2,2-disubstituted 4-acylthio-3-oxobutyl groups as esterase- and thermolabile protecting groups of phosphodiesters

Article abstract of DOI:10.1021/jo302421u

Five different 2,2-disubstituted 4-acylthio-3-oxobutyl groups have been introduced as esterase-labile phosphodiester protecting groups that additionally are thermolabile. The phosphotriesters 1-3 were prepared to determine the rate of the enzymatic and no

Full text of DOI:10.1021/jo302421u

Article
pubs.acs.org/joc
2,2-Disubstituted 4‑Acylthio-3-oxobutyl Groups as Esterase- and
Thermolabile Protecting Groups of Phosphodiesters
Emilia Kiuru,*,† Zafar Ahmed,† Harri Lonnberg,† Leonid Beigelman,‡ and Mikko Ora*,†
̈
†Department of Chemistry, University of Turku, FIN-20014 Turku, Finland
‡AliosBiopharma, 260 East Grand Avenue, Second Floor, South San Francisco, California 94080, United States
S
* Supporting Information
ABSTRACT: Five different 2,2-disubstituted 4-acylthio-3-oxobutyl groups
have been introduced as esterase-labile phosphodiester protecting groups
that additionally are thermolabile. The phosphotriesters 1−3 were
prepared to determine the rate of the enzymatic and nonenzymatic
removal of such groups at 37 °C and pH 7.5 by HPLC-ESI-MS.
1
Additionally, H NMR spectroscopic monitoring was used for structural
characterization of the intermediates and products. When treated with hog
liver esterase, these groups were removed by enzymatic deacylation
followed by rapid chemical cyclization to 4,4-disubstituted dihydrothio-
phen-3(2H)-one. The rate of the enzymatic deprotection could be tuned
by the nature of the 4-acylthio substituent, the benzoyl group and acetyl
groups being removed 50 and 5 times as fast as the pivaloyl group. No
alkylation of glutathione could be observed upon the enzymatic deprotection. The half-life for the nonenzymatic deprotection
varied from 0.57 to 35 h depending on the electronegativity of the 2-substituents and the size of the acylthio group. The acyl
group evidently migrates from the sulfur atom to C3-gem-diol obtained by hydration of the keto group and the exposed mercapto
group attacks on C1 resulting in departure of the protecting group as 4,4-disubstituted 3-acyloxy-4,5-dihydrothiophene with
concomitant release of the desired phosphodiester.
have to be protected in a single molecule. The group of
Beaucage has introduced several purely thermolytic groups,
including (N-formyl-N-methyl)-2-aminoethyl,15 4-hydroxybu-
tyl,16 and several from ω-(alkylthio)alkyl groups,17 for the
protection of immunostimulatory phosphoromonothioate
oligomers. All these groups are removed by cyclization, the
most appropriate being 5-(methylthio)pentyl, 5-(isopropyl-
thio)pentyl, 4-(methoxymethylthio)butyl, and 2-(methylthio-
methoxy)ethyl groups, which exhibit half-lives of removal
ranging from 6 to 35 h at 37 °C. Cyclosaligenyl group has, in
turn, shown some promise as a thermolabile protection of
phosphate monoesters.18−21
We now introduce a set of 2,2-disubstituted 4-acylthio-3-
oxobutyl groups as novel protecting groups for phosphodiester
linkages. These groups are esterase-labile, but they undergo
thermolytic removal in case the enzymatic reaction becomes
severely retarded. The groups are released by cyclization to
substituted tetrahydrothiophenones which do not form adducts
with glutathione and, hence, most likely are not markedly
alkylating. The rate of the esterase-catalyzed thioester
hydrolysis may be varied by the size of the S-acyl group, and
the thermolytic stability may be tuned by the polar nature of 2-
substituents. The following compounds were prepared as
model compounds to determine the rate of the enzymatic and
INTRODUCTION
■
Most of the prodrug approaches are based on a strategy
consisting of an initial enzymatic activation, usually deacylation
by esterases, followed by nonenzymatic removal of the
remaining linker attached to phosphate function.1,2 The
protecting groups falling in this esterase-labile category include
acyloxymethyl,3 alkoxycarbonyloxymethyl,4 S-acyl-2-thioethyl,5
4-acyloxybenzyl,6 2,2-bis(substituted)-3-acyloxypropyl,7,8 2,2-
bis(substituted)-3-acyloxymethoxypropyl,9 and 1-acyloxypro-
pan-1,3-diyl10 groups. In addition, the conversion of extensively
studied O-aryl-N-[2-(alkoxycarbonyl)alkyl]phosphoramidates
to phosphate monoesters is trigged enzymatically by esterases,
but hydrolysis of the deprotected phosphoramidate to
phosphate monoester depends on another enzyme, phosphor-
amidase.11,12 Esterases accept a wide range of neutral molecules
as substrates, and hence, the first phosphate protecting group is
usually removed without difficulty. The cleaving activity of
esterases is, however, dramatically reduced upon accumulation
of negative charge on the substrate.9,10,13,14 For this reason,
removal of esterase-labile protecting groups from molecules
containing more than one phosphodiester linkages still is
problematic. Another shortcoming is that the nonenzymatic
removal of the remnant of the protecting group often produces
potentially toxic products, such as episulfide, formaldehyde,
enones, phenols or quinoid structures. In fact, thermolabile
protecting groups appear to offer an attractive alternative for
esterase-labile groups as long as several phosphodiester linkages
Received: November 2, 2012
Published: December 28, 2012
© 2012 American Chemical Society
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nonenzymatic removal of such groups at physiological pH: 3′-
O-levulinoylthymidine 5′-methylphosphate protected with 4-
acetylthio-2-ethoxycarbonyl-3-oxo-2-methylbutyl (1a), 4-ben-
zoylthio-2-ethoxycarbonyl-3-oxo-2-methylbutyl (1b) groups,
2′,3′-O-isopropylideneuridine 5′-methylphosphate protected
with 4-acetylthio-2,2-dimethyl-3-oxobutyl (2a), 4-benzoylthio-
2,2-dimethyl-3-oxobutyl (2b), and 2-ethoxycarbonyl-2-methyl-
3-oxo-4-pivaloylthiobutyl (2c) groups, and bis(2′-C-methylur-
idin-5′-yl) phosphate protected with 4-acetylthio-2,2-dimethyl-
3-oxobutyl group (3). The progress of the deprotection of 3,
containing potentially antiviral 2′-C-methyl nucleosides, was
additionally followed in a whole cell extract of human prostate
carcinoma.
noates (5a−c) were obtained by bromination of commercial
ethyl 2-methyl-3-oxobutanoate,22 followed by treatment with
thioacetic, thiobenzoic, or thiopivalic acid in the presence of
triethylamine to give thioesters 4a−c, and these were finally
subjected to triethylamine-promoted hydroxymethylation23 to
5a−c. S-(4-Hydroxy-3,3-dimethyl-2-oxobutyl)thioacetate (7a)
and thiobenzoate (7b) were, in turn, prepared by trifluoro-
acetoxymethylation of 3-methylbutan-2-one and subsequent
alkaline hydrolysis to 4-hydroxy-3,3-dimethylbutan-2-one24 (6),
which then was converted to 7a and 7b by bromination and
subsequent displacement of the bromo substituent with
thioacetic or thiobenzoic acid.
Phosphitylation of 3′-O-levulinoylthymidine25 with 1-chloro-
N,N-diisopropyl-1-methoxyphosphinamine and subsequent
tetrazole-promoted displacement of the diisopropylamino
ligand by alcohols 5a and b gave after oxidation triesters 1a,b
(Scheme 2). Similarly, 2′,3′-O-isopropylideneuridine was
converted to triesters 2a−c with the aid of alcohols 7a,b and
5c. All of the triesters were mixtures of RP and SP
diastereomers. In addition, the protecting group of triesters
1a,b and 2c contained a stereogenic center which increased the
number of stereoisomers from two to four. No attempt was
made to assign the absolute configuration of the diastereomers.
Phosphotriester 3 was prepared by displacing first the two
chloro ligands of 1,1-dichloro-N,N-diisopropylphosphinamine
with 2′,3′-O-isopropylidene-2′-C-methyluridine26 (8) and the
diisopropylamino ligand subsequently with S-(4-hydroxy-3,3-
dimethyl-2-oxobutyl) thioacetate (7a), using tetrazole as an
activator (Scheme 3). The phosphite triester formed was
oxidized to phosphate ester (9) with iodine in aqueous THF
containing 2,6-lutidine. The isopropylidene groups were then
removed by treatment with aq 80% AcOH, giving 4-acetylthio-
2,2-dimethyl-3-oxobutyl bis[2′-C-methyluridin-5′-yl] phosphate
(3).
