Synthesis of Terminal Ribose Analogues of Adenosine 5′-Diphosphate Ribose as Probes for the Transient Receptor Potential Cation Channel TRPM2

Article abstract of DOI:10.1021/acs.joc.9b00338

TRPM2 (transient receptor potential cation channel, subfamily M, member 2) is a nonselective cation channel involved in the response to oxidative stress and in inflammation. Its role in autoimmune and neurodegenerative diseases makes it an attractive pharmacological target. Binding of the nucleotide adenosine 5′-diphosphate ribose (ADPR) to the cytosolic NUDT9 homology (NUDT9H) domain activates the channel. A detailed understanding of how ADPR interacts with the TRPM2 ligand binding domain is lacking, hampering the rational design of modulators, but the terminal ribose of ADPR is known to be essential for activation. To study its role in more detail, we designed synthetic routes to novel analogues of ADPR and 2′-deoxy-ADPR that were modified only by removal of a single hydroxyl group from the terminal ribose. The ADPR analogues were obtained by coupling nucleoside phosphorimidazolides to deoxysugar phosphates. The corresponding C2″-based analogues proved to be unstable. The C1″- and C3″-ADPR analogues were evaluated electrophysiologically by patch-clamp in TRPM2-expressing HEK293 cells. In addition, a compound with all hydroxyl groups of the terminal ribose blocked as its 1″-β-O-methyl-2″,3″-O-isopropylidene derivative was evaluated. Removal of either C1″ or C3″ hydroxyl groups from ADPR resulted in loss of agonist activity. Both these modifications and blocking all three hydroxyl groups resulted in TRPM2 antagonists. Our results demonstrate the critical role of these hydroxyl groups in channel activation.

Full text of DOI:10.1021/acs.joc.9b00338

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Article
Synthesis of terminal ribose analogues of adenosine 5'-
diphosphate ribose (ADPR) as probes for the Transient
Receptor Potential (TRP) cation channel TRPM2
Ondrej Baszczy#ski, Joanna M Watt, Monika D Rozewitz,
Andreas H. Guse, Ralf Fliegert, and Barry V. L. Potter
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00338 • Publication Date (Web): 12 Apr 2019
Downloaded from http://pubs.acs.org on April 13, 2019
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Synthesis of terminal ribose analogues of adenosine 5'-diphosphate ribose (ADPR)
as probes for the Transient Receptor Potential (TRP) cation channel TRPM2
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§2ǂ
Ondřej Baszczyňski , Joanna M. Watt§1,2, Monika D. Rozewitz , Andreas H. Guse , Ralf Fliegert
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and Barry V. L. Potter1,2,¶
*
1Medicinal Chemistry & Drug Discovery, Department of Pharmacology, University of Oxford,
Mansfield Road, Oxford, OX1 3QT, UK
2Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology,
University of Bath, Bath, BA2 7AY, UK
3The Calcium Signalling Group, Department of Biochemistry and Molecular Cell Biology, University
Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany
*
Corresponding author Email: barry.potter@pharm.ox.ac.uk
ǂPresent address: Department of Organic Chemistry, Faculty of Science, Charles University,
Hlavova 2030/8, 128 43 Prague 2, Czech Republic.
