Synthesis of α- L -threose nucleoside phosphonates via regioselective sugar protection

Article abstract of DOI:10.1021/jo400907g

A new synthesis route to α-l-threose nucleoside phosphonates via 2-O and 3-O selectively protected l-threose is developed. The key intermediates 2-O-benzoyl-l-threonolactone and 1-O-acetyl-2-O-benzoyl-3-O-t- butyldiphenylsilyl-l-threofuranose were functionalized to synthesize 2′-deoxy-2′-fluoro- and 3′-C-ethynyl l-threose 3′-O-phosphonate nucleosides. The key intermediates developed are important intermediates for the synthesis of new l-threose-based nucleoside analogues, TNA phosphoramidites, and TNA triphosphates.

Full text of DOI:10.1021/jo400907g

Article
pubs.acs.org/joc
Synthesis of α‑L‑Threose Nucleoside Phosphonates via
Regioselective Sugar Protection
Shrinivas G. Dumbre, Mi-Yeon Jang, and Piet Herdewijn*
Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven,
Belgium
S
* Supporting Information
ABSTRACT: A new synthesis route to α-L-threose nucleoside
phosphonates via 2-O and 3-O selectively protected L-threose is
developed. The key intermediates 2-O-benzoyl-L-threonolac-
tone and 1-O-acetyl-2-O-benzoyl-3-O-t-butyldiphenylsilyl-L-
threofuranose were functionalized to synthesize 2′-deoxy-2′-
fluoro- and 3′-C-ethynyl L-threose 3′-O-phosphonate nucleo-
sides. The key intermediates developed are important
intermediates for the synthesis of new L-threose-based
nucleoside analogues, TNA phosphoramidites, and TNA
triphosphates.
activity.16 This has generated our interest in synthesizing the 3′-
INTRODUCTION
■
C-ethynyl analogue of PMTA (8). Furthermore, 2′-deoxy-2′-
The modification of the nucleobase and/or sugar moiety of a
fluoro nucleosides were found to possess interesting biological
natural nucleoside is an obvious choice for developing new
properties because fluorine mimics both H or OH to some
antiviral compounds, and threose-based nucleosides could serve
extent.17 In particular, the cytosine-based 2′-deoxy-2′-fluoro-
this purpose.1−4 Moreover, (3′,2′)-α-L-threose nucleic acid
threose phosphonate (7) analogue is an attractive target
(TNA) is a possible RNA progenitor because of the chemical
because several bioactive nucleosides are cytosine nucleo-
simplicity of threose relative to that of ribose and the ability of
sides4,18 with a 2′-mono or 2′,2′-difluoro modification (e.g., 1−
TNA to form thermally stable duplexes with DNA and RNA
3), with the latter showing potent inhibition of HCV
that are comparable to those of natural nucleic acid
replication. 2′-Fluoro-2′-methyl-substituted nucleosides have
become leading compounds in the development of an anti-
HCV therapy, and these analogues are currently being
investigated under phase II and III clinical trials.4 Herein, we
describe an easy chemical synthesis route to develop new α-L-
threose nucleoside analogues via differentially protected 2-O-
and 3-O-L-threose (17, Scheme 2).
associations.5 α-L-Threose nucleosides have been synthesized
and assembled chemically5,6 to generate TNA; recently, all four
threo-nucleoside triphosphates (tNTPs) were successfully
assembled into TNA oligomers with high fidelity by DNA
polymerases.7−9 There are convincing reasons to develop new
efficient routes for the synthesis of L-threose nucleosides and to
try to discover new α-L-threose nucleoside analogues that can
target viral polymerase as a triphosphate.10−15
Recently, we have demonstrated that the L-2′-deoxythreose
nucleoside phosphonates PMDTA (5, EC50 = 2.5 μM) and
PMDTT (6) selectively inhibit HIV without affecting normal
cell proliferation (Figure 1).10 The 4′-C-ethynyl substitution of
natural nucleosides (e.g., 4) has a beneficial effect on anti-HIV
RESULTS AND DISCUSION
■
Our initial synthesis plan for the synthesis of 7 is shown in
Scheme 1. Eschenmoser et al.6 have described an efficient
strategy for the synthesis of compounds 9 and 10. The
stereoselective Vorbruggen glycosylation of silylated nucleo-
̈
bases requires the availability of 1-O- and 2-O-acylated sugars.
To fulfill this requirement, the synthesis of the PMDT analogue
was accomplished via multistep protection, deprotection, and
reprotection procedures to obtain compound 1410 via 11 and
12 (Scheme 1). For the nucleophilic fluorination at the 2′
position, the epimerization of the 2′-hydroxyl group in threose
to an erythrose is needed, which was successfully accomplished
via Dess−Martin periodinane (DMP) oxidation followed by
sodium borohydride reduction to afford compound 15.
Figure 1. Structures of biologically active nucleoside analogues (1−6)
and current L-threose nucleoside phosphonate targets (7, 8).
Received: May 14, 2013
Published: July 3, 2013
© 2013 American Chemical Society
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Scheme 1. Previous Synthesis Strategies for α-L-Threose Nucleosides and Our Initial Synthesis Work toward the Target
a
Compound 7 via 15.
a
Reagents and conditions: (a) DMP (15% in CH2Cl2), 0 °C to rt, 1 h, 98% and (b) NaBH4, MeOH, 1 h, 0 °C to rt, 80%. Note that the attempts to
form the anhydro from 14 were unsuccessful.
a
Scheme 2. Synthesis of 2′-Deoxy-2′-fluoro L-Threose Analogue
a
Reagents and conditions: (a) benzoyl chloride (1 equiv), imidazole (Im, 2 equiv), CH3CN, −5 °C to rt, 8 h, 95%; (b) TBDPSCl, cat. DMAP, Im (2
equiv), 0 °C to rt, 10 h, 98%; (c) DIBAL-H (1.2 M in toluene, 2 equiv), dry THF, −70 to −60 °C, 3 h, 94%; (d) acetic anhydride, Et3N, dry CH2Cl2,
0 °C to rt, 8 h, 92%; (e) O4-methyluracil or uracil, N,O-bis(trimethylsilyl)acetamide (BSA, 2 equiv), TMSOTf (3 equiv), 60 °C, 2.5 h, 82−88%; (f)
LiOH, H2O/MeOH, rt, 1 h, 83%; (g) DMP (15% in CH2Cl2), 0 °C to rt, 1 h, 98%; (h) NaBH4, MeOH, 1 h, 0 °C to rt, 80%; (i) TBAF (1 M in
THF), 0 °C to rt or Et3N·3HF, 15 h, 0 °C to rt, 84−90%; (j) NaH, CH3CN, −5 to −10 °C, (iPrO)2POCH2OTf, 45 min, 85%; (k) DAST, Py-HF,
CH2Cl2, −5 °C to rt, 12 h, 29% of 23a; (l) DAST (4 equiv), Et3N·HF (0.05 equiv), CH2Cl2, −10 °C to rt, 10 h, 46%; (m) sat. NH3 in MeOH, 60
°C, 6 h, 81%; and (n) TMSBr, Et3N, rt, 30 h, 71%.
This synthesis approach is cumbersome, and we have
investigated ways to shorten this synthesis pathway. All of the
effort to 3-O-phosphonomethylate 12 in the presence of
threonolactone (9) was successful. The addition of benzoyl
chloride to a solution of 9 and imidazole in acetonitrile at −5
°C to room temperature (rt) for 8 h furnished 16 exclusively in
excellent yield (95%, Scheme 2). Compound 16 was silylated at
the 3′-hydroxyl group with TBDPSCl (98%) followed by the
DIBAL-H-mediated reduction of the lactone to the lactol and
acetylation, affording key intermediate 18 in 87% yield.
hindered bases (e.g., 2,6-di-tert-butyl-4-methylpyridine, Hunig’s
̈
base) as well as the Lewis acid-catalyzed benzylation of the 3′-
hydroxyl group with benzyl trichloroacetimidate were un-
successful.19 However, the regioselective benzoylation of L-
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a
The Vorbruggen glycosylation of silylated uracil using
̈
Scheme 3. Synthesis of the 3′-Ethynyl Analogue of PMTA
trimethylsilyl trifluoromethanesulfonate (TMSOTf) at 60 °C
in acetonitrile furnished 18 in 80% yield. Unfortunately, the 2′-
O-debenzoylation of 18 and the oxidation followed by
reduction of the 2′-hydroxy function resulted in the formation
of a mixture of 19a and 19b resulting from TBDPS migration
under reducing conditions. Alternatively, compound 17 was
glycosylated with O4-methyluracil to obtain 20 in 82% yield.
Several reaction conditions were tested for the sodium hydride-
mediated 3′-O-phosphomethylation of desilylated compound
20 with diisopropylphosphonomethyl triflate to avoid un-
desired side reactions resulting from the presence of the 2′-O-
benzoyl moiety. The addition of 1 equiv of sodium hydride to a
equimolar mixture of desilylated compound 20 and diisopro-
pylphosphonomethyl triflate in acetonitrile at −5 to −10 °C for
45 min afforded 3′-phonsphonomethylated 21 in 85% yield.