Hydrolytic Stability of Phosphotriesters 1−3. Non-
enzymatic removal of the substituted butyl protecting groups
from phosphotriesters derived from 3′-O-levulinoylthymidine
(1a,b) or 2′,3′-O-isopropylideneuridine (2a−c and 3) was
studied in a 2-[4-(2-hydroxyethyl)piperazin-1-yl)]-
ethanesulfonic acid (HEPES) buffer at pH 7.5 and at 37.0
0.1 °C. The composition of the aliquots withdrawn from the
RESULTS AND DISCUSSION
■
Syntheses. The preparation of various substituted butanols
required for preparation of compounds 1−3 is outlined in
Scheme 1. 4-Acylthio-2-hydroxymethyl-2-methyl-3-oxobuta-
a
Scheme 1
a
Conditions: (i) Br2, DCM; (ii) RSH, TEA, Et2O; (iii) H2CO, TEA, dioxane; (iv) H2CO, TFA, reflux; (v) 15% aq NaHCO3.
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a
spectrometric analysis (HPLC/ESI-MS). Diastereomeric mix-
tures were used as starting materials, but the rate constants
were calculated separately for the disappearance of individual
diastereomers that exhibited different HPLC signals. With all
the phosphotriesters studied, the desired phosphodiester was
the main product: 3′-O-levulinoylthymidine 5′-methylphos-
phate (10, [M + H]+ at m/z 435.6) from 1a,b, 2′,3′-O-
isopropylideneuridine 5′-methylphosphate (11, [M + H]+ at
m/z 379.6) from 2a−c, and bis[2′-C-methyluridin-5′-yl]
phosphate (12, [M + H]+ at m/z 621.2) from 3. In fact, the
only marked side products were formed from 2b and 2c that
underwent demethylation in 15% and 5% yield, respectively,
giving phosphodiesters 13b,c still bearing the original
protecting group (Scheme 4). Compound 2b additionally
yielded an unidentified byproduct, the amount of which was
less than 5%. Otherwise, more than 95% of the triester was
converted to the diester. The RP and SP diastereomers reacted
at very similar rates. For example, the more slowly eluting
diastereomers of 2a and 2b reacted 2% and 4% faster,
Scheme 2
a
Scheme 4
a
Conditions: (i) TEA, DCM; (ii) TetH, MeCN; (iii) I2, THF, H2O,
2,6-lutidine.
a
Scheme 3
a
Conditions: (i) TEA, DCM; (ii) TetH, MeCN; (iii) I2, THF, H2O,
2,6-lutidine; (iv) 80% AcOH.
reaction mixture at appropriate time intervals was analyzed by
RP-HPLC and the products were identified by mass
a
Conditions: HEPES buffer, pH 7.5.
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respectively, than the faster eluting ones. Table 1 records the
rate constants and half-lives for the departure of the protecting
groups at pH 7.5 and 37 °C. At pH 6.5 in a MES buffer, the
reaction was 1 order of magnitude slower.
Esterase-Triggered Deprotection of Triesters 1−3. To
elucidate the susceptibility of triesters 1−3 to esterase-triggered
deprotection, they were treated with hog liver carboxyesterase
(HLE; 2.6 units/mL) in a HEPES buffer at pH 7.5 and 37 °C.
Under these conditions, the conversion of triesters 1−3 to the
corresponding phosphodiesters (10−12) is nearly quantitative.
The half-life for the disappearance of 1a,b and 2a−c ranged
from 0.17 to 14 min, the deacylation of the 4-benzoylthio-2,2-
dimethyl-3-oxobutyl and 2-ethoxycarbonyl-2-methyl-3-oxo-4-
(pivaloylthio)butyl group being the fastest and slowest
reactions, respectively. Triesters bearing the deacylated 4-
mercapto group were accumulated as intermediates. With the
4-benzoylthio-2,2-dimethyl-3-oxobutyl-protected triester, 2b,
and its 4-acetylthio analogue, 2a, the maximal accumulation
level of the intermediate (15, [M + H]+ at m/z 509.7) was 80%
and 25%, respectively (Figure 1). Accordingly, the rate
constants for the chemical removal of deacylated protecting
group were 7.2 × 10−3 s−1 (τ1/2 1.6 min) and 9.6 × 10−3 s−1
(τ1/2 1.2 min), respectively. With their 4-pivaloylthio analogue,
2c, the accumulation of the mercapto intermediate remained,
owing to slow deacylation, at a low level. The removal of 2-
ethoxycarbonyl-4-mercapto-2-methyl-3-oxobutyl group ap-
peared, in turn, to be so fast that the mole fraction of the
mercapto intermediate ([M + H]+ at m/z 623.4) remained
below 0.02 with 1a,b and no accumulation was detected with
2c. This means that the lower limit for the rate constants of the
removal of the deacylated group is 1 s−1 (τ1/2 < 1s). Table 2
records the rate constants and half-lives for the esterase-
catalyzed deacylation.
Table 1. First-Order Rate Constants and Half-Lives for the
Departure of Various 2,2-Disubstituted 4-Acylthio-3-
oxobutyl Groups from Nucleoside 5′-Phosphtriesters in
HEPES Buffer at pH 7.5 and 37 °C (I = 0.1 mol L−1 with
NaCl)
a
group RS
2-substituents
triester
k (10−6 s−1
)
τ1/2 (h)
AcS
BzS
PivS
BzS
AcS
AcS
AcS
Me, COOEt
Me, COOEt
Me, COOEt
Me, Me
1a
1b
2c
2b
2a
2a
3
338
121
2
3
0.57
1.59
12.9
35.3
11.2
79.6
6.9
b
14.9 0.1
c
5.45 0.05
17.2 0.3
2.42 0.03
Me, Me
d
Me, Me
e
Me, Me
28 0.5
a
Mean of the rate constants of various diastereomers, which differed
b
by less than 5%. Contains a 5% contribution of side reactions not
giving the desired diester. Contains a 20% contribution of side
reactions not giving the desired diester. At 20 °C. 32% of the diester
c
d
e
obtained had one of the sugar hydroxyl groups acetylated.
The removal of the 2,2-dimethyl-4-acetylthio-3-oxobutyl
group from the internucleosidic phosphodiester bond of
bis(2′-C-methyluridin-5′-yl) phosphate (3) was also studied.
The phosphodiester linkage was exposed 60% faster than with
the similarly protected 2′,3′-O-isopropylideneuridine 5′-meth-
ylphosphate (2a), but unexpectedly, 32% of the phosphodiester
formed was still acetylated. Prolonged treatment with HEPES
buffer resulted in slow deacetylation of the side product ([M +
H]+ at m/z 621.2) to the desired phosphodiester (12, [M +
H]+ at m/z 578.9). Most likely, the acetyl group of the
phosphate protecting group had migrated to the 3′-OH
function of the sugar moiety. To ensure this assumption, the
kinetic analysis was also carried out with the 2′,3′-O-
isopropylidene-protected counterpart (9). The only product
observed to be accumulated was bis[2′,3′-O-isopropylidene-2′-
C-methyluridin-5′-yl] phosphate, the half-life for the depro-
tection being 11.4 h (k = 1.69 × 105 s−1).
2,2-Dimethyl-2-(4-acetylthio-3-oxo)butyl bis[2′-C-methylur-
idin-5′-yl] phosphate (3) was converted by HLE to diester (12)
more slowly than 2a, having one of the nucleoside moieties
replaced with a methyl group. No intermediate accumulated,
Figure 1. Time-dependent product distribution for the HLE triggered deprotection of diastereomeric (RP/SP) 5′-phosphotriesters of 2′,3′-O-
isopropylideneuridine at pH 7.5 and 37 °C (I = 0.1 mol L−1 with NaCl). (A) Removal of the (4-benzoylthio-2,2-dimethyl-3-oxo)butyl group: ( )
■
□
■
□
○
○
2b, ( ) 15, ( ) 11. (B) Removal of the (4-acetylthio-2,2-dimethyl-3-oxo)butyl group: ( ) 2a, ( ) 15, ( ) 11.
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Consistent with the suggested removal of the protecting
group by intramolecular cyclization, the ESI−-MS signal of 16a
([M − H]− at m/z 187.0 see the Supporting Information,
Figure S46) was detected upon enzymatic deprotection of a
single (HPLC-separated) diastereomer of 1a. To obtain
additional evidence for the structure of this species, the course
Table 2. First-Order Rate Constants and Half-Lives for the
Esterase-Catalyzed Deacylation of Phosphotriesters 1−3 in a
HEPES Buffer (pH 7.5, 37 °C, I = 0.1 mol L−1) Containing
HLE 2.6 Units/mL
group RS 2-substituents triester deacylation k (10−3 s−1
)
τ1/2 (min)
1
AcS
BzS
PivS
BzS
AcS
AcS
Me, COOEt
Me, COOEt
Me, COOEt
Me, Me
1a
1b
2c
2b
2a
3
4.05 0.06
39.1 1.6
0.81 0.02
67.4 3.4
2.45 0.10
0.14 0.01
2.9
0.30
14
of the reaction was followed by H NMR spectroscopy in a 10
mmol L−1 phosphate buffer (pH 7.0) in a mixture of D2O and
CD3CN (10:1 v/v) (see the Supporting Information, Figure
S47). Appearance of 1H resonaces at 3.77 (q, 2H, J = 7.0 Hz, 4-
OCH2CH3), 3.33 (d, 1H, J = 11.5 Hz, H5) and 2.89 (d, 1H, J =
11.5 Hz, H5), 1.28 (q, 3H, J = 7.0 Hz, 4-OCH2CH3), and 1.25
(s, 3H, 4-CH3) was consistent with the assumed structure, but
no resonances referring to the protons at C2 could be identified
because of rapid deuteration as a consequence of keto−enol
tautomerism. When the experiment was repeated with 2b
(D2O/CD3CN 4:1), containing a methyl group instead of the
ethoxycarbonyl group, all four ring proton resonances of 16b
were observed. On the basis of HSQC and HMBC correlations,
C2 protons correlated at 3.39 and 2.98 ppm and C4 protons at
2.49 and 2.34 ppm.