§ = Equal contribution, ¶= Equal contribution
Keywords: TRPM2, ADPR, calcium, NUDT9, pyrophosphate
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TOC Graphic
NH2
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R = OH or H
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TRPM2
Abstract
TRPM2 (transient receptor potential cation channel, subfamily M, member 2) is a non-selective cation
channel involved in the response to oxidative stress and in inflammation. Its role in autoimmune and
neurodegenerative diseases makes it an attractive pharmacological target. Binding of the nucleotide
adenosine 5'-diphosphate ribose (ADPR) to the cytosolic NUDT9 homology (NUDT9H) domain
activates the channel. A detailed understanding of how ADPR interacts with the TRPM2 ligand
binding domain is lacking, hampering the rational design of modulators, but the terminal ribose of
ADPR is known to be essential for activation. To study its role in more detail we designed synthetic
routes to novel analogues of ADPR and 2′-deoxy-ADPR that were modified only by removal of a
single hydroxyl group from of the terminal ribose. The ADPR analogues were obtained by coupling
nucleoside phosphorimidazolides to deoxysugar phosphates. The corresponding C2″-based analogues
proved to be unstable. The C1″- and C3″-ADPR analogues were evaluated electrophysiologically by
patch-clamp in TRPM2-expressing HEK293 cells. In addition, a compound with all hydroxyl groups
of the terminal ribose blocked as its 1ʺ-α-methylfuranoside-2ʺ, 3ʺ-isopropylidene derivative was
evaluated. Removal of either C1ʺ or C3ʺ hydroxyl groups from ADPR resulted in loss of agonist
activity. Both these modifications, and blocking all three hydroxyl groups resulted in ADPR
antagonists. Our results demonstrate the critical role of these hydroxyl groups in channel activation.
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Introduction
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The non-selective cation channel TRPM2 (transient receptor potential cation channel,
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subfamily M, member 2) is activated in a Ca -dependent manner after binding of the nucleotide ADP-
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ribose (ADPR) to its cytosolic C-terminal NUDT9H domain – that shares homology with a
1
mitochondrial nucleotide pyrophosphatase NUDT9. While earlier studies indicated that the NUDT9H
domain also has a low pyrophosphatase activity, hydrolyzing ADPR to AMP and ribose 5-phosphate
2
(
R5P), a recent study showed that the Nudix box motif in TRPM2 does not support catalysis and the
production of AMP might have been due to spontaneous hydrolysis of ADPR at alkaline pH.3
Reactive oxygen species (ROS) and genotoxic stress can result in release of ADPR from the
nucleus due to the activation of the poly(ADP-ribose) polymerase-1 and poly(ADP-ribose)
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,5
glycohydrolase pathway. The ADPR so generated can then activate TRPM2, resulting in prolonged
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Ca -entry, mitochondrial Ca -overload and apoptosis, thereby contributing to cell damage in post
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ischemic reperfusion injury during myocardial infarction and stroke.
Besides this role in cell death, TRPM2 also participates in physiological processes like
inflammation.9 In neutrophil granulocytes and dendritic cells TRPM2 contributes to chemotaxis.10–12
The chemotaxis of murine neutrophils in response to fMLP is independent of PARP-1 but can be
inhibited by 8-Br-ADPR, a compound that inhibits activation of TRPM2 by ADPR, and by knock-out
1
0
of CD38, a glycohydrolase that produces ADPR from NAD. In macrophages and monocytes TRPM2
is involved in secretion of chemokines and cytokines in response to ROS and pro-inflammatory
cytokines,13,14 whereas in effector T cells it plays a role in proliferation and secretion of pro-
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inflammatory cytokines. Inhibition of TRPM2 has been shown to reduce tissue damage after stroke
by preventing invasion of neutrophils16 and knock-out of TRPM2 ameliorates the symptoms of
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experimentally induced autoimmune encephalomyelitis, a model for multiple sclerosis. TRPM2 is
thus an attractive pharmacological target for the treatment of neurodegenerative and autoimmune
diseases.17,18
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Further investigations into the role of TRPM2 in physiology and pathophysiology require
specific modulators of channel function and their rational design will benefit from a structure of the
binding site and a better understanding of its structure activity relationship (SAR). Recently, TRPM2
structures from three different species were elucidated by cryoEM. TRPM2 from the sea anemone
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(Nematostella vectensis) NvTRPM2 lacks the NUDT9H domain, probably due to the flexibility of this
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part of the molecule. TRPM2 from zebrafish (Danio rerio) drTRPM2 has been solved in the apo state
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and in an ADPR-bound state. While ADPR could not be localized in the NUDT9H domain, electron
density corresponding to ADPR was found in the N-terminal MHR1/2 domain. Comparison of the
conformations of the apo and the ADPR-bound state as well as mutational analysis indicate that in
drTRPM2 gating of the channel occurs via binding of ADPR to the MHR1/2 domain instead
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NUDT9H. Structures of human TRPM2 (hTRPM2) have been resolved for the apo state, an ADPR
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bound (primed) state and an open conformation bound to ADPR and Ca . Mutagenesis of the
MHR1/2 domain and removal of the NUDT9H domain show that in human TRPM2 channel gating
occurs after binding of ADPR to the NUDT9H domain, but again poor resolution prevents the
placement of ADPR in the presumed binding pocket. Structure-based drug design therefore still awaits
a high resolution structure of either the full-length channel or the isolated NUDT9H domain of
TRPM2.
