4-(Acetylthio)-2,2-dimethyl-3-oxobutyl and 4-(tert-Butyldisulfanyl)-2,2-dimethyl-3-oxobutyl as Protecting Groups for Nucleoside 5′-Phosphoramidates Derived from L -Alanine Methyl Ester

Article abstract of DOI:10.1002/ejoc.201500531

Phosphoramidates 1 and 2 were synthesized by H-phosphonate methodology and subsequent oxidative amination with L-alanine methyl ester. The removal of the protecting groups at pH = 7.5 and 37°C in the absence and presence of porcine liver esterase (PLE) or glutathione (GSH) was monitored by HPLC. The stability of phosphoramidate 1 was additionally studied at pH = 9 and 10. The reduction of the disulfide bond with glutathione from 2 triggers the removal of the protecting group by cyclization releasing quantitatively nucleoside 5′-{N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate} (7) as the desired product. With 1, enzymatic deacetylation or acetyl migration from the sulfur atom to the adjacent hydrated oxo group followed by chemical cyclization produces 7. The S-S-bond-mediated dimerization (8) competes as a side reaction. Prolonged treatment, however, resulted in the conversion of the S-S dimer 8 into 7 that undergoes slow alanine methyl ester hydrolysis to form 10.

Full text of DOI:10.1002/ejoc.201500531

FULL PAPER
DOI: 10.1002/ejoc.201500531
4-(Acetylthio)-2,2-dimethyl-3-oxobutyl and 4-(tert-Butyldisulfanyl)-2,2-
dimethyl-3-oxobutyl as Protecting Groups for Nucleoside 5Ј-Phosphoramidates
Derived from -Alanine Methyl Ester
L
Vyankat A. Sontakke,*[a,b] Harri Lönnberg,[a] and Mikko Ora*[a]
Keywords: Phosphoramidates / Protecting groups / Prodrugs / Kinetics / Nucleosides / Amino acids
Phosphoramidates 1 and 2 were synthesized by H-phos-
nucleoside
5Ј-{N-[(1S)-2-oxo-2-methoxy-1-methylethyl]-
phonate methodology and subsequent oxidative amination
with L-alanine methyl ester. The removal of the protecting
phosphoramidate} (7) as the desired product. With 1, enzy-
matic deacetylation or acetyl migration from the sulfur atom
to the adjacent hydrated oxo group followed by chemical cy-
clization produces 7. The S–S-bond-mediated dimerization
(8) competes as a side reaction. Prolonged treatment, how-
ever, resulted in the conversion of the S–S dimer 8 into 7 that
undergoes slow alanine methyl ester hydrolysis to form 10.
groups at pH = 7.5 and 37 °C in the absence and presence of
porcine liver esterase (PLE) or glutathione (GSH) was moni-
tored by HPLC. The stability of phosphoramidate 1 was ad-
ditionally studied at pH = 9 and 10. The reduction of the di-
sulfide bond with glutathione from 2 triggers the removal of
the protecting group by cyclization releasing quantitatively
treatment, have advanced to clinical trials.[4] It has been
suggested that the carboxylic ester linkages of amino-acid-
derived aryloxyphosphoramidates of nucleoside 5Ј-mono-
phosphates undergo hydrolysis triggered by esterase or ca-
thepsin A,[5] and that this is followed by intramolecular dis-
placement of the phenoxide ion by the carboxylate group.
The resulting cyclic mixed anhydride then undergoes hy-
drolysis to give an N-acylated phosphoramidate, and finally
intracellular phosphoramidase or HINT-1[6] releases the nu-
cleoside 5Ј-monophosphate.[7] Another prodrug strategy for
nucleoside 5Ј-phosphoramidates derived from l-amino acid
methyl esters is based on protection of the phosphoramid-
ate oxygen atom with an S-pivaloyl-2-thioethyl group
(SATE).[8] In this case, carboxyesterase-mediated hydrolysis
of the pivaloyloxy group is followed by a non-enzymatic
departure of the remaining 2-mercaptoethyl linker in a cy-
clization to give the episulfide.[2] Esterase-labile 2,2-disub-
stituted 3-(acyloxy)propyl and 3-(acyloxymethoxy)propyl
groups have, in turn, been shown to release the amino acid
phosphoramidate by an esterase-triggered retroaldol con-
densation that gives an enone-like by-product.[9] It has also
been shown that amino-acid-derived phosphoramidates
may themselves become incorporated into DNA by HIV
reverse transcriptase.[10]
Introduction
The antiviral and anticancer activity of structurally