Compound 24 could readily be prepared by the functional-
group manipulation of 21 in a manner similar to the one
described for the conversion of 14 to 15 (64% over three
steps). The difluorination of 2′-keto nucleoside 22 was
attempted under various reaction conditions with diethylami-
nosulfur trifluoride (DAST) and morpholinodifluorosulfinium
tetraborate20 (XtalFluoro-M) with or without a promoter, such
as Et3N·3HF, Py-HF, or DBU. Required difluoro compound
23b could not be obtained. Under DAST and Py-HF-DBU
conditions, only a trace of required compound 23b was formed.
All of the efforts to improve the yields failed in our hands. TLC
of the reaction mixture showed the formation of a second
product that, however, disappeared to starting material after an
aqueous sat. NaHCO3 workup. Thus, the reaction mixture was
purified (elution with 98:2 CH2Cl2/MeOH) without a previous
reaction workup to afford 23a in 29% yield. The configuration
of 2′-OMe in 23a was confirmed by HMBC analysis.
a
Reagents and conditions: (a) N6-benzoyladenine, N,O-bis-
(trimethylsilyl)acetamide (BSA, 2 equiv), TMSOTf (1 equiv), 70
°C, 12 h, 65%; (b) p-TSA, toluene, reflux; (c) TBAF (1 M in THF), 0
°C to rt, 78%; (d) DMP (15% in CH2Cl2), 0 °C to rt, 1 h then rt, 10 h,
97%; (e) CeCl3, ethynylmagnesium bromide (0.5 M in THF), dry
THF, −78 °C, 6 h, 32%; (f) NaH, CH3CN, −10 °C,
(iPrO)2POCH2OTf, 1 h, 88%; (g) sat. NH3 in MeOH, 24 h, rt,
86%; (h) TMSI, HMDS, 0 °C, 2 h, 60%; (i) Bu3N, N,N-
carbonyldiimidazole, tris(tetra-butylammonium)hydrogen pyrophos-
phate, DMF, 16 h, 31%; (j) phenyl chloroformate, DMAP, CH3CN,
rt, 2 h, 91%; and (k) Bu3SnH, AIBN, toluene, 75 °C.
The fluorination of 24 was carried out with DAST and
Et3N·3HF as a promoter from −10 °C to rt to furnish 2′-
fluorinated nucleoside 25 in 46% yield. The smooth conversion
of the O4-methyluracil moiety to a cytosine base was achieved
by treatment of 25 with sat. methanolic ammonia at 60 °C to
obtain analogue 25m in 81% yield. The final trimethylsilyl
bromide-mediated hydrolysis of the diisopropylesters furnished
the target cytosyl nucleoside phosphonate 7.
3′-ethynyl analogue of PMTA (8). Diphosphate-phosphonate
30 was prepared by the reaction with pyrophosphate, following
a literature procedure.22,23
To synthesize the 3′-C-ethynyl analogue of PMTA (8),
silylated N6-benzoyladenine was glycosylated with 17 using
TMSOTf as a Lewis acid catalyst to give a mixture of 26 and
26b (1.7:1, Scheme 3). N7 isomer 26b was isomerized to N9
isomer 26 under acidic conditions. N7 nucleoside 26b was
refluxed in toluene in the presence of p-toluenesulfonic acid to
furnish 26 in 45% yield.21 The TBDPS group was removed by
treatment with TBAF in THF, and the oxidation of the 3′-
hydroxyl with DMP provided ketone 27. The ethynyl group
was introduced using ethynylmagnesium bromide in the
presence of CeCl3 in THF at −78 °C. As expected, the
addition reaction showed no selectivity, yielding 3′-ethynyl
nucleoside 28 and its epimer as a 1:1 mixture that can be
separated by silica gel chromatography. The configuration of
both epimers was confirmed by 2D-NMR experiments. In the
ROESY measurement, a cross peak was observed between 3′-
OH and 4′-Hb (28) and 4′-Ha (epimer), respectively. The
phosphonate function was introduced using diisopropylphos-
phonomethyl triflate and NaH in THF at −15 °C. The benzoyl
group was removed with ammonia in methanol, and the
hydrolysis of the phosphonate ester function of 29 was carried
out with TMSI in the presence of HMDS at 0 °C to furnish the
Biellmann et al. described a modified Barton−McCombie
reaction for the 2′-deoxygenation of 3′-C-ethynyl nucleoside
analogues.24 However, in our hands the attempts to 2′-
deoxygenate compound 29 were not successful. When we
followed Biellmann’s method no reaction had taken place.
Under standard conditions, a complex mixture of byproducts
was obtained.
CONCLUSION
■
An efficient synthesis route to L-threose nucleoside analogues is
developed. Key intermediates 2-O-benzoyl-L-threonolactone
(16) and 1-O-acetyl-2-O-benzoyl-3-O-tert-butyldiphenylsilyl-L-
threofuranose (17) were functionalized to obtain the 2′-deoxy-
2′-fluoro and 3′-C-ethynyl (PMTA) L-threose 3′-O-phospho-
nate nucleosides (7 and 8) and diphosphate-phosphonate
compound 30. Unfortunately, none of the synthesized
compounds showed either any significant in vitro activity
against HIV, HCV, and RSV or cytotoxicity at concentrations
up to 100 μM. Diphosphate-phosphonate compound 30 did
not inhibit HCV polymerase (IC50 > 200 μM) and was not
incorporated into mtRNA. Nevertheless, key intermediate 17 is
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0.4) to obtain 16b (52.2 g, 113 mmol, 98% yield). [α]2D0 −20.2 (c =
an important synthon for the synthesis of new L-threose-base
nucleoside analogues. Until now,25 a mixture of 2′-O-DMTr-
and 3′-O-DMTr-protected L-threose nucleosides needed to be
separated6 to obtain TNA phosphoramidite and TNA
triphosphate for the construction of TNA oligomers. The
accessibility of intermediates such as 16 and 17 allows for a
more straightforward synthesis of TNA monomers.
1
0.47, CH2Cl2). H NMR (300 MHz, CDCl3): δ 7.18−7.89 (m, 15H,
Ph), 6.05 (d, J = 7.8 Hz, 1H, H2′), 4.95 (dd, J = 7.8, 16.8 Hz, 1H,
H3′), 4.35 (appt t, J = 7.8 Hz, 1H, H4a′), 4.27 (appt t, J = 8.2 Hz, 1H,
H4b′), 0.97 (s, 9H, C(CH3)3). 13C NMR (75 MHz, CDCl3): δ 170.9
(C1′), 165.4 (PhCO), 136.0, 135.9, 135.4, 135.0, 133.0, 132.6, 131.1,
130.9, 130.4, 129.7, 129.0, 128.9, 128.9, 128.7, 128.3, 75.8 (C2′), 72.5
(C3′), 69.7 (C4′), 27.3 (CH3), 19.5 (C(CH3)3). HRMS (ESI+): [M +
Na]+ calcd for C27H28O5SiNa, 483.1598; found, 483.1581.
2-O-Benzoyl-3-O-tert-butyldiphenylsilyl-L-threofuranose
(16c). To a solution of 16b (27.7 g, 60.1 mmol) in 350 mL of dry
THF at −78 °C, a 1.2 M DIBAL-H (100 mL, 120 mmol) solution in
toluene was added dropwise via cannula. The mixture was stirred at
−78 to −60 °C for 3 h (the starting material was consumed in 3 h).
The reaction was quenched by the dropwise addition of 15 mL of
MeOH at −78 °C. The cooling bath was removed, the reaction
mixture was diluted with EtOAc, 300 mL of a sat. aq. sodium
potassium tartrate solution was added, and the mixture was stirred
vigorously for 2 h. The reaction mixture was filtered, and the organic
phase was washed with water and sat. NaCl solution and dried over
MgSO4. The organic phase was concentrated under reduced pressure
and coevaporated with 2 × 200 mL of toluene. This crude residue was
passed through a short silica column (1:1 EtOAc/hexane) to obtain
16c as a diastereomeric mixture at anomeric position (26 g, 56.2
mmol, 94% yield). HRMS (ESI+): [M + Na]+ calcd for
C27H30O5SiNa, 485.1755; found, 485.1740.
EXPERIMENTAL SECTION
■
1H, 13C, and 31P NMR spectra were recorded on a 300 (1H NMR, 300
MHz; 13C NMR, 75 MHz; 31P NMR, 121 MHz) or 500 MHz (1H
NMR, 500 MHz; 13C NMR, 125 MHz) spectrometer. Two-
dimensional NMRs (H−COSY, NOESY, ROESY, HSQC, and
HMBC) were used for the assignment of all of the intermediate and
final compounds. The specific optical rotation was measured at 589
nm, and c is given in g/100 mL. The mass spectra were acquired on a
quadrupole orthogonal acceleration time-of-flight mass spectrometer.
The column chromatographic separations were carried out by gradient
elution with a suitable combination of two or three solvents and silica
gel (100−200 mesh or 230−400 mesh). The solvents for the reactions
were distilled prior to use (THF and toluene from Na/benzophenone;
CH2Cl2 and CH3CN from CaH2; Et3N and pyridine from KOH).
