Total synthesis of a cyclic adenosine 5′-diphosphate ribose receptor agonist

Article abstract of DOI:10.1021/jo202319f

Stable cyclic adenosine 5′-diphosphate ribose (cADPR) analogues are chemical biology tools that can probe the Ca²⁺ release mechanism and structure-activity relationships of this emerging potent second messenger. However, analogues with an intac

Full text of DOI:10.1021/jo202319f

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pubs.acs.org/joc
Total Synthesis of a Cyclic Adenosine 5′-Diphosphate Ribose
Receptor Agonist
Joanna M. Swarbrick and Barry V. L. Potter*
Wolfson Laboratory of Medicinal Chemistry, University of Bath, Department of Pharmacy and Pharmacology, Claverton Down, Bath
BA2 7AY, UK
S
* Supporting Information
ABSTRACT: Stable cyclic adenosine 5′-diphosphate ribose
(cADPR) analogues are chemical biology tools that can probe
the Ca2+ release mechanism and structure−activity relationships
of this emerging potent second messenger. However, analogues
with an intact “northern” ribose have been inaccessible due to
the difficulty of generating the sensitive N1-ribosyl link. We
report the first total synthesis of the membrane permeant,
hydrolytically stable, cADPR receptor agonist 8-Br-N1-cIDPR
via regio- and stereoselective N1-ribosylation of protected 8-
bromoinosine.
derivative by Aplysia californica cyclase and the correct
orientation of substrate within the active site to close the 18-
membered macrocycle. This limitation is demonstrated by
adenine base modifications in NAD+ that generate biologically
inactive, N7-cyclized products.13,14 Total synthetic approaches
have included the preparation of analogues with a carbocyclic
“northern” ribose (cADPcR15 and cIDPcR16,17), replacement of
the “northern” ribose, or both riboses, by an alkyl or ether
bridge (cIDPRE and cIDPDE)18,19 and attaching the “north-
ern” ribose though C-2′′.20 Thus far, all such reported routes
have required considerable modification of the “northern”
ribose. In Jurkat T-cells, replacement of the “northern” furanose
oxygen as in cADPcR or replacement of the entire ribose with
an alkyl or ether bridge, gave considerably weaker ago-
nists.6,18,19,21 These results, and ligand−protein crystal
structures obtained using N1-cIDPR,11 both suggest that the
“northern” ribose provides key interactions with the binding
site. In contrast, more minor changes to the “northern” ribose,
such as modification of a single hydroxyl (e.g., 2′′-NH2-
cADPR6) and changes to the “southern” ribose (e.g., cyclic
aristeromycin diphosphoribose22) both generated analogues
that are equipotent or slightly more active than cADPR and
illustrated key SAR features.6,7 Since the N1-ribose motif is the
locus of both cADPR formation and degradation it is likely that
retaining the complete “northern” ribose motif is crucial for
optimal cADPR analogue activity.
INTRODUCTION
■
Intracellular Ca2+ signaling controls a diverse range of highly
regulated cellular processes, from gene transcription and muscle
contraction to fertilization, cell proliferation and apoptosis.1
Cyclic adenosine 5′-diphosphate ribose (cADPR, 1, Figure 1) is
an emerging principal second messenger,2−5 like the well
characterized IP3, that mobilizes intracellular Ca2+. The
cADPR/Ca2+ signaling system is active in diverse mammalian
cellular systems such as smooth, skeletal and cardiac muscle,
acinar cells, as well as in protozoa and plant cells.5 cADPR is a
cyclic dinucleotide that is produced enzymatically from
nicotinamide adenine dinucleotide (NAD+) by ADP-ribosyl
cyclases (Figure 1). It is readily hydrolyzed at the labile N1 link
to give inactive linear adenosine 5′-diphosphoribose in both
neutral aqueous solution and under physiological conditions.6,7
Therefore, the synthesis of stable analogues is particularly
important. We have previously reported a chemo-enzymatic
route to cyclic inosine 5′-diphosphate ribose (N1-cIDPR, 2) a
chemically and biologically stable cADPR analogue.8 N1-cIDPR
acts as an agonist with equivalent potency to cADPR in
permeablized T-cells. In order to generate this desired N1
analogue using the enzyme Aplysia californica cyclase, N1-
cIDPR had to be accessed via its 8-bromo derivative, 8-Br-
cIDPR 3, later shown to be the first membrane permeant
agonist of the cADPR receptor.9,10
The retention of activity and the chemical and biological
stability afforded by the 6NH2→O substitution make the
cIDPR scaffold a key template for exploring cADPR structure−
activity relationships (SAR) and for structure-based inhibitor
design. Indeed, it has been used in cocrystallization studies with
native human CD38 to explore the mechanism of cADPR
hydrolysis11 and to engineer potent inhibitors in a rational
fashion.12 However, the chemo-enzymatic route used to
prepare these analogues relies on the recognition of an NAD+
A total synthesis that retains an intact “northern” ribose is
synthetically challenging, as it requires regio- and stereospecific
generation of the sensitive N1 glycosidic link. Chemical
ribosylation of inosine is limited to basic conditions, under
which both the N1 and O6 positions are nucleophilic.23 Under
Received: November 9, 2011
Published: January 25, 2012
© 2012 American Chemical Society
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Figure 1. Formation of cADPR 1 by ADP-ribosyl cyclases and the structure of stable analogues cIDPR 2 and 8-Br-cIDPR 3.
Scheme 1. Application of Modified Vorbruggen Glycosylation Conditions to Introduce a “Northern” Ribose
̈
Mitsunobu conditions, ribosylation of inosine gave a 5:1
mixture of two products, favoring the undesired O6 regioisomer
and, under phase transfer conditions, ribosylation of inosine
with 2,3,5-tri-O-benzoyl-1-bromoribose was also reported to
form a mixture of both N1 and O6 regioisomers.24 To address
this unmet need, we report here the first total synthesis of the
cADPR receptor agonist, 8-Br-N1-cIDPR 3, via regio- and
stereoselective N1-ribosylation of a protected inosine derivative.
13% of a ribosylated product, 6 (Scheme 1). During their
studies into the mechanism of glycosylation for the synthesis of
adenosine, Framski et al. reported initial kinetic ribosylation of
N6-protected adenine at N1 under similar conditions.28 We
were encouraged by the observation that, when TMSOTf was
added to deprotonated 5 and 1,2,3,5-tetra-O-acetyl-β-D-
ribofuranose at −78 °C, 6 was formed as a single product
upon warming to rt.
