Biodegradable protections for nucleoside 5′-monophosphates: Comparative study on the removal of O-acetyl and O-acetyloxymethyl protected 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl groups

Article abstract of DOI:10.1021/jo9005987

(Chemical Equation Presented) The applicability of 3-acetyloxy-2,2- bis(ethoxycarbonyl)propyl and 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl) propyl groups as biodegradable phosphate protecting groups for nucleoside 5′-monophosphates has been studied in a HEPES buffer at pH 7.5. Enzymatic deacetylation with porcine carboxyesterase triggers the removal of the resulting 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl and 3-hydroxymethoxy-2,2- bis(ethoxycarbonyl)propyl groups by retro-aldol condensation and consecutive half acetal hydrolysis and retro-aldol condensation, respectively. The kinetics of these multistep deprotection reactions have been followed by HPLC, using appropriately protected thymidine 5′-monophosphates as model compounds. The enzymatic deacetylation of the 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl) propyl 5′-triester (2) is 25-fold faster than the deacetylation of its 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl-protected counterpart 1, and the difference in the deacetylation rates of the resulting diesters, 12b and 12a, is even greater. With 2, conversion to thymidine 5′-monophosphate (5′-TMP) is quantitative, while conversion of 1 to 5′-TMP is accompanied by formation of thymidine. Consistent with the preceding observations, quantitative release of 5′-TMP from 2 has been shown to take place in a whole cell extract of human prostate cancer cells.

Full text of DOI:10.1021/jo9005987

Biodegradable Protections for Nucleoside 5′-Monophosphates:
Comparative Study on the Removal of O-Acetyl and
O-Acetyloxymethyl Protected
3-Hydroxy-2,2-bis(ethoxycarbonyl)propyl Groups
Mikko Ora,*,† Sharmin Taherpour,† Risto Linna,† Anna Leisvuori,† Emilia Hietama¨ki,†
Pa¨ivi Poija¨rvi-Virta,† Leonid Beigelman,‡ and Harri Lo¨nnberg†
Department of Chemistry, UniVersity of Turku, FIN-20014 Turku, Finland, and AliosBiopharma,
260 East Grand AVe, 2nd Floor, South San Francisco, California 94080
mikko.ora@utu.fi
ReceiVed April 1, 2009
The applicability of 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl and 3-acetyloxymethoxy-2,2-bis(ethoxy-
carbonyl)propyl groups as biodegradable phosphate protecting groups for nucleoside 5′-monophosphates
has been studied in a HEPES buffer at pH 7.5. Enzymatic deacetylation with porcine carboxyesterase
triggers the removal of the resulting 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl and 3-hydroxymethoxy-
2,2-bis(ethoxycarbonyl)propyl groups by retro-aldol condensation and consecutive half acetal hydrolysis
and retro-aldol condensation, respectively. The kinetics of these multistep deprotection reactions have
been followed by HPLC, using appropriately protected thymidine 5′-monophosphates as model compounds.
The enzymatic deacetylation of the 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl 5′-triester (2) is
25-fold faster than the deacetylation of its 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl-protected counterpart
1, and the difference in the deacetylation rates of the resulting diesters, 12b and 12a, is even greater.
With 2, conversion to thymidine 5′-monophosphate (5′-TMP) is quantitative, while conversion of 1 to
5′-TMP is accompanied by formation of thymidine. Consistent with the preceding observations, quantitative
release of 5′-TMP from 2 has been shown to take place in a whole cell extract of human prostate cancer
cells.
Introduction
that triggers a chemical reaction by which the remnants of the
protecting groups are removed. The groups employed for this
purpose include acyloxymethyl,2 alkoxycarbonyloxymethyl,3
Protected nucleoside monophosphates and their congeners
constitute a promising class of therapeutic agents.1 A viable
pronucleotide candidate is reasonably stable in plasma and
sufficiently hydrophobic to penetrate into cells, where it releases
the parent nucleotide drug by cleavage of the phosphate
protecting groups to nontoxic products. The deprotection of the
pronucleotide is usually initiated by an enzymatic transformation
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† University of Turku.
‡ AliosBiopharma.
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10.1021/jo9005987 CCC: $40.75  2009 American Chemical Society
Published on Web 05/22/2009

Biodegradable Protections for 5′-Monophosphates
S-acyl-2-thioethyl,4 2-(2-hydroxyethyldisulfanyl)ethyl,5 4-acyloxy-
benzyl,2a,6 1-acyloxypropan-1,3-diyl,7 and 1-arylpropan-1,3-diyl8
functional groups. In addition, cyclosaligenyl nucleotides,9
phosphoramidates,8b,10 diamidates,11 lipid esters,12 nitrofura-
nylmethylamidates,13 and bis-ketol nucleotide triesters14 have
been studied as pro-drugs. Despite the fact that some pronucle-
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otides have been accepted for clinical use and quite many have
advanced into clinical phase studies,1a many questions have still
to be answered to achieve a proper compromise between
chemical stability, cellular uptake, rate of biodegradation and
byproduct toxicity.
Bis(acyloxymethyl) pro-drugs, constituting one of the most
commonly used pro-drug categories, have been suggested to
undergo an enzyme-triggered conversion to nucleoside mono-
phosphates via a monoanionic diester intermediate.2g In spite
of the wide interest in this approach, the kinetic data on the
consecutive esterase-catalyzed cleavage of the neutral triester
pro-drugs to monoanionic diesters and eventually to nucleoside
phosphates is surprisingly scarce, being limited to the early
studies of Farquhar et al.15 on the cleavage of bis(acyloxym-
ethyl)esters of benzyl and phenyl phosphates. According to these
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different acyl groups, but the second step remains slow due to
the short distance between the enzyme cleavage site and the
negative charge of the phosphodiester intermediate. The studies
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phonate,16a phosphonoacetate,16b and 5′-monophosphate of
azidothymidine,6b in turn, suggest that the benzene ring as a
spacer between the acyl group and the anionic phosphate
accelerates the cleavage of both the neutral triester and monoan-
ionic diester by porcine liver carboxyesterase.
We have recently introduced 2,2-bis(substituted)-3-acyloxy-
propyl groups as biodegradable phosphate protecting groups.
Studies with dinucleoside phosphoromonothioates17 and oligo-
nucleotides18 have shown that such groups are stable under
physiological conditions, but are cleaved by retro-aldol con-
densation after enzymatic deacylation (Scheme 1).
The hydrolytic stability of the deacylated intermediate may
be tuned within wide limits by the polar nature of the
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J. Org. Chem. Vol. 74, No. 14, 2009 4993

Ora et al.