0.17
4.7
Me, Me
a
Me, Me
83
a
Contains a 10% contribution of formation of a diester having one of
the sugar hydroxyl groups acetylated.
but sugar-acetylated diester 14 appeared as a side product,
representing about 10% of the disappearance of 3. Depro-
tection of 3 was additionally studied in a whole cell extract of
human prostate carcinoma PC3 cells, obtained by disrupting 3
× 106 cells in 1 mL of RIPA buffer (pH 7.5) and diluting to
triple volume with a HEPES buffer. The 5′,5′-phosphodiester
(12) was released, the half-life being 8.3 h. The sugar acetylated
diester (14) was again observed as a side product, now
representing 25% of the overall disappearance of 3. No
indication of the desired subsequent hydrolysis to 2′-C-
methyluridine and 2′-C-methyluridine-5′-monophosphate was
obtained.
Mechanism of Esterase-Triggered Departure of the
2,2-Disubstituted 4-Acylthio-3-oxobutyl Groups. As
indicated above, the carboxyesterase-mediated conversion of
phosphotriesters 1−3 to phosphodiesters by departure of the
2,2-disubstituted 4-acylthio-3-oxobutyl group is initiated by
deacylation that exposes the mercapto group. The 4-mercapto
intermediate clearly accumulates when the protecting group is
2,2-dimethylated and the removable acyl group is benzoyl or
acetyl. The exposed mercapto group then attacks the
phosphate-bound carbon atom resulting in departure of the
group as 4,4-disubstituted dihydrothiophen-3(2H)-one (16 in
Scheme 5). When one of the methyl groups at C2 is replaced
with a more electronegative ethoxycarbonyl group, the electron
density at the neighboring methylene group is reduced and the
intramolecular attack of the mercapto function is facilitated.
Accordingly, accumulation of the deacylated intermediate can
hardly be detected with the 2-ethoxycarbonyl substituted
compounds 1a,b and 2c.
Mechanism of Nonenzymatic Departure of the
Phosphate Protecting Groups. The nonenzymatic con-
version of triesters 1−3 to the corresponding diesters 10−12 is
too fast to be initiated by hydroxide ion catalyzed hydrolysis of
the thioester linkage. The rate of hydroxide ion catalyzed
hydrolysis of thioesters is comparable to that of oxoesters,27
and the half-life for the hydroxide ion catalyzed deacetylation of
thymidine 5′-bis-[3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl]-
phosphate, for example, is 20 days (k = 4.0 × 10−7 s−1) at
pH 7.5 and 37 °C.7 The keto group at C3 seems to play a
decisive role by allowing migration of the acyl group from sulfur
atom to the neighboring oxygen atom. Two alternative
mechanisms may be proposed: either a hydroxyl group of the
gem-diol formed by hydration of the keto group attacks as
oxyanion on the carbonyl carbon of the sulfur-bound acyl
group, as depicted in Scheme 6, or the attacking neighboring
oxyanion is obtained by enolization of the starting material. The
exposed mercapto group may then attack on C1 resulting in
removal of the protecting group by cyclization. The reaction
through hydration of the keto group appears more attractive for
the reason that an ESI+-MS signal at 313.3, possibly referring to
the Na+ adduct of 17, was observed during the course of the
deprotection of 2c. This intermediate then undergoes
dehydration to a 4,4-disubstituted 3-acyloxy-4,5-dihydrothio-
phen (18b). Formation of ethyl 4-acetyloxy-3-methyl-2,3-
Scheme 5
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Scheme 6
dihydrothiophene-3-carboxylate (18a, [M + H]+ at m/z 231.3
and [M − H]− at m/z 229.1) from 1a was detected by ESI-MS
(see the Supporting Information, Figure S46). When 2a was
used as a starting material, a HEPES adduct of 4,4-dimethyl-4,5-
dihydrothiophen-3-yl acetate (18c) was observed.
exhibited the [M + H]+ signal at m/z = 667.6, i.e., two units
higher than the authentic 1a. For this reason, the 1H resonance
of H4 on 18a was not detected.
Deprotection in the Presence of Glutathione. To
evaluate the potential of the released protecting group as an
alkylating agent, triester 2b was treated with HLE in the
presence of glutathione. The only indication of glutathione
adduct formation was the appearance of a weak HPLC signal
exhibiting a m/z value of 814.4 on positive mode. This signal,
representing a few percent of the starting material, most likely
refers to debenzoylated 2b that has formed a disulfide linkage
with the mercapto group of glutathione. No alkylation products
were detected. The results were similar when the glutathione
was added after 2b had already debenzoylated.
As mentioned in the foregoing, triesters 2b and 2c
unexpectedly underwent demethylation as a minor side reaction
concurrent with the removal of the protecting group.
Conversion to diesters bearing the original protecting group
instead of methyl group may also be explained by attack of the
C3-oxyanion (formed from the C3-gem-diol or -enol) on
phosphorus with concomitant displacement of methoxide ion.
The cyclic phosphotriester intermediate then collapses to
phosphodiester by hydrolysis of the CO bond involved in the
half acetal of vinyl ether structure obtained. It is worth noting
that a related mechanism, viz. nucleophilic attack of hydrated 2-
oxopropyl group on phosphorus, has been proposed for the
hydrolysis of bis(ketol) pro-drugs of nucleoside 5′-phos-
phates.28
CONCLUSIONS
■
2,2-Disubstituted 4-acylthio-3-oxobutyl groups have been
introduced as applicable protection groups for phosphodiesters.
These groups resemble other esterase-labile protecting groups
in the sense that deacylation by carboxyestarases triggers rapid
removal of the remnants of the group. The rate of the
enzymatic deprotection may, hence, be tuned by the nature of
the 4-acylthio substituent. The benzoyl group, for example, is
removed 50 times as fast as the pivaloyl group. Exposure of the
mercapto function results in removal of the group by
cyclization to 4,4-disubstituted dihydrothiophen-3(2H)-one. It
is worth noting that this product is not markedly alkylating and
was not observed to form glutathione adduct. The novel feature
of these esterase-labile groups, however, is that they additionally
are thermolabile. The nonenzymatic stability may be tuned
within wide limits be electronegativity of the 2-substituents.
The acyl group evidently migrates from the sulfur atom to C3-
The removal of the phosphate protecting group was also
1
followed by H NMR spectroscopy. Experiments with 1a in
D2O/CD3CN (10:1, v/v) buffered with potassium phosphate
(0.010/0.010 mol L−1; pH 7.0) revealed the release of a
product assigned as ethyl 4-acetyloxy-3-methyl-2,3-dihydrothio-
1
phene-3-carboxylate (18a). H resonances at 4.35 (q, 2H, J =
7.0 Hz, 4-CO2CH2CH3), 3.65 (d, 1H, J = 12.0 Hz, H2), 3.13
(d, 1H, J = 12.0 Hz, H2), 2.06 (s, 3H, AcO), 1.54 (s, 3H, CH3)
1.36 (t, 3H, J = 7.0 Hz, 4-CO2CH2CH3) (see the Supporting
Information, Figure S47). It is worth noting that the methylene
protons adjacent to keto group of 1a are subject to rapid
deuteration under the experimental conditions, as evidenced by
the fact that the aliquots withdrawn from the reaction mixture
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4.21 (q, 2H, J = 7.21 Hz, CH3CH2O); 3.90 (d, 1H, J = 16.43 Hz,
CH2S); 3.82 (d, 1H, J = 16.43 Hz, CH2S); 3.74 (q, 1H, J = 7.21, CH);
1.39 (d, 3H, J = 7.21 Hz, CH3CH); 1.36 (s, 3H, CH3 of Piv); 1.29 (t,
3H, J = 7.21 Hz, CH3CH2O); 1.26 (s,6H, CH3 of Piv). 13C NMR (101
MHz, CDCl3): δ = 205.25 (CO); 199.89 (SCO); 170.07 (OC
O); 61.58 (CH3CH2O); 51.76 (CHCH3); 46.39 (spiro C); 37.99
(SCH2); 27.25 (CH3 of Piv); 14.05 (CH3CH2O), and 12.86
(CHCH3). ESI+-HRMS: m/z [M + Na]+ calcd for C12H20NaO4S
283.0975, found 283.0968.
gem-diol obtained by hydration of the keto group and the
exposed mercapto group attacks on C1 resulting in departure of
the protecting group as 4,4-disubstituted 3-acyloxy-4,5-dihy-
drothiophene. The advantage of thermolability is that these
groups will be removed even when the enzymatic reaction
becomes severely retarded. This seems to be the case with
molecules containing several phosphodiester linkages; accumu-
lation of negative charge in the molecule makes it an
increasingly poor substrate for esterases.