Starting from 2′,3′-O-isopropylidine adenosine 7, an 8-bromo
substituent was introduced using a solution of bromine in
aqueous sodium hydrogen phosphate buffer (Scheme 2).29
Direct bromination of identically protected inosine 5 was
unsuccessful. Subsequent treatment with a large excess of
sodium nitrite afforded 2′,3′-O-isopropylidene-8-bromoinosine,
8. After protection of the 5′-OH as a silyl ether, the fully
protected 8-bromoinosine 9 was subjected to the modified
RESULTS AND DISCUSSION
■
The N1 link of previously reported carbocyclic analogues was
prepared using substitution of N1-2,4-dinitrophenyl purines by
alkylamines.16,17,25 Both a protected N1-2,4-dinitrophenylino-
sine, and the reportedly more reactive N1-nitroinosine, were
prepared. However, neither of these analogues reacted with a
protected ribosylamine. Subsequently, alternative conditions to
introduce an intact ribose were sought from the area of
nucleoside synthesis. Glycosylation of purine bases at N9 to
generate a variety of nucleosides is widely reported, and it was
hypothesized that these conditions could be exploited to effect
a second glycosylation of inosine in the N1-position. Therefore,
we prepared protected inosine 5 by silylation of the 5′-OH,
followed by introduction of an 2′,3′-O-isopropylidene ketal,
Scheme 1.
Vorbruggen conditions described above. This time, a single
̈
product could be isolated in 91% yield (10, Scheme 2).
Scheme 2. Introduction of 8-Br and Glycosylation
The reaction of 5 both with a protected α-chlororibofur-
anose, and under phase transfer glycosylation conditions,26 gave
a complex mixture which included both N1 and O6 products
and unreacted starting material. In all cases, despite utilizing a
wide variety of bases and solvents, both N1 and O6 mixtures
were obtained. Therefore, further optimization was not
attempted, and we sought alternative conditions that would
render only the N1 position nucleophilic. We predicted that
treatment of 5 under modified Vorbruggen conditions27 would
̈
effect deprotonation at N1 and that under silylating conditions
this would generate the O6 silyl ether, therefore favoring N1,
rather than O6, alkylation. However, treatment of protected
inosine 5 with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),
followed by trimethylsilyl triflate (TMSOTf) and 1,2,3,5-
tetra-O-acetyl-β-D-ribofuranose, afforded a maximum of only
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Scheme 3. Introduction of Phosphate Triesters
The regiospecificity of alkylation to generate an N1-
ribosylated product, rather than the unwanted O6-product,
was confirmed by 2D-NMR experiments. HMBC interactions
were observed between the anomeric H-1′′ of the “northern”
ribose and the adenine C-2/C-6, and between the adenine H-2
and “northern” ribose C-1′′. Furthermore, 1D-NOE irradiation
of H-2 excited both H-1′′ and H-2′′. The β-configuration of the
newly formed N1 link at the “northern” ribose anomeric center
was confirmed by the presence of a doublet (J = 4.2 Hz) in the
1H NMR spectrum and an NOE between H-1′′ and H-4′′,
confirming that these two protons lie on the same face of the
ribose ring.
In the chemo-enzymatic route, substitution at purine C-8 was
employed to predispose the linear precursor to cyclize at N1,
rather than N7.8 Introduction of a bulky C-8 group likely
reorientates the purine relative to the “southern” ribose,30 so
that it lies in the syn-conformation prior to interaction with the
active site, and the N1 product is generated. In previous
synthetic routes, cyclization was carried out by promoting
formation of the pyrophosphate bond, and C-8 substitution was
initially employed as it was assumed that a syn-orientation
would facilitate colocalization of the two phosphate groups.16
However, it was later demonstrated that while this substitution
gave slightly improved yields, it was not necessary for successful
formation of the macrocycle.17 We have demonstrated that,
protected 12, and a higher Rf product 12a (see Supporting
Information), that was isolated by column chromatography and
identified as the 2′′,3′′-O-isopropylidene-5′′-O-(2-methoxypro-
pan-2-yl) derivative. In subsequent reactions, this unwanted
side product was converted to 12 by stirring the crude reaction
mixture with methanol containing DOWEX H+ resin for 30
min, to selectively cleave the 5′′-O-hemiacetal before
purification.
The isolated “northern” 5′′-OH could now be used for
introduction of the first phosphate triester. In our hands,
phosphorylation of 12 with di(anilino)phosphorochloridate31
was unreliable. The preparation of the P(V) reagent was low
yielding (maximum ∼15%) and the phosphorylation of 12 did
not go to completion, leaving an inseparable mixture of
unreacted starting material and phosphorylating reagent debris.
However, introduction of a tert-butyl-protected phosphate
triester proceeded in high yield using di-tert-butyl N,N-
diisopropylphosphoramidite and 5-phenyl-1H-tetrazole fol-
lowed by oxidation under basic conditions with Et3N/
H2O2.32 We found that oxidation of the intermediate phosphite
with mCPBA, or purification of the reaction mixture using silica
gel, led to unreliable yields due to partial cleavage of the tert-
butyl phosphate esters (yields from 17 to 49%). However,
when oxidation was carried out using Et3N/H2O2 followed by
purification on basified silica with eluting solvents containing
0.5% pyridine, the yield was significantly improved (94%).
The “southern” ribose 5′-OH was then revealed by treatment
of 13 with TBAF under neutral conditions to afford 14, and a
diphenylphosphorodithioate triester was introduced using
cyclohexylammonium S,S-diphenylphosphorodithioate (PSS)33
with 5-phenyl-1H-tetrazole and 2,4,6-triisopropylbenzenesul-
fonyl chloride (TPS-Cl) as activating agents.34 If TPS-Cl was
used as the sole activating agent, we observed partial
substitution of the 8-bromo substituent by chlorine, which
generated an inseparable mixture of the two 8-substituted
products (the identity of which was confirmed by mass
spectrometry).
when using modified Vorbruggen conditions, substitution at C-
̈
8 alters the reactivity of inosine for an entirely different reason,
unrelated to the conformation of the purine relative to the
“southern” ribose. The 8-bromo substitution appears to
increase the nucleophilicity of the deprotonated purine
sufficiently to attack the acyloxonium ion and generate a
glycosylated product. It has not previously been possible to
prepare a scaffold containing both intact riboses in a selective
manner.
Treatment of 10 with methanolic ammonia effected
simultaneous removal of the three acetyl esters from the
“northern” ribose, and the resulting triol 11 was stirred with
acetone/2,2-dimethoxypropane (4:1 v/v) in the presence of p-
TsOH, Scheme 3. Surprisingly, initially this generated two
distinct products; the desired 2′′,3′′-O-isopropylidene ketal
The phosphate and phosphorodithioate esters of 15 were
sequentially deprotected (Scheme 4). Treatment at 0 °C with
50% aqueous TFA for 4 h effected simultaneous deprotection
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Scheme 4. Deprotection and Intramolecular Cyclization
EXPERIMENTAL SECTION
■
General Experimental Procedures. All reagents and solvents
were of commercial quality and were used without further purification,
unless described otherwise. Unless otherwise stated, all reactions were
carried out under an inert atmosphere of argon. H and 13C chemical
1
1
shifts (δ) were internally referenced to the residual solvent peak. H
and 13C NMR assignments are based on gCOSY, gHMBC, gHSQC,
and DEPT-135 experiments. Abbreviations for splitting patterns are as
follows: br, broad; s, singlet; d, doublet; t, triplet; m, multiplet etc.