SCHEME 2a
a Conditions: (i) Et3N, DCM; (ii) TetH, MeCN; (iii) I2, THF, H2O; (iv) NH2NH2, AcOH, Py with 6 and NH2NH2OAc, DCM, MeOH with 7.
expectedly is much more difficult. The enzymatic deacetylation
may be retarded and dianionic nucleoside monoester is a poor
leaving group compared to a monoanionic diester. To facilitate
the enzymatic deacetylation of the diester monoanion, the
distance between the phosphate moiety and the ester function
has been increased by inserting an extra hydroxymethyl fragment
in the structure, giving a 3-acetyloxymethoxy-2,2-bis(ethoxy-
carbonyl)propyl group. Accordingly, thymidine 5′-bis[3-acetyl-
oxy-2,2-bis(ethoxycarbonyl)propyl]phosphate (1) and 5′-bis[3-
acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl]phosphate (2)
have been prepared and their conversion to thymidine 5′-
monophosphate (5′-TMP) by porcine liver carboxyesterase has
been studied. In addition, the hydrolysis of 1 in human serum
and the hydrolysis of 2 in a whole cell extract of human prostate
carcinoma cells have been studied.
Results and Discussion
Preparation of Protected Thymidine 5′-Monophosphates
(1, 2). Protected thymidine 5′-monophosphates 1 and 2 were
prepared by stepwise alcoholysis of bis(diethylamino)phospho-
rochloridite with 3′-O-levulinoylthymidine (5) and the appropri-
ate protecting group reagent, either diethyl 2-acetyloxymethyl-
2-hydroxymethylmalonate(3)ordiethyl2-acetyloxymethoxymethyl-
2-hydroxymethylmalonate (4), as outlined in Scheme 2 in more
detail. Oxidation to phosphate triester and removal of the
levulinoyl protection completed the synthesis. Alcohol 3 was
obtained by conversion of commercially available diethyl 2,2-
bis(hydroxymethyl)malonate to orthoacetate (8) and its subse-
quent hydrolysis (Scheme 3).21 Alcohol 4 was, in turn,
synthesized by protecting one of the hydroxyl functions of
diethyl 2,2-bis(hydroxymethyl)malonate with a tert-butyldi-
methylsilyl group (9) and subjecting the other one to methylthio-
methylation (10) followed by displacement of the methylthio
group with acetate ion22 (11) and removal of the tert-butyldi-
methylsilyl group by Et3N·3HF treatment.
Hydrolytic Stability of Protected Thymidine 5′-Monophos-
phates (1, 2). The nonenzymatic hydrolysis of thymidine 5′-
phosphotriesters 1 and 2 was first studied by analyzing the
composition of the aliquots withdrawn from the reaction mixture
(21) Guzaev, A.; Salo, H.; Azhayev, A.; Lo¨nnberg, H. Bioconj. Chem. 1996,
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3869–3872.
4994 J. Org. Chem. Vol. 74, No. 14, 2009

Biodegradable Protections for 5′-Monophosphates
SCHEME 3a
a Conditions: ( i) (EtO)3CCH3, H2SO4, THF; (ii) 80% AcOH; (iii) TBDMSCl, Py; (iv) DMSO, AcOH, Ac2O; (v) SO2Cl2, AcOK, DCM, 18-crown-6; (vi)
Et3N·3HF, THF.
SCHEME 4
at appropriate time intervals by HPLC. The reactions were
carried out in HEPES buffers at pH 7.5 and 37 ( 0.1 or 25 (
0.1 °C. The products were identified by mass spectrometric
analysis (HPLC/ESI-MS) and by spiking with authentic samples.
Triester 1 is hydrolytically quite stable. The reaction is first
order in hydroxide ion concentration and it in all likelihood
proceeds by hydroxide ion-catalyzed deacetylation, followed by
hydroxide ion-catalyzed loss of formaldehyde and concomitant
FIGURE 1. Time-dependent product distribution for the PLE-catalyzed
hydrolysis of thymidine 5′-bis[3-acetyloxy-2,2-bis(ethoxycarbonyl)]-
propylphosphate (1) at pH 7.5 and 25.0 °C (I ) 0.1 mol L-1 with NaCl).
Notation: (b) 1; (0) thymidine 5′-[3-acetyloxy-2,2-bis(ethoxycarbon-
elimination of diethyl 2-methylenemalonate (cf. Scheme 1). At
pH 7.5 and 25 °C, the half-life for the conversion of 1 to the
corresponding diester (12a in Scheme 4) is about 20 days (k )
4.0 × 10-7 s-1). The subsequent hydrolysis of the diester to
5′-TMP is almost 1 order of magnitude slower (k ) 5.0 × 10-8
s-1). In fact, the nonenxymatic hydroloysis of 12a is so slow
that cleavage of the ethoxycarbonyl groups to carboxylate
functions starts to compete.
Triester 2 is hydrolytically almost 1 order of magnitude less
stable than 1. The first-order rate constant for the conversion
of 2 to diester 12b is 6.1 × 10-6 s-1 (τ1/2 ) 32 h) at pH 7.5 and
37 °C, the subsequent hydrolysis of 12b to 5′-TMP being 5
times slower (k ) 1.3 × 10-6 s-1). Evidently, hydroxide ion-
catalyzed deacetylation is followed by rapid hydroxide ion-
catalyzed loss of formaldehyde. The resulting 3-hydroxy-2,2-
bis(ethoxycarbonyl)propyl group is then cleaved by a similar
retro-aldol condensation mechanism as with 1. Besides 12b, no
intermediates appear.
Enzymatic Removal of the 3-Acetyloxy-2,2-bis(ethoxycar-
bonyl)propyl Groups from Thymidine 5′-Phosphotriester 1.
Figure 1 shows the time-dependent product distribution for the
treatment of triester 1 with porcine liver esterase (PLE; 26.0
units mL-1) in a HEPES buffer at pH 7.5 (I ) 0.1 mol L-1
with NaCl) and 25 °C. The identification and quantification of
the intermediates indicated is based on HPLC-ESI-MS. PLE-
catalyzed deacetylation first gives a monodeacetylated triester
(13a; [M + H]+ 769.5; reaction A in Scheme 5), which then
undergoes two subsequent reactions: PLE-catalyzed hydrolysis
yl)]propyl 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl]phosphate (13a);
(9) thymidine 5′-bis[3-hydroxy-2,2-bis(ethoxycarbonyl)propyl]phos-
phate (14); and (O) mixture of thymidine 5′-[3-acetyloxy-2,2-
bis(ethoxycarbonyl)propyl]phosphate (12a) and thymidine 5′-[3-hydroxy-
2,2-bis(ethoxycarbonyl)propyl]phosphate (15). For the structures, see
Scheme 5.
to the fully deacetylated triester (14; [M + H]+ 727.7; reaction
E) and breakdown to acetylated diester 12a ([M + H]+ 567.5)
by removal of the deacetylated protecting group by retro-aldol
condensation (reaction B). The deacetylated triester (14) un-
dergoes a similar retro-aldol condensation giving deacetylated
diester 15 ([M + H]+ 525.4; reaction F). The subsequent
reactions of the diesters 12a and 15 are slow compared to their
formation. At the high PLE concentration employed, the diesters
are accumulated in approximately equimolar amounts, but at a
lower PLE concentration, the acetylated diester (12a) predomi-
nates. For example, at [PLE] ) 4.3 units mL-1, the ratio [12a]/
[15] is 10:1. Most likely, deacetylation of triester 13a (E) is
not fast enough in the intracellular environment to compete with
the nonenzymatic conversion of 13a to 12a. In other words,
the desired nucleoside 5′-monophosphate should be formed by
deacetylation of 12a to 15, which then releases the monophos-
phate by retro-aldol condensation.