Ethyl 4-(Acetylthio)-2-(hydroxymethyl)-2-methyl-3-oxobuta-
noate (5a). Compound 4a (2.02 g, 8.62 mmol) was dissolved in
dioxane (9.0 mL). The solution was cooled on an ice bath, and 20% aq
formaldehyde (9.50 mmol) and TEA (1 M in THF, 0.17 mmol, 0.17
mL) were added. The mixture was stirred for 20 min. The water bath
was removed, and TEA (0.17 mmol, 0.17 mL) was added. The acidity
of the mixture was kept at pH 7.5−8.5 by addition of triethylamine
twice in small portions (0.17 mL, 0.17 mmol) within next 4 h. The
solvent was removed by evaporation under reduced pressure, and the
residue was coevaporated from toluene. The product was purified by
silica gel chromatography using DCM containing 2% MeOH as eluent.
Compound 5a was obtained as an oil in 72% yield (1.56 g). 1H NMR
(400 MHz, CDCl3): δ = 4.27 (qd, 2H, J = 7.20 and 0.4 Hz,
CH3CH2O); 4.05 (d, 2H, J = 17.19 Hz, CH2S); 4.00 (dd, 1H, J = 6.40
and 5.60 Hz, CH2OH); 3.96 (d, 1H, J = 17.19 Hz, CH2S); 3.85 (dd,
1H, J = 8.00 and 7.60 Hz, CH2OH); 2.84 (dd, 1H, J = 8.00 and 7.60
Hz, OH); 2.39 (s, 3H, CH3CO); 1.47 (s, 3H, CH3) 1.31 (t, 3H, J =
7.20 Hz, CH3CH2O). 13C NMR (101 MHz, CDCl3): δ = 201.62 (C
O); 194.74 (SCO); 171.85 (OCO); 66.56; (CH2OH); 61.97
(OCH2CH3); 61.37 (spiro C); 37.35 (CH2S); 30.09 (CH3CO);
17.49 (CH3); 14.01 (CH3CH2O). ESI+-HRMS: m/z [M + Na]+ calcd
for C10H16NaO5S 271.0611, found 271.0627.
EXPERIMENTAL SECTION
■
General Methods. 2′,3′-O-Isopropylideneuridine was a product of
Carbosynth. The preparation of 3′-O-levulinoylthymidine25 and 2′,3′-
O-isopropylidene-2′-C-methyluridine (8)26,29 has been described
previously. Reactions requiring anhydrous conditions were carried
out under argon or nitrogen gas. The solvents were dried over 4 Å
molecular sieves. Triethylamine was dried by refluxing over CaH2 and
distilled before use. For column chromatography, silica gel 60 (230−
400 mesh) was used. 2D spectra (COSY and HSQC) have been used
1
in assigning H and 13C signals.
Ethyl 4-(Acetylthio)-2-methyl-3-oxobutanoate (4a). Ethyl 2-
methyl-3-oxobutanoate (4.00 mL, 28.3 mmol) was dissolved in 25
mL of dry DCM. The solution was cooled on an ice bath, and bromine
(28.3 mmol, 1.45 mL) in DCM (3 mL) was added. The ice bath was
removed, and the reaction mixture was stirred for 12 h at room
temperature. The solvent was removed under reduced pressure, and
the residue was coevaporated from dry DCM. Ethyl 4-bromo-2-
methyl-3-oxobutanoate (28.27 mmol, 6.3 g, dried over P2O5
overnight), used without further purification, was dissolved in dry
diethyl ether (60 mL). The solution was cooled on an ice bath, and
thioacetic acid (42.4 mmol, 3.03 mL) was added. Triethylamine (4.60
mL, 33.1 mmol) in diethyl ether (14 mL) was added dropwise. The ice
bath was removed, and the reaction mixture was stirred for 3.5 h at
room temperature. Diethyl ether (40 mL) was added, and the organic
phase was washed twice with water, 5% aq NaHCO3, and saturated aq
NaCl, dried over Na2SO4, and evaporated to dryness. The product was
purified by silica gel chromatography using DCM as eluent.
Compound 6a was obtained as oil in 33% yield from ethyl 2-
methyl-3-oxobutanoate (2.08 g). 1H NMR (500 MHz, CDCl3) δ: 4.22
(q, 2H, J = 7.50 Hz, CH3CH2O); 3.92 and 3.89 (d, 2H, J = 17.00 and
16.50 Hz, CH2S); 3.75 (q, 2H, J = 7.50 Hz, CHCH3); 2.39 (s, 3H,
CH3CO); 1.39 (d, 3H, J = 7.50 Hz, CHCH3); 1.30 (t, 3H, J = 7.50
Hz, CH3CH2O). 13C NMR (126 MHz, CDCl3): δ = 199.46 (CO);
194.18 (SCO); 169.94 (OCO); 61.67 (OCH2CH3); 51.85
(CHCH3); 38.44 (CH2S); 30.08 (CH3CO); 14.05 (CH3CH2);
12.81 (CH3CH). ESI+-HRMS: m/z [M + Na]+ calcd for C9H14NaO4S
241.0505, found 241.0509.
Ethyl 4-(Benzoylthio)-2-(hydroxymethyl)-2-methyl-3-oxobuta-
noate (5b). Compound 5b was prepared as described for 5a. The
product was not purified by silica gel chromatography owing to its
lability. The reaction mixture was evaporated to dryness. After addition
of water (60 mL), the mixture was extracted with DCM (3 × 40 mL).
The organic phase was dried over Na2SO4 and evaporated to dryness.
1
Compound 5b was obtained as solid in 90% yield (0.17 g). H NMR
(500 MHz, CDCl3): δ = 7.99 (dd, 2H, J = 8.50 and 1.00 Hz, Bz); 7.62
(t, 1H, J = 7.50 Hz, Bz), 7.48 (t, 2H, J = 7.50 Hz, Bz), 4.32 (q, 2H, J =
7.00 Hz, CH3CH2O); 4.26 (d, 1H, J = 17.00 Hz, CH2S); 4.18 (d, 1H, J
= 17.50 Hz, CH2S); 4.09 (d, 1H, J = 11.50, CH2O); 3.92 (d, 1H, J =
11.50, CH2O); 1.55 (s, 3H, CH3CH); 1.35 (t, 3H, J = 7.00 Hz,
CH3CH2O). 13C NMR (126 MHz, CDCl3): δ = 201.67 (CO);
190.89 (SCO); 171.95 (OCO); 136.10, 134.00, 129.37, 128.83,
127.55, and 127.44 (Bz); 67.10 and 66.66 (CH3CH2O); 62.00 and
61.50 (CH2OH); 37.29 and 36.74 (SCH2); 17.52 (CH3CH2O) and
14.04 (CHCH3). ESI+-MS: m/z [M + H]+ calcd for C15H19O5S 311.4,
found 311.5. ESI+-HRMS: m/z [M + Na]+ calcd for C15H18NaO5S
333.0767, found 333.0771.
Ethyl 4-(Benzoylthio)-2-methyl-3-oxobutanoate (4b). Compound
4b was prepared in a similar manner as 4a, but by using thiobenzoic
acid instead of thioacetic acid. The product was purified by silica gel
chromatography using DCM as eluent. The yield of 4b, obtained as a
Ethyl 2-(Hydroxymethyl)-2-methyl-3-oxo-4-(pivaloylthio)-
butanoate (5c). Compound 5c was prepared as described for 5a.
Compound 5c was obtained as a solid in quantitative yield (4.13 g).
1H NMR (400 MHz, CDCl3): δ = 4.27 (q, 2H, J = 7.20 Hz,
1
solid, was 21% (3.2 g) from ethyl 2-methyl-3-oxobutanoate. H NMR
CH3CH2O); 4.01 (d, 1H, J = 11.60 Hz, CH2OH); 4.00 (d, 1H, J =
17.19 Hz, CH2S); 3.92 (d, 1H, J = 17.19 Hz, CH2S); 3.86 (d, 1H, J =
12.00 Hz, CH2OH); 1.48 (s, 3H, CH3); 1.40 (s, 3H, CH3 of Piv); 1.33
(t, 3H, J = 7.20 Hz, CH3CH2O); 1.26 (s, 6H, CH3 of Piv). 13C NMR
(101 MHz, CDCl3): δ = 205.89 (CO); 201.85 (SCO); 171.92
(OCO); 66.62 (CH2OH); 61.90 (CH3CH2O); 61.40 (spiro C);
46.43 (spiro C of Piv); 36.93 (SCH2); 27.29 (CH3 of Piv); 17.52
(CHCH3) and 14.02(CH3CH2O). ESI+-HRMS: m/z [M + Na]+ calcd
for C13H12NaO5S 313.1080, found 313.1081.