Coupling constants are given in hertz (Hz). Synthetic phosphates were
assayed and quantified by the Ames phosphate test.36
5′-O-(tert-Butyldiphenylsilyl)inosine. Triethylamine (518 μL, 3.72
mmol) and TBDPS-Cl (1.06 mL, 4.09 mmol) were added to inosine
(500 mg, 1.86 mmol) in DMF (10 mL). After 30 h of stirring at rt, all
solvents were evaporated. The crude material was purified by column
chromatography on silica gel eluting with DCM/MeOH (9:1 → 4:1 v/
v) to afford the title compound (669 mg, 71%): Rf = 0.33 (DCM/
MeOH 9:1 v/v); 1H NMR (270 MHz, DMSO-d6) δ 8.22 (s, 1H), 8.00
(s, 1H), 7.60 (dd, 4H, J = 7.7, 1.4), 7.39−7.34 (m, 6H) (10 × Ar−H),
5.91 (d, 1H, J = 4.7, H-1′), 5.62 (d, 1H, J = 5.8, −OH, ex), 5.29 (d, 1H,
J = 5.5, −OH, ex), 4.54 (q, 1H, J = 5.0, ex → t, H-2′), 4.30 (q, 1H, J =
4.9, ex → t, H-3′), 4.05−4.02 (m, 1H), 3.89 (dd, 1H, J = 11.3, 3.4, H-
t
5′a), 3.79 (dd, 1H, J = 11.3, 4.7, H-5′b), 0.94 (s, 9H, Bu) ppm.
5′-O-(tert-Butyldiphenylsilyl)-2′,3′-O-isopropylideneinosine (5). p-
TsOH (28 mg, 0.148 mmol) was added to 5′-O-(tert-
butyldiphenylsilyl)inosine (75 mg, 0.148 mmol) in acetone/2,2-
dimethoxypropane (4:1 v/v, 5 mL). After 30 min of stirring at rt, all
solvents were removed under reduced pressure, and the resulting
material was purified by column chromatography on silica gel eluting
with DCM/MeOH (1:0 → 4:1 v/v) to afford the title compound (74
mg, 91%) as a white solid: Rf = 0.85 (DCM/MeOH 9:1 v/v); mp
of the tert-butyl phosphate esters and both isopropylidene
ketals to reveal the 5′′-O-phosphomonoester. At higher
temperatures, or with longer reaction times, we observed
partial cleavage of the N9 glycosidic bond.35 Notably, the
inosine N1-glycosidic bond was stable to this and all other
chemical transformations that were required during the
synthesis. Selective basic hydrolysis of one thiophenol group
has reportedly been carried out using H3PO2.34 However, we
found this reaction difficult to monitor, and the removal of
excess reagent during purification was complex.
1
256−258 °C; H NMR (500 MHz, CDCl3) δ 13.17 (bs, 1H, NH),
8.19 (s, 1H, H-8), 8.07 (s, 1H, H-2), 7.59−7.55 (m, 4H), 7.38−7.28
(m, 6H), 6.10 (d, 1H, J = 2.4, H-1′), 5.20 (dd, 1H, J = 6.1, 2.4, H-2′),
4.87 (dd, 1H, J = 6.1, 2.8, H-3′), 4.40 (ddd, 1H, J = 5.2, 4.1, 2.8, H-4′),
3.88 (dd, 1H, J = 11.5, 4.1, H-5′a), 3.78 (dd, 1H, J = 11.5, 5.2, H-5′b),
t
1.60 (s, 3H, CH3), 1.35 (s, 3H, CH3), 0.99 (s, 9H, Bu) ppm; 13C
NMR (100 MHz, CDCl3) 159.4, 148.4, 145.2, 139.0, 135.6 (2C),
135.5 (2C), 132.8, 132.7, 130.0 (2C), 127.83 (2C), 127.78 (2C),
125.4, 114.4, 91.3, 87.1, 84.8, 81.3, 64.0, 27.2, 26.9 (3C), 25.4, 19.2
ppm; HRMS (ESI+) found m/z [M + H]+ 547.2354, C29H35N4O5Si
requires 547.2371.
We sought alternative conditions using a phosphorus-free
base, so that the progression of the reaction could be
monitored by 31P NMR. Treatment of 16 with 50% 0.1 M
NaOH in dioxane effected rapid (15−30 min) selective
deprotection to give 17 (Scheme 4). The resulting substrate
for intramolecular cyclization did not require any further
purification. After neutralizing the solution with 0.1 M HCl, the
resulting sodium salt of 17 was converted to the triethylammo-
nium salt. Intramolecular cyclization of 17 was carried out by a
modified Hata condensation. A dilute pyridine solution of 17
added over 15 h by syringe pump to a solution of iodine and
molecular sieves17 to promote metaphosphate formation,
followed by intramolecular cyclization to generate the
pyrophosphate linkage of the target 8-Br-cIDPR. Synthetic 8-
N1-(2′′,3′′,5′′-Tri-O-acetyl-β-D-ribofuranosyl)-5′-O-(tert-butyldi-
phenylsilyl)-2′,3′-O-isopropylideneinosine (6). 5′-O-TBDPS-2′,3′-O-
isopropylideneinosine (5, 200 mg, 0.366 mmol) was taken up in
MeCN (1.0 mL) and DBU (164 μL, 1.098 mmol) added. After 30
min, 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (128 mg, 0.402 mmol)
was added and the solution cooled to −78 °C. Trimethylsilyl
trifluoromethanesulfonate (136 μL, 1.464 mmol) was added dropwise
and the solution stirred for a further 45 min before warming to rt.
After 1 h, NaHCO3 (satd aq) was added and the crude material
extracted into DCM (×3). The combined organic fractions were dried
(Na2SO4), and solvent was evaporated under reduced pressure. The
residue was purified by column chromatography on silica gel eluting
with DCM/acetone (1:0 → 0:1 v/v) to afford the title compound (41
1
Br-cIDPR was identical (by H, 13C, 31P NMR and HPLC) to
that prepared by our previous chemo-enzymatic route.8,9
In summary, the potent, chemically and biologically stable
membrane permeant cADPR receptor agonist 8-Br-cIDPR has
been synthesized using a novel total synthetic route. The key
feature of this route is the early introduction of an 8-bromo
substituent to promote entirely regio- and stereoselective N1-
glycosylation with an intact “northern” ribose. The resultant
N1-ribosyl inosine can be sequentially phosphorylated and the
product cyclized after appropriate deprotection using the
phosphorodithioate method. This procedure is amenable to
nucleosides with modifications to the “southern” ribose and will
facilitate generation of analogues of cADPR for SAR studies
with an intact “northern” ribose that have previously been
inaccessible by the chemo-enzymatic approach.