modified nucleosides depends on their uptake into cells and
their intracellular conversion into nucleotides. To bypass
the initial, usually rate-limiting, phosphorylation,[1] nucleo-
tides protected with biodegradable protecting groups are
often used instead of nucleosides as drug candidates.[2] One
such class of prodrugs are the 5Ј-O-aryl-N-[2-(alkoxycarb-
onyl)alkyl]phosphoramidates, which have shown higher
antiviral potency than their parent nucleoside analogs.[3] 2Ј-
Fluoro-2Ј-deoxy-2Ј-C-methyluridine 5Ј-(O-phenyl-N-isop-
ropoxyalaninyl)phosphoramidate (Sofosbuvir) has even
been approved for the treatment of HCV (Hepatitis C). Ad-
ditionally, 2Ј,3Ј-didehydro-2Ј,3Ј-dideoxythymidine 5Ј-[O-(4-
bromophenyl)-N-methoxyalaninyl]phosphoramidate (Stam-
pidine) and 5-(2-bromovinyl)-2Ј-deoxyuridine 5Ј-(O-phenyl-
N-methoxyalaninyl)phosphoramidate (Thymectacin), devel-
oped as anti-HIV and anticancer drugs, respectively, as well
as O6-methyl-2Ј-C-methylguanosine 5Ј-(O-naphth-1-yl-N-
neopentoxyalaninyl)phosphoramidate (INX-08189) and
O6-methyl-2Ј-C-methylguanosine 5Ј-(O-phenyl-N-isopro-
poxyalaninyl)phosphoramidate (PSI-353661) for HCV
[a] University of Turku, Department of Chemistry,
20014 Turku, Finland
We now report on the thermally and enzymatically labile
2,2-disubstituted 4-(acylthio)-3-oxobutyl group[11] and the
reductively cleavable 4-(tert-butyldisulfanyl)-2,2-dimethyl-3-
oxobutyl group[12] as alternative protecting groups for nu-
cleoside 5Ј-phosphoramidates. For this purpose, 2Ј,3Ј-O-
isopropylideneuridine 5Ј-{O-[4-(acetylthio)-2,2-dimethyl-3-
oxobutyl]-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phos-
phoramidate} (1) and 2Ј,3Ј-O-isopropylideneuridine 5Ј-
E-mail: mikora@utu.fi
http://www.utu.fi/en/units/sci/units/chemistry
[b] University of Pune, Department of Chemistry, Garware
Research Centre
Pune 411007, India
E-mail: sontakkevyankat@gmail.com
http://www.unipune.ac.in/dept/science/chemistry/
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500531.
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Protecting Groups of Nucleoside 5Ј-Phosphoramidates
{O-[4-(tert-butyldisulfanyl)-2,2-dimethyl-3-oxobutyl]-N- ethyl]phosphoramidate} (1) and 2Ј,3Ј-O-isopropylideneuri-
[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate} (2) dine 5Ј-{O-[4-(tert-butyldisulfanyl)-2,2-dimethyl-3-oxo-
were prepared as model compounds (Figure 1). The pro- butyl]-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]-phosphor-
tecting groups introduced are released after deacylation or amidate} (2) were prepared by H-phosphonate methodol-
reduction by chemical cyclization to give 4,4-disubstituted ogy with a subsequent amination with an amino acid
dihydrothiophen-3(2H)-one, which, as expected, does not methyl
ester
under
Atherton–Todd
conditions
show significant alkylation activity,[11] unlike the enone and (Scheme 1).[13] Accordingly, treatment of diphenyl phos-
episulfide by-products mentioned above. The cleavage rate phite with commercially available 2Ј,3Ј-O-isopropylideneur-
of the 2,2-disubstituted 4-(acylthio)-3-oxobutyl group may idine in pyridine at room temperature, followed by addition
be tuned by the size of the 4-(acylthio) group and the size of a mixture of H2O and Et3N gave the triethylammonium
and electronegativity of the 2-substituents.[11] Deprotection salt of 2Ј,3Ј-O-isopropylideneuridine 5Ј-(H-phosphonate)
of 1 and 2 was monitored by HPLC in the absence and (3; Scheme 1). Compound 3 was esterified with 1-(tert-bu-
presence of enzyme or glutathione at 37 °C and pH = 7.5.
tyldisulfanyl)-4-hydroxy-3,3-dimethylbutan-2-one (4)[12] or
S-(4-hydroxy-3,3-dimethyl-2-oxobutyl) ethanethioate (5)[11]
in pyridine using pivaloyl chloride as activator. The prod-
uct, the 4-(acetylthio)-2,2-dimethyl-3-oxobutyl or 4-(tert-
butyldisulfanyl)-2,2-dimethyl-3-oxobutyl ester of 2Ј,3Ј-O-
isopropylidene 5Ј-(H-phosphonate), was not isolated, but
subjected to oxidative amination with l-alanine methyl es-
ter in the presence of CCl4 and Et3N in a mixture of MeCN
and pyridine. The overall yields of 1 and 2 from 3 were 21
and 23%, respectively.