The hydrolytic stability of the monodeacetylated triester (13a)
was determined by applying the rate law of two consecutive
first-order reactions to the time-dependent concentration of 13a.
The rate constant obtained on this basis for the disappearance
of 13a (7.5 × 10-4 s-1) was then divided into contributions of
J. Org. Chem. Vol. 74, No. 14, 2009 4995

Ora et al.
SCHEME 5
TABLE 1. First-Order Rate Constants for the Partial Reactions
retro-aldol condensation (reaction B) and deacetylation (reaction
E) with the aid of concentration ratio [12a]/([14] + [15]) at
early stages of their appearance. It should be noted that 12a is
considerably more stable than 14, and hence, at early stages of
the appearance of 12a, 14, and 15, the latter compound is formed
predominantly via 14. In this manner, a value of 3.7 × 10-4
s-1 was obtained for the first-order rate constant of the retro-
aldol condensation of 13a (reaction B; τ1/2 ) 31 min). Owing
to more complicated kinetics, the rate constant for the conversion
of triester 14 to diester 15 (reaction F) could not be determined
as accurately, but it appeared to be of the same order of
magnitude.
To determine the rate constant for the retro-aldol condensation
of the deacetylated diester (15, reaction D), triester 1 was first
deacetylated with porcine liver esterase (PLE) in a dilute HEPES
buffer (pH 7.5, 25 °C) and after 3 h, the enzyme was denaturated
by adjusting the pH to 2 with aqueous hydrogen chloride. At
this stage, the starting material had been fully converted to a
mixture of the acetylated (12a) and deacetylated (15) diester.
The diesters gave separate HPLC signals, but no attempt to
isolate them was made. The pH of the filtered solution (1 mL)
was then adjusted to 7.5 with a more concentrated HEPES
solution (0.1 mol L-1), and the conversion of the deacetylated
diester (15) to 5′-TMP was followed by HPLC. The rate constant
for the process was 8.5 × 10-6 s-1 (τ1/2 ) 23 h). In other words,
the 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl group is cleaved
from a phosphotriester (13a) 45 times as fast as from a
phosphodiester (15). It should be noted that the nonenzymatic
deacetylation of 12a to 15 (reaction C) is so much slower than
the conversion of 15 to 5′-TMP (reaction D) that the concentra-
tion of 12a remains constant, within the limits of experimental
errors, during the disappearance of 15. Table 1 records the rate
constants for the partial reactions indicated in Scheme 5.
The problem from the point of view of pro-drug applications
is the very slow deacetylation of diester 12a. While the half-
lives for the deacetylation of 1 to 13a and subsequently to 14
are 4.3 (reaction A) and 31 min (reaction E) at 25 °C,
respectively (Table 1), the half-life for the deacetylation of
diester 12a (reaction C) is 190 h. Conversion of the resulting
deacetylated diester 15 to thymidine 5′-monophosphate is
Involved in the PLE-Triggered Hydrolysis of Thymidine
5′-Bis[3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl]phosphate (1) and
Thymidine 5′-Bis[3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)-
propyl]phosphate (2) to 5′-TMP at pH 7.5 (I ) 0.1 mol L-1 with
NaCl)a
reaction
T/oC
k/10-4 s-1
t1/2/min
1 f 13a (A)
25.0
37.0
25.0
25.0
25.0
25.0
25.0
37.0
37.0
37.0
37.0
37.0
37.0
37.0
37.0
37.0
27
36
3.8
3.8
4.3
3.2
31
31
11500
≈30
1360
0.17
≈0.14
10.4
0.90
210
13a f 12a (B)
13a f14 (E)
12a f 15 (C)
14 f 15 (F)
15 f 5′-TMP (D)
2 f 16 (G)
16 f 13b (H)
13b f 12b (I)
13b f 17 (J)
12b f 18 (K)
17 f 14 (L)
14 f 15 (F)
18 f 15 (M)
15 f 5′-TMP (D)
0.010
≈4
0.085
680
≈800
11
130
0.54
≈800
≈10
fast
≈0.14
≈10
fast
0.31
370
a For the reactions, see Schemes 5 and 6. [PLE] ) 26 units mL-1
.
somewhat faster, although still 45 times slower than the
corresponding reaction of 13a to 12a (or 14 to 15), the half-
lives for reactions B and D being 0.5 and 23 h, respectively
(Table 1). However, as indicated above, 13a is at a low
carboxyesterase activity converted rather to 12a than to 14, and
hence, deacetylation of 12a constitutes the rate-limiting step
for the release of thymidine 5′-monophosphate. Consistent with
this, still after 2 weeks, about 20% of the starting material (1)
was present as diester 12a. Thymidine 5′-monophosphate
accumulated as an intermediate at maximally 15% level (at t )
5 d) and was largely dephosphorylated to thymidine (Figure
2).
The hydrolytic stability of 1 was additionally determined in
human serum at 25 °C. The half-live for the disappearance of
1 was 24 h. In addition to diester 12a, a considerable amount
of thymidine accumulated (25% of the products) during two
half-lives of the breakdown of the starting material. In summary,
while the 3-acyloxy-2,2-bis(ethoxycarbonyl)propyl group shows
4996 J. Org. Chem. Vol. 74, No. 14, 2009

Biodegradable Protections for 5′-Monophosphates
SCHEME 6
potential as a protecting group for phosphodiester linkages, it
does not show promise as a protecting group for phosphomo-
noesters, such as nucleoside 5′-monophosphates.
EnzymaticRemovalofthe3-acetyloxymethoxy-2,2-bis(ethoxy-
carbonyl)propyl Groups from Thymidine 5′-Phosphotriester
2. As indicated above, the 3-acetyloxy-2,2-bis(ethoxycarbon-
yl)propyl group is not appropriate for protection of nucleoside
5′-monophosphates for the reason that enzymatic deacetylation
of the negatively charged phosphodiester is too slow. To
elongate the distance between the scissile ester bond and the
phosphate moiety, and to diminish the steric hindrance caused
by the bulky ethoxycarbonyl substituents, the 3-acetyloxy group
was replaced with a 3-acetyloxymethoxy group. Thymidine 5′-
phosphotriester bearing two such groups (2) was treated with
PLE at 37 °C and the progress of the reaction was followed by
HPLC-ESI-MS. Figure 3 gives as illustrative examples the
chromatograms obtained at an early stage, in the middle of and
at the end of the cleavage of 2. The time-dependent product
distribution after the deacetylation of 2 with PLE is shown in
Figure 4.