1
mg, 13%) as a colorless glass: Rf = 0.74 (DCM:Acetone 3:1 v/v); H
NMR (400 MHz, CDCl3) δ 8.13 (s, 1H, H-8), 7.96 (s, 1H, H-2), 7.61
(dd, 2H, J = 8.0, 1.5), 7.59 (dd, 2H, J = 8.0, 1.5), 7.42−7.30 (m, 6H),
6.37 (d, 1H, J = 4.2, H-1′′), 6.09 (d, 1H, J = 2.8, H-1′), 5.45−5.43 (m,
2H), 5.07 (dd, 1H, J = 6.3, 2.9), 4.86 (dd, 1H, J = 6.3, 3.1), 4.40−4.36
(m, 4H), 3.88 (dd, 1H, J = 11.0, 4.0, H-5′a), 3.82 (dd, 1H, J = 11.0, 4.9,
H-5′b), 2.13, 2.12, 2.09 (each s, 3H, 3 × OAc), 1.61 (s, 3H, CH3), 1.36
(s, 3H, CH3), 1.03 (s, 9H, tBu) ppm; HRMS (ESI+) found m/z [M +
Na]+ 827.2917, C40H48N4NaO12Si requires 827.2930.
2′,3′-O-Isopropylidene-8-bromoadenosine.37 Na2HPO3 (30 g)
was dissolved in Milli-Q (300 mL). The resulting solution was
covered in foil and bromine (0.6 mL) added. In a separate flask, 2′,3′-
O-isopropylidene-adenosine (7, 3.05 g) was taken up in dioxane (300
mL) and covered in foil. The bromine solution was decanted into this
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solution, and the resulting solution stirred in the dark for 16 h. The
solution was thoroughly extracted with CHCl3, and the combined
organic layers washed with NaHSO3 (satd aq), then H2O, dried
(MgSO4) and evaporated to dryness. The residue was crystallized from
83.3, 81.4, 80.3, 74.3, 70.1, 64.1, 63.0, 27.3, 26.7 (3C), 25.5, 20.6, 20.5,
20.4, 19.2 ppm; HRMS (ESI+) found m/z [M + H]+ 883.2190,
885.2178, C40H48N4O1279BrSi requires 883.2216, C40H48N4O1281BrSi
requires 885.2195.
1
EtOH to yield the title compound (3.19 g, 83%) as needles; H (400
N1-(β-D-Ribofuranosyl)-5′-O-(tert-butyldiphenylsilyl)-2′,3′-O-iso-
propylidene-8-bromoinosine (11). N1-(2,3,5-Tri-O-acetyl-β-D-ribo-
furanosyl)-2′,3′-O-isopropylidene-5′-O-TBDPS-8-bromoinosine (10,
500 mg, 0.57 mmol) was taken up in MeOH (5 mL) in a pressure
tube. The solution was saturated with NH3 (g) at 0 °C and then
stirred at rt for 12 h. The solvent was evaporated and the residue
purified by column chromatography on silica gel eluting with PE/
EtOAc (1:0 → 0:1 v/v) to afford the title compound (349 mg, 81%)
MHz, CDCl3) δ 8.26 (s, 1H, H-2), 6.36 (d, 1H, J = 10.8, 5′−OH), 6.10
(d, 1H, J = 5.3, H-1′), 6.01 (bs, 2H, NH2), 5.28 (dd, 1H, J = 5.7, 5.3,
H-2′), 5.08 (dd, 1H, J = 5.7, 1.1, H-3′), 4.53 (d, 1H, J = 1.1, H-4′), 3.97
(d, 1H, J = 13.1, H-5′a), 3.78 (dd, 1H, J = 13.1, 10.8, H-5′b), 1.67 (s,
3H, CH3), 1.38 (s, 3H, CH3) ppm; HRMS (ESI+) found m/z [M +
H]+ 386.0450, 388.0433; C13H17N5O479Br requires 386.0458,
C13H17N5O481Br requires 388.0438.
2′,3′-O-Isopropylidene-8-bromoinosine16 (8). 2′,3′-O-Isopropyli-
dene-8-bromoadenosine (2.00 g, 5.18 mmol) was taken up in acetic
acid−water (57.5 mL, 20:3 v/v). NaNO2 (4.29 g, 62.14 mmol) was
added in one portion and the resulting solution stirred for 16 h. All
solvents were evaporated and the residue taken up in EtOH and
evaporated. The residue was partitioned between CHCl3 and H2O and
the organic layer washed with NaHCO3 (satd aq) and then brine,
dried (MgSO4), and evaporated to dryness. The residue was
crystallized from aqueous EtOH to yield the title compound (1.66
1
as a white amorphous solid: Rf = 0.24 (PE:EtOAc 1:3 v/v); H NMR
(400 MHz, CDCl3) δ 8.06 (s, 1H, H-2), 7.61 (dd, 2H, J = 8.0, 1.4),
7.53 (dd, 2H, J = 8.0, 1.4), 7.41−7.31 (m, 4H), 7.23 (t, 2H, J = 7.4),
6.12 (d, 1H, J = 2.3, H-1′), 5.79 (d, 1H, J = 4.6, H-1′′), 5.43 (dd, 1H, J
= 6.4, 2.3, H-2′), 5.00 (dd, 1H, J = 6.4, 3.9, H-3′), 4.49 (t, 1H, J = 4.6,
H-2′′), 4.36−4.32 (m, 2H, H-3′′ and H-4′), 4.25−4.22 (m, 1H, H-4′′),
3.91−3.85 (m, 2H, H-5′a and H-5′′a), 3.79−3.73 (m, 2H, H-5′b and H-
5′′b), 1.60 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.00 (s, 9H, tBu) ppm; 13
C
NMR (100 MHz, CDCl3) δ 155.9, 148.4, 145.3, 135.6 (2C), 135.5
(2C), 133.5, 132.9, 129.8 (2C), 127.7 (2C), 127.5 (2C), 126.7, 124.8,
114.6, 94.2 91.3, 87.7, 86.4, 83.3, 81.6, 74.7, 70.7, 64.0, 62.0, 27.3, 26.8,
25.5, 19.2 ppm; HRMS (ESI+) found m/z [M + H]+ 757.1877 and
759.1864, C34H42N4O979BrSi requires 757.1899, C34H42N4O981BrSi
requires 759.1878.
1
g, 83%) as crystals: H NMR (400 MHz, CDCl3) δ 12.99 (bs, 1H,
NH), 8.34 (s, 1H, H-2), 6.10 (d, 1H, J = 5.5, H-1′), 5.24 (t, 1H, J = 5.5,
H-2′), 5.06 (dd, 1H, J = 5.5, 1.9, H-3′), 4.48 (dd, 1H, J = 1.9, 1.6, H-
4′), 3.94 (dd, 1H, J = 12.1, 1.6, H-5′a), 3.79 (d, 1H, J = 12.1, H-5′b),
1.66 (s, 3H, CH3), 1.38 (s, 3H, CH3) ppm; HRMS (ESI+) found m/z
[M + H]+ 387.0311, 389.0289, C13H16N4O579Br requires 387.0299,
C13H16N4O581Br requires 389.0278.