Phosphorus trichloride was also used as a phosphitylat-
ing agent instead of diphenyl phosphite. Accordingly, 2Ј,3Ј-
O-isopropylideneuridine was treated with PCl3 in CH2Cl2
at –20 °C. Reaction of the resulting 5Ј-dichlorophosphite
with S-(4-hydroxy-3,3-dimethyl-2-oxobutyl) ethanethioate
and subsequent hydrolysis in aq. THF gave the 4-(acet-
ylthio)-2,2-dimethyl-3-oxobutyl ester of 2Ј,3Ј-O-isopropyl-
ideneuridine 5Ј-(H-phosphonate). Oxidative amination was
then carried out as described above. The yield (10%) was,
however, lower than that obtained using diphenyl phosphite
as the phosphitylating agent. Attempts to prepare phos-
phoramidate 1 by oxidative coupling,[12,14] i.e., by iodin-
ation of the 5Ј-[4-(acetylthio)-2,2-dimethyl-3-oxobutyl H-
phosphonate] and subsequent displacement of iodine
Figure 1. Structures of phosphoramidates 1 and 2.
Results and Discussion
Synthesis
2Ј,3Ј-O-Isopropylideneuridine 5Ј-{O-[4-(acetylthio)-2,2- from the iodophophate with l-alanine methyl ester,
dimethyl-3-oxobutyl]-N-[(1S)-2-oxo-2-methoxy-1-methyl- failed.
Scheme 1. Preparation of phosphoramidates 1 and 2. Reaction conditions: (i) diphenyl phosphite, py; (ii) PivCl; (iii) py/MeCN, CCl4,
Et3N, H-Ala-OMe·HCl.
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Deprotection of 4-(Acetylthio)-2,2-dimethyl-3-oxobutyl- and
4-(tert-Butyldisulfanyl)-2,2-dimethyl-3-oxobutyl-Protected
Phosphoramidates 1 and 2
The removal of the protecting groups from disastereo-
meric (RP/SP) phosphoramidates 1 and 2 was studied at
37 °C by analysing the composition of aliquots withdrawn
as a function of time by reverse-phase HPLC. The deprotec-
tion of 1 was monitored in the presence of porcine liver
esterase (PLE; 2.6 UmL–1) at pH = 7.5, and in the absence
of PLE over a pH range from 7.5 to 10, whereas the depro-
tection of 2 was carried out in the presence of glutathione
(GSH; 5 mm) at pH = 7.5 (see Table 1).
Table 1. First-order rate constants and half-lives for the decomposi-
tion of diastereomeric nucleoside 5Ј-phosphoramidates 1 and 2 at
37 °C (I = 0.1 m with NaCl).
Figure 2. Time-dependent product distribution for the hydrolysis
of diastereomeric 2Ј,3Ј-O-isopropylideneuridine 5Ј-{O-[4-(tert-
butyldisulfanyl)-2,2-dimethyl-3-oxobutyl]-N-[(1S)-2-oxo-2-meth-
oxy-1-methylethyl]phosphoramidate} (2) in the presence of GSH at
pH = 7.5 and 37 °C. Notation: 2 (filled squares), 7 (circles), and 6
(empty squares).
Compound k [10–4 s–1] t1/2 [min]
Reaction solution
1
1
1
1
2
0.134
6.75
862
17.1
3.2
pH = 7.5
pH = 9.0
pH = 10.0
36.3
ca. 12.0[a]
2.03
ca. 10[a] pH = 7.5 (PLE 2.6 UmL–1)
56.9
pH = 7.5 (GSH 5 mm)
[a] 50% of 1 was converted into deacetylated intermediate 6.
Enzymatic Deprotection of the Phosphoramidate 1
In the presence of PLE, compound 1 (tR = 25.7 min)
undergoes PLE-triggered deprotection to give 7 (82% of the
Reductive Deprotection of Phosphoramidate 2
In the presence of glutathione, the disulfide bond of 2 products; Scheme 3) via deacetylated intermediate 6, which
(tR = 28.6 min) was reductively cleaved. The half-life for the is also converted into its S–S dimer 8 (18% of the products;
disappearance of the starting material was 56.9 min (k = tR = 28.6 min; m/z = 1179.3154 [M + Na]+). This observa-
2.03ϫ10–4 s–1). 2Ј,3Ј-O-Isopropylideneuridine 5Ј-{O-(4- tion is consistent with earlier findings with oligomeric phos-
mercapto-2,2-dimethyl-3-oxobutyl)-N-[(1S)-2-oxo-2-meth- phodiesters, which undergo an intrachain S–S-bond-medi-
oxy-1-methylethyl]phosphoramidate} (6; tR = 23.5 min; m/z ated reaction between two deacetylated 4-(acetylthio)-2,2-
= 602.1 [M + Na]+) accumulated as an intermediate and dimethyl-3-oxobutyl protecting groups.[15] As seen from
underwent quantitative conversion into 2Ј,3Ј-O-isopropyl- Figure 3, the enzymatic hydrolysis of 1 is initially rapid, but
ideneuridine-5Ј-{N-[(1S)-2-oxo-2-methoxy-1-methylethyl]- then becomes slower. For example, 50% of the starting ma-
phosphoramidate} (7; tR = 16.7 min; m/z = 448.1104 [M – terial was converted into deacetylated intermediate 6 within
H]–; Figure 2 and Scheme 2), the rate constant being 10 min, but to reach 90% conversion took 125 min. Most
6.50ϫ10–4 s–1 (t1/2 = 17.8 min). Concurrently, GSH was ox- probably, one of the two diastereomers (i.e., RP-1 or SP-1)
idized to glutathione disulfide (GSSG; tR = 3.7 min).
reacts more readily than the other.