4-Hydroxy-3,3-dimethylbutan-2-one (6). Compound 6 was
prepared as described previously.24 A mixture of 3-methylbutan-2-
one (5.00 g, 58.0 mmol), paraformaldehyde (1.74 g, 58.0 mmol), and
TFA (8.90 mL, 116.1 mmol) was heated at reflux for 7 h. Saturated aq
NaHCO3 (100 mL) was added, and the resulting suspension was
stirred at room temperature for 1 d. The mixture was extracted with
DCM (8 × 50 mL), after which the organic phase was dried over
(400 MHz, CDCl3): δ = 7.97 (dd, 2H, J = 7.21 and 1.00 Hz, Bz); 7.79
(t, 1H, J = 7.61 Hz, Bz), 7.46 (t, 2H, J = 7.61 Hz, Bz), 4.23 (q, 2H, J =
7.20 Hz, CH3CH2O); 4.14 (d, 1H, J = 16.39 Hz, CH2S); 4.05 (d, 1H, J
= 16.79 Hz, CH2S); 3.84 (q, 1H, J = 7.20, CH); 1.43 (d, 3H, J = 8.00
Hz, CH3CH); 1.29 (t, 3H, J = 7.20 Hz, CH3CH2O). 13C NMR (100.5
MHz, CDCl3): δ = 199.63 (CO); 190.31 (SCO); 170.04 (OC
O); 136.04 (Bz); 133.89 (Bz) ; 128.75 (Bz); 127.47 (Bz); 61.66
(CH3CH2O); 51.85 (CHCH3); 38.26 (SCH2); 14.07 (CH3CH2O),
and 12.88 (CHCH3). ESI+-HRMS: m/z [M + Na]+ calcd for
C14H16NaO4S 303.0662, found 303.0662.
Ethyl 2-Methyl-3-oxo-4-(pivaloylthio)butanoate (4c). Compound
4c was prepared in a similar manner as 4a, but by using
trimethylthioacetic S-acid instead of thioacetic acid. The product was
purified by silica gel chromatography using DCM as eluent.
Compound 4c was obtained as a solid in 62% yield (6.92 g) from
1
ethyl 2-methyl-3-oxobutanoate. H NMR (400 MHz, CDCl3): δ =
956
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The Journal of Organic Chemistry
Article
Na2SO4 and evaporated to dryness. The product was purified by silica
gel chromatography using a 6:4 mixture of hexane and EtOAc as
eluent. Compound 6 was obtained as a yellow oil in 73% yield (4.91
diastereomer pairs were separated on a SunFire C18 column (250 ×
10 mm, 5 μm) eluting with a mixture of water and MeCN (65:35, v/
v). 1H NMR (500 MHz, CDCl3): δ = 8.18 (s, 1H, NH); 7.48 (m, 1H,
H6); 6.37 (dd, 1H, J = 9.00 and 5.50 Hz, H1′); 5.29 (1H, H3′), 4.47
(m, 1H, H5″); 4.39 (m, 1H, H5′); 4.31 (m, 2H, POCH2); 4.26 (m,
2H, OCH2CH3); 4.17 (m, 1H, H4′); 3.95 (m, 2H, CH2S); 3.81 and
3.79 (s, 3H, POCH3); 2.78 (m, 2H CH2 Lev); 2.59 (m, 2H, CH2 Lev);
2.42 (dd, 1H J = 14.00 and 5.50 Hz, H2′); 2.36 (d, 3H, J = 1.50 Hz,
CH3CO); 2.22−2.18 (m, 4H, H2″ and CH3 Lev); 1.95 (s, 3H, CH3
Thy); 1.58, 1.57, 1.56, and 1.56 (s, 3H, CCH3); 1.29 (m, 3H,
CH3CH2O). 13C NMR (126 MHz, CDCl3): δ = 206.33 (CO);
198.56 (CO Lev); 193.75 (SCO); 172.38 (OCO Lev), 169.52
(OCO), 163.52 (C4); 150.40 (C2); 135.05 (C6); 111.84 and
111.79 (C5); 84.51 (C1′); 82.67 and 82.61 (C4′); 74.50 (H3′); 69.46
and 69.33 (C5′); 67.50 and 67.46 (POCH2); 62.46 (OCH2CH3);
60.07 and 60.00 (spiro C); 54.82 and 54.77 (CH3OP); 37.79 (CH2
Lev); 37.02 (C2′); 36.73 and 36.66 (CH2S); 30.07 (CH3CO);
29.76 (CH3 Lev); 27.86 (CH2 Lev); 17.85 (CH3); 13.96 (CH3CH2O);
12.37 (CH3 Thy). 31P NMR (202 MHz, CD3CN): δ = −0.30, −0.35,
−0.52, and −0.58 ppm. ESI+-HRMS: m/z [M + Na]+ calcd for
C26H37N2NaO14PS 687.1595, found 687.1585.
3′-O-Levulinoylthymidine 5′-(Methyl 4-benzoylthio-2-ethoxycar-
bonyl-2-methyl-3-oxobutyl) Phosphate (1b). The phosphotriester 1b
was prepared in a similar manner as 1a, but by using ethyl 4-
(benzoylthio)-2-(hydroxymethyl)-2-methyl-3-oxobutanoate (5b) in-
stead of ethyl-4-(acetylthio)-2-(hydroxymethyl)-2-methyl-3-oxobuta-
noate (5a). The crude product 1b was purified on a silica gel column
eluting with a mixture of ethyl acetate and methanol (95:5, v/v). The
product (1b) was obtained as solid foam in 42% yield (0.36 g). The
diastereomers were separated on a SunFire C18 column (250 × 10
mm, 5 μm) eluting with a mixture of water and MeCN (57:43, v/v).
1H NMR (500 MHz, CDCl3): δ = 8.17 (s, 1H, H6); 7.91 (d, 2H, J =
7.00 Hz, Bz); 7.60 (t, 1H, J = 7.50 Hz, Bz); 7.46 (m, 2H, Bz); 6.37
(dd, 1H, J = 9.00 and 5.50 Hz, H1′); 5.29 (d, 1H, J = 6.50 Hz, H3′),
4.53 (dd, 1H, J = 10.00 and 4.50 Hz, POCH2); 4.44 (dd, 1H, J = 10.00
and 4.50 Hz, POCH2); 4.34−4.26 (m, 4H, H5″, H5′ and OCH2CH3);
4.20−4.10 (m, 3H, H4′ and CH2S); 3.82 and 3.82 and 3.80 (s, 3H,
POCH3); 2.77 (m, 2H CH2 Lev); 2.58 (m, 2H, CH2 Lev); 2.41 (dd,
1H J = 13.50 and 5.50 Hz, H2′); 2.22−2.17 (m, 4H, H2″ and CH3
Lev); 1.94 (d, 3H, J = 0.50 Hz, CH3 Thy); 1.63−1.61 (m, 3H, CCH3);
1.31 (t, 3H, J = 7.00 Hz, CH3CH2O). 13C NMR (126 MHz, CDCl3): δ
= 206.30 (CO); 198.56 (CO Lev); 189.89 (SCO); 172.38
(OCO Lev), 169.52 (OCO), 163.20 (C4); 150.17 (C2); 136.00
(H6); 135.07 (Bz); 133.93 (Bz); 128.78 (Bz); 127.41 (Bz); 111.76
(C5); 84.33 (H1′); 82.72 and 82.71 (C4′); 74.61 (C3′); 69.46 and
69.42 (POCH2); 67.50 and 67.46 (C5′); 62.49 (OCH2CH3); 60.13
and 60.05 (spiro C); 54.84 and 54.79 (CH3OP); 37.79 (CH2 Lev);
37.07 (C2′); 36.65 (CH2S); 29.78 (CH3 Lev); 27.85 (CH2 Lev);
17.71 (CH3); 14.01 (CH3CH2O); 12.38 (CH3 Thy). 31P NMR (202
MHz, CD3CN): δ = −0.26, −0.32, −0.50, and −0.56 ppm. ESI+-
HRMS: m/z [M + H]+ calcd for C31H39N2NaO14PS 749.1752, found
749.1785.
2′,3′-O-Isopropylideneuridine 5′-(Methyl 4-benzoylthio-2,2-di-
methyl-3-oxobutyl) Phosphate (2b). To a solution of dried 2′,3′-O-
isopropylidineuridine (0.333 g, 1.17 mmol) in dry DCM (5.0 mL)
were added anhydrous triethylamine (0.81 mL, 5.85 mmol) and
methyl-N,N-diisopropylchlorophosphoramidite (0.249 mL, 1.29
mmol), and the reaction mixture was stirred for 1 h 15 min under
argon. The product was filtered through a short silica gel column
eluting with a mixture of anhydrous EtOAc and triethylamine (99.5:0.5
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 (1.5
mL) and S-(4-hydroxy-3,3-dimethyl-2-oxobutyl) benzothioate (7b)
(0.325 g, 1.29 mmol) in dry MeCN (1.5 mL) and 0.45 mol L−1 of
tetrazole solution in MeCN (7.8 mL, 3.51 mmol) were added under
argon. The reaction mixture was stirred for 1 h at room temperature.
The phosphite ester formed was oxidized with I2 (0.3 g) in a mixture
of THF, H2O, and lutidine (4:2:1, v/v/v; 10.5 mL) by stirring
overnight at room temperature. The solvent was removed under
1
g). H NMR (500 MHz, CDCl3): δ = 3.49 (s, 2H, CH2OH); 2.09 (s,
3H, CH3CO); 1.09 (s, 6H, C(CH3)2). 13C NMR (126 MHz,
CDCl3): δ = 215.10 (CO); 69.31 (CH2OH); 49.28 C(CH3)2);
25.56 (CH3CO); 21.53 (C(CH3)2). ESI+-MS: m/z [M + Na]+ calcd
for C6H12NaO2 139.15, found 139.13.