N1-(2′′,3′′-O-Isopropylidene-β-D-ribofuranosyl)-5′-O-(tert-butyl-
diphenylsilyl)-2′,3′-O-isopropylidene-8-bromoinosine (12). p-TsOH
(55 mg, 0.29 mmol) was added to N1-(β-D-ribofuranosyl)-5′-O-
TBDPS-2′,3′-O-isopropylidene-8-bromoinosine (11, 220 mg, 0.29
mmol) in acetone-2,2-dimethoxypropane (10 mL, 4:1 v/v). After 30
min, DCM and NaHCO3 (satd aq) were added, and the organic layer
was dried (Na2SO4) and evaporated. The residue was taken up in
MeOH (5 mL) and DOWEX H+ resin (50 mg) added to convert any
unwanted 5′′-O-hemiacetal side product into 12. After 30 min, the
resin was removed by filtration under gravity and the solvent
evaporated to obtain the title compound (229 mg, 99%) as a white
amorphous solid: Rf = 0.74 (PE/EtOAc 1:3 v/v); 1H NMR (400 MHz,
CDCl3) δ 7.61 (dd, 2H, J = 7.9, 1.3), 7.57 (s, 1H, H-2), 7.53 (dd, 2H, J
= 7.9, 1.3), 7.43−7.32 (m, 4H), 7.23 (t, 2H, J = 7.7), 6.12 (d, 1H, J =
2.2, H-1′), 5.61 (d, 1H, J = 2.9, H-1′′), 5.41 (dd, 1H, J = 6.4, 2.2, H-2′),
5.15 (dd, 1H, J = 6.4, 3.0, H-2′′), 5.07 (dd, 1H, J = 6.4, 3.4, H-3′′), 5.02
(dd, 1H, J = 6.4, 3.9, H-3′), 4.36−4.33 (m, 2H, H-4′ and H-4′′), 3.91
(dd, 1H, J = 12.3, 2.5, H-5′′a), 3.85 (dd, 1H, J = 10.9, 5.6, H-5′a), 3.79
(dd, 1H, J = 12.3, 3.6, H-5′′b), 3.76 (dd, 1H, J = 10.9, 6.4, H-5′b), 1.61
(s, 3H, CH3), 1.59 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.35 (s, 3H,
5′-O-(tert-Butyldiphenylsilyl)-2′,3′-O-isopropylidene-8-bromoino-
sine38 (9). Imidazole (228 mg, 3.36 mmol) and TBDPSCl (435 μL,
1.68 mmol) were added to a solution of 2′,3′-O-isopropylidene-8-
bromoinosine (8, 500 mg, 1.29 mmol) in DMF (10 mL). After 16 h,
EtOAc (100 mL) and H2O (50 mL) were added. The organic layer
was washed with brine, dried (MgSO4), and evaporated to dryness.
The residue was purified by column chromatography on silica gel
eluting with DCM/MeOH (1:0 → 1:19 v/v) to afford the title
compound (808 mg, 98%) as a white foam: Rf = 0.39 (PE/EtOAc, 1:3
v/v); 1H (400 MHz, CDCl3) δ 13.08 (bs, 1H, NH), 7.91 (s, 1H, H-2),
7.59 (dd, 2H, J = 8.0, 1.4), 7.54 (dd, 2H, J = 8.0, 1.4), 7.40−7.31 (m,
4H), 7.26 (t, 2H, J = 7.3), 6.18 (d, 1H, J = 2.1, H-1′), 5.57 (dd, 1H, J =
6.4, 2.1, H-2′), 5.10 (dd, 1H, J = 6.4, 3.7, H-3′), 4.39 (ddd, 1H, J = 6.5,
5.6, 3.7, H-4′), 3.82 (dd, 1H, J = 11.5, 5.6, H-5′a), 3.74 (dd, 1H, J =
11.5, 6.5, H-5′b), 1.63 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.02 (s, 9H,
tBu) ppm; HRMS (ESI+) found m/z [M + H]+ 625.1447, 627.1431,
C29H34N4O5Si79Br requires 625.1482, C29H34N4O5Si81Br requires
627.1461.
t
N1-(2′′,3′′,5′′-Tri-O-acetyl-β-D-ribofuranosyl)-5′-O-(tert-butyldi-
phenylsilyl)-2′,3′-O-isopropylidene-8-bromoinosine (10). 5′-O-
TBDPS-2′,3′-O-isopropylidene-8-bromoinosine (9, 160 mg, 0.255
mmol) was taken up in DCM (1.6 mL) and DBU (114 μL, 0.765
mmol) added. After 30 min, 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose
(89 mg, 0.281 mmol) was added and the solution cooled to −78 °C.
Trimethylsilyl trifluoromethanesulfonate (185 μL, 1.020 mmol) was
added dropwise and the solution stirred for a further 45 min before
warming to rt. After 1 h, NaHCO3 (satd aq) was added and the crude
material extracted into DCM (×3). The combined organic fractions
were dried (Na2SO4), and solvent was evaporated under reduced
pressure. The residue was purified by column chromatography on
silica gel eluting with PE/EtOAc (1:0 → 0:1 v/v) to afford the title
compound (203 mg, 90%) as a colorless glass: Rf = 0.85 (DCM/
CH3), 1.01 (s, 9H, Bu) ppm; 13C NMR (100 MHz, CDCl3) δ 155.1,
148.1, 146.2, 135.6 (2C), 135.2 (2C), 133.6, 132.8, 129.8, 129.7, 127.7
(2C), 127.5 (2C), 126.8, 125.5, 114.5, 114.3, 96.9, 91.3, 87.87, 87.86,
83.39, 83.36, 81.5, 80.6, 63.9, 62.9, 27.4, 27.3, 26.8, 25.5, 25.3, 19.2
ppm; HRMS (ESI+) found m/z [M + H]+ 797.2186 and 799.2192,
and [M + Na]+ 819.1975 and 821.1995, C37H46N4O979BrSi requires
797.2212, C37H46N4O981BrSi requires 799.2192, C37H45N4O979BrSiNa
requires 819.2031, C37H45N4O981BrSiNa requires 821.2011.