Scheme 2. Hydrolysis of diastereomeric phosphoramidates 2 in the presence of GSH.
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Protecting Groups of Nucleoside 5Ј-Phosphoramidates
Scheme 4. Mechanism of PLE- and GSH-triggered deprotection.
Non-Enzymatic Deprotection of Phosphoramidate 1
In the absence of PLE at pH = 7.5, 7 was observed as
the major product (see Figure 4) and disulfide 8 as an inter-
mediate. In addition, two side-products were formed: 2Ј,3Ј-
O-isopropylideneuridine (9; tR = 18.0 min; 10%; m/z =
283.0 [M – H]–) and the deprotected phosphoramidate with
the alanyl ester function hydrolysed (i.e., 10; 10%; tR
=
15.5 min; m/z = 436.1121 [M + H]+). The rate constant for
the disappearance of the starting material (i.e., 1) was
1.34ϫ10–5 s–1 (t1/2 = 14.4 h). Prolonged treatment (4 d) in
a HEPES buffer resulted in an accumulation of 7 (70%)
and 10 (20%) as the main products.
Scheme 3. Enzymatic hydrolysis of diastereomeric phosphoramid-
ates 1.
Figure 4. Time-dependent product distribution for hydrolysis of
diastereomeric 2Ј,3Ј-O-isopropylideneuridine 5Ј-{O-[4-(acetylthio)-
2,2-dimethyl-3-oxobutyl]-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]-
phosphoramidate} (1) at pH = 7.5 and 37 °C. Notation: 1 (filled
squares), 7 (empty circles),8 (filled triangles), 9 (filled circles), and
10 (empty triangles).
Figure 3. Time-dependent product distribution for hydrolysis of
diastereomeric 2Ј,3Ј-O-isopropylideneuridine 5Ј-{O-[4-(acetylthio)-
2,2-dimethyl-3-oxobutyl]-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]-
phosphoramidate} (1) in the presence of PLE at pH = 7.5 and
37 °C. Notation: 1 (filled squares), 6 (empty squares), 7 (circles),
and 8 (triangles).
Under basic conditions (pH = 9–10), the product distri-
bution was more complicated. In addition to the products
mentioned above, 6 was observed (at pH = 10), and also
the 4-mercapto-2,2-dimethyl-3-oxobutyl group was con-
verted into a 4-mercapto-2,2-dimethylbut-3-yn-1-yl group
As discussed previously,[11] the mercapto intermediate (to give 11b) or a 2-methyl-2-(thiiren-2-yl)propyl group (to
(i.e., 6), obtained by the esterase-catalysed hydrolysis of 1 give 11a) (Scheme 5). Most probably, a peak observed by
or thiol/disulfide exchange reaction of 2 with GSH, releases HPLC/ESI-MS (tR = 21.2 min) with m/z = 562.2 is due to
the phosphoramidate monoanion (i.e., 7) by departure of the molecular ion [M + H]+ of 11b, the thiirene 11a being
the remnant of the protecting group in a cyclization reac- unstable. Two unidentified intermediates were also detected.
tion to give 4,4-dimethyl-dihydrothiophen-3(2H)-one The rate constants for the disappearance of the starting ma-
(Scheme 4).
terial (i.e., 1) were 6.75ϫ10–4 s–1 and 3.63ϫ10–3 s–1 at pH
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Scheme 5. Non-enzymatic hydrolysis of phosphoramidate 1.
= 9 and 10, respectively. As an example, the product distri-
bution at pH = 10 is shown in Figure 5. Under these condi-
tions, 7 finally underwent hydrolysis to 10. After 2 d, 7
(15%) and 10 (75%) accumulated as the main products.
In all likelihood, hydration of the oxo group at C-3 initi-
ates the non-enzymatic decomposition of 1, allowing mi-
gration of the acetyl group from the sulfur atom to the adja-
cent oxygen atom, as described recently (see Schemes 5 and
6).[11] Intermediate I does not accumulate, but breaks down
as follows: (i) removal of the protecting group [Route (A)]
by intramolecular attack of the sulfur atom on C-1, which
results in the release of 7,[11] (ii) cleavage of the P–O-5Ј
bond [Route (B)] by intramolecular attack of the oxyanion
of the hydrated oxo group onto the phosphorus atom[11] to
give 9, and (iii) departure of acetic acid to give 6
[Route (C)], which is further converted into 7. The S–S-
bond-mediated dimerization to give 8, most probably also
takes place via 6 [Route (C)/(D)] after the loss of AcOH,
although disulfide bond formation before the elimination
of AcOH [Route (D) or Route (G)/(D)] cannot strictly be
excluded. No evidence for the appearance of an S–S dimer
Figure 5. Time-dependent product distribution for hydrolysis of
diastereomeric 2Ј,3Ј-O-isopropylideneuridine 5Ј-{O-[4-(acetylthio)-
2,2-dimethyl-3-oxobutyl]-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]-
phosphoramidate} (1) at pH = 10 and 37 °C. Notation: 1 (filled
squares), 7 (empty circles), 8 (filled triangles), 9 (filled circles), 11
(stars), 10 (empty triangles), 6 (empty squares), and two unknown
bearing acetyl group(s) was obtained. Application of the products (filled and empty diamonds).