S-(4-Hydroxy-3,3-dimethyl-2-oxobutyl)ethanethioate (7a). The
synthesis of 7a was performed in a similar manner as described
below for 7b, but by using thioacetic acid instead of thiobenzoic acid.
The product was purified by silica gel chromatography using a 6:4
mixture of hexane and EtOAc as eluent. Compound 7a was obtained
as oil in 53% yield (1.33 g) from 4-hydroxy-3,3-dimethylbutan-2-one
1
(6). H NMR (500 MHz, CDCl3): δ = 3.91 (s, 2H, CH2S); 3.59 (s,
2H, CH2OH); 2.70 (s, 1H, OH); 2.34 (s, 3H, CH3CO); 1.20 (s,
6H, 2 × CH3). 13C NMR (126 MHz, CDCl3): δ = 208.49 (CO);
195.39 (SCO); 69.83 (CH2OH); 49.97 (spiro C); 36.65 (CH2S);
30.15 (CH3CO) and 21.56 (2 × CH3). ESI+-HRMS: m/z [M +
Na]+ calcd for C8H14NaO3S 213.0556, found 213.0562.
S-(4-Hydroxy-3,3-dimethyl-2-oxobutyl)benzothioate (7b). Com-
pound 7b was prepared from 6 essentially as described for 4b and 5b.
4-Hydroxy-3,3-dimethylbutan-2-one (6) (1.08 g, 9.3 mmol) was
dissolved in 13 mL of dry DCM. The solution was cooled on an ice
bath, and bromine (0.476 mL, 9.3 mmol) in DCM (3 mL) was added.
The reaction mixture was stirred for 1 h at 0 C. The solvent was
removed under reduced pressure, and the residue was coevaporated
from dry DCM. 1-Bromo-4-hydroxy-3,3-dimethylbutan-2-one (1.80 g,
9.23 mmol), used without further purification, was dissolved in dry
diethyl ether (23 mL). The solution was cooled on an ice bath, and
thiobenzoic acid (1.62 mL, 13.84 mmol) was added. Triethylamine
(1.5 mL, 10.79 mmol) in diethyl ether (5 mL) was added dropwise.
The ice bath was removed, and the reaction mixture was stirred for 3.5
h at room temperature. Diethyl ether (15 mL) was added, and the
organic phase was washed twice with water, 5% aq NaHCO3 and
saturated aq NaCl and dried over Na2SO4 and evaporated to dryness.
The product was purified by silica gel chromatography using a 6:4
mixture of hexane and EtOAc as eluent. Compound 7b was obtained
as a yellow solid in 85% yield (2.08 g) from 4-hydroxy-3,3-
1
dimethylbutan-2-one (6): H NMR (500 MHz, CDCl3): δ = 7.97
(d, 2H, J = 8.00 Hz, Bz); 7.58 (t, 1H, J = 7.50 Hz, Bz); 7.45 (t, 2H, J =
7.50 Hz, Bz); 4.11 (s, 2H, CH2S); 3.68 (s, 2H, CH2OH); 2.62 (s, 1H,
OH); 1.29 (s, 6H, 2 × CH3). 13C NMR (126 MHz, CDCl3): δ =
208.54 (CO); 191.47 (SCO); 136.19 (Bz), 133.87 (Bz); 128.72
(Bz); 127.47 (Bz); 70.08 (CH2OH); 50.16 (spiro C); 36.64 (CH2S)
and 21.67 (2 × CH3). ESI+-HRMS: m/z [M + H]+ calcd for
C13H17O3S 253.0893, found 253.0897.
3′-O-Levulinoylthymidine 5′-(Methyl 4-acetylthio-2-ethoxycar-
bonyl-2-methyl-3-oxobutyl) Phosphate (1a). To a solution of dried
3′-O-levulinoylthymidine25 (0.39 g, 1.15 mmol) in dry DCM (4.0 mL)
were added anhydrous triethylamine (0.80 mL, 5.73 mmol) and
methyl-N,N-diisopropylchlorophosphoramidite (0.244 mL, 1.26
mmol), and the reaction mixture was stirred for 1 h 15 min under
nitrogen. The product was filtered through a short silica gel column
eluting with a mixture of anhydrous ethyl acetate and triethylamine
(99.5:0.5 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
(1.5 mL), and compound 5a (0.31 g, 1.26 mmol) in dry MeCN (1.5
mL) and 0.45 mol L−1 tetrazole solution in MeCN (1.83 mmol, 4.0
mL) were added under nitrogen. The reaction mixture was stirred for
1 h at room temperature. The phosphite ester formed was oxidized
with I2 (0.3 g) in a mixture of THF, H2O, and lutidine (4:2:1, v/v/v;
10.5 mL) by stirring overnight at room temperature. The solvent was
removed under reduced pressure, and the residue was dissolved in
DCM and washed twice with saturated 5% aq NaHSO3. The organic
phase was dried over Na2SO4 and evaporated to dryness. The product
was purified by silica gel chromatography, eluting with a mixture of
EtOAc containing 5% MeOH. Compound 1a (diastereomeric
mixture) was obtained as as solid foam in 71% yield (0.54 g). The
957
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The Journal of Organic Chemistry
Article
reduced pressure, and the residue was dissolved in DCM and washed
twice with saturated 5% aq NaHSO3. The organic phase was dried
over Na2SO4 and evaporated to dryness. The product was purified by
silica gel chromatography, eluting with a mixture of EtOAc containing
5% MeOH. Compound 2b was obtained as solid foam in 73% yield
5476, 54.72, and 54.71 (CH3OP); 46.37 (spiro C of Piv); 38.40, 36.31,
36.28, and 36.26 (CH2S); 27.22; 25.25 and 25.23 (CH3 of Piv); 27.10
(CH3 of isopropylidene); 17.55, 17.53, and 17.51 (CHCH3); 13.95
(CH3CH2O). 31P NMR (202 MHz, CD3CN): δ = −0.41, −0.44,
−0.57, and −0.60 ppm. ESI+-HRMS: m/z [M + Na]+ calcd for
C26H39N2NaO13PS 673.1803, found 673.1832.
1
(0.52 g). H NMR (500 MHz, CDCl3): δ = 9.15 (br, 1H, NH); 7.96
(dd, 2H, J = 10.00 Hz, J = 2.00 Hz, Bz); 7.58 (t, 1H, J = 10.00 Hz, Bz);
7.45 (t, 2H, J = 10.00 Hz, Bz); 7.35 (d, 1H, J = 10.00 Hz, H6); 5.73
(m, 2H, H5 and H1′); 4.93 (m, 1H, H2′); 4.86 (m, 1H, H3′); 4.32
(m, 1H, H4′), 4.29−4.24 (m, 2H, H5″ and H5′); 4.16−4.12 (m, 4H,
CH2O and CH2S); 3.79 and 3.76 (s, 3H, POCH3); 1.55 (s, 3H, CH3);
1.34 (s, 3H, CH3); 1.33 (s, 6H, 2 × CH3). 13C NMR (126 MHz,
CDCl3): δ = 205.40 (CO); 190.61 (SCO); 163.23 (4′CO);
149.98 (C2′CO); 141.97 and 141.91 (H6); 136.23 (Bz); 133.81
(Bz); 128.74 (Bz); 127.42 (Bz); 114.70 (spiro C of isopropylidene);
102.76 and 102.73 (C5); 93.98 and 93.94 (C1′); 85.34 and 85.28
(C4′); 84.33 (C2′); 80.61 and 80.55 (C3′); 73.07 and 73.02
(CH2OH); 67.13 and 67.08 (C5′); 54.77 and 54.73 (CH3OP);
48.84 and 48.82 (spiro C); 36.42 (CH2S); 27.11; 25.27 and 25.25
(CH3) ; 21.71 and 21.67 (CH3); 21.65 (CH3). 31P NMR (202 MHz,
CDCl3): δ = −0.28 and −0.40 ppm. ESI+-HRMS: m/z [M + H]+ obsd
613.1617, calcd for C26H34N2O11PS 613.1615. ESI+-HRMS: m/z [M +
Na]+ calcd for C26H33N2NaO11PS 635.1435, found 635.1424.