1′α-(Uracil-1-yl)-3′-O-diisopropylphosphonomethyl-L-eryth-
rose (15). Following the procedure described for 24, the 2′-hydroxyl
1
moiety in compound 18 was epimerized to 15. H NMR (acetone-d6,
1-O-Acetyl-2-O-benzoyl-3-O-tert-butyldiphenylsilyl-L-threo-
furanose (17). To a solution of lactol 16c (26.0 g, 56.2 mmol) and
triethylamine (39 mL, 281 mmol) in 200 mL of dry dichloromethane
at 0 °C was added acetic anhydride (10.6 mL, 112 mmol) dropwise.
The reaction mixture was stirred at rt for 8 h and given a water and
brine wash. The organic layer was dried over MgSO4 and concentrated
under reduced pressure to obtain the crude product. The residue was
purified by column chromatography (95:5 EtOAc/hexane; Rf = 0.15)
to obtain 17 as a diastereomeric mixture at anomeric position (26 g,
51.5 mmol, 92% yield). C1′ β-isomer: 1H NMR (300 MHz, CDCl3): δ
7.89 (d, J = 7.1 Hz, 2H, Bz-H), 7.68−7.63 (m, 4H, Ph-H), 7.56 (t, J =
7.5 Hz, 1H, Bz-H), 7.43−7.30 (m, 8H, Ph-H, Bz-H), 6.17 (s, 1H,
H1′), 5.43 (d, J = 1.9 Hz, 1H, H2′), 4.50 (dt, J = 2.0, 5.2 Hz, 1H, H3′),
4.06−3.96 (m, 2H, H4′), 2.18 (s, 3H, CH3CO), 1.10 ((s, 9H,
C(CH3)3). 13C NMR (75 MHz, CDCl3): δ 169.9, 165.3, 135.9, 135.8,
133.6, 133.3, 132.8, 130.2, 130.2, 130.0, 129.9, 128.6, 128.1, 128.03,
100.0, 83.5, 75.9, 75.0, 26.9, 21.3, 19.3. HRMS: [M + Na]+ calcd for
500 MHz): δ 11.25 (s, 1H, NH), 7.67 (d, J = 8.0 Hz, 1H, H6), 5.99 (d,
J = 5.8 Hz, 1H, H1′), 5.56 (d, J = 5.3 Hz, 1H, OH), 5.47 (dd, J = 8.1,
2.2 Hz, 1H, H5), 4.62 [m, 2H, P(OCH(CH3)2)2], 4.15 (q, J = 5.2 Hz,
1H, H-2′), 3.97 (dd, J = 9.4, 4.4 Hz, 1H, H4a′), 3.95 (dd, J = 13.9, 8.3
Hz, 1H, PCHa), 3.88 (dd, J = 13.9 Hz, 9.1 Hz, 1H, PCHb), 3.83 (dd, J
= 9.4, 4.8 Hz, 1H, H4b′), 1.25 [m, 12H, P(OCH(CH3)2)2]. 13C NMR
(acetone-d6, 125 MHz): δ 163.3 (C4), 150.8 (C2), 143.3 (C6), 100.0
2
(C5), 84.1 (C1′), 79.5 (d, JP,C = 11.9 Hz, C3′), 70.4 and 70.4
1
(CH(CH3)2), 69.4 (C2′), 68.9 (C4′), 64.2 (PCH2, JP,C = 165.5 Hz),
23.8 and 23.9 [P(OCH(CH3)2)2]. HRMS (ESI+): [M + Na]+ calcd
for C15H25N2O8PNa, 415.1246; found, 415.1240.
2-O-Benzoyl-L-threonolactone (16). To a solution of L-
threonolactone (9, 11.20 g, 95 mmol) and imidazole (12.91 g, 190
mmol) in dry acetonitrile at 0 to −5 °C, benzoyl chloride (11.02 mL,
95 mmol) was slowly added over a period of 10 min. The reaction
mixture was stirred at rt for 8 h. The acetonitrile was removed under
reduced pressure, the residue was taken up into 150 mL of ethyl ether,
and 100 mL of water was added. The resulting mixture was
sequentially washed with 2 × 100 mL of ice-cold 1 M HCl soln.,
100 mL of water, 100 mL of sat. NaHCO3 soln., and 100 mL of sat. aq.
NaCl soln. The organic layer was dried over MgSO4 and concentrated
to obtain the crude product. The residue was purified by column
chromatography (9:1 to 7:3, hexane/EtOAc; Rf = 0.4, 6:1 hexane/
EtOAc) to obtain 16 (20 g, 90 mmol, 95% yield). [α]2D0 −21.5 (c =
1
C29H32O6SiNa, 527.1860; found, 527.1863. C1′ α-isomer: H NMR
(300 MHz, CDCl3): δ 7.92 (d, J = 7.1 Hz, 2H, Bz-H), 7.67−7.56 (m,
4H, Ph-H), 7.45 (t, J = 7.5 Hz, 1H, Bz-H), 7.43−7.25 (m, 8H, Ph-H,
Bz-H), 6.50 (d, J = 4.5 Hz, 1H, H1′), 5.44 (t, J = 5.3 Hz, 1H, H2′),
4.75 (q, J = 6.0 Hz, 1H, H3′), 3.95 (dd, J = 6.5, 9.3 Hz, 1H, H4′), 3.77
(dd, J = 4.7, 9.3 Hz, 1H, H4′), 1.87 (s, 3H, CH3CO), 1.06 (s, 9H,
C(CH3)3). 13C NMR (75 MHz, CDCl3): δ 169.6, 165.5, 135.8, 133.5,
133.3, 132.8, 130.3, 130.1, 129.9, 129.3, 128.5, 128.1, 127.9, 94.5, 79.4,
74.3, 72.5, 26.9, 21.0, 19.2. HRMS (ESI+): [M + Na]+ calcd for
C29H32O6SiNa, 527.1860; found, 527.1859.
1
0.065, CH2Cl2). H NMR (500 MHz, CDCl3): δ 8.05 (d, J = 7.4 Hz,
2H, Ph), 7.73 (t, J = 7.4 Hz, 1H, Ph), 7.60 (t, J = 7.9 Hz, 2H, Ph), 6.9
(d, J = 4.9 Hz, 1H, 3′-OH), 5.74 (d, J = 8.0 Hz, 1H, H2′), 4.75 (ddd, J
= 5.21, 8.0 Hz, 1H, H3′), 4.55 (appt t, J = 8.3 Hz, 1H, H4a′), 4.10
(appt, J = 8.3 Hz, 1H, H4b′). 13C NMR (125 MHz, CDCl3): δ 172.0
(C1′), 165.7 (CO), 135.0 (Ph), 130.5 (Ph), 129.9 (Ph), 129.4 (Ph),
76.0 (C2′), 70.6 (C4′ and C3′). HRMS (ESI+): [M + Na]+ calcd for
C11H10O5Na, 245.0421; found, 245.0414.
2-O-Benzoyl-3-O-tert-butyldiphenylsilyl-L-threonolactone
(16b). To a solution of lactone (16, 25.7 g, 116 mmol), cat. DMAP
(0.1 g), and imidazole (15.75 g, 231 mmol) in dry acetonitrile at 0 °C,
tert-butyldiphenylchlorosilane (29.7 mL, 116 mmol) was added
dropwise. The reaction mixture was allowed to stir at rt for 10 h.
The acetonitrile was removed under reduced pressure, the residue was
taken up into 400 mL of ethyl ether, and 200 mL of water was added.
The resulting mixture was sequentially washed with 2 × 200 mL of ice-
cold 1 M HCl soln., 200 mL of water, 100 mL of sat. NaHCO3 soln.,
and 100 mL of sat. aq. NaCl soln. The organic layer was dried over
MgSO4 and concentrated to obtain the crude product. The residue
was purified by column chromatography (95:5 hexane/EtOAc; Rf =
1′α-(Uracil-1-yl)-2′-O-benzoyl-3′-O-tert-butyldiphenylsilyl-
L-threose (18). Following the procedure described for 20, compound
18 was obtained in 87% yield.
1H NMR (500 MHz, CDCl3): δ 7.93 (dd, J = 1.2, 8.4 Hz, 2H, Ph),
7.90 (d, J = 8.1 Hz, 1H, H6), 7.60−7.64 (m, 4H, Ph), 7.54−7.58 (m,
1H, Ph), 7.34−7.48 (m, 8H, Ph), 6.10 (d, J = 1.5 Hz, 1H, H1′), 5.76
(dd, J = 2.0, 8.1 Hz, 1H, H5), 5.46 (d, J = 1.0 Hz, 1H, H2′), 4.40 (dt, J
= 1.0, 3.4 Hz, 1H, H3′), 4.12 (bd, J = 10.4 Hz, 1H, H4a′), 3.94 (dd, J =
3.4, 10.4 Hz, 1H, H4′), 1.10 (s, 9H, C(CH3)3). 13C NMR (125 MHz,
CDCl3): δ 164.2 (C4), 162.0 (PhCO), 149.7 (C3), 140.4 (C6),
135.40, 135.38, 133.3, 132.1, 131.3, 130.3, 129.5, 128.4, 128.1, 127.8,
127.7, 100.1 (C5), 89.5 (C1′), 82.1 (C2′), 75.5 (C4′), 75.4 (C3′),
26.5 (CH3), 18.7 [SiC(CH3)3]. HRMS (ESI+): [M + Na]+ calcd for
C31H32N2O6SiNa, 579.1922; found, 579.1890.