Recently, we synthesised ADPR analogues to explore the role of the adenosine, pyrophosphate
and terminal ribose motifs in activation of TRPM2.22,23 It is of note that the pyrophosphate-forming
couplings used generally employed morpholidate- or CDI –mediated methodologies . Yields were
generally low and reaction times were generally long and improvements are warranted. To our surprise
the majority of structural modifications of ADPR led to compounds that do not activate TRPM2,
2
3
indicating that all three moieties are required for channel opening. These studies also revealed that
hydroxyl group removal at C2ʹ resulted, in the case of ADPR, in a compound that is a TRPM2 agonist
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with significantly higher efficacy than ADPR itself and also improved the antagonist 8-Ph-ADPR
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IC50 11µmol/L compared to 8-Ph-2ʹ-deoxy-ADPR, IC50 2 µmol/L).22 We also highlighted the
(
importance of the terminal ribose for TRPM2 activation by evaluating a series of modified ADPR
analogues.23 While simple ADP neither activated nor antagonized the channel, introduction of
substituents at the -phosphate that increasingly resembled the terminal ribose (Figure 1) returned
antagonist properties, but none of the analogues exhibited agonist activity. The fact that β-
(tetrahydrofuran-2-yl)methyl-ADP, an analogue with the ribofuranose backbone, but lacking the
hydroxyl groups, did not activate the channel but instead antagonized ADPR indicated that one or more
of the hydroxyl groups might be important for the gating of TRPM2. Previous studies have shown
hydroxyl group deletion to be a valuable tool in SAR elucidation for complex bioactive
molecules.24,25,26 While such endeavors may be synthetically protracted in order to effect a desired
precision edit to the parent molecule, they can divulge key mechanistic information.27,28
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Figure 1. The structure of ADPR and known terminal ribose analogues29
NH2
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terminal ribose
adenosine
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OH
pyrophosphate
NH2
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NH2
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THF-ADP
ADP-glucose
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NH2
NH2
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1''-O-Methyl-ADPR
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We report here the synthesis and electrophysiological evaluation of a series of novel terminal-ribose-
modified analogues of the TRPM2 agonists ADPR and 2ʹ-deoxy-ADPR to study the role of the
individual hydroxyl groups of the terminal ribose in TRPM2 activation. The critical step of analogue
formation was achieved by combining a sugar phosphate with a P-activated nucleotide to form a single
molecule linked by a pyrophosphate bond. A recently reported improved procedure for the preparation
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of NDP-sugars from nucleoside phosphorimidazolides uses 2-4 equivalents of magnesium chloride
to achieve high reaction yields and short reactions times for nucleoside 5-phosphorimidazolide
coupling to sugar phosphates.