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Protecting Groups of Nucleoside 5Ј-Phosphoramidates
Scheme 6. Mechanism of the non-enzymatic removal of the protecting group, cleavage of the P–O bond, and S–S-bond-mediated dimeriza-
tion.
rate-law of parallel consecutive first-order reactions for 8 hydrolysis of the hemiacetal-like structure takes place to
suggests that the S–S-bond-mediated dimerization to give 8 form 7 and 1,1Ј-disulfanediylbis(4-hydroxy-3,3-dimethyl-
represents 74 and 62% of the overall disappearance of 1 at butan-2-one) (13; m/z = 295.0 [M + H]+; Scheme 7). The
pH = 7.5 and 9, respectively. At pH = 10, 6 accumulated as conversion of 8 into 9 and 10 is also possible after the
an intermediate; so the rate constant for the formation of 8 alaninyl methyl ester hydrolysis by intramolecular displace-
could not be reliably determined. The intermediate accumu- ment of 2Ј,3Ј-isopropylideneuridine (9) or the protecting
lation of 11a or 11b [Route (E)] can, in turn, be explained group by the carboxylate ion as described earlier with thym-
by attack of the sulfur atom of 6 on the adjacent carbonyl idine 5Ј-{O-phenyl-N-[(1S)-2-oxo-2-methoxy-1-methyleth-
group, and subsequent elimination of water (see Schemes 5 yl]phosphoramidate}.[7h] In the latter case, hydrolysis of the
and 6). The resulting thiirene (i.e., 11a) is then converted resulting cyclic mixed anhydride may then produce 10. This
into 7 via ethynylthiol derivative 11b [Route (H)] by a ring- pathway does not, however, play a significant role. As seen
opening reaction followed by attack of the sulfur atom on from Figure 5, only a small amount of 9 (Ͻ3%) and 10
C-1.
(Ͻ6%), which may also be formed via 12 and/or I, is ob-
The thioester hydrolysis [Route (G); 1 Ǟ 6] and the alani- tained upon decomposition of 8 at pH = 10. Moreover, the
nyl methyl ester hydrolysis [Route (F); 1 Ǟ 12] represent decomposition of 8 takes place eight times as fast as the
only minor pathways, as evidenced by the following facts. alaninyl methyl ester hydrolysis of 2Ј-O-methylcytidine 5Ј-
The rate of hydroxide-ion-catalysed hydrolysis of the thioes-
ter linkage has been reported to be comparable to that of
the ester linkage.[16] Moreover, earlier studies indicate that
the acetate ester hydrolysis of 2Ј-O-methylcytidine 5Ј-{O-3-
(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl-N-[(1S)-2-meth-
oxy-1-methyl-2-oxoethyl]phosphoramidate} is 11 and 19
times slower than the disappearance of 1 at pH = 9 and 10,
respectively.[9] Under these conditions, the hydrolysis of the
alaninyl methyl ester group of 2Ј-O-methylcytidine 5Ј-{O-3-
(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl-N-[(1S)-2-meth-
oxy-1-methyl-2-oxoethyl]phosphoramidate} is known to
take place only 1.5 times faster than the acetate ester hy-
drolysis.[9] As a conclusion, at most 5–9% and 8–14% of
the overall degradation of 1 can be estimated to take place
by Routes (G) and (F), respectively.
Dimer 8 breaks down to give mainly 7 [Route (I) in
Scheme 5]. Hydration of the oxo group and subsequent at-
tack of the oxyanion of the geminal diol onto the phos-
phorus atom results in a cleavage of the P–O bond. Finally, Scheme 7. Mechanism of the conversion of 8 into 7.