4-Acetylthio-2,2-dimethyl-3-oxobutyl Bis(2′,3′-isopropylidene-2′-
C-methyluridin-5′-yl) Phosphate (9). 2′,3′-O-Isopropylidene-2′-C-
methyluridine26,29 (0.60 mmol; 0.18 g) was dried over P2O5 for 2
days and dissolved in dry DCM (4 mL) under nitrogen. Dry Et3N (5.0
mmol; 0.70 mL) and 1,1-dichloro-N,N-diisopropylphosphinamine
(0.50 mmol; 93 μL) were added. After the reaction mixture was
stirred at rt for 1 h, the mixture was passed through a short silica gel
column eluting with EtOAc that contained 0.5% Et3N. The solvents
were removed under reduced pressure. The residue was coevaporated
three times from dry MeCN and dissolved in dry MeCN (1.5 mL), (4-
hydroxy-3,3-dimethyl-2-oxobutyl)ethanethioate (7a) (1.50 mmol, 0.29
g) in dry MeCN (1.5 mL) and 1-H-tetrazole (1.50 mmol, 3.33 mL of
0.45 mol L−1 solution in MeCN) were added under nitrogen, and the
reaction mixture was stirred at rt for 2.5 h. The phosphite ester formed
was oxidized with I2 in a 4:2:1 mixture of THF, H2O, and 1,2-lutidine
by stirring overnight at room temperature. The product was isolated
by conventional 5% aq NaHSO3/DCM workup. The crude product
was purified on a silica gel column eluting with 2 to 6% MeOH in
DCM. Compound 9 was obtained as a white solid in 10% yield (52
2′,3′-O-Isopropylideneuridine 5′-(Methyl 4-acetylthio-2,2-di-
methyl-3-oxobutyl) Phosphate (2a). Phosphotriester 2a was
prepared in a similar manner as 2b, but by using S-(4-hydroxy-3,3-
dimethyl-2-oxobutyl) ethanethioate (7a) instead of S-(4-hydroxy-3,3-
dimethyl-2-oxobutyl) benzothioate (7b). The crude product 2a was
purified on a silica gel column eluting with a mixture of ethyl acetate
and methanol (95:5, v/v). The product 2a was obtained as solid foam
1
mg). H NMR (500 MHz, CDCl3): δ = 9.56 (s, 2H, NH); 7.53 (d,
1H, J = 5.00 Hz, H6); 7.51 (d, 1H, J = 5.00 Hz, H6); 6.02 (s, 1H,
H1′); 6.01 (s, 1H, H1′); 5.74 (d, 2H, J = 5.00 Hz, H5); 4.43−4.41 (m,
2H, H3′), 4.38−4.32 and 4.18−4.12 (m, 8H, H4′, H5′, H5″ and
CH2O); 3.94 (s, 2H, CH2S); 2.35 (s, 3H, CH3CO); 1.31 (s, 3H,
CH3); 1.30 (s, 3H, CH3); 1.28 (s, 3H, CH3); 1.26 (s, 3H, CH3). 13C
NMR (126 MHz, CDCl3): δ = 205.13 (CO); 194.46 (SCO);
163.28 (C4); 150.11 (C2); 140.67 (C6); 114.92 (spiro C of
isopropylidene); 102.22 (C5); 93.29 (C1′); 89.80 and 89.77 (C2′);
85.37 and 85.34 (C3′); and 81.68 (C4′); 73.30 and 73.26 (CH2O);
67.38 and 67.33 (C5′); 48.66 and 48.60 (spiro C); 36.26 (CH2S);
30.23 (CH3 of Ac); 28.46, 28.44, 27,57 and 27.55 (CH3 of
isopropylidene); 21.64 and 21.55 (CH3); 19.38 and 19.36 (2′-CH3).
31P NMR (202 MHz, CDCl3): δ = −1.21 ppm. ESI+-HRMS: m/z [M
1
in 29% yield (325 mg). H NMR (500 MHz, CDCl3): δ = 10.04 (br,
1H, NH); 7.33 (d, 1H, J = 8.00 Hz, H6); 5.68 (m, 2H, H5 and H1′);
4.90 (m, 1H, H2′); 4.79 (m, 1H, H3′); 4.25 (m, 1H, H4′), 4.21−4.15
(m, 2H, H5″ and H5′); 4.04 (m, 2H, CH2O); 3.89 (d, 2H, J = 3.00
Hz, CH2S); 3.70 and 3.68 (d, 3H, J = 1.00 Hz, POCH3); 2.29 (2s,
CH3CO); 1.49 (s, 3H, CH3); 1.28 (s, 3H, CH3); 1.21 (s, 3H, CH3)
1.20 (s, 3H, CH3). 13C NMR (126 MHz, CDCl3): δ = 205.34 (C
O); 194.44 and 194.43 (SCO); 163.68 (C4); 150.22 (C2); 142.17
(C6); 114.55 (spiro C of isopropylidene); 102.66 and 102.64 (C5);
93.97 and 93.90 (C1′); 85.36, 85.34, 85.31, and 85.28 (C4′); 84.28
(C2′); 80.63 and 80.58 (C3′); 72.91 and 72.89 (CH2OH); 67.18 and
67.14 (C5′); 54.73, 54.68, and 54.64 (CH3OP); 48.67, 48,65, 48,60
and 48.58 (spiro); 36. 33 (CH2S); 30.15 (CH3); 27.06 (CH3); 25.23
and 25.21 (CH3); 21.52, 21.50, 21.49 (CH3). 31P NMR (202 MHz,
CDCl3): δ = −0.40 and −0.50 ppm. ESI+-MS: m/z [M + H]+ obsd
551.1454, calcd for C21H32N2O11PS 551.1459. ESI+-MS: m/z [M +
Na]+ calcd for C21H31N2NaO11PS 573.1278 found 573.1282.
+ H]+ calcd for C34H48N4O16PS 831.2518, found 831.2487.
4-Acetylthio-2,2-dimethyl-3-oxobutyl Bis(2′-C-methyluridin-5′-yl)
Phosphate (3). Compound 9 was dissolved in 80% aqueous AcOH (5
mL) and stirred at 90 °C for 36 h. The solvent was removed under
reduced pressure and the residue was coevaporated two times from
water. The crude product was purified on a silica gel column with
gradient elution from 5% to 10% MeOH in DCM. Compound 3 was
obtained as solid in 42% yield (20 mg). 1H NMR (500 MHz,
CD3OD): δ = 7.65 (d, 1H, J = 5.00 Hz, H6); 7.63 (d, 1H, J = 5.00 Hz,
H6); 5.97 (s, 2H, H1′); 4.53−4.47 (m, 2H, H3′); 4.43−4.37 (m, 2H,
CH2O); 4.30−4.20 (m, 2H, H4′); 4.13−4.11 (m, 2H, H5″), 4.07 (s,
2H, CH2S); 3.81 (t, 2H, J = 10.00 Hz, H5′); 2.34 (s, 3H, CH3 of Ac);
1.30 (s, 6H, 2 × 2′-CH3); 1.18 (s, 6H, 2 × CH3). 13C NMR (126
MHz, CDCl3): δ = 205.84 (CO); 194.86 (SCO); 164.44 (4′C
O); 150.87 (C2′CO); 140.62 (C6); 101.69 (C5); 92.5 (C1′); 80.04
and 80.05, 78.19 (C4′, C3′ and C2′); 73.17, 72.89, and 72.84
(CH2O); 66.66 (C5′); 48.13 (spiro C); 35.89 (CH2S); 28.65 (CH3 of
Ac); 20.42 (CH3); 18.83 (2′-CH3). 31P NMR (202 MHz, CDCl3): δ =
−1.88. ESI+-HRMS: m/z [M + H]+ calcd for C28H40N4O16PS
751.1892, found 751.1880.
Kinetic Measurements. The reactions were carried out in sealed
tubes immersed in a thermostated water bath (37.0 0.1 °C). The
hydronium ion concentration of the reaction solutions (3.0 mL) was
adjusted with sodium hydroxide and N-[2-hydroxyethyl]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 the buffer acid under the
experimental conditions. The initial substrate concentration was ca. 0.2
mmol L−1.
2′,3′-O-Isopropylideneuridine 5′-(Methyl 2-ethoxycarbonyl-2-
methyl-3-oxo-4-pivaloylthiobutyl) Phosphate (2c). Phosphotriester
2c was prepared in a similar manner as 1a, but by using ethyl 2-
(hydroxymethyl)-2-methyl-3-oxo-4-(pivaloylthio)butanoate (5c) in-
stead of ethyl-4-(acetylthio)-2-(hydroxymethyl)-2-methyl-3-oxobuta-
noate (5a). The crude product was purified on a silica gel column
eluting with a mixture of EtOAc and MeOH (95:5, v/v). The product
1
2c was obtained as solid foam in 13% yield (0.10 g). H NMR (500
MHz, CDCl3): δ = 9.75 (s br. 1H, NH); 7.34 (d, 1H, J = 8.00 Hz,
H6); 5.73−5.71 (m, 2H, H1′ and H5); 4.93−4.91 (m, 1H, H2′),
4.84−4.82 (m, 1H, H3′); 4.43−4.35 (m, 1H,); 4.32−4.27 (dd, 1H,
H4′); 4.26−4.19 (m, 4H, H5″, H5′ and OCH2CH3); 3.93−3.83 (m,
3H, H4′ and CH2S); 3.74−3.72 (4s, 3H, POCH3); 1.53 (s, 3H,
CCH3); 1.53 (s, 3H, CH3 of isopropylidene); 1.32 (s, 3H, CH3 of
Piv); 1.26 (t, 3H, J = 7.00 Hz, CH3CH2O); 1.21 (s, 3H, CH3 of Piv);
1.85 (s, 3H, CH3 of isopropylidene). 13C NMR (126 MHz, CDCl3): δ
= 204.79 and 204.77 (CO); 198.91,198.86, 198.85, and 198.83
(SCO); 169.52 and 169.49 (OCO), 163.59 (C4); 150.16 and
150.14 (C2); 142.08 and 142.04 (H6); 114.68 and 114.65 (C of
isopropylidene); 102.74 and 102.72 (C5); 93.92 and 93.89 (C1′);
85.30, 85.24, and 85.18 (C4′); 84.29 and 84.27 (C2′); 80.55 (C3′);
69.46 and 69.43 (POCH2); 67.22 and 67.19 (C5′); 62.34
(OCH2CH3); 60.15−60.03 (spiro C of protecting group); 54.80,
958
dx.doi.org/10.1021/jo302421u | J. Org. Chem. 2013, 78, 950−959

The Journal of Organic Chemistry
Article
The enzymatic and nonenzymatic hydrolysis was carried out in a
HEPES buffer at pH 7.5 (0.036/0.024 mol L−1) at 37 °C. The acyl
group was removed with hog liver esterase (2.6 units mL−1). The
samples (200 μL) withdrawn at appropriate intervals were made acidic
(pH 2) with 1 mol L−1 aqueous hydrogen chloride or 1 mol L−1
AcOH to inactivate enzyme and to quench the hydrolysis, cooled in an
ice bath, and filtered with RC4 syringe (0.2 μm). The composition of
the samples was analyzed on an C18 column (4 × 250 mm 5 μm, flow
rate 0.95 mL min−1), using a mixture of MeCN and 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
mixture of 1a, 2a, 2b, 2c, and 3 was obtained on using a 5 min isocratic
elution with the buffer containing 2% MeCN, followed by a 30 min
linear gradient (40 min with 2c) up to 50% MeCN (75% with 2c).