As seen from Figure 4, the disappearance of the starting
material is accompanied by formation of four phosphotriesters
formed in the order 16 f 13b f 17 f 14 (Scheme 6). Among
these, 16, 13b, and 17 may occur as RP- and SP-diastereomers,
but the diastereomers could not be separated by the gradient
HPLC system applied. The triesters are subsequently hydrolyzed
to three nucleoside phosphodiesters (12b, 18, and 15), and
finally, the desired 5′-TMP is released. The time-dependent
appearance of the signals reveals that 5′-TMP is formed along
the two pathways depicted in Scheme 6. All the intermediates
FIGURE 2. Time-dependent product distribution for the prolonged
PLE-promoted hydrolysis of 1 at pH 7.5 and 25.0 °C (I ) 0.1 mol L-1
with NaCl). Notation: (2) (1), (4) thymidine 5′-[3-acetyloxy-2,2-
bis(ethoxycarbonyl)]propyl 3-[hydroxy-2,2-bis(ethoxycarbonyl)propyl]-
phosphate (13a) and thymidine 5′-bis[3-hydroxy-2,2-bis(ethoxycarbo-
nyl)propyl]phosphate (14), (O) thymidine 5′-[3-acetyloxy-2,2-bis(eth-
oxycarbonyl)propyl]phosphate (12a), (b) thymidine 5′-[3-hydroxy-2,2-
bis(ethoxycarbonyl)propyl]phosphate (15), (0) 5′-TMP, and (9) thy-
midine. For the structures, see Scheme 5.
J. Org. Chem. Vol. 74, No. 14, 2009 4997

Ora et al.
FIGURE 4. Time-dependent product distribution for the PLE-catalyzed
hydrolysis of thymidine 5′-bis[3-acetyloxymethoxy-2,2-bis(ethoxycarbo-
nyl)]propylphosphate (2) at pH 7.5 and 37.0 °C (I ) 0.1 mol L-1 with
NaCl). Notation: (0) 2; (9) thymidine 5′-[3-acetyloxymethoxy-2,2-
bis(ethoxycarbonyl)]propyl 3-[hydroxymethoxy-2,2-bis(ethoxycarbonyl)-
propyl]phosphate (16); (b) thymidine 5′-[3-acetyloxymethoxy-2,2-bis(eth-
oxycarbonyl)]propyl 3-[hydroxy-2,2-bis(ethoxycarbonyl)propyl]phosphate
(13b); (O) thymidine 5′-[3-hydroxymethoxy-2,2-bis(ethoxycarbonyl)]pro-
pyl 3-[hydroxy-2,2-bis(ethoxycarbonyl)propyl]phosphate (17); (2) thymi-
dine 5′-bis[3-hydroxy-2,2-bis(ethoxycarbonyl)propyl]phosphate (14), (4)
thymidine 5′-[3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)]propylphos-
phate (12b), and ()) thymidine 5′-[3-hydroxy-2,2-bis(ethoxycarbonyl)]pro-
pylphosphate (15). For the structures, see Scheme 6.
FIGURE 3. RP-HPLC profiles for the PLE-catalyzed hydrolysis of
thymidine 5′-bis[3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)]propyl-
phosphate (2) at pH 7.5 and 37.0 °C (I ) 0.1 mol L-1 with NaCl).
Notation: (a) 2, (b) thymidine 5′-[3-acetyloxymethoxy-2,2-bis(ethoxy-
carbonyl)]propyl 3-hydroxymethoxy-2,2-bis(ethoxycarbonyl)propyl]-
phosphate (16), (c) thymidine 5′-[3-acetyloxymethoxy-2,2-bis(ethoxy-
carbonyl)]propyl 3-[hydroxy-2,2-bis(ethoxycarbonyl)propyl]phosphate
(13b), (d) thymidine 5′-[3-hydroxymethoxy-2,2-bis(ethoxycarbonyl)]-
propyl 3-[hydroxy-2,2-bis(ethoxycarbonyl)propyl]phosphate (17), (e)
thymidine 5′-bis[3-hydroxy-2,2-bis(ethoxycarbonyl)propyl]phosphate
(14), (f) thymidine 5′-[3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)]-
propylphosphate (12b), (g) thymidine 5′-[3-hydroxy-2,2-bis(ethoxy-
carbonyl)]propylphosphate (15), (h) thymidine 5′-[3-hydroxymethoxy-
2,2-bis(ethoxycarbonyl)]propylphosphate (18), (i) thymidine, and (j)
5′-TMP. For detailed chromatographic conditions, see the Experimental
Section. For the structures, see Scheme 6.
(reaction I) 12 times slower than the esterase-catalyzed deacety-
lation (reaction J). At low esterase activity, the route via 12b
may, however, predominate and the enzymatic deacetylation
of 12b (reaction K) most likely becomes rate-limiting. Alter-
natively, conversion by 15 to 5′-TMP by retroaldol condensation
(reaction D) may constitute the rate-limiting step. The half-life
for this reaction is 6 h at 37 °C.
were identified on the basis of their m/z values: [M + H]+ 799.6
(13b), [M + H]+ 727.7 (14), [M + Na]+ 851,6 (17), [M +
Na]+ 779.8 (16), [M - H]- 595.5 (12b), [M + H]- 553.2 (18),
and [M - H]- 523.4 (15). Accordingly, enzymatic removal of
one of the acetyl groups from 2 (reaction G) gives triester 16
still bearing the deacetylated hydroxymethoxy group, which is
then rather rapidly cleaved (reaction H) giving triester 13b. This
triester then undergoes breakdown by two parallel routes: a retro-
aldol condensation produces diester 12b (reaction I) and
enzymatic deacetylation yields triester 17 (reaction J). Both of
these products are eventually cleaved to diester 15, diester 12b
by enzymatic deacetylation (reaction K) and hydroxide ion-
catalyzed loss of formaldehyde (reaction M) and triester 17 by
loss of formaldehyde (reaction L) followed by retro-aldol
condensation (reaction F). In the final step, diester 15 undergoes
another retro-aldol condensation to 5′-TMP (reaction D).
Prolonged treatment with PLE resulted in partial degradation
of 5′-TMP to thymidine.
The initial deacetylation of 2 is much faster than the
corresponding reaction of 1: the half-lives on using 26 units of
PLE in 1 mL are 10 s and 3.2 min at 37 °C (Table 1),
respectively. Intermediary accumulation of the resulting hy-
droxymethoxy substituted triester (16) is clearly detected,
although the loss of formaldehyde giving triester 13b is fast,
approximatively as fast as the deacetylation. The next intermedi-
ate, triester 13b, accumulated as a main product at the early
stages of the reaction. At the high PLE concentration employed,
this compound undergoes retro-aldol condensation to diester 12b
The rate-accelerating effect that insertion of a -CH2O- group
between the ester function and C3 of the 2,2-disubstituted propyl
group exerts on the esterase-catalyzed deacetylation of both the
starting material (2 compared to 1) and the monoanionic
phosphodiester (12b compared to 12a) is remarkable. At 37 °C,
the half-lives for the deacetylation of 1 and 2 are 194 s [k )
(3.6 ( 0.1) × 10-3 s-1] and 10 s [k ) (6.8 ( 2.0) × 10-2 s-1],
respectively (see Table 1). The acceleration is thus 20-fold.