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to proceed at room temperature for 3 h. A mixture of Et3N (3 mL)
and H2O (3 mL) was added. The mixture was stirred for 15 min,
then the solvent was removed under reduced pressure, and the resi-
due was coevaporated twice with toluene (15 mL). The crude prod-
uct was purified by column chromatography eluting with dichloro-
methane containing 2–10% methanol to give 3 (1.43 g, 90%) as a
{O-3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl-N-[(1S)-2-
methoxy-1-methyl-2-oxoethyl]phosphoramidate}, at pH =
9.[9]
Conclusions
1
solid. H NMR (500 MHz, CD3OD): δ = 7.83 (d, J = 8.10 Hz, 1
H, 6-H), 6.79 (d, J = 622.31 Hz, 1 H, PH), 5.95 (d, J = 2.90 Hz, 1
H, 1-H), 5.76 (d, J = 8.10 Hz, 1 H, 5-H), 4.95 (dd, J = 6.25 and
2.95 Hz, 1 H, 2Ј-H), 4.92 (dd, J = 6.30 and 2.85 Hz, 1 H, 3ЈЈ-H),
4.36 (m, 1 H, 4Ј-H), 4.08–4.05 (m, 2 H, 5Ј-H and 5ЈЈ-H), 3.22 (q,
J = 7.30 Hz, 6 H, Et3N), 1.57 (s, 3 H, CH3), 1.37 (s, 3 H, CH3), 1.33
(t, J = 7.30 Hz, 9 H, Et3N) ppm. 13C NMR (126 MHz, CD3OD): δ
= 164.72 (C-4), 150.73 (C-2), 142.10 (C-6), 113.77 (spiro C), 101.64
(C-5), 92.1 (C-1Ј), 85.16 and 85.10 (C-4Ј), 84.21 (C-2Ј), 81.11 (C-
3Ј), 63.38 and 63.35 (C-5Ј), 46.37 (CH2 of Et3N), 26.1 and 24.14
(CH3), 7.81 (CH3 of Et3N) ppm. 31P NMR (202 MHz, CD3OD): δ
= 4.39 ppm. HRMS (ESI–): calcd. for C12H16N2O8P [M – H]–
347.0650; found 347.0650.
Thermally and enzymatically labile (1) and reductively
labile (2) phosphoramidates were prepared by the diphenyl-
phosphite-based H-phosphonate strategy and subsequent
oxidative amination with l-alanine methyl ester. Glutathi-
one-triggered deprotection of 2 produced exclusively, and
enzyme-triggered deprotection of 1 mainly, diester-like
phosphoramidate 7 via intermediate 6, formed by reduction
or deacetylation, respectively. In the absence of enzyme, mi-
gration of the acetyl group from the sulfur atom to the adja-
cent hydrated oxo group triggers the deprotection of 1 to
give 7. The S–S-bond-mediated dimerization to give 8 com-
petes with the removal of the protecting group from 1. In
all cases, an attack of the exposed sulfur atom onto C-1
results in the removal of the protecting group by cycliza-
tion. Finally, dimer 8 also undergoes degradation to give
mainly 7.
2Ј,3Ј-O-Isopropylideneuridine 5Ј-{O-[4-(Acetylthio)-2,2-dimethyl-3-
oxobutyl]-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphor-
amidate} (1)
Procedure A: 2Ј,3Ј-O-Isopropylideneuridine 5Ј-(H-phosphonate) (3;
150 mg, 0.334 mmol) was dissolved in pyridine (3 mL), and pivaloyl
chloride (83 μL, 0.668 mmol) was added dropwise. The mixture was
stirred at room temperature for 5 min, then S-(4-hydroxy-3,3-di-
methyl-2-oxobutyl) ethanethioate (5; 70 mg, 0.367 mmol) was
added dropwise. The reaction mixture was stirred for 30 min. The
resulting 2Ј,3Ј-O-isopropylideneuridine 5Ј-(H-phosphonate) 4-
(acetylthio)-2,2-dimethyl-3-oxobutyl ester was not isolated, but
CCl4 (0.25 mL) and Et3N (0.75 mL) were added. The mixture was
stirred for 5 min, then a solution of l-alanine methyl ester hydro-
chloride (70 mg, 0.501 mmol) in a mixture of pyridine (0.5 mL) and
MeCN (1.5 mL) was added dropwise. The mixture was stirred for
2 h, then EtOAc (50 mL) was added, and the organic phase was
washed with water (2ϫ 10 mL). The organic phase was dried with
Na2SO4, and the solvents were evaporated. The crude product was
purified by column chromatography eluting with EtOAc to give 1
Experimental Section
General Remarks: 1-(tert-Butyldisulfanyl)-4-hydroxy-3,3-dimeth-
ylbutan-2-oneethanethioate (5)[11] were prepared as described pre-
viously. Pyridine and dichloromethane were dried with 4 Å molecu-
lar sieves. Pivaloyl chloride was distilled before use. 1H, 13C, 31P,
and 2D NMR spectra were recorded with a Bruker Avance 500
spectrometer. High-resolution mass spectra were recorded with a
Bruker Daltonics microTOF-Q instrument using electrospray ion-
ization. The composition of the samples was analysed with a Merck
Hitachi LaChrom D7000 HPLC with an l-7455 UV detector and
an l-7100 pump using an ODS Hypersil C18 column (4ϫ250 mm,
5 μm).