Results & Discussion
The terminal ribose of ADPR is essential for the ligand-driven activation of the cation channel
29
TRPM2. To investigate the SAR of this part of ADPR in more detail, we focused upon the synthesis
of all three possible 1ʺ-deoxy, 2ʺ-deoxy and 3ʺ-deoxy terminal ribose ADPR derivatives 2, 3, 4 by
selective deletion of the appropriate hydroxyl group (Figure 2). The similar reactivity of the ribose
hydroxyl groups and potential for ring opening of the cyclic hemi-acetal required development of
multi-step synthetic routes with selective masking and deprotection. The modified ribose building
blocks were then coupled to either AMP or 2ʹ-deoxy-AMP via pyrophosphate coupling reaction
(
Figure 2). We also synthesized a corresponding analogue 1 with all hydroxyl groups present, but
blocked with small alkyl groups to interrogate the H-bond donating capability of the terminal ribose
Figure 2). The chemically stable analogues were evaluated regarding their agonist and antagonist
(
activity by patch clamp experiments in whole cell configuration using HEK cells with stable expression
of human TRPM2.
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Figure 2. Pyrophosphate coupling reaction and ADPR analogues prepared in this study
NH2
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P
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ONa
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O OHO OH
OH
NH2
NH2
NH2
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OH
O
O
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HO
OH
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O
O
O
O
O
3"
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P
P
P
P
P
O
O OHO OH
OH
HO
O OHO OH
OH
HO
O OHO OH
OH
1''-O-Me-2'', 3''-iPr-ADPR, 1
1''-dADPR, 2
2''-dADPR, 3
NH2
NH2
NH2
N
N
N
1
N
1
1
N
N
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N
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9
N
HO
HO
N
1"
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'
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OH
HO
HO
O
O
O
O
O
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HO
O
O
O
P
P
P
P
P
P
3"
3"
O OHO OH
OH
HO
O OHO OH
OH
O OHO OH
OH
3''-dADPR, 4
1'', 2'-di-dADPR, 5
3'', 2'-di-dADPR, 6
At first, routes to the required terminal ribose 5-phosphates were designed. It was envisaged that such
phosphates would be ideal coupling partners for activated nucleotide imidazolides. Protected ribose
monophosphate 9 was chosen to exploit the ligand space around the terminal ribose, H-bond donating
capability, and due to its good susceptibility for coupling with activated nucleosides. The precursor of
9
, compound 8, was obtained from protected ribose30 7 by phosphitylation with dibenzyl N,N-
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diisopropylphosphoramidite and subsequent oxidation using hydrogen peroxide to afford 8 (79%),
(
Scheme 1 A). Compound 8 was then hydrogenated using Pd/C to afford 9 (82%). Ribose-5-
monophosphate 9 was converted to its tributylammonium salts and freeze-dried. Synthesis of 1-
deoxyribose-5-phosphate 10, to explore the role of the anomeric OH group of terminal ribose, started
from 2, 3-isopropylidene ribose 13 that was protected at position 5 by a trityl group, to afford 14 (65%)
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(
Scheme 1 B). Reduction of 14 by sodium borohydride led to compound 15 (85%). Reaction of 15
with tosyl chloride in pyridine gave no significant result at room temperature, but heating the reaction
mixture up to 60 °C helped to afford the protected 1-deoxyribose 16 (80%). However, deprotection of
1
6 with acetic acid failed after multiple attempts, as both the 5-O-trityl and 2-3-O-isopropylidene
protecting groups were cleaved, leading to the undesired fully deprotected 1-deoxyribose or to complex
mixtures. Using the alternative deprotection of 16 with formic acid in diethylether32 afforded
compound 17 (32%).33 Phosphitylation and subsequent oxidation of 17 with di-tert-butyl N, N-
diisopropylphosphoramidite gave the phosphate derivative 18 (41%). Careful deprotection of 18 with
aqueous trifluoroacetic acid at low temperature afforded the target 1-deoxyribose-5-phosphate 10
(49%) that was subsequently converted to its tributylammonium salt.