Deacetylation of the diester accelerated even more markedly.
The half-life for the disappearance of 12b is 2.7 h [k ) (5.4 (
0.1) × 10-5 s-1], while the half-life for the disappearance of
the corresponding 3-acetyloxy derivative (12a) is 150 h at this
temperature. Since the latter reaction most likely limits the rate
of release of nucleoside 5′-monophosphate from its triester pro-
drug, it appears clear that the 3-acetyloxymethoxy-2,2-
bis(ethoxycarbonyl)propyl group is much more appropriate than
its 3-acetyloxy counterpart as a protecting group of nucleoside
monophosphates.
Removal of the 3-Acetyloxymethoxy-2,2-bis(ethoxycarbon-
yl)propyl Groups from Thymidine 5′-Phosphotriester 2 in a
Whole-Cell Extract. The deprotection of thymidine 5′-bis[3-
acetyloxymetoxy-2,2-bis(ethoxycarbonyl)propyl]phosphate (2)
(Figure 5) was additionally studied in a whole cell extract of
human prostate carcinoma. The starting material was added into
2 mL of the PC3 cell extract obtained by disrupting 3 × 107
cells in 10 mL of RIPA-buffer (pH 7.5) and diluted in 1:2 ratio
with a HEPES buffer. The progress of the deprotection was
4998 J. Org. Chem. Vol. 74, No. 14, 2009

Biodegradable Protections for 5′-Monophosphates
2H), 2.22 (s, 3H), 1.92 (s, 3H); 13C NMR (126 MHz, CDCl3) δ
206.79, 172.61, 164.11, 150.59, 136.59, 111.32, 86.04, 85.08, 74.96,
62.47, 37.83, 37.15, 29.81, 27.95, 12.55; ESI+-MS m/z obsd 341.6
[M + H]+, calcd 341.1.
Diethyl 2-Ethoxy-2-methyl-1,3-dioxane-5,5-dicarboxylate (8).
Concentrated H2SO4 (1.3 mmol; 71 µL) was added to a mixture of
diethyl 2,2-bis(hydroxymethyl)malonate (43.5 mmol, 9.6 g) and
triethyl orthoacetate (65.2 mmol; 11.9 mL) in dry THF (15 mL).
The reaction was allowed to proceed overnight and the mixture
was then poured into an ice-cold solution of 5% NaHCO3 (50 mL).
The product was extracted with diethyl ether (2 × 50 mL), washed
with saturated aqueous NaCl (2 × 50 mL), and dried over Na2SO4.
The solvent was evaporated and the crude product was purified on
a silica gel column eluting with a mixture of dichloromethane and
methanol (95:5, v/v). The product was obtained as a clear oil in
89% yield (11.3 g). 1H NMR (500 MHz, CDCl3) δ 4.30-4.36 (m,
6H), 4.18 (q, J ) 7.1 Hz, 1H), 3.54 (q, J ) 7.10 Hz, 2H), 1.46 (s,
3H), 1.32 (t, J ) 7.10 Hz, 3H), 1.27 (t, J ) 7.1 Hz, 3H), 1.26 (t,
J ) 7.1 Hz 3H); 13C NMR (126 MHz, CDCl3) δ 168.0 and 167.0,
111.1, 62.0 and 61.9, 61.6, 58.7, 52.3, 22.5, 15.1, 14.0, and 13.9;
ESI+-MS m/z [M + Na]+ obsd 313.1241, calcd 313.1264.
Diethyl 2-(Acetyloxymethyl)-2-(hydroxymethyl)malonate (3).
Compound 8 (17.9 mmol; 5.2 g) was dissolved in 80% aqueous
acetic acid (30 mL) and left for 2 h at room tempertature. The
solution was evaporated to dryness and the residue was coevapo-
rated three times with water. The product was purified by silica
gel colum chromatogaphy eluting with ethyl acetate in dichlo-
romethane (8:92, v/v). The product was obtained as a yellowish
FIGURE 5. RP-HPLC profiles for the PLE-catalyzed hydrolysis of
thymidine 5′-bis[3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)]propyl-
phosphate (2) in a whole-cell extract of human prostate carcinoma at
pH 7.5 and 37.0 °C (I ) 0.1 mol L-1 with NaCl). Notation: (a) 2, (f)
thymidine 5′-[3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)]propylphos-
phate (12b), (g) thymidine 5′-[3-hydroxy-2,2-bis(ethoxycarbonyl)]pro-
pylphosphate (15), (i) thymidine, and (j) 5′-TMP. For detailed
chromatographic conditions, see the Experimental Section. For the
structures, see Scheme 6.
1
oil in 75% yield (3.6 g). H NMR (500 MHz, CDCl3) δ 4.76 (s,
followed at 37 ( 0.1 °C. Deacetylation of triester 2 was much
slower in the cell extract than at the high concentration of PLE
(26 units mL-1), but the expected diester intermediates and 5′-
TMP appeared. The acetylated diester 12b accumulated and was
converted to 5′-TMP via the deacetylated intermediate 18.
In summary, the present study indicates that the 3-acetyloxy-
2,2-bis(ethoxycarbonyl)propyl group, shown previously to be
a viable biodegradable protecting group for phosphodiesters,
does not seem appropriate for the protection of nucleoside 5′-
monophosphates as triesters. The diester obtained by departure
of the first proteting group undergoes esterase triggered removal
very slowly. By contrast, introduction of an additional -CH2O-
group between the enzyme labile ester function and the 2,2-
disubstituted propyl group markedly accelerates the enzymatic
deacetylation of the triester, but what is more important, the
deacetylation of the diester. The latter reaction experiences a
50-fold acceleration by this structural modification. It has
additionally been shown that the thymidine 5′-triester released
5′-TMP in a whole cell extract of human prostate carcinoma.
2H), 4.26 (q, J ) 7.10 Hz, 4H), 4.05 (d, J ) 7.10 Hz, 2H), 2.72 (t,
J ) 7.1 Hz, 1H), 2.08 (s, 3H), 1.27 (t, J ) 7.10 Hz, 6H); 13C NMR
(126 MHz, CDCl3) δ 170.9, 168.1, 62.3, 62.2, 61.9, 59.6, 20.7,
14.0; ESI+-MS m/z [M + Na]+ obsd 285.0942, calcd 285.0951.
Thymidine 5′-Bis[3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl]-
phosphate (1). Compound 3 was coevaporated once from dry
pyridine and three times from dry MeCN after which it was dried
over P2O5 overnight. To a solution of dried 3 (2.9 mmol; 0.76 g)
in dry DCM (2 mL) were added anhydrous triethylamine (14.4
mmol, 2 mL) and bis(diethylamino)chlorophosphine (4.0 mmol;
850 µL), and the reaction mixture was stirred for 1 h under nitrogen.