1′α-(Uracil-1-yl)-3′-O-tert-butyldiphenylsilyl-L-threose (18f).
To a solution of compound 18 (1.0 g, 1.790 mmol) in 6 mL of
methanol was added a LiOH (0.107 g, 4.47 mmol) solution in 3 mL of
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water, and the reaction mixture was stirred at rt for 2 h. The solvent
was removed, and the residue was subjected to column chromatog-
raphy (elution with 1:1 hexane/EtOAc; Rf = 0.5, 1:1 hexane/EtOAc)
172.4 (C2), 166.2 (PhCO), 155.8 (C4), 145.6 (C6), 145.6, 133.8,
129.9, 128.6, 96.2 (C5), 96.1 (C1′), 84.1 (C2′), 76.1 (C3′), 75.1
(C4′), 55.2 (CH3O). HRMS (ESI+): [M + Na]+ calcd for
C16H16N2O6Na, 355.0906; found, 355.0898.
1
to obtain compound 18f (0.7 g, 15.40 mmol, 86% yield). H NMR
(500 MHz, CDCl3): δ 7.77 (dd, J = 8.0 Hz, 1H, H6), 7.52−7.61 (m,
4H, Ph), 7.33−7.48 (m, 6H, Ph), 5.74 (d, J = 8 Hz, 1H, H5), 5.72 (s,
1H, H1′), 4.35 (s, 1H, H2′), 4.27 (d, J = 2.7, 1H, H3′), 4.09−4.17 (m,
2H, H4), 1.02 (s, 9H, C(CH3)3). 13C NMR (75 MHz, CDCl3): δ
164.0 (C4), 151.0 (C2), 140.7 (C6), 135.66, 135.57, 132.58, 132.41,
130.20, 130.16, 127.9, 100.5 (C5), 94.0 (C1′), 81.4 (C2′), 76.9 (C3′),
77.5 (C4′), 26.8 (CH3), 19.0 (SiC(CH3)3). HRMS (ESI+): [M + Na]+
calcd for C24H28N2O5SiNa, 475.1660; found, 475.1657.
1′α-(O4-Methyluracil-1-yl)-2′-O-benzoyl-3′-O-diisopropyl-
phosphonomethyl-L-threose (21). To a solution of 20i (3.20 g,
9.63 mmol) in dry acetonitrile (10 mL) was added triflate
diisopropylphosphonomethanol (6.32 g, 19.26 mmol). The reaction
mixture was cooled to 5−10 °C (ice−salt bath) under argon before the
addition of NaH (0.463 g, 11.56 mmol, 60% suspension). After 45 min
of stirring at 0 °C, the reaction was quenched with 5 mL of EtOAc
containing 0.1 mL of acetic acid. The content was further diluted with
20 mL of EtOAc, and 10 mL of water was added, the mixture was
stirred well, extracted, dried over MgSO4, and concentrated under
reduced pressure. Purification by column chromatography (3% MeOH
in CH2Cl2) afforded product 21 as a colorless liquid (4.2 g, 8.23 mmol,
A Dess−Martin oxidation reaction of compound 18f in CH2Cl2 (2
h, rt) followed by the sodium borohydride-mediated reduction of the
ketone resulted in mixture 19a and 19b (6:4).
Diisopropylphosphonomethyl Trifluoromethanesulfonate.
To a solution of (hydroxymethyl)diisopropylphosphonate (2.9 g,
14.78 mmol) in 50 mL of dry diethyl ether at −78 °C was added a
solution of 2.5 M n-BuLi (6.12 mL, 15.29 mmol). This reaction
mixture was allowed to stir at this temperature for 5 min, and
trifluoromethanesulfonyl chloride (1.628 mL, 15.29 mmol) was added
dropwise over 5 min. The reaction mixture was stirred at this
temperature for 1 h. The reaction was quenched with sat. NH4Cl. The
organic layer was given a wash with water and brine, dried over
MgSO4, and concentrated under reduced pressure at rt to obtain
diisopropylphosphonomethyl triflate (4.77 g, 14.53 mmol, 98% yield).
The crude product was pure, without any traces of phosphonate dimer
or any other reactant. 1H NMR (300 MHz, CDCl3): δ 1.35−1.40 (m,
12H, POCH(CH3)2), 4.55 (d, 1JP,H = 8.97 Hz, 2H, PCH2), 4.82 (sept,
J = 6.21 Hz, 2H, POCH(CH3)2). 31P NMR (CDCl3, 125 MHz): δ
10.01.
1
85% yield). H NMR (500 MHz, CDCl3): δ 8.06 (d, J = 7.4 Hz, 2H,
Ph), 7.61 (d, J = 7.6 Hz, 1H, H6), 7.62 (t, J = 7.6 Hz, 1H, Ph), 7.48 (t,
J = 7.6 Hz, 2H, Ph), 6.35 (s, 1H, H1′), 5.95 (d, J = 7.6 Hz, 1H, H5),
5.46 (s, 1H, H2′), 4.66−4.80 (m, 2H, CH(CH3)3), 4.46 (bd, J = 9.17
Hz, 1H, H4a′), 4.24−4.29 (m, 2H, H4b′ and H3), 3.98 (s, 3H), 3.93
(dd, J = 9.17, 13.6 Hz, 1H, PCHa), 3.87 (dd, J = 9.17, 13.6 Hz, 1H,
PCHb), 1.30−1.35 (m, 12H, CH3). 13C NMR (150 MHz, CDCl3): δ
171.7 (C2), 164.9 (PhCO), 155.4 (C4), 142.8 (C6), 135.3, 129.6,
3
128.5, 128.2, 95.2 (C5), 90.2 (C1′), 83.1 (C3′, JP,C = 10.4 Hz), 79.4
(C2′), 73.6 (C4′), 71.0 (CH(CH3)2, 2JP,C = 6.4 Hz), 70.9 (CH(CH3)2,
1
2JP,C = 6.4 Hz), 64.1 (PCH2, JP,C = 169.3 Hz), 54.1 (OMe), 23.6
[CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 17.9. HRMS (ESI+):
[M + H]+ calcd for C23H32N2O9P, 511.1840; found, 511.1847.
1′α-(O4-Methyluracil-1-yl)-3′-O-diisopropylphosphono-
methyl-L-threose (21f). A solution of 21 (4.70 g, 9.21 mmol) in 10
mL of acetonitrile was treated with LiOH (0.220 g, 9.21 mmol) in 4
mL of water and 10 mL of MeOH. The reaction mixture was stirred at
rt. The reaction was found to be complete in 1 h, and the reaction
mixture was neutralized with acetic acid. The solvent was removed and
purified by column chromatography (elution with 7% MeOH in
CH2Cl2; Rf = 0.2, 5% MeOH in CH2Cl2) to obtain 21f as a colorless
solid (3.11 g, 7.65 mmol, 83% yield). [α]2D0 +33.3 (c = 0.045 CH2Cl2).
1H NMR (300 MHz, CDCl3): δ 7.80 (d, J = 7.6 Hz, 1H, H6), 5.92 (d,
1′α-(O4-Methyluracil-1-yl)-2′-O-benzoyl-3′-O-tert-butyldi-
phenylsilyl-L-threose (20). A solution of 17 (12.88 g, 25.5 mmol)
and O4-methyluracil (3.22 g, 25.5 mmol) in 120 mL of dry acetonitrile
was treated with BSA (13.94 mL, 56.2 mmol) and stirred at 60 °C for
30 min. TMSOTf (13.86 mL, 77 mmol) was added, and the heating
was continued of another 2 h, after which time the Vorbruggen
̈
glycosylation was found to be complete. The reaction mixture was
cooled to rt, diluted with 200 mL of EtOAc, and poured into 100 mL
of a 10% NaHCO3 solution with stirring. The organic layer was
separated and washed with water and brine, dried over MgSO4, and
concentrated under reduced pressure. The oily residue was purified by
column chromatography (6:4 hexane/EtOAc; Rf = 0.4) to obtain light-
J = 7.6 Hz, 1H, H5), 5.75 (d, J = 1.3 Hz, 1H, H1′), 4.61−4.85 (m, 2H,
CH(CH3)2), 4.22−4.37 (m, 3H, H3′ and H4′), 4.09 (s, 1H, H2′), 3.90
(s, 3H, CH3O), 3.68 (d, J = 8.7 Hz, 1H, PCH2), 1.15−1.42 (m, 12H,
P[CH(CH3)2]2). 13C NMR (75 MHz, CDCl3): δ 172.1 (C2), 156.6
(C4), 143.1 (C6), 95.1 (C5), 94.2 (C1′), 85.6 (C3′, 3JP,C = 11.2 Hz),
1
yellow low-melting solid 20 (12 g, 21.03 mmol, 82% yield). H NMR
2
(500 MHz, CDCl3): δ 8.05 (d, J = 7.5, Hz 1H, H6), 7.96 (dd, J = 1.9,
8.2 Hz, 2H, Ph), 7.51−7.64 (m, 4H, Ph), 7.51−7.56 (m, 1H, Ph),
7.34−7.46 (m, 8H, Ph), 6.22 (d, J = 1.0 Hz, 1H, H1′), 5.92 (d, J = 7.5
Hz, 1H, H5), 5.60 (d, J = 1.0 Hz, 1H, H2′), 4.36 (dt, J = 1.0, 3.5 Hz,
1H, H3′), 4.12 (d, J = 9.9 Hz, 1H, H4′), 4.00 (dd, J = 3.5, 10.2 Hz, 1H,
H4′), 3.98 (s, 3H, CH3O), 1.05 (s, 9H, C(CH3)3). 13C NMR (125
MHz, CDCl3): δ 172.0 (C2′), 164.5 (PhCO), 155.9 (C3), 143.4 (C6),
135.9, 135.8, 133.5, 132.7, 131.8, 130.34, 130.32, 130.0, 129.2, 128.5,
128.4, 128.1, 128.0, 95.5 (C5), 91.0 (C1′), 82.2 (C2′), 76.4 (C4′),
75.9 (C3′), 54.6 (OMe), 26.7 (CH3), 19.1 (SiC(CH3)3). HRMS (ESI
+): [M + Na]+ calcd for C32H34N2O6SiNa, 593.2078; found, 593.2076.