N1-[2′′,3′′-O-Isopropylidene-5′′-O-(di-tert-butyl)phosphoryl-β-D-
ribofuranosyl]-5′-O-(tert-butyldiphenylsilyl)-2′,3′-O-isopropylidene-
8-bromoinosine (13). 5-Phenyl-1H-tetrazole (103 mg, 0.70 mmol)
and N,N-diisopropyldibutylphosphoramidite (166 μL, 0.53 mmol)
were added to a solution of N1-(2′′,3′′-O-isopropylidene-β-D-
ribofuranosyl)-5′-O-TBDPS-2′,3′-O-isopropylidene-8-bromoinosine
(12, 280 mg, 0.35 mmol) in DCM (2 mL). After 2 h, the solution was
cooled to 0 °C, and Et3N (367 μL, 2.63 mmol) and H2O2 (96 μL, 1.09
mmol) were added. The solution was allowed to warm to rt and stirred
for a further 2 h, after which time DCM (20 mL) and H2O were
added. The organic layer was washed with NaHCO3 (satd aq) and
then brine, dried (Na2SO4), and evaporated to dryness. The residue
was purified by column chromatography on silica gel eluting with PE/
EtOAc (1:0 → 0:1 v/v), where both solvents contained 0.5% v/v
pyridine, to afford the title compound (323 mg, 94%) as a colorless
glass: Rf = 0.41 (PE/EtOAc 1:3 v/v); 1H NMR (400 MHz, CDCl3) δ
1
acetone 3:1 v/v); H NMR (400 MHz, CDCl3) δ 7.90 (s, 1H, H-2),
7.61 (dd, 2H, J = 8.0, 1.4), 7.56 (dd, 2H, J = 8.0, 1.4), 7.42−7.33 (m,
4H), 7.31−7.26 (m, 2H), 6.20 (d, 1H, J = 4.2, H-1′′), 6.17 (d, 1H, J =
2.4, H-1′), 5.45−5.41 (m, 3H, H-2′, H-2′′ and H-3′′), 5.00 (dd, 1H, J =
6.4, 4.3, H-3′), 4.43−4.38 (m, 3H, H-4′′, 2 × H-5′′), 4.36−4.33 (m, 1H,
H-4′), 3.90 (dd, 1H, J = 11.0, 5.2, H-5′a), 3.82 (dd, 1H, J = 11.0, 6.6,
H-5′b), 2.15, 2.12, 2.04 (each s, 3H, 3 × OAc), 1.64 (s, 3H, CH3), 1.40
t
(s, 3H, CH3), 1.04 (s, 9H, Bu) ppm; 13C NMR (100 MHz, CDCl3)
170.2, 169.6, 169.4, 154.5, 147.8, 144.3, 135.6, 133.3, 132.9, 129.77,
129.76, 127.7 (2C), 127.5 (2C), 126.2, 124.9, 114.7, 90.9, 88.6, 87.5,
4195
dx.doi.org/10.1021/jo202319f | J. Org. Chem. 2012, 77, 4191−4197

The Journal of Organic Chemistry
Featured Article
7.63 (s, 1H, H-2), 7.60 (dd, 2H, J = 8.0, 1.4), 7.53 (dd, 2H, J = 8.0,
1.4), 7.41−7.31 (m, 4H), 7.23 (t, 2H, J = 7.9), 6.12 (d, 1H, J = 2.2, H-
1′), 5.89 (d, 1H, J = 1.8, H-1′′), 5.41 (dd, 1H, J = 6.4, 2.2, H-2′), 4.99
(dd, 1H, J = 6.4, 4.1, H-3′), 4.97−4.92 (m, 2H, H-2′′ and H-3′′), 4.39
(ddd, 1H, J = 5.8, 4.6, 4.2, H-4′′), 4.33 (ddd, 1H, J = 6.6, 5.4, 4.1, H-4′),
4.24 (ddd, 1H, J = 11.1, 7.0, 4.6, H-5′′a), 4.15 (ddd, 1H, J = 11.1, 7.5,
5.8, H-5′′b), 3.84 (dd, 1H, J = 10.9, 5.4, H-5′a), 3.74 (dd, 1H, J = 10.9,
154.4, 147.9, 146.3, 135.3 (d, 2C, J = 5.2), 135.2 (d, 2C, J = 5.2),
129.69, 129.67, 129.4−129.3 (m, 4C), 126.2, 125.6 (d, J = 6.7), 125.5
(d, J = 6.7), 125.2, 114.8, 114.5, 93.6, 91.0, 86.5 (d, J = 7.8), 85.3 (d, J
= 8.4), 84.9, 83.4, 83.1 (d, J = 7.4), 83.0 (d, J = 7.4), 81.2, 80.9, 66.3 (d,
J = 6.0), 66.1 (d, J = 8.2), 29.81 (3C), 29.77 (3C), 27.2, 27.1, 25.3,
25.2 ppm; 31P NMR (162 MHz, 1H decoupled, CDCl3) δ 50.5, −10.4
ppm; HRMS (ESI+) found m/z [M + Na]+1037.1563 and 1039.1539,
C41H53N4O1379BrNaP2S2 requires 1037.1601, C41H53N4O1381BrNaP2S2
requires 1039.1581.
t
6.6, H-5′b), 1.61 (s, 3H, CH3), 1.59 (s, 3H, CH3), 1.45 (s, 9H, Bu),
t
1.44 (s, 9H, Bu), 1.36 (s, 3H, CH3), 1.34 (s, 3H, CH3), 1.01 (s, 9H,
tBu) ppm; 13C NMR (100 MHz, CDCl3) δ 154.4, 147.8, 145.7, 135.5
(2C), 135.4 (2C), 133.4, 132.8, 129.74, 129.67, 127.6 (2C), 127.5
(2C), 126.2, 125.2, 114.47, 114.46, 93.7, 91.0, 87.7, 86.5 (d, J = 7.8),
84.7, 83.5, 82.56 (d, J = 7.2), 82.48 (d, J = 7.2), 81.5 (2C), 66.3 (d, J =
6.2), 64.0, 29.8 (3C), 29.7 (3C), 27.2, 27.1, 26.7 (3C), 25.4, 25.3, 19.1
N1-[5′′-O-Phosphoryl-β-D-ribofuranosyl]-5′-O-[(diphenylthio)-
phosphoryl]-8-bromoinosine (16). N1-(2′′,3′′-O-Isopropylidene-(di-
tert-butyl)phosphoryl-β-D-ribofuranosyl)-5′-O[(diphenylthio) phos-
phoryl]-2′,3′-O-isopropylidene-8-bromoinosine (15, 150 mg, 0.059
mmol) was stirred in 50% TFA (2 mL) at 0 °C for 4 h. All solvents
were evaporated, and the residue was coevaporated with MeOH (×4).
The residue was purified by column chromatography on silica gel
eluting with EtOAc/MeOH/H2O (1:0:0 → 4:2:0 → 7:2:1 v/v/v) to
afford the title compound (100 mg, 82%) as a colorless glass: Rf = 0.28
1
ppm; 31P NMR (162 MHz, H decoupled, CDCl3) δ −10.1 ppm;
HRMS (ESI+) found m/z [M + Na]+ 1011.2914 and 1013.2935,
C45H62N4O1279BrSiNaP requires 1011.2947, C45H62N4O1281BrSiNaP
requires 1013.2926.