1
(42 mg, 21%) as a white solid foam. H NMR (500 MHz, CDCl3):
Kinetic Measurements: The breakdown of the mixtures of RP and
SP diastereomers of phosphoramidates 1 and 2 in HEPES (0.036/
0.024 m; 3 mL; pH = 7.5) and glycine (0.02/0.04 and 0.05/0.01 m;
pH = 9 and 10, respectively) buffers at 37 °C was monitored by
reverse-phase HPLC (UV detection at 260 nm, flow rate
0.95 mL min–1) in the absence and presence of porcine liver esterase
(PLE; 2.6 UmL–1) or glutathione (GSH; 5 mm). The ionic strength
of the solutions was adjusted to 0.1 m with sodium chloride. The
initial concentration of the starting material was 0.15 mm. Samples
(200 μL) were withdrawn from the reaction solution at appropriate
time intervals, and were made acidic (pH = 3) with HCl (1 m, aq.;
10 μL) to deactivate the enzyme and quench the hydrolysis. The
solution was filtered with Minisart RC 4 filters (0.2 μm). Products
were separated using 7 min isocratic elution with AcOH/AcONa
buffer (0.045/0.015 m) containing NH4Cl (0.1 m) and 0.5% MeCN,
followed by a 35 min linear gradient up to 70.0% MeCN. The prod-
ucts detected were identified by HPLC/ESI-MS, or samples were
collected and identified by MS analysis.
δ = 9.50 (br. s, 1 H, NH), 7.42 and 7.38 (d, J = 8.10 and 8.10 Hz,
1 H, 6-H), 5.79 and 5.72 (d, J = 2.50 and 2.40 Hz, 1 H, 1Ј-H), 5.77
and 5.75 (d, J = 8.05 and 8.05 Hz, 1 H, 5-H), 4.94–4.92 (m, 1 H,
2-H), 4.90 and 4.85 (dd, J = 6.45 and 3.70 Hz, 1 H, 3-H), 4.31–
4.29 (m, 1 H, 4-H), 4.24–4.17 (m, 2 H, 5Ј-H and 5ЈЈ-H), 4.06–4.00
(m, 2 H, OCH2), 3.96–3.87 (3 H, SCH2 and Hα), 3.73 (s, 3 H,
OCH3), 2.37 (s, 3 H, SAc), 1.57, 1.56, 1.36 and 1.35 (s, 6 H, CH3
of isopropylidene), 1.39 and 1.38 (d, J = 1.80 and 1.95 Hz, 3 H, β
CH3), 1.28, 1.25, 1.23, 1.22 and 1.20 (s, 6 H, 2 CH3) ppm. 13C
NMR (126 MHz, CDCl3): δ = 205.56 and 205.51 (C=O), 194.81
and 194.78 (AcS), 174.25, and 174.20 (C=O of amino acid), 170.68
(EtOAc), 163.35 and 135.32 (C-4), 150.16 and 150.10 (C-2), 142.01
and 141.86 (C-6), 114.64, 114.59 and 114.56 (spiro C), 102.82,
102.72 and 102.67 (C-5), 94.02 and 93.35 (C-1), 85.41, 85.34, 84.95,
and 84.89 (C-4), 84.38 and 84.20 (C-2), 80.64 and 80.39 (C-3),
71.93, 71.89, 71.84 and 71.79 (OCH2), 65.98, 65.94, 65.82 and 65.78
(C-5), 52.55 (OCH3), 49.99 and 49.95 (CH amino acid), 48.73,
48.71, 48.66 and 48.63 (spiro C), 46.14 (spiro C), 36.43 (SCH2),
30.23 (SAc), 27.29, 27.19, 27.15, 25.32 and 25.30 (CH3 of isoprop-
ylidene), 21.69, 21.64 and 21.60 (CH3 of the protecting group),
21.00 and 20.98 (β CH3) ppm. 31P NMR (202 MHz, CDCl3): δ =
7.15, 7.04 ppm. HRMS (ESI+): calcd. for C24H36N3NaO12PS [M +
Na]+ 644.1655; found 644.1650.
2Ј,3Ј-O-Isopropylidene 5Ј-(H-Phosphonate) (3): 2Ј,3Ј-O-Isopropylid-
eneuridine (1.0 g, 3.5 mmol) was dissolved in dry pyridine (10 mL)
while cooling with an ice bath. Diphenyl phosphite (1.16 mL,
5.2 mmol) was added dropwise, and the mixture was stirred for
15 min. The ice bath was removed, and the reaction was allowed
5010
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Eur. J. Org. Chem. 2015, 5004–5012

Protecting Groups of Nucleoside 5Ј-Phosphoramidates
Precedure B: 2Ј,3Ј-O-Isopropylideneuridine (284 mg, 1.0 mmol)
was dissolved in CH2Cl2 (10 mL), and the resulting solution was
added dropwise to a solution of PCl3 (0.9 mL, 10 mmol) in CH2Cl2
(10 mL) at –20 °C over a period of 5 min. The mixture was stirred
for 1 h, then the cooling bath was removed, and the reaction mix-
ture was stirred at room temperature for 6 h. The solvent was re-
moved under reduced pressure, and the residue was coevaporated
twice with CH2Cl2 (2ϫ 20 mL). The 2Ј,3Ј-O-isopropylideneuridine
5Ј-dichlorophosphite obtained was not isolated, but it was dis-
solved in CH2Cl2 (5 mL), and a solution of S-(4-hydroxy-3,3-di-
methyl-2-oxobutyl) ethanethioate (5; 190 mg, 1.0 mmol) in a mix-
ture of CH2Cl2 (2.5 mL) and pyridine (2.5 mL) was added drop-
wise. The resulting mixture was stirred for 1 h, then a mixture of
THF (2 mL) and water (2 mL) was added, and the reaction was
allowed to proceed for 30 min. The solvent was removed under
reduced pressure, and the residue was coevaporated twice with an-
hydrous toluene (2ϫ 20 mL). The resulting 2Ј,3Ј-O-isopropylidene-
uridine 5Ј-(H-phosphonate) was not isolated, but was dissolved in
anhydrous MeCN (5 mL), and a mixture of CCl4 (1.5 mL) and
Et3N (0.5 mL) was added. The mixture was stirred for 5 min, then
(OCH3), 49.94 (CH amino acid), 48.38–47.61 (3 spiro C and
SCH2), 29.85 (tBu), 27.18, 27.15, 25.34 and 25.31 (CH3 of isoprop-
ylidene), 21.65, 21.58 and 21.56 (CH3 of the protecting group),
21.07, 21.02 and 20.98 (β CH3) ppm. 31P NMR (202 MHz, CDCl3):
δ = 7.10, 7.01 ppm. HRMS (ESI+): calcd. for C26H43N3O11PS2 [M
+ H]+ 668.2077; found 668.2076.