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Scheme 1. Synthesis of modified terminal riboses 9 and 10.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
A)
O 1
O
O
a, b)
9%
c, d)
82%
O
O
O
O
2
O
O
O
OH
OP(O)(OBn)2
O P OH
O-
7
3
5
5
5
O
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HNBu3
7
8
9
B)
HO
HO
HO
OTr
OTr
O
O
O
e)
65%
f)
OTr
g)
80%
2
HO
5
O
OH
O
O
3
5
85%
5
O
O
O
O
O
O
1
3
14
15
16
OP(O)(OtBu)2
OH
O P OH
O
O
O
O-
5
5
5
k, l)
i, j)
41%
h)
32%
HNBu3
O
O
HO
49%
O
O
OH
10
18
1
7
Reagents and conditions: a) 5-Ph-1-H-tetrazole, dibenzyl N,N-diisopropylphosphoramidite, DCM, 20 °C, 1h; b)
, 0-20 °C, 1h; c) hydrogen (balloon), Pd/C, 5h, TEAB (1M), 20 °C; d) Dowex® D50 (H+),
2 2
triethylamine, H O
tributylamine; e) tritylchloride, pyridine, 20 °C, 16h; f) sodium borohydride, ethanol, 0-20 °C, 2h; g) tosylchloride,
pyridine, 60 °C, 16h; h) HCOOH/diethylether, 20 °C, 16h; i) 5-Ph-1-H-tetrazole, di-tert-butyl N,N-
2 2
diisopropylphosphoramidite, DCM, 20 °C, 1h; j) triethylamine, H O , 0-20 °C, 1h; k) aqueous TFA, 0-20 °C, 4h; l)
+
Dowex® D50 (H ), tributylamine.
Synthesis of the 2-deoxy version of the terminal ribose (Scheme 2) started from commercially available
2
-deoxyribose-5-phosphate sodium salt 19 (Sigma Aldrich) that was converted to its corresponding
+
mono-tributylammonium salt 11 by using Dowex® resin (H form) followed by titration with
tributylamine to pH~7 (Scheme 2A). Partial decomposition (20-30%) of 19 was observed during the
transformation to 11 via 31PNMR, suggesting that 2-deoxyribose-5-monophosphate is less stable
towards changes in pH (see Supplementary Information, S14-16) compared to its 1-deoxy- and 3-
deoxy-counterparts. Synthesis of 3-deoxyribose-5-monophosphate 12 started from 1, 2-O-
34
isopropylidene xylose 20, that was protected at the 5-hydroxyl group as the TBDPS ether to give 21
(83%) (Scheme 2B). Reaction of 21 with 1,1ʹ-thiocarbonyldiimidazole in DCM afforded compound
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2 (63%). Reductive deoxygenation of 22 with tributyltin hydride led to the compound 23 (76%) which
was subsequently deprotected in buffered TBAF to give 1, 2-O-isopropylidene-3-deoxyribose 24
(82%). Phosphitylation and subsequent oxidation of 24 gave the desired di-tert-butyl phosphate
derivative 25 (50%). However, several attempts to deprotect compound 25 with aqueous TFA led to a
complex mixture suggesting that di-tert-butyl protected phosphate 25 was not the optimal precursor
for 12. Therefore, 24 was phosphitylated and subsequently oxidized to its dibenzylphosphate derivative
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
6 (72%) that was successfully deprotected to give 27 (68%). Hydrogenation of 27 followed by
neutralization of the free phosphate using tributylamine afforded the target analogue 3-deoxyribose-5-
phosphate as its mono-tributylammonium salt 12 (67%).
Scheme 2. Synthesis of modified terminal riboses 11 and 12.