1
(EtOAc/MeOH/H2O 7:2:1 v/v/v); H NMR (500 MHz, MeOD-d4)
N1-[2′′,3′′-O-Isopropylidene-5′′-O-(di-tert-butyl)-phosphoryl-β-D-
ribofuranosyl]-2′,3′-O-isopropylidene-8-bromoinosine (14). Acetic
acid (29 μL, 0.51 mmol) and TBAF·3H2O (153 mg, 0.49 mmol) were
stirred in DMF (1 mL) for 30 min, after which the solution was cooled
to 0 °C and N1-(2′′,3′′-O-isopropylidene-5′′-O-(di-tert-butyl)-
phosphoryl-β-D-ribofuranosyl)-5′-O-TBDPS-2′,3′-O-isopropylidene-8-
bromoinosine (13, 160 mg, 0.16 mmol) in DMF (1.5 mL) added. The
resulting solution was allowed to warm to rt and stirred for a further 4
h. The solution was diluted with ether, and NaHCO3 (satd aq) and
NH4Cl (satd aq) were added. The organic layer was separated and the
aqueous layer extracted with ether (×3). The combined organic layers
were dried (Na2SO4) and evaporated to dryness, and the residue was
purified by column chromatography on silica gel eluting with DCM/
acetone (1:0 → 0:1 v/v) to afford the title compound (96 mg, 79%) as
δ 8.58 (s, 1H, H-2), 7.44−7.39 (m, 6H), 7.36−7.32 (m, 4H) (10 ×
Ar−H), 6.33 (d, 1H, J = 3.0, H-1′), 6.24 (d, 1H, J = 1.5, H-1′′), 5.72
(dd, 1H, J = 6.5, 1.5, H-2′), 5.21 (dd, 1H, J = 6.0, 3.0, H-3′), 4.95 (dd,
1H, J = 6.5, 3.0, H-3′′), 4.90 (dd, 1H, J = 6.5, 3.0, H-2′′), 4.45−4.37 (m,
4H), 4.15−4.08 (m, 2H) (H-4′, H-4′′, both H-5′ and H-5′′), 1.60 (s,
3H, CH3), 1.56 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.27 (s, 3H, CH3)
ppm; 13C NMR (125 MHz, MeOD-d4) δ 156.6, 149.5, 147.8, 136.6 (d,
2C, J = 5.4), 136.4 (d, 2C, J = 5.0), 131.1 (d, J = 2.9), 130.9 (d, J =
2.9), 130.6 (d, 4C, J = 2.3), 130.1 (d, J = 10.5), 126.7, 126.6 (d, J =
5.9), 125.5, 115.6, 115.4, 93.1, 92.0, 87.8 (d, J = 8.1), 86.9 (d, J = 8.3),
86.6, 85.0, 82.5, 82.4, 68.7 (d, J = 9.3), 65.8 (d, J = 5.3), 27.6, 27.4,
1
25.59, 25.56 ppm; 31P NMR (202 MHz, H decoupled, d4-MeOD) δ
51.7, 1.0 ppm; HRMS (ESI−) found m/z [M − H]− 901.0389 and
903.0376, C3 3 H3 6 N4 O1 3 7 9 BrP2 S2 requires 901.0384,
C33H36N4O1381BrP2S2 requires 903.0364.
1
a colorless glass: Rf = 0.73 (DCM/acetone 1:1 v/v); H NMR (400
MHz, CDCl3) δ 8.15 (s, 1H, H-2), 6.07 (d, 1H, J = 4.3, H-1′), 5.97 (d,
1H, J = 1.8, H-1′′), 5.23 (dd, 1H, J = 6.0, 4.3, H-2′), 5.05 (dd, 1H, J =
6.0, 2.1, H-3′), 5.02 (dd, 1H, J = 6.4, 1.8, H-2′′), 4.92 (dd, 1H, J = 6.4,
3.9, H-3′′), 4.43−4.38 (m, 2H, H-4′ and H-4′′), 4.21 (ddd, 1H, J = 11.2,
6.5, 4.2, H-5′′a), 4.12 (ddd, 1H, J = 11.2, 7.2, 5.4, H-5′′b), 3.81 (dd, 1H,
J = 12.2, 3.3, H-5′a), 3.70−3.68 (m, 1H, H-5′b), 1.61 (s, 3H, CH3),
N1-(5′′-O-Phosphoryl-β-D-ribofuranosyl)-5′-O-(phenylthio)-
phosphoryl-8-bromoinosine (17). N1-(5′′-O-Phosphate-β-D-ribofura-
nosyl)-5′-O-(diphenylthio)phosphoryl-8-bromoinosine (16, 20 mg,
0.024 mmol) was taken up in dioxane/H2O (1 mL, 1:1 v/v).
NaOH (100 μL, 1 M) was added and the solution stirred for 30 min at
rt before addition of HCl (100 μL, 1 M). The solution was diluted
with H2O and washed with hexane (×3) before evaporation of all
solvents to give a colorless glass which was converted to the TEA salt
t
t
1.55 (s, 3H, CH3), 1.45 (s, 9H, Bu), 1.42 (s, 9H, Bu), 1.34 (s, 3H,
CH3), 1.32 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3) δ 154.4,
147.7, 145.8, 126.1, 125.7, 114.3, 114.2, 94.4, 92.9, 86.9 (d, J = 7.9),
86.3, 85.1, 83.0, 82.83 (d, J = 8.7), 82.78 (d, J = 8.7), 81.4, 81.1, 66.3
(d, J = 6.3), 62.6, 29.8 (d, 3C, J = 4.2), 29.7 (d, 3C, J = 4.2), 27.4, 27.0,
25.3, 25.2 ppm; 31P NMR (162 MHz, 1H decoupled, CDCl3) δ −10.7
ppm; HRMS (ESI+) found m/z [M + Na]+ 773.1736 and 775.1777,
C29H44N4O1279BrNaP requires 773.1769, C29H44N4O1281BrNaP re-
quires 775.1748.