The product was filtered through a short silica gel column eluting
with a mixture of anhydrous ethyl acetate and triethylamine in
hexane (60:0.5:39.5, v/v/v). The solvent was removed under reduced
pressure and the residue was coevaporated three times from dry
MeCN to remove the traces of triethylamine. The residue was
dissolved in dry MeCN (2.0 mL) and 3 (2.9 mmol; 0.77 g) in dry
MeCN (2.0 mL) and tetrazole (7.2 mmol; 16.0 mL of 0.45 mol
L-1 solution in MeCN) were added under nitrogen. The reaction
mixture was stirred for 2.5 h at room temperature. 3′-O-Levuli-
noylthymidine (5; 2.9 mmol; 1.0 g), dried over phosphorus
pentoxide, and 1-H-tetrazole (2.9 mmol; 6.4 mL of 0.45 mol L-1
solution in MeCN) were added and the stirring was continued for
1.5 h. The phosphite ester formed was oxidized with I2 (0.1 mol
L-1) in a mixture of THF, H2O, and lutidine (4:2:1, v/v/v; 10 mL).
The crude product (6) was isolated by DCM/aq NaHSO3 workup,
and purified on a silica gel column eluted with a mixture of hexane
and ethyl acetate (40:60, v/v). The purification was repeated with
ethyl acetate and a mixture of DCM and MeOH (90:10, v/v) as an
Experimental Section
3′-O-Levulinoylthymidine (5). 5′-O-(4,4′-dimethoxytrityl)thymi-
dine (16.2 mmol, 8.8 g), prepared by established synthetic
procedures, was dissolved in anhydrous 1,4-dioxane (100 mL). A
solution of levulinic anhydride, prepared from levulinic acid (49.0
mmol, 5.70 g) in pyridine (60 mL) with 1,3-dicyclohexylcarbodi-
imide (48.4 mmol, 10.0 g) as a condensing agent, was filtered onto
the nucleoside. After being stirred for 4 h at room temperature, the
reaction mixture was evaporated to dryness and a conventional aq
NaHCO3/CH2Cl2 was carried out. The organic phase was evaporated
to dryness. The dimethoxytrityl protecting group was removed with
80% aqueous acetic acid (80 mL; 9 h). The reaction mixture was
evaporated to dryness and the residue was purified on a silica gel
column eluted with a mixture of DCM and MeOH (90:10, v/v).
The product was obtained as a white powder in 51% yield (2.8 g).
1H NMR (500 MHz, CDCl3) δ 9.36 (s, 1H), 7.73 (s, 1H), 6.25 (dd,
1H, J ) 6.5 and 2.0 Hz), 5.37 (m, 1H), 4.11 (d, 1H, J ) 2.0 Hz),
3.90 (m, 2H), 2.80 (m, 2H), 2.59 (t, 2H, J ) 6.0 Hz), 2.42 (m,
1
eluent. H NMR (500 MHz, CDCl3) δ 8.75 (s, 1H), 7.48 (d, J )
1.0 Hz, 1H), 6.37 (dd, J ) 7.0 and 5.5 Hz, 1H), 5.25 (d, J ) 6.5
Hz, 1H), 4.65-4.48 (m, 19H), 2.78 (t, 2H), 2.60 (t, 2H), 2.42 (dd,
1H), 2.22 (dd, 1H), 2.21 (s, 3H), 2.063 (s, 3H), 2.056 (s, 3H), 1.97
(d, 3H), 1.27 (m, 12H); ESI+-MS m/z obsd 909.8 [M + H]+, calcd
909.8.
A mixture of NH2NH2 ·H2O (0.5 mol L-1), 6 (0.14 mmol, 0.12 g),
pyridine (0.9 mL), and acetic acid (0.2 mL) was stirred for 80 min
at 0 °C. The ice-water bath was removed and the solution was
stirred for an additional 11 h at room temperature. The crude product
was isolated by DCM/aq NaHCO3 workup and purified on a silica
J. Org. Chem. Vol. 74, No. 14, 2009 4999

Ora et al.
gel column eluted with a mixture of DCM and MeOH (90:10%,
v/v) and by reversed phase chromatography on a Lobar RP-18
column (37 × 440 mm, 40-63 µm), eluting with a mixture of water
and acetonitrile (60:40%, v/v). The product was obtained as a clear
gel chromatography with DCM containing 2-5% MeOH as an
eluent. The product was obtained as an oil in 74% yield. 1H NMR
(500, CDCl3) δ 5.25 (s, 2H), 4.16-4.29 (m, 6H), 4.13 (s, 2H),
2.10 (s, 3H), 1.81 (br s, 1H), 1.26 (t, J ) 9.0 Hz, 6H); 13C NMR
(126 MHz, CDCl3) δ 170.41, 168.55, 89.10, 68.95, 63.96, 61.83,
60.29, 20.97, 13.99; MS [M + Na]+ obsd 315.3, calcd 315.1; ESI+-
MS m/z [M + Na]+ obsd 315.1058, calcd 315.1056.
1
oil in 55% yield. 31P NMR (202 MHz, D2O) δ -1.96 ppm. H
NMR (500 MHz, CDCl3) δ 9.30 (s, 1H), 7.35 (s, 1H), 6.33 (dd, J
) 6.5 Hz, 1H), 4.64-4.48 (m, 9H), 4.25 (m, 10H), 4.40 (br. s,
1H), 2.40 (m, 1H), 2.20 (dd, 1H), 2.06 (s, 6H), 1.94 (s, 3H), 1.25
(t, 12H); 13C NMR (126 MHz, CDCl3) δ 170.3, 166.3, 163.9, 150.5,
135.5, 111.4, 84.7, 84.3, 84.2, 70.5, 67.1, 65.3, 62.4, 58.8, 39.7,
20.6, 13.9, 12.4; ESI+-MS m/z [M + Na]+ obsd 833.7, calcd 833.2;
ESI+-MS m/z [M + H]+ obsd 811.2517, calcd 811.2460.
Thymidine 5′-Bis[3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)-
propyl]phosphate (2). 3′-O-Levulinoylthymidine (5; 0.47 mmol;
0.166 g) was coevaporated once from dry pyridine and three times
from dry MeCN and dissolved in dry DCM (1.2 mL) under nitrogen.
Triethylamine (2.35 mmol; 0.34 mL) and bis(diethylamino)chlo-
rophosphine (0.68 mmol; 0.145 mL) were added and the mixture
was stirred under nitrogen for 2 h. The product was isolated by
passing the mixture through a short silica gel column with a 4:1
mixture of ethyl acetate and hexane containing 0.5% triethylamine.