A)
HO
HO
O
O
O
a)
quant. with
20-30% decomposition
see SI, S14-16
2
2
OP(O)(ONa)2
O P OH
5
5
O-
HO
HO
HNBu3
1
9
11
B)
TBDPSO
O
O
TBDPSO
5
O
TBDPSO
O
O
O
O
HO
O
O
O
b)
c)
63%
d)
3
O
5
S
5
5
O
83%
3
76%
3
O
3
HO
N
HO
23
22
82% e)
20
21
N
HO
O
O
O
h)
(tBuO)2(O)PO
OH
O
(
HO) (O)PO
O
f, g)
0%
2
O
5
5
5
3
5
3
3
O
OH
2
5
24
complex mixture
7
2% i, j)
O
HO P O
O-
O
OH
OH
(BnO) (O)PO
(BnO) (O)PO
O
O
O
l, m)
2
O
OH
OH
k)
68%
2
5
5
5
67%
3
3
3
Bu NH
3
1
2
27
26
+
Reagents and conditions: a) Dowex® D50 (H ), tributylamine; b) TBDPS chloride, DMAP, Pyridine, 20 °C, 16 h; c) 1,1ʹ-
thiocarbonyldiimidazole, DCM, reflux, 2h; d) tributyltinhydride, AIBN, toluene, 116 °C, 3h; e) TBAF.3H
2
O, acetic acid,
0 °C, 3h; f) 5-Ph-1-H-tetrazole, di-tert-butyl N,N-diisopropylphosphoramidite, DCM, 20 °C, 1h; g) triethylamine, H
-20 °C, 1h; h) various conditions with aqueous TFA; i) 5-Ph-1-H-tetrazole, dibenzyl N,N-diisopropylphosphoramidite,
2
0
2 2
O ,
1
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
DCM, 20 °C, 1h; j) triethylamine, H
2
O
2
, 0-20 °C, 1h; k) aqueous TFA, 0 °C, 1.5h; l) hydrogen (balloon), Pd/C, 4h, 20 °C;
m) tributylamine.
In previous work we used morpholidate chemistry or 1,1ʹ-carbonyldiimidazole-based methodology in
coupling reactions, but these had been less than satisfactory during ADPR analogue preparation To
find the most suitable conditions here for pyrophosphate bond formation between the corresponding
ribose-5-monophosphate and either AMP or 2ʹ-dAMP, two different methods were tried. Activation
of AMP tributylammonium salt with 1,1ʹ-carbonyldiimidazole35 followed by addition of the
triethylammonium salt of ribose 9 did show formation of the desired pyrophosphate product 1 by
HPLC. However, several byproducts were also observed (see Supplementary Information Figure 1a,
S2). In contrast, following the Dabrowski-Tumanski procedure36 activation of the AMP
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TM
tributylammonium salt using imidazole, 2,2ʹ-dithiodipyridine (Aldrithiol ) and triphenyl phosphine
cleanly afforded the AMP-imidazolide that was isolated by precipitation with a cold solution of NaI in
acetone. When stirred with the triethylammonium salt of 9 and magnesium chloride in DMF, the AMP-
imidazolide gave almost exclusively the desired compound 1 (see Supplementary Information Figure
1b, S2-S3). Finally, purified target ribose derivatives 9, 10, 11 and 12 were individually coupled to
imidazolide-activated AMP or 2ʹ-deoxy-AMP (Scheme 3). Adenosine-5ʹ-monophosphate 28 and 2ʹ-
deoxy-adenosine-5ʹ-monophosphate 29 were transformed to their imidazolides 30 and 31, by reaction
of the corresponding mono-tributylammonium salt of 28 and 29 and imidazole using the AldrithiolTM
36
and triphenylphosphine condensation protocol. Imidazolides 30 and 31 were precipitated from the
reaction mixture by addition of a 0.1 M solution of sodium iodide in cold, anhydrous acetone, and were
directly used for coupling. The target modified ADPR analogues 1 - 6 were then prepared by coupling36
the corresponding imidazolide 30 or 31 and mono-tributylammonium salt of the modified ribose-5-
phosphate 9, 10, 11 or 12 to give variable yields of the desired ADPR analogues 1 (24%), 2 (66%), 3
(8%), 4 (17%), 5 (42%), 6 (27%). Final compounds 1-6 were purified by semi-preparative HPLC using
triethylammonium bicarbonate buffer and isolated as the corresponding triethylammonium salts.