1′α-(O4-Methyluracil-1-yl)-2′-O-benzoyl-L-threose (20i). To a
solution of compound 20 (0.330 g, 0.578 mmol) in 10 mL of THF at
0 °C was added tetra-n-butylammonium fluoride (0.578 mL, 0.578
mmol) dropwise. The reaction mixture was stirred in an ice bath for 1
h and found to be complete (i.e., clean reaction). The solvent was
removed and purified by column chromatography with 1:1 hexane/
EtoAc followed by 100% EtOAc to obtain 20i (0.162 g, 0.487 mmol,
84% yield). Note that the deprotection of TBDPS with triethylamine
trihydrofluoride (2 equiv) in dichloromethane for 15 h at rt gave 20i in
79.0 (C2′), 73.6 (C4′), 71.3 (CH(CH3)2, JP,C = 4.7 Hz), 71.2
2
1
(CH(CH3)2, JP,C = 4.7 Hz), 64.2 (PCH2, JP,C = 169.3 Hz), 54.4
(OMe), 24.0 (CH(CH3)2). 31P NMR (121 MHz, CDCl3): δ 18.3.
HRMS (ESI+): [M + Na]+ calcd for C16H27N2O8PNa, 429.1403;
found, 429.1408.
(1′α,2′S)-1′-(O4-Methyluracil-1-yl)-2′-fluoro-2′-O-methyl-3′-
O-diisopropylphosphonomethyl-L-threose (23a). A solution of
22 (0.033 g, 0.082 mmol) in dry CH2Cl2 was cooled in an ice−salt
bath to maintain temperature from −5 to −10 °C. To this was added
(diethylamino)sulfur trifluoride (0.219 mL, 1.632 mmol) and Py-HF
(0.809 mg, 8.16 μmol) as a solution in 1 mL of dry CH2Cl2. The
reaction mixture was allowed to stir at rt for 12 h. The volatiles were
removed under reduced pressure to obtain a crude oil. This residue
was purified by silica gel flash column chromatography (98:2 CH2Cl2/
MeOH) to obtain 23a as a light-yellow liquid (0.01 g, 0.023 mmol,
1
29% yield). H NMR (500 MHz, CDCl3): δ 7.89 (d, J = 7.6 Hz, 1H,
H6), 6.44 (d, 2JFH = 16.4 Hz, 1H, H1′), 5.86 (d, J = 7.6 Hz, 1H, H5),
4.70−4.81 (m, 2H, CH(CH3)2), 4.33 (d, J = 11.5 Hz, 1H, H4a′), 4.14
(dd, J = 2.4, 7.9 Hz, 1H, H3′,), 4.00−4.07 (m, 1H, H4b′), 3.97 (s, 3H,
OCH3), 3.86 (d, J = 9.0 Hz, 2H, PCH2), 3.52 (s, 3H, 2′-OCH3), 1.30−
1.38 (m, 12 H, P[CH(CH3)2]2). 13C NMR (125 MHz, CDCl3): δ
90% yield. [α]2D0 −34.5 (c = 0.185, CH2Cl2). H NMR (500 MHz,
1
1
CDCl3): δ 8.05 (dd, J = 1.5, 8.4 Hz, 2H, Ph), 7.60−7.64 (m, 2H, H6
and Ph), 7.46−7.50 (appt t, J = 7.8 Hz, 2H, Ph), 5.98 (d, J = 7.5 Hz,
1H, H5), 5.65 (d, J = 1.8 Hz, 1H, H1′), 5.47 (s, 1H, H2′), 4.54 (m,
1H, H3′), 4.30 (bd, J = 10.0 Hz, 1H, H4a′), 4.24 (dd, J = 4.3, 10.0 Hz,
1H, H4b′), 3.99 (s, 3H, CH3O). 13C NMR (125 MHz, CDCl3): δ
171.4 (C2), 155.8 (C4), 144.9 (C6), 119.0 (C2′, JF,C = 227.2 Hz),
2
3
2
95.3 (C5), 86.6 (C1′, JF,C = 48.6), 82.3 (C3′, JP,C = 11.1 Hz, JF,C
=
2
43.1), 71.1 and 71.0 (CH(CH3)2, JP,C = 6.4 Hz), 69.1 (C4′), 64.1
(PCH2, JP,C = 171.0 Hz), 54.1 (OCH3), 53.27 (2′-OCH3, JF,C = 4.5
Hz), 29.6 (CH(CH3)2). 31P NMR (121 MHz, CDCl3): δ 17.6. HRMS
1
2
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(ESI+): [M + Na]+ calcd for C17H28FN2O8PNa, 461.1465; found,
461.1457.
155.1 (C4), 141.3 (C6), 95.4 (C2′, 1JF,C = 181.0 Hz), 93.4 (C5), 90.3
2
3
2
(C1′, JF,C = 36.1 Hz), 82.3 (C3′, JP,C = 10.8 Hz, JF,C = 28.7, Hz),
1′α-(O4-Methyluracil-1-yl)-3′-O-diisopropylphosphono-
methyl-L-erythrose (24). A solution of 21f (0.129 g, 0.317 mmol) in
1 mL of dichloromethane was treated with a 15% solution of Dess−
Martin periodinane (DMP) in CH2Cl2 (1.8 mL, 0.635 mmol). A drop
of water was added to accelerate the reaction, and the reaction was
completed in 1 h. The reaction mixture was diluted with diethyl ether
and filtered off. The organic solvent was removed under reduced
pressure to obtain crude keto 22 (Rf = 0.4, 7% MeOH in CH2Cl2).
HRMS (ESI+): [M + H]+ calcd for C16H26N2O8, 405.1421; found,
405.1386.
73.1 (C4′), 71.1 (CH(CH3)2, JP,C = 7.4 Hz), 64.2 (PCH2, JP,C
=
2
1
169.0 Hz), 23.7 [CH(CH3)2]. 31P NMR (121 MHz, MeOD): δ 17.6.
HRMS (ESI+): [M + H]+ calcd for C15H26FN3O6P−, 394.1538; found,
394.1555.
(1′α,2′R)-1′-(Cytosin-1-yl)-2′-deoxy-2′-fluoro-3′-O-phospho-
nomethyl-L-threose (7). A solution of 25m (5.00 mg, 0.014 mmol)
and triethylamine (0.019 mL, 0.137 mmol) in 2 mL of dry CH2Cl2 was
treated with bromotrimethylsilane (0.018 mL, 0.137 mmol) at rt for 30
h. The reaction was quenched with 1 M TEAB solution, and the
reaction mixture was concentrated to dryness. The residue was purified
by flash column chromatography (50:25:3 CH2Cl2/MeOH/1 M
TEAB) to obtain target compound 7 (3 mg, 9.70 μmol, 70.9% yield)
as an off-white solid. 1H NMR (500 MHz, MeOD): δ 7.76 (d, J = 7.6
Crude ketone 22 was taken up in 2 mL of methanol and treated
with sodium borohydride (0.635 mmol, 0.024g) at 0 °C. The reaction
mixture was stirred at rt for 1 h, quenched by the dropwise addition of
brine solution (1 mL), and stirred at rt for 30 min. The reaction
mixture was diluted in 20 mL of CH2Cl2 and washed with 5 mL of
water and 5 mL of brine. The organic layer was dried and concentrated
under reduced pressure. The residue was purified by column
chromatography (elution with 7% MeOH in CH2Cl2; Rf = 0.3) to
obtain 24 (0.1 g, 0.247 mmol, 78% yield). 1H NMR (300 MHz,
CDCl3): δ 7.76 (d, J = 7.6, Hz 1H, H6), 6.20 (d, J = 3.4 Hz, 1H′), 5.91
(d, J = 7.6 Hz, 1H, H5), 4.61−4.83 (m, 2H, CH(CH3)2), 4.60 (t, J =
3.9 Hz, 1H, H2′), 4.20−4.32 (m, 1H, H3′), 4.11 (d, J = 5.8 Hz, 2H,
H4′), 3.90−4.00 (m, CH3O and PCHa), 3.77 (dd, J = 8.6, 14.1 Hz, 1H,
PCHa,) 1.18−1.45 (m, 12H, P[CH(CH3)2]2). 13C NMR (75 MHz,
CDCl3): δ 171.6 (C2), 156.2 (C4), 144.1 (C6), 94.3 (C5), 86.7 (C1′),
1
Hz, 1H, H6), 6.08 (d, JF,H = 20.5 Hz, 1H, H1′), 6.02 (d, J = 7.6 Hz,
1H, H5), 5.22 (d, 1JF,H = 47.8 Hz, 1H, H2), 4.50 (d, J = 11.2 Hz, 1H,
H4a′), 4.39 (bd, J = 12.8, 1H, H3′), 4.19 (bd, J = 11.2 Hz, 1H, H4b′),
3.47 (bd, J = 8.6 Hz, 2H, PCH2). 13C NMR (125 MHz, MeOD): δ
166.0 (C2), 157.1 (C4), 142.0 (C6), 96.1 (C2′, 1JF,C = 181.6 Hz), 95.8
(C5), 89.7 (C1′, 2JF,C = 39.0 Hz), 81.3 (C3′, 3JP,C = 9.0 Hz, 2JF,C = 27.0
Hz), 72.9 (C4′), 66.6 (PCH2, 1JP,C = 152.4 Hz). 31P NMR (121 MHz,
MeOD): δ 13.4. HRMS (ESI-): [M − H]− calcd for C9H12FN3O6P−,
308.0453; found, 308.0450.