The solvent was removed under reduced pressure and the residue
was coevaporated three times from dry MeCN to remove the traces
of triethylamine. The residue was dissolved in dry MeCN (1.0 mL)
and 4 (1.68 mmol; 0.49 g) in dry MeCN (1.0 mL) and tetrazole
(2.91 mmol; 6.46 mL of 0.45 mol L-1 solution in MeCN) were
added under nitrogen. The reaction was allowed to proceed for 6 h
and then iodine (0.73 mmol; 0.185 g) in a mixture of THF (4.0
mL), H2O (2.0 mL), and 2,6-lutidine (1.0 mL) was added. The
oxidation was allowed to proceed overnight. The excess of iodine
was destroyed with 5% NaHSO3. The mixture was extracted three
times with DCM. The organic phase was washed with brine, dried
on Na2SO4, and evaporated to dryness. The crude product was
purified on a silica gel column eluting with DCM containing 5-10%
MeOH. The yield of the 3′-O-levulinoyl ester derivative (7) of 2
was 15% from 5.
Diethyl 2-(tert-Butyldimethylsilyloxymethyl)-2-hydroxymethyl-
malonate (9). Diethyl 2,2-bis(hydroxymethyl)malonate (28.3 mmol;
6.23 g) was coevaporated twice from dry pyridine and dissolved
in the same solvent (20 mL). tert-Butyldimethylsilyl chloride (25.5
mmol; 3.85 g) in dry pyridine (10 mL) was added portionwise.
The reaction was allowed to proceed for 4 days. The mixture was
evaporated to give solid foam, which was then equilibrated between
water (200 mL) and DCM (4 × 100 mL). The organic phase was
dried on Na2SO4. The product was purified by silica gel chroma-
tography eluting with 10% ethyl acetate in DCM. The product was
1
obtained as a clear oil in 78% yield. H NMR (500 MHz, CDCl3)
δ 4.18-4.25 (m, 4H), 4.10 (s, 2H), 4.06 (s, 2H), 2.63 (br s, 1H),
1.26 (t, J ) 7.0 Hz, 6H), 0.85 (s, 9H), 0.05 (s, 6H); 13C NMR (126
MHz, CDCl3) δ 169.2, 63.3, 62.8, 61.6, 61.4, 25.6, 18.0, 14.0, -3.6;
MS [M + H]+ obsd 335.7, calcd 335.2; [M + Na] obsd 357.6,
calcd 357.2; ESI+-MS m/z [M + Na]+ obsd 357.1699, calcd
357.1710.
Diethyl2-(tert-Butyldimethylsilyloxymethyl)-2-methylthiomethyl-
oxymethylmalonate (10). 9 (19.7 mmol; 6.59 g) was dissolved into
a mixture of acetic anhydride (40 mL), acetic acid (12.5 mL), and
DMSO (61 mL), and the mixture was stirred overnight. The reaction
was stopped by dilution with cold aq Na2CO3 (290 mL, 10% aq
solution) and the product was extracted in diethyl ether (4 × 120
mL). The combined organic phase was dried on Na2SO4. The
product was purified by silica gel chromatography with DCM as
an eluent. The product was obtained as a clear oil in 91% yield. 1H
NMR (400 MHz, CDCl3) δ 4.61 (s, 2H), 4.14-4.19 (m, 4H), 4.06
(s, 2H), 4.00 (s, 2H), 2.06 (s, 3H), 1.22 (t, J ) 7.0 Hz, 6H), 0.83
(s, 9H), 0.02 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 168.3, 75.6,
65.7, 61.4, 61.2, 60.9, 25.6, 18.0, 14.0, 13.7, -3.6 (Si-CH3); MS
[M + H]+ obsd 395.4, calcd 395.2, [M + Na]+ obsd 417.6, calcd
417.2; ESI+-MS m/z [M + Na]+ obsd 417.1732, calcd 417.1743.
Diethyl 2-Acetyloxymethyloxymethyl-2-(tert-butyldimethylsilyl-
oxymethyl)malonate (11). 10 (17.9 mmol; 7.08 g) was dissolved
in dry DCM (96 mL) under nitrogen. Sulfuryl chloride (21.5 mmol;
21.5 mL of 1.0 mol L-1 solution in DCM) was added in three
portions and the mixture was stirred for 70 min under nitrogen.
The solvent was removed under reduced pressure and the residue
was dissolved into dry DCM (53 mL). Potassium acetate (30.9
mmol; 3.03 g) and dibenzo-18-crown-6 (13.5 mmol; 4.85 g) in
DCM (50 mL) were added and the mixture was stirred for 1.5 h.
Ethyl acetate (140 mL) was added, and the organic phase was
washed with water (2 × 190 mL) and dried on Na2SO4. The product
was purified by silica gel chromatography with DCM as an eluent.
The 3′-O-levulinoylthymidine 5′-bis[3-acetyloxymethoxy-2,2-
bis(ethoxycarbonyl)propyl]phosphate obtained (0.071 mmol; 69 mg)
was dissolved in dry DCM (2.0 mL) and hydrazine acetate (0.12
mmol; 11 mg) in dry MeOH (0.20 mL) was added. After 1 h,
hydrazinium acetate (0.05 mmol; 4.6 mg) in a mixture of DCM
(100 µL) and MeOH (20 µL) was added. The reaction was allowed
to proceed for 2 h and the addition of hydrazinium acetate was
repeated. The reaction was quenched with acetone and the mixture
was evaporated to dryness. The product was purified on a silica
gel column eluting first with ethyl acetate and then with DCM
containing 15% MeOH. The product was obtained as a solid in
1
quantitative yield (15% from 5). H NMR (500 MHz, CDCl3) δ
8.91 (s, 1H), 7.34 (s, 1H), 6.31 (dd, J ) 6.0 and 6.0 Hz, 1H), 5.25
(s, 4H), 4.54 (m, 5H), 4.24 (m, 10H), 4.05 (s, 1H), 3.61 (br s, 1H),
2.42 (m, 1H), 2.24 (m, 1H), 2.11 (s, 6H), 1.95 (s, 3H), 1.27 (m,
12H); 13C NMR (126 MHz, CDCl3) δ 170.6, 166.6, 163.7, 150.3,
135.5, 111.4, 88.8, 84.8, 84.4, 70.7, 67.2, 67.0, 65.3, 62.3, 58.8,
39.6, 20.9, 13.9, 12.4; 31P NMR (202 MHz, CDCl3) δ -2.1 ppm;
ESI+-MS m/z [M + Na]+ obsd 893.8, calcd 893.3; ESI+-MS m/z
[M + Na]+ obsd 893.2567, calcd 893.2569.
Preparation of the Cell Extract. A sample of 3 × 107 human
prostate carcinoma cells (PC3) was treated with 10 mL of RIPA-
buffer [15 mmol L-1 Tris-HCl, pH 7.5, 120 nmol L-1 NaCl, 25
mmol L-1 KCl, 2 mmol L-1 EDTA, 2 mmol L-1 EGTA, 0.1%
deoxycholic acid, 0.5% Triton X-100, 0.5% PMSF supplemented
with Complete Protease Inhibitor Cocktail (Roche Diagnostics
GmBH, Germany)] at 0 °C for 10 min. Most of the cells were
disrupted mechanically. The cell extract obtained was centrifuged
(900 rpm, 10 min) and the pellet was discarded. The extract was
stored at -20 °C.