1
as described below: H NMR (500 MHz, D2O) δ 8.51 (s, 1H, H-2),
7.14 (d, 2H, J = 7.6), 7.09 (t, 1H, J = 7.6), 6.97 (t, 2H, J = 7.6) (5 ×
Ar−H), 6.10 (d, 1H, J = 3.4, H-1′′), 6.05 (d, 1H, J = 5.5, H-1′), 5.59 (t,
1H, J = 5.5, H-2′), 4.57 (t, 1H, J = 3.8, H-3′), 4.31−4.01 (m, 8H) ppm;
13C NMR (125 MHz, D2O) δ 156.1, 148.8, 145.0, 132.1 (d, 2C, J =
5.3), 129.5 (d, J = 5.2), 128.7, 128.6 (2C), 127.6, 123.7, 90.5, 88.7,
84.2 (d, J = 10.7), 83.1 (d, J = 8.6), 74.7, 70.9, 70.2, 69.2, 65.7 (d, J =
5.7, 63.2 (d, J = 3.9) ppm; 31P NMR (202 MHz, D2O, 1H-decoupled)
δ 17.3, 3.3 ppm; HRMS (ESI−) calcd for C21H24N4O14P2S79Br
N1-[2′′,3′′-O-Isopropylidene-5′′-O-(di-tert-butyl)phosphoryl-β-D-
ribofuranosyl]-5′-O-[(diphenylthio)phosphoryl]- 2′,3′-O-isopropyli-
dene-8-bromoinosine (15). N1-(2′′,3′′-O-Isopropylidene-5′′-O-(di-
tert-butyl)phosphoryl-β-D-ribofuranosyl)-2′,3′-O-isopropylidene-8-bro-
moinosine (14, 80 mg, 0.11 mmol) was evaporated from pyridine (3 ×
1 mL) and taken up in pyridine (1.5 mL). This solution was added to
PSS (122 mg, 0.32 mmol), which had also been evaporated from
pyridine (3 × 1 mL). 5-Phenyl-1H-tetrazole (47 mg, 0.32 mmol) and
TPS-Cl (64 mg, 0.21 mmol) were added, and the solution was stirred
at rt for 5 h. DCM and H2O were added, the organic layer was
separated, and the aqueous layer was washed with DCM (×2). The
combined organic layer was washed with brine, dried (Na2SO4), and
evaporated to dryness. The residue was purified by column
chromatography on silica gel eluting with PE:EtOAc (1:0 → 0:1 v/
v) to afford the title compound (108 mg, 100%) as a white foam: Rf =
728.9674 [(M
−
H)− ], found 728.9663; calcd for
C21H24N4O14P2S81Br 730.9653 [(M − H)−], found 730.9640.
Conversion to TEA salt: The Na+ salt was passed through prewashed
DOWEX H+ resin. Acidic fractions were neutralized with TEAB (2
mL, 1M). All solvents were evaporated and the residue coevaporated
with H2O to remove excess buffer. The colorless glass obtained was
used directly for cyclization.
Cyclic-8-bromoinosine 5′-Diphosphate Ribose8,9 (8-Br-cIDPR) (3).
N1-(5′′-O-Phosphoryl-β-D-ribofuranosyl)-5′-O-(phenylthio)-
phosphoryl-8-bromoinosine (17, 0.024 mmol) was evaporated from
pyridine (2 mL, × 2). The residue was taken up in pyridine (10 mL)
and added over 15 h to a solution of iodine (140 mg, 0.591 mmol) and
3 Å molecular sieves (0.5 g) in pyridine (20 mL), in the dark. The
solution was filtered through Celite and washed with H2O. After
addition of TEAB (2 mL), all solvents were evaporated and the residue
partitioned between H2O and CHCl3. The aqueous layer was washed
with CHCl3 and evaporated to dryness. The residue was purified by
semipreparative reversed-phase HPLC eluted at 5 mL/min with
acetonitrile/0.1 M TEAB (1:0 → 13:7 v/v) over 25 min. Fractions
1
0.61 (EtOAc); H NMR (400 MHz, CDCl3) δ 7.99 (s, 1H, H-2),
7.45−7.25 (m, 10H, Ar-H), 6.16 (d, 1H, J = 2.1, H-1′), 5.98 (d, 1H, J =
1.8, H-1′′), 5.46 (dd, 1H, J = 6.4, 2.1, H-2′), 5.09 (dd, 1H, J = 6.4, 3.6,
H-3′), 5.00 (dd, 1H, J = 6.5, 1.8, H-2′′), 4.90 (dd, 1H, J = 6.5, 4.3, H-
3′′), 4.45−4.12 (m, 6H, H-4′, H-4′′, both H-5′ and both H-5′′), 1.60 (s,
3H, CH3), 1.55 (s, 3H, CH3), 1.46 (s, 9H, tBu), 1.45 (s, 9H, tBu), 1.36
(s, 3H, CH3), 1.28 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3) δ
4196
dx.doi.org/10.1021/jo202319f | J. Org. Chem. 2012, 77, 4191−4197

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were analyzed by analytical RP-HPLC eluted at 1 mL/min with ion-
pair buffer: 0.17% (m/v) cetrimide and 45% (v/v) phosphate buffer
(pH 6.4) in MeOH. Appropriate fractions were collected and
evaporated under vacuum to give the title compound (5.3 mg, 35%
over two steps): UV (H2O, pH 7), λmax 255 nm (ε 11,100); 1H NMR
(500 MHz, D2O) δ 8.88 (s, 1H, H-2), 6.06 (d, 1H, J = 6.1, H-1′), 6.01
(s, 1H, H-1′′), 5.31 (dd, 1H, J = 6.1, 2.4, H-2′), 4.62 (d, 1H, J = 2.4, H-
3′), 4.49 (dd, 1H, J = 10.8, 5.6, H-5′a), 4.40−4.30 (m, 5H), 4.11 (d,
1H, J = 11.9, H-5′′a), 4.02 (d, 1H, J = 10.8, H-5′b) ppm; 13C NMR
(125 MHz, D2O) δ 156.7, 149.3, 144.3, 128.5, 123.9, 91.9, 90.8, 84.8
(d, J = 11.0, 83.1 (d, J = 9.4), 75.5, 72.4, 70.5, 67.4, 64.9 (d, J = 3.7),
(17) Fukuoka, M.; Shuto, S.; Minakawa, N.; Ueno, Y.; Matsuda, A. J.
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Jin, H.; Li, Q.; Carrier, L.; Zhang, L. J. Biol. Chem. 2005, 280, 15952.
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1
62.1 (d, J = 3.2) ppm; 31P NMR (202 MHz, D2O, H-decoupled) δ
B. V. L FEBS Lett. 1996, 379, 227.
−10.15 (d, J = 12.1), −11.17 (d, J = 12.1) ppm; HRMS (ESI−) calcd
for C15H18N4O14P279Br 618.9484 [(M − H)−], found 618.9508, and
calcd for C15H18N4O14P281Br 620.9463 [(M − H)−], found 620.9467.
(23) De Napoli, L.; Di Fabio, G.; Messere, A.; Montesarchio, D.;
Piccialli, G.; Varra, M. J. Chem. Soc., Perkin Trans. 1 1999, 23, 3489.
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Nucleotides 1996, 15, 1.
ASSOCIATED CONTENT
(25) Galeone, A.; Mayol, L.; Oliviero, G.; Piccialli, G.; Varra, M.
Tetrahedron 2002, 58, 363.
■
S
* Supporting Information
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2006, 62, 10123.
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1H, 13C, and 31P NMR for compounds 3, 5, 6, and 10−17. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: b.v.l.potter@bath.ac.uk.
■
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ACKNOWLEDGMENTS
■
We thank the Wellcome Trust for Project Grant No. 084068
and Programme Grant No. 082837 (to B.V.L.P.) and Dr. C.
Moreau for useful discussions and for proofreading this
manuscript. Professor Barry V. L. Potter and Drs. Joanna
Swarbrick, Stephen Tovey, and Mark Thomas are thanked for
their contributions to the cover art.
2006, 1127.
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Guse, A. H.; Zhang, L. Org. Biomol. Chem. 2010, 8, 4705.
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