1′α-(N6-Benzoyladenin-9-yl)-2′-O-benzoyl-3′-O-tert-butyldi-
phenylsilyl-L-threose (26). To a solution of N6-benzyladenine (3.98
g, 16.7 mmol) in CH3CN (80 mL) was added BSA (8.14 mL, 33.3
mmol), and the mixture was stirred for 20 min at rt. To the mixture, a
solution of 17 in CH3CN (10 mL) was added. After cooling to 0 °C,
TMSOTf (3.01 mL, 16.7 mmol) was added dropwise, and the reaction
mixture was stirred for 1 h at rt and held at 70 °C overnight. The
reaction mixture was poured into a mixture of CH2Cl2 and sat.
NaHCO3 solution (5:1 v/v). The CH2Cl2 layer was removed, and the
water layer was extracted with CH2Cl2. The combined CH2Cl2 extracts
were dried over Na2SO4. After the solvent was removed, the residue
was purified by column chromatography (CH2Cl2/MeOH 50:1) on
silica gel to afford 26 (2.71 g, 51%) and 26b (1.71 g, 30%) as a light-
yellow foam.
3
2
81.1 (C3′, JP,C = 7.6 Hz), 71.5 (CH(CH3)2, JP,C = 6.9 Hz), 71.3
2
(CH(CH3)2, JP,C = 6.9 Hz), 69.4 (C4′), 69.30 (C2′), 65.3 (PCH2,
1JP,C = 169.0 Hz), 54.4 (CH3O), 23.7 (CH(CH3)2). 31P NMR (121
MHz, CDCl3): δ 19.3. HRMS (ESI+): [M + Na]+ calcd for
C16H27N2O8PNa, 429.1403; found, 429.1408.
(1′α,2′R)-1′-(O4-Methyluracil-1-yl)-2′-deoxy-2′-fluoro-3′-O-
diisopropylphosphono-methyl-L-threose (25). A solution of 24
(0.111 g, 0.273 mmol) in 1 mL of dry CH2Cl2 at −10 °C was treated
with diethylaminosulfur trifluoride (DAST) (0.147 mL, 1.093 mmol).
The reaction mixture was allowed to warm to rt and was stirred for 10
h. The resulting solution was then carefully poured into an ice-cold sat.
solution of sodium bicarbonate (5 mL) and extracted with diethyl
ether (4 × 20 mL). The combined organic extracts were washed with
brine (50 mL), dried over MgSO4, filtered, and concentrated to a
crude oil that was purified by silica gel flash column chromatography
(elution with 3% MeOH in CH2Cl2; Rf = 0.3) to obtain 25 as a light-
yellow liquid (0.051 g, 0.125 mmol, 45.7% yield). Note that the
N7 to N9 isomerization was carried out as follows. To a solution of
26b (1.71 g, 2.50 mmol) in toluene (25 mL), a catalytic amount of p-
TsOH-H2O (48 mg, 0.25 mmol) was added and the mixture was held
at 130 °C overnight. After the volatiles were removed, the residue was
diluted with CH2Cl2 and washed with a sat. NaHCO3 solution. The
CH2Cl2 layer was removed, and the water layer was extracted with
CH2Cl2. The combined CH2Cl2 extracts were dried over Na2SO4.
After the solvent was removed, the residue was purified by column
chromatography (CH2Cl2/MeOH 50:1) on silica gel to afford 26
(0.77 g, 45%) as a light-yellow foam. [α]2D0 −115.4 (c = 0.42 CH2Cl2).
1H NMR (300 MHz, CDCl3): δ 9.17 (s, 1H, NH), 8.84 (s, 1H, H2),
8.55 (s, 1H, H8), 8.04 (d, J = 7.1 Hz, 2H, Bz-H), 7.95 (d, J = 7.1 Hz,
2H, Bz-H), 7.66−7.29 (m, 16H, Ph-H, Bz-H), 6.37 (d, J = 1.4 Hz, 1H,
H1′), 5.94 (s, 1H, H2′), 4.60−4.57 (m, 1H, H3′), 4.22 (dd, J = 2.3,
10.0 Hz, 1H, H4′), 4.11 (dd, J = 4.4, 10.0 Hz, 1H, H4′), 1.03 (s, 9H,
C(CH3)3). 13C NMR (75 MHz, CDCl3): δ 165.0, 164.7, 153.1, 151.8,
149.7, 141.8, 135.9, 135.8, 134.0, 133.9, 132.9, 132.6, 131.9, 130.5,
130.4, 130.1, 129.0, 128.8, 128.7, 128.2, 128.1, 128.0, 123.2, 88.5, 82.8,
76.3, 75.8, 27.0, 19.1. HRMS: [M + H]+ calcd for C39H38N5O5Si,
684.2637; found, 684.2642.
1
reaction yield is poor if the reaction begins at rt or 0 °C. H NMR
2
(300 MHz, CDCl3): δ 7.70 (d, J = 7.6 Hz, 1H, H6), 6.10 (d, JFH
=
1
17.1 Hz, 1H, H1′), 5.89 (d, J = 7.6 Hz, 1H, H5), 5.14 (d, JFH = 47.8
Hz, 1H, H2′), 4.62−4.76 (m, 2H, CH(CH3)2), 4.45 (d, J = 10.3 Hz,
1H, H4a′), 4.25−4.32 (m, 2H, H4b′ and H3′), 3.95 (s, 3H, CH3O),
3.69 (d, J = 1.48, 8.69 Hz, 2H, PCH2), 1.27−1.32 (m, 12H,
P[CH(CH3)2]2). 13C NMR (75 MHz, CDCl3): δ 172.1 (C2), 155.8
(C4), 142.7 (C6), 95.5 (C2′, 1JF,C = 186.2 Hz), 95.2 (C5), 90.8 (C1′,
2JF,C = 36.5 Hz), 82.3 (C3′, 3JP,C = 9.6 Hz, 2JF,C = 26.9 Hz), 73.7 (C4′),
71.4 (CH(CH3)2, 2JP,C = 4.6 Hz), 64.5 (PCH2, 1JP,C = 169.0 Hz), 54.4
(OCH3), 29.6 (CH(CH3)2). 31P NMR (121 MHz, CDCl3): δ 17.5.
HRMS (ESI+): [M + Na]+ calcd for C16H26FN2O7PNa, 431.1360;
found, 431.1365.
(1′α,2′R)-1′-(Cytosin-1-yl)-2′-deoxy-2′-fluoro-3′-O-diisopro-
pylphosphonomethyl-L-threose (25m). Compound 25 (0.051 g,
0.125 mmol) was taken up in 2 mL of sat. ammonia in methanol and
refluxed for 6 h. The volatiles were removed and purified by column
chromatography (elution with 7% MeOH in CH2Cl2; Rf = 0.2) to
obtain compound 25m (0.04 g, 0.102 mmol, 81% yield) as a light-
yellow liquid. 1H NMR (500 MHz, MeOD): δ 7.65 (d, J = 7.6 Hz, 1H,
H6), 5.99 (d, 1JF,H = 18.3 Hz, 1H, H1), 5.89 (d, J = 7.6 Hz, 1H, H5),
1′α-(N6-Benzoyladenin-9-yl)-2′-O-benzoyl-L-threose (26c).