1
The product was obtained as an oil in 71% yield. H NMR (400
MHz, CDCl3) δ 5.24 (s, 2H), 4.15-4.22 (m, 4H), 4.13 (s, 2H),
4.08 (s, 2H), 2.08 (s, 3H), 1.26 (t, J ) 8.0 Hz, 6H), 0.85 (s, 9H),
0.04 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 170.2, 168.0, 89.3,
67.5, 61.4, 61.1, 60.2, 25.6, 21.0, 18.1, 14.0, -5.7; MS [M + Na]+
obsd 429.6, calcd 429.2; ESI+-MS m/z [M + Na]+ obsd 429.2013,
calcd 429.1921.
Diethyl 2-Acetyloxymethyloxymethyl-2-hydroxymethylmalonate
(4). 11 (7.2 mmol; 2.93 g) was dissolved in dry THF (23 mL) and
triethylamine trihydrofluoride (8.64 mmol; 1.42 mL) was added.
The mixture was stirred for 1 week. Aqueous triethylammonium
acetate (13 mL of 2.0 mol L-1 solution) was added. The mixture
was evaporated to dryness and the residue was purified by silica
Kinetic Measurements. The reactions were carried out in sealed
tubes immersed in a thermostated water bath (25.0 and/or 37.0 (
0.1 °C). The oxonium ion concentration of the reaction solutions
(2.85 mL) was adjusted with sodium hydroxide and N-[2-hydroxy-
ethyl]piperazine-N-[2-ethanesulfonic acid] (HEPES) buffer. The
ionic strength of the solutions was adjusted to 0.1 mol L-1 with
sodium chloride. The hydronium ion concentration of the buffer
solutions was calculated with the aid of the known pKa values of
5000 J. Org. Chem. Vol. 74, No. 14, 2009

Biodegradable Protections for 5′-Monophosphates
the buffer acid under the experimental conditions. The initial
the formation of this species was so much faster than its breakdown
that the intermediary accumulation was virtually quantitative.
substrate concentration was ca. 0.3 mmol L-1
.
The enzymatic hydrolysis was carried out at pH 7.5. The acetyl
group was removed with Porcine Liver Esterase (26 units mL-1
[13a]t
[1]0
kA
)
)
[exp(-kAt) - exp(-kB+Et)] (1)
in a HEPES buffer at pH 7.5 (0.01/0.01 mol L-1 at 25 °C; 0.040/
0.024 mol L-1 at 37 °C). The samples (200 µL) withdrawn at
appropriate intervals were made acidic (pH 2) with 1 mol L-1
aqueous hydrogen chloride to inactivate enzyme, cooled in an ice
bath to quench the hydrolysis, and filtered with SPARTAN 13A
filters (0.2 µm). The composition of the samples was analyzed on
an ODS Hypersil C18 column (4 × 250 mm 5 µm, flow rate 1 mL
min-1), using a mixture of acetic acid/sodium acetate buffer (0.045/
0.015 mol L-1) and MeCN, containing ammonium chloride (0.1
mol L-1). A good separation of the product mixtures of 2 was
obtained on using a 5 min isocratic elution with the buffer
containing 2% MeCN, followed by a linear gradient (23 min) up
to 40.0% MeCN. Signals were recorded on a UV-detector at a
wavelength of 267 nm. The reaction products were identified by
the mass spectra (LC/MS), using a mixture of water and MeCN
containing a formic acid (0.1%) as an eluent.
kB+E - kA
To determine the rate constant, kD, for the disappearance of the
deacetylated diester, 15, triester 1 was deacetylated with porcine
liver esterase (PLE) in a dilute HEPES buffer (pH 7.5, 25 °C), and
after 3 h, the enzyme was denaturated by adjusting the pH to 2
with aqueous hydrogen chloride. In this manner, a mixture of
the acetylated (12a) and deacetylated (15) diester was obtained.
The pH of the filtered solution was readjusted to 7.5 with a more
concentrated HEPES solution (0.1 mol L-1), and the disappearance
of 15 was followed. The rate constant for the disappearance was
obtained by the first-order rate law. The nonenzymatic deacetylation
of 12a to 15 (reaction C) was so slow that the concentration of
12a remained constant during the disappearance of 15.
The PLE-triggered formation of triester 13b was so much faster
than its breakdown that 13b was still largely accumulated after
complete disappearance of 2 and 16 (cf. Scheme 6). Accordingly,
the first-order rate constants for the disappearance of 13b (kI+J),
conversion of 13b to diester 12b (kI), and disappearance of 12b
(kK) could be obtained by three-parameter fitting of the rate law of
parallel and consecutive first-order reactions (eq 2) to the time-
dependent concentration of 12b. In eq 2, [13b]0 stands for the initial
concentration of the triester 13b and [12b]t for the concentration
of 12b at moment t. The rate constant, kJ, for the deacetylation of
13b is then obtained as a difference of kI+J and kI.
Calculation of the Rate Constants. The first-order rate constants
for the nonenzymatic hydrolysis of triesters 1 and 2 to diesters 12a
and 12b, respectively (Scheme 4), were obtained by applying first-
order rate law to the diminution of the concentration of the starting
material. The first-order rate constants for the hydrolysis of 12a
and 12b to 5′-TMP were then calculated with the aid of the rate
equation of two consecutive first-order reactions.
The enzymatic deacetylations obeyed first-order kinetics at the
high PLE concentrations employed. The pseudo-first-order rate
constants, kA and kG, for the disappearance of 1 and 2 (reaction A
in Scheme 4 and reaction G in Scheme 5, respectively) were
obtained by applying the integrated first-order rate equation to the
time-dependent diminution of the concentration of the starting
material. The first-order rate constant, kB+E, for the disappearance
of the deacetylation product of 1 (triester 13 in Scheme 5) was
then obtained by least-squares fitting of the time-dependent
concentration of 13 with the rate law of two consecutive first-order
reactions (eq 1). Here, [1]0 stands for the initial concentration of 1
and [13a]t for the concentration of 13a at moment t. Breakdown of
kB+E to contributions of two parallel first-order reactions with the
aid of the product distribution ([12a]/[14]) at early stages of their
formation then gave kB and kE. The rate constant, kC, for the
disappearance of 12a was obtained by first-order rate law, since
[12b]t
[13b]0
kI
)
[exp(-kI+Jt) - exp(-kKt)] (2)
kK - kI+J
Acknowledgment. The authors thank Dr. Tiina Laita-
Leinonen, University of Turku, for the generous gift of the
whole-cell extract and Ms. Anna-Maria Ylioikarainen for her
skillful assistance with HPLC analyses.
Supporting Information Available: Copies of 1H NMR and
13C NMR spectra of compounds 1-5 and 8-11 and copies of
MS spectra of compounds 1-4 and 8-11. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO9005987
J. Org. Chem. Vol. 74, No. 14, 2009 5001