Supporting Information (see footnote on the first page of this arti-
1
cle): Copies of H, 13C, and 31P NMR spectra of compounds 1, 2,
and 3.
Acknowledgments
The Eramus Mundus Experts III program is gratefully acknowl-
edged for financial support (Fellowship for V. A. S.).
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solution of l-alanine methyl ester hydrochloride (166 mg,
1.2 mmol) in a mixture of MeCN (4 mL) and pyridine (1 mL) was
added dropwise at 0 °C. The mixture was stirred for 2 h, then
EtOAc (50 mL) was added, and the organic phase was washed with
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2Ј,3Ј-O-Isopropylideneuridine 5Ј-{O-[4-(tert-Butyldisulfanyl)-2,2-di-
methyl-3-oxobutyl]-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phos-
phoramidate} (2): 2Ј,3Ј-O-Isopropylideneuridine 5Ј-(H-phos-
phonate) (3; 0.50 g, 1.11 mmol) was dissolved in pyridine (5 mL),
and pivaloyl chloride (272 μL, 2.22 mmol) was added dropwise.
The mixture was stirred at room temperature for 5 min, then
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1-(tert-butyldisulfanyl)-4-hydroxy-3,3-dimethylbutan-2-one
(4;
290 mg, 1.22 mmol) was added dropwise. The reaction mixture was
stirred for 30 min. The resulting 2Ј,3Ј-O-isopropylideneuridine 5Ј-
(H-phosphonate) 4-(tert-butyldisulfanyl)-2,2-dimethyl-3-oxobutyl
ester was not isolated, but CCl4 (1.5 mL) and Et3N (0.5 mL) were
added. The mixture was stirred for 5 min, then a solution of l-
alanine methyl ester hydrochloride (232 mg, 1.67 mmol) in a mix-
ture of pyridine (1 mL) and acetonitrile (3 mL) was added drop-
wise. After 15 min, EtOAc (50 mL) was added, and the organic
phase was washed with water (2ϫ 10 mL). The organic phase was
dried with Na2SO4, and the solvents were evaporated. The crude
product was purified by column chromatography eluting with
EtOAc to give 2 (165 mg, 23%) as a white solid foam. 1H NMR
(500 MHz, CDCl3): δ = 9.45 (br. s, 1 H, NH), 7.42 and 7.37 (d, J
= 8.05 and 8.10 Hz, 1 H, 6-H), 5.80 and 5.72 (d, J = 2.55 and
2.25 Hz, 1 H, 1Ј-H), 5.78 and 5.75 (d, J = 8.05 and 8.05 Hz, 1 H,
5-H), 4.96–4.93 (m, 1 H, 2-H), 4.89 and 4.86 (dd, J = 6.40 and
3.75 Hz, 1 H, 3-H), 4.31–4.28 (m, 1 H, 4-H), 4.23–4.16 (m, 2 H,
5Ј-H, 5ЈЈ-H), 4.08–3.99 (m, 2 H, OCH2), 3.93–3.80 (m, 3 H, Hα
and SCH2), 3.74 and 3.73 (s, 3 H, OCH3), 1.57, 1.56 1.36 and 1.35
(s, 3 H, CH3 of isopropylidene), 1.40 and 1.38 (d, J = 2.30 and
2.45 Hz, 3 H, β CH3), 1.34 (s, 9 H, tBu), 1.26 (EtOAc), 1.23, 1.22,
1.21 and 1.19 (s, 6 H, 2 CH3) ppm. 13C NMR (126 MHz, CDCl3):
δ = 207.16 and 207.12 (C=O), 174.24, 174.19 and 174.14 (C=O of
amino acid), 163.32 (C-4), 150.16 and 150.09 (C-2), 142.07 and
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