To a solution of 26 (4.3 g, 6.29 mmol) in THF (65 mL) were
added a 1 M solution of TBAF in THF (7.55 mL, 7.55 mmol) and
AcOH (0.5 mL), and the solution was stirred for 2 h at rt. After
removing the volatiles, the residue was diluted with CH2Cl2, washed
with water and brine, and dried over Na2SO4. After evaporation, the
crude residue was purified by silica gel chromatography (40:1 CH2Cl2/
MeOH) to afford 26c (2.18 g, 78%) as a white foam. [α]2D0 −75.2 (c =
0.34 CH2Cl2). 1H NMR (300 MHz, MeOD): δ 8.68 (s, 1H, H2), 8.66
(s, 1H, H8), 8.08−8.02 (m, 4H, Bz-H), 7.64−7.45 (m, 6H, Bz-H),
6.44 (d, J = 1.1 Hz, 1H, H1′), 5.68 (s, 1H, H2′), 4.59 (s, 1H, H3′),
4.35 (br s, 2H, H4′). 13C NMR (75 MHz, MeOD): δ 166.7, 153.2,
1
5.16 (d, JF,H = 47.8 Hz, 1H, H2), 4.62−4.73 (m, 2H, CH(CH3)2),
4.50 (d, J = 10.9 Hz, 1H, H4a′), 4.35 (dd, J = 3.6, 11.2 Hz, 1H, H4b′),
4.25 (ddd, J = 10.9, 3.6, 2.1 Hz, 1H, H3′), 3.95 (dd, J = 9.7, 13.6 Hz,
1H, PCHa), 3.88 (dd, J = 9.7, 13.6 Hz, 1H, PCHb), 1.28−1.34 (m,
12H, P[CH(CH3)2]2). 13C NMR (125 MHz, MeOD): δ 165.3 (C2),
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The Journal of Organic Chemistry
Article
152.9, 151.2, 144.7, 135.0, 134.9, 133.9, 130.8, 130.3, 129.7, 129.4,
124.9, 90.0, 84.0, 77.0, 75.0. HRMS: [M + H]+ calcd for C23H20N5O5,
446.1459; found, 446.1459.
(0.35 g, 86%) as a white solid. 1H NMR (300 MHz, CDCl3): δ 8.27 (s,
1H, H2), 8.23 (s, 1H, H8), 7.29 (s, 2H, NH2), 6.19 (s, 1H, H1′),
4.79−4.69 (m, 2H, CH(CH3)2), 4.53 (s, 1H, H2′), 4.47 (d, J = 9.9 Hz,
1H, H4a′), 4.18 (d, J = 9.9 Hz, 1H, H4b′), 3.95 (quint, J = 9.9 Hz, 2H,
PCH2), 2.93 (s, 1H, CH), 1.34−1.24 (m, 12H, CH3). 13C NMR (75
1′α-(N6-Benzoyladenin-9-yl)-2′-O-benzoyl-3′-oxo-L-threose
(27). Under argon, to 26c (1.87 g, 4.2 mmol) was added a solution of
Dess−Martin periodinane (15% in CH2Cl2, 13.4 mL, 6.3 mmol) in dry
CH2Cl2 (100 mL) at 0 °C. The reaction mixture was stirred overnight
at rt (a white solid formed). To the reaction mixture, a solution of sat.
aqueous sodium thiosulfate and sat. aqueous NaHCO3 (1:1, 100 mL)
was added, and the mixture was stirred for 20 min at rt. After
separation, the organic layer was brined, dried over Na2SO4, filtered,
and concentrated under reduced pressure to afford crude product 27
(1.8 g, 97%) as a white foam. 1H NMR (300 MHz, CDCl3): δ 9.00 (s,
1H, NH), 8.79 (s, 1H, H2), 8.20 (s, 1H, H8), 8.04 (d, J = 7.2 Hz, 4H,
Bz-H), 7.66−7.44 (m, 6H, Bz-H), 6.46 (d, J = 5.5 Hz, 1H, H1′), 6.18
(d, J = 5.5 Hz, 1H, H2′), 4.76 (d, J = 16.9 Hz, 1H, H4a′), 4.58 (d, J =
16.9 Hz, 1H, H4b′). 13C NMR (75 MHz, CDCl3): δ 204.5, 165.5,
165.0, 152.9, 151.9, 150.1, 142.3, 134.3, 133.5, 132.9, 130.2, 128.9,
128.7, 128.1, 127.7, 123.8, 86.3, 73.9, 72.0. HRMS: [M + H]+ calcd for
C23H18N5O5, 444.1302; found, 444.1307.
MHz, CDCl3): δ 156.1, 152.9, 149.1, 140.0, 119.4, 90.6, 83.8 (d, 3JP,C
13.9 Hz), 80.6, 80.4, 76.4, 74.4, 71.9 (d, 2JP.C = 3.5 Hz), 71.8 (d, 2JP,C
=
=
1
3
2.8 Hz), 60.4 (d, JP,C = 171.4 Hz), 24.2 (d, JP,C = 3.5 Hz), 24.1 (d,
3JP,C = 4.4 Hz). 31P NMR (121 MHz, CDCl3, 25 °C): δ 18.4. HRMS:
[M + H]+ calcd for C18H27N5O6P1, 440.1693; found, 440.1687.
(1′α,3′S)-1′-(Adenin-9-yl)-3′-ethynyl-3′-O-(phosphonometh-
yl)-L-threose (8). Compound 29 (0.15 g, 0.34 mmol) in dry CH2Cl2
(2 mL) was treated with HMDS (1.07 mL, 5.12 mmol), and TMSI
(0.29 mL, 2.05 mmol) was added dropwise under stirring at 0 °C. The
mixture was stirred for 2 h at 0 °C and quenched with 1 M TEAB (1
mL). The mixture was concentrated, and the residue was purified by
RP-HPLC running a gradient of CH3CN in 0.1 M TEAB buffer
solution to afford 8 (0.115 g, 60%, triethylamine salt) as a white solid.
[α]2D0 −65.9 (c = 0.22 H2O). H NMR (300 MHz, D2O): δ 8.41 (s,
1
1H, H2), 8.07 (s, 1H, H8), 6.04 (s, 1H, H1′), 4.68 (s, 1H, H2′), 4.58
(d, J = 10.0 Hz, 1H, H4a′), 4.21 (d, J = 10.0 Hz, 1H, H4b′), 3.74 (d, J
= 10.5 Hz, 2H, PCH2), 3.27 (s, 1H, CH). 13C NMR (75 MHz, D2O):
δ 154.9, 152.2, 148.1, 140.5, 117.8, 88.9, 82.4 (CH(CH3)2, 3JP,C = 13.8
(1′α,3′S)-1′-(N6-Benzoyladenin-9-yl)-2′-O-benzoyl-3′-ethyn-
yl-L-threose (28). A suspension of anhydrous CeCl3 (5.10 g, 20.7
mmol) in THF (80 mL) under argon was stirred overnight. The
solution was cooled to −78 °C. Ethynylmagnesium bromide (0.5 M in
THF, 40.6 mL, 20.3 mmol) was added over 20 min. The suspension
was stirred at −78 °C for 1.5 h. A solution of 27 (1.8 g, 4.06 mmol) in
THF (10 mL) was added over 10 min and stirred at −78 °C for 4 h.
The reaction was quenched with sat. NH4Cl (10 mL). After being
allowed to warm to rt, the reaction mixture was filtered and extracted
with CH2Cl2, washed with brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by silica gel
chromatography (60:1 CH2Cl2/MeOH) to afford 28 (0.61 g, 32%) as
1
Hz), 80.4, 79.2, 75.8, 74.3, 61.7 (PCH2, JP,C = 154.6 Hz). 31P NMR
(121 MHz, D2O): δ 14.1. HRMS: [M − H]− calcd for C12H13N5O6P,
354.0609; found, 354.0613.
(1′α,3′S)-1′-(Adenin-9-yl)-3′-ethynyl-3′-O-(diphosphate-
phosphonomethyl)-L-threose (30). Compound 8 (70 mg, 0.13
mmol) was dissolved in anhydrous DMF (1 mL), and Bu3N (0.09 mL,
0.38 mmol) was added. The mixture was concentrated in vacuo,
redissolved in anhydrous DMF (1 mL), and treated with N,N-
carbonyldiimidazole (0.2 g, 1.25 mmol). The mixture was stirred for
30 min and tris(tetrabutylammonium) hydrogen pyrophosphate (0.9
g, 1.0 mmol) in DMF (1 mL) was added. The mixture was stirred
overnight. Excess aqueous ammonia (25% in water, 2 mL) was added,
and the mixture was concentrated by lyophilization. The residue was
purified by RP-HPLC running a gradient of CH3CN in 0.1 M TEAB
1
a white foam. H NMR (500 MHz, DMSO): δ 11.24 (s, 1H, NH),
8.76 (s, 1H, H2), 8.59 (s, 1H, H8), 8.09−8.05 (m, 4H, Bz-H), 7.73 (tt,
J = 1.3, 7.5 Hz, 1H, Bz-H), 7.66 (tt, J = 1.2, 7.4 Hz, 1H, Bz-H), 7.59 (t,
J = 8.1 Hz, 2H, Bz-H), 7.56 (t, J = 8.0 Hz, 2H, Bz-H), 7.00 (s, 1H,
OH), 6.47 (d, J = 2.2 Hz, 1H, H1′), 5.88 (d, J = 1.9 Hz, 1H, H2′), 4.48
(d, J = 9.1 Hz, 1H, H4a′), 4.27 (d, J = 9.1 Hz, 1H, H4b′), 3.70 (s, 1H,
CH). 13C NMR (75 MHz, CDCl3): δ 165.7, 164.3, 151.9, 151.8, 150.5,
142.7, 134.1, 133.4, 132.5, 129.7, 129.0, 128.5, 125.5, 87.9, 81.2, 78.5,
78.2, 73.4. HRMS: [M + H]+ calcd for C25H20N5O5, 470.1459; found,
470.1458.