Signals were recorded on a UV-detector at a wavelength of 267 or 260
nm. In case of 1b, an isocratic eluation with the buffer containing 35%
MeCN was used. 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.
(3) Farquhar, D.; Khan, S.; Srivastva, D. N.; Saunders, P. P. J. Med.
Chem. 1994, 37, 3902−3909.
(4) Armirilli, M. N.; Kim, C. U.; Dougherty, J.; Mulato, A.; Oliyai, R.;
Shaw, J.-P.; Cundy, K. C.; Bischofberger, N. Antiviral Chemother. 1997,
8, 557−564.
(5) Perigaud, C.; Gosselin, G.; Lefebvre, I.; Girardet, J. L.; Benzaria,
S.; Barber, I.; Imbach, J. L. Bioorg. Med. Chem. Lett. 1993, 3, 2521−
2526.
(6) Glazier, A.; Kwong, C.; Rose, J.; Buckheit, R. Antiviral Res. 1992,
17 (Suppl. 1), 66.
(7) Poijarvi, P.; Heinonen, P.; Virta., P.; Lonnberg, H. Bioconjugate
̈
̈
Chem. 2005, 16, 1564−1571.
(8) Poijarvi, P.; Oivanen, M.; Lonnberg, H. Lett. Org. Chem. 2004, 1,
̈
̈
183−188.
(9) Ora, M.; Taherpour, S.; Linna, R.; Leisvuori, A.; Hietamaki, E.;
Poijarvi-Virta, P.; Beigelman, L.; Lonnberg, H. J. Org. Chem. 2009, 74,
̈
4992−5001.
(10) Farquhar, D.; Chen, R.; Khan, S. J. Med. Chem. 1995, 38, 488−
495.
Esterase-Triggered Deprotection in the Presence of Gluta-
thione. The hydrolytic reactions of 2b were carried out in the
presence of glutathione (GSH; 5.0 mmol L−1) and carboxyesterase
(HLE; 2.6 units mL−1) in a 2-(N-morpholino)ethanesulfonic acid
(MES) buffer (0.005/0.005 mol L−1) at pH 6.1 and 37 °C. The initial
substrate concentration was ca. 0.5 mmol L−1. Glutathione was present
in the reaction mixture from the beginning or it was added 20 min
after addition of 2b.The samples (250 μL) withdrawn at appropriate
intervals were made acidic (pH 2) with 1 mol L−1 aq. HCl, cooled in
an ice-bath and filtered with minisart RC4 filters (0.2 μm). The
reaction products were identified by mass spectra (LC/MS) on a
Phenomenex Gemini C18 column (2.0 × 150 mm 5 μm; flow rate 0.4
mL min−1). A good separation of the products was obtained using 5
min isocratic elution with 0.1% formic acid containing 4% MeCN,
followed by a linear gradient to 100% MeCN in 40 min. The signals
were recorded on a UV detector at a wavelength of 220 and 260 nm.
In 4 days, the starting material was converted to phosphodiester 11
([M + H]+ = 379.3 and tR = 15.6 min). The mixture additionally
contained debenzoylated 2b (15; [M + H]+ = 509.4 and tR = 24.3
min) and a disulfide adduct of 15 with glutathione ([M + H]+ = 814.4
and tR = 19.3 min). In addition, glutathione was partially converted to
its oxidized form, glutathione disulfide (GSSG; [M + H]+ = 613.2 and
tR = 3.8 min). A minor signal of an unidentified byproduct was
detected at m/z value of 316.1 (tR = 17.8).
(11) McGuigan, C.; Pathirana, R. N.; Balzarini, J.; De Clercq, E. J.
Med. Chem. 1993, 36, 1048−1052.
(12) McGuigan, C.; Camarasa, M.-J.; Egberink, H.; Hartmann, K.;
Karlsson, A.; Perno, C. F.; Balzarini, J. Int. Antiviral News 1997, 5, 19−
21.
(13) Kiuru, E.; Ora, M.; Beigelman, L.; Blatt, L.; Lonnberg, H. Chem.
̈
Biodivers 2011, 8, 266−286.
(14) Kiuru, E.; Ora, M.; Beigelman, L.; Blatt, L.; Lonnberg, H. Chem.
̈
Biodivers 2012, 9, 669−688.
(15) Grajkowski, A.; Pedras-Vasconcelos, J.; Wang, V.; Ausin, C.;
Hess, S.; Verthelyi, D.; Beaucage, S. Nucleic Acids Res. 2005, 33, 3550−
3560.
(16) Grajkowski, A.; Ausin, C.; Kauffman, J. S.; Snyder, J.; Hess, S.;
Lloyd, J. R.; Beaucage, S. J. Org. Chem. 2007, 72, 805−815.
(17) Ausin, C.; Kauffman, J. S.; Duff, R. J.; Shivaprasad, S.; Beaucage,
S. L. Tetrahedron 2010, 66, 68−79.
(18) Gisch, N.; Pertenbreiter, F.; Balzarini, J.; Meier, C. J. Med. Chem.
2008, 51, 8115−8123.
(19) Gisch, N.; Balzarini, J.; Meier, C. J. Med. Chem. 2008, 51, 6752−
6760.
(20) Ducho, C.; Gorbig, U.; Jessel, S.; Gisch, N.; Balzarini, J.; Meier,
̈
C. J. Med. Chem. 2007, 50, 1335−1346.
(21) Gisch, N.; Balzarini, J.; Meier, C. J. Med. Chem. 2007, 50, 1658−
1667.
(22) Svendsen, A.; Boll, P. M. Tetrahedron 1973, 29, 4251−4258.
ASSOCIATED CONTENT
(23) Guzaev, A.; Lonnberg, H. Synthesis 1997, 1281−1284.
̈
■
(24) Boeykens, M.; De Kimpe, N.; Tehrani, K. A. J. Org. Chem. 1994,
59, 6973−6985.
S
* Supporting Information
HPLC chromatograms of compounds 1−3 and NMR spectral
(25) Ora, M.; Mantyvaara, A.; Lonnberg, H. Molecules 2011, 16,
̈
̈
1
data for compounds 1−7 and 9. ESI-MS and H NMR spectra
552−566.
of compounds 16a and 18a. This material is available free of
(26) Jenkinson, S. F.; Jones, N. A.; Moussa, A.; Stewart, A. J.; Heinz,
T.; Fleet, G. W. J. Tetrahedron Lett. 2007, 48, 4441−4444.
(27) Yang, W.; Drueckhammer, D. G. J. Am. Chem. Soc. 2001, 123,
11004−11009.
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
(28) Calvo, K. C.; Wang, X.; Koser, G. F. Nucleosides Nucleotides
Nucleic Acids 2004, 23, 637−646.
(29) Leisvuori, A.; Ahmed, Z.; Ora, M.; Blatt, L.; Beigelman, L.;
Corresponding Author
*E-mail: emilia.kiuru@utu.fi mikko.ora@utu.fi.
Notes
Lonnberg, H. ARKIVOC 2012, 5, 226−243.
̈
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Ms. Johanna Kunttu and Ms. Marjukka Elmeranta for
the synthesis of compounds 2c and 1b, respectively. We also
thank Dr. Pasi Virta for fruitful discussions.
REFERENCES
■
(1) Hecker, S. J.; Erion, M. D. J. Med. Chem. 2008, 51, 2328−2345.
(2) Poijarvi-Virta, P.; Lonnberg, H. Curr. Med. Chem. 2006, 13,
̈
̈
3441−3465.
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dx.doi.org/10.1021/jo302421u | J. Org. Chem. 2013, 78, 950−959