 8C, TMS): d=8.56 (s, 1H; NH),
FULL PAPER
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H1’), 4.46 (m, 1H; H3’), 4.08 (m, 1H; H4’), 3.92–3.86 (m, 2H; H5’, H5’’),
3.47 (d, J(H,H)=7.0 Hz, 1H; Ha), 2.31–2.23 (m, 2H; H2’, H2’’), 1.86 (s,
A
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Received: April 24, 2007
Published online: July 25, 2007
Chem. Eur. J. 2007, 13, 8591 – 8599
ꢀ 2007 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
www.chemeurj.org
8599