1
buffer solution to afford 30 (0.02 g, 31%) as a white solid. H NMR
(500 MHz, D2O): δ 8.37 (s, 1H, H2), 8.06 (s, 1H, H8), 5.97 (d, J =
3.3 Hz, 1H, H1′), 4.60 (s, 1H, H2′), 4.45 (d, J = 10.0 Hz, 1H, H4a′),
4.07 (d, J = 10.0 Hz, 1H, H4b′), 3.87 (d, J = 10.5 Hz, 2H, PCH2), 3.12
(s, 1H, CH). 13C NMR (125 MHz, D2O): δ 155.3, 152.4, 148.5, 140.5,
3
(1′α,3′S)-1′-(N6-Benzoyladenin-9-yl)-2′-O-benzoyl-3′-ethyn-
yl-3′-O-(diisopropylphosphonomethyl)-L-threose (28f). To a
solution of 28 (0.60 g, 1.28 mmol) and diisopropylphosphonomethyl
trifluoromethanesulfonate (0.42 g, 1.28 mmol) in dry THF was added
NaH (60% in oil, 0.13 g, 3.3 mmol) at −15 °C. The reaction mixture
was stirred at −15 °C for 1 h. The reaction was quenched with sat.
NH4Cl (5 mL) and concentrated. The residue was partitioned
between water and EtOAc. The organic layer was washed with water
and brine, dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by chromatography on a silica gel
column (50:1 CH2Cl2/MeOH) to afford 28f (0.73 g, 88%) as a white
solid. 1H NMR (300 MHz, CDCl3): δ 9.33 (s, 1H, NH), 8.70 (s, 1H,
H2), 8.66 (s, 1H, H8), 8.01 (d, J = 7.1 Hz, 2H, Bz-H), 7.95 (d, J = 7.1
Hz, 2H, Bz-H), 7.55−7.48 (m, 2H, Bz-H), 7.44−7.38 (m, 4H, Bz-H),
6.47 (d, J = 2.1 Hz, 1H, H1′), 5.79 (s, 1H, H2′), 4.77−4.70 (m, 2H,
CH(CH3)2), 4.54 (d, J = 10.5 Hz, 1H, H4a′), 4.17 (d, J = 10.5 Hz, 1H,
H4b′), 4.01−3.96 (m, 2H, PCH2), 2.71 (s, 1H, CH), 1.31−1.26 (m,
12H, CH3). 13C NMR (75 MHz, CDCl3): δ 164.7, 164.6, 152.9, 151.6,
149.6, 142.5, 133.9, 132.6, 130.0, 128.8, 128.6, 127.9, 122.6, 87.9, 82.6
(d, 3JP,C = 15.5 Hz), 80.9, 80.5, 75.4, 74.3, 71.7 (d, 2JP,C = 6.5 Hz), 60.5
118.1, 88.7, 82.5 (d, JP,C = 15.4 Hz), 80.9, 79.2, 74.8, 74.3, 61.2 (d,
1JP,C = 165.7 Hz). 31P NMR (200 MHz, D2O): δ 7.5 (d, J = 24 Hz,
Pα), −7.3 (br s, Pγ), −22.2 (t, J = 21 Hz, Pβ). HRMS: [M − H]− calcd
for C12H16N5O12P3, 513.9935; found, 513.9933.
ASSOCIATED CONTENT
■
S
* Supporting Information
NMR data for compounds 7, 8, 15−21, and 23−30. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: Piet.Herdewijn@rega.kuleuven.be.
Author Contributions
S.G.D. and M.-Y.J. contributed equally.
Notes
1
3
(d, JP,C = 170.8), 24.1 (d, J = 4.2 Hz), 24.0 (d, JP,C = 5.5 Hz). 31P
NMR (125 MHz, CDCl3, 25 °C): δ 17.7. HRMS: [M + H]+ calcd for
C32H35N5O8P1, 648.2218; found, 648.2218.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(1′α,3′S)-1′-(N6-Benzoyladenin-9-yl)-3′-ethynyl-3′-O-(diiso-
propylphosphonomethyl)-L-threose (29). A solution of 28f (0.6 g,
0.93 mmol) in 7 N NH3 in MeOH (5 mL) was stirred at rt for 24 h.
After removing the volatiles, the crude residue was purified by
chromatography on silica gel (20:1 CH2Cl2/MeOH) to afford 29
We thank Richard Mackman (Gilead Sciences) for providing
the antiviral data. We thank Profs. Eveline Lescrinier and Jef
Rozenski for spectrometric analysis and Luc Baudemprez and
Chantal Biernaux for technical assistance. Mass spectrometry
7143
dx.doi.org/10.1021/jo400907g | J. Org. Chem. 2013, 78, 7137−7144

The Journal of Organic Chemistry
Article
was made possible by the support of the Hercules Foundation
of the Flemish Government (grant 20 100 225-7).
REFERENCES
■
(1) Field, H. J.; Vere Hodge, R. A. Antiviral Agents. In Encyclopedia of
Virology, 3rd ed.; Mahy, B. W. J., Van Regenmortel, M. H. V., Eds.;
Academic Press: Boston, MA, 2008, p 142.
(2) Yang, P. L.; Gao, M.; Lin, K.; Liu, Q.; Villareal, V. A. Curr. Opin.
Virol. 2011, 1, 607.
(3) Antonelli, G.; Turriziani, O. Int. J. Antimicrob. Agents 2012, 40, 95.
(4) De Clercq, E. Med. Res. Rev. 2012, 1.
(5) Schoning, K.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy, R.;
Eschenmoser, A. Science 2000, 290, 1347.
(6) Schoning, K. U.; Scholz, P.; Wu, X. L.; Guntha, S.; Delgado, G.;
Krishnamurthy, R.; Eschenmoser, A. Helv. Chim. Acta 2002, 85, 4111.
(7) Ichida, J. K.; Horhota, A.; Zou, K.; McLaughlin, L. W.; Szostak, J.
W. Nucleic Acids Res. 2005, 33, 5219.
(8) Yu, H. Y.; Zhang, S.; Chaput, J. C. Nat. Chem. 2012, 4, 183.
(9) Chaput, J. C.; Ichida, J. K.; Szostak, J. W. J. Am. Chem. Soc. 2003,
125, 856.
(10) Wu, T.; Froeyen, M.; Kempeneers, V.; Pannecouque, C.; Wang,
J.; Busson, R.; De Clercq, E.; Herdewijn, P. J. Am. Chem. Soc. 2005,
127, 5056.
(11) Vina, D.; Wu, T. F.; Renders, M.; Laflamme, G.; Herdewijn, P.
Tetrahedron 2007, 63, 2634.
(12) Huang, Q.; Herdewijn, P. Nucleosides, Nucleotides Nucleic Acids
2009, 28, 337.
(13) Toti, K. S.; Derudas, M.; McGuigan, C.; Balzarini, J.; Van
Calenbergh, S. Eur. J. Med. Chem. 2011, 46, 3704.
(14) Huang, Q.; Herdewijn, P. J. Org. Chem. 2011, 76, 3742.
(15) Mathe, C.; Gosselin, G. Antiviral Res. 2006, 71, 276.
(16) Kodama, E. I.; Kohgo, S.; Kitano, K.; Machida, H.; Gatanaga,
H.; Shigeta, S.; Matsuoka, M.; Ohrui, H.; Mitsuya, H. Antimicrob.
Agents Chemother. 2001, 45, 1539.
(17) Pankiewicz, K. W. Carbohydr. Res. 2000, 327, 87.
(18) Hewish, M.; Martin, S. A.; Elliott, R.; Cunningham, D.; Lord, C.
J.; Ashworth, A. Br. J. Cancer 2013, 108, 983.
(19) Iversen, T.; Bundle, D. R. J. Chem. Soc., Chem. Commun. 1981,
1240.
(20) L’Heureux, A.; Beaulieu, F.; Bennett, C.; Bill, D. R.; Clayton, S.;
Laflamme, F.; Mirmehrabi, M.; Tadayon, S.; Tovell, D.; Couturier, M.
J. Org. Chem. 2010, 75, 3401.
(21) Framski, G.; Gdaniec, Z.; Gdaniec, M.; Boryski, J. Tetrahedron
2006, 62, 10123.
(22) Koh, Y. H.; Shim, J. H.; Wu, J. Z.; Zhong, W. D.; Hong, Z.;
Girardet, J. L. J. Med. Chem. 2005, 48, 2867.
(23) Mackman, R. L.; Zhang, L. J.; Prasad, V.; Boojamra, C. G.;
Douglas, J.; Grant, D.; Hui, H.; Kim, C. U.; Laflamme, G.; Parrish, J.;
Stoycheva, A. D.; Swaminathan, S.; Wang, K. Y.; Cihlar, T. Bioorg. Med.
Chem. 2007, 15, 5519.
(24) Jung, P. M.; Burger, A.; Biellmann, J. F. J. Org. Chem. 1997, 62,
8309.
(25) Zhang, S.; Chaput, J. C. Bioorg. Med. Chem. Lett. 2013, 23, 1447.
7144
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