1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 3. Synthesis of target terminal-modified ADPR analogues
A)
O
O
N
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-HO P
N
P
O
NH2
O
NH2
9
a), b)
9
O
N
O
N
Bu NH HO
NaO
3
1
1
2'
N
2'
N
N
N
R
R
HO
28. R = OH
9. R = H
HO
30. R = OH hygroscopic
31. R = H not quantified
2
B)
2
Et NH
3
NH2
N
1
O
O
N
9
O
O
N
N
O
c)
O
P OH
O-
O
O
2"
2'
O
R
O
O
O
O
30. R = OH
P
P
HNBu3
-
-
9
O
O O O O
OH
1. R = OH, 24%
2Et NH
3
NH2
N
1
N
1
9
N
O
O
1
"
N
c)
HO
O
P OH
O-
O
O
2'
HO
R
O
O
O
HO
30. R = OH
P
P
HNBu3
-
or
-
HO
O O O O
OH
1
0
3
1. R = H
2
. R = OH, 66%
5
. R = H, 42%
2Et3NH
NH2
N
1
N
HO
9
N
O
O
HO
N
2
c)
O
P OH
O-
O
O
2'
2"
R
O
O
O
HO
30. R = OH
P
P
HNBu3
-
11
O
- O O
HO
O
OH
2Et NH
3
. R = OH, 8%, sensitive,
3
NH2
see SI, Fig. 2, S4
N
1
N
HO
HO
9
N
O
O
HO
HO
N
c)
O
P OH
O-
O
O
2'
3
R
O
O
O
3
0. R = OH
P
P
3"
HNBu3
12
or
-
-
O O O O
OH
31. R = H
4
. R = OH, 17%
6
. R = H, 27%
Reagents and conditions: a) AldrithiolTM, imidazole, triethylamine, triphenylphosphine, 20 °C, 16h; b) NaI, acetone
(precipitation), 0 °C; c) MgCl , dimethylformamide, 20 °C, 3-16h.
2
Stability of terminal-modified ADPR analogues 1 - 6
The stability of ADPR analogues 1 - 6 was evaluated using analytical HPLC. Aqueous solutions of 1
- 6 stored at 5 °C were compared with freshly prepared standard solutions of 1 – 6 (from solid samples
1
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stored at -20 °C). All compounds except 3 were stable in aqueous solutions for several days when
stored in the fridge (5 °C). Compound 3, 2ʺ-deoxy-ADPR was found to be unstable under these
conditions. We also observed decomposition of 3 during transport and sample preparation, so we could
not do electrophysiological experiments with 3 (Supplementary Information Figure 2, S4). The
instability of 3 may correspond with the lower stability of 2-deoxyribose-5-monophosphate 11 that
was sensitive towards changes in pH during conversion of the commercially available sodium salt to
the tributylammonium salt (conversion of 19 to 11). This decomposition led to hydrolysis of the 5ʹ-
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
1
phosphate (approx. 20-30% de-phosphorylation of 11 was observed via PNMR). The corresponding
decomposition by de-phosphorylation in 2ʺ-deoxy-ADPR 3 might cleave the pyrophosphate bond, and
would be predicted to lead to inactive fragments.
Evaluation of novel analogues against TRPM2
Recently, we investigated the importance of the ADPR terminal ribose for the activation of TRPM2.29
This can be investigated by whole cell patch clamp experiments. During these experiments a glass
pipette with a diameter much smaller than a cell and filled with a solution mimicking the ionic
composition of the cytosol is attached to the plasma membrane. By applying suction to the pipette the
membrane patch underneath the pipette is ruptured. In voltage-clamp experiments a voltage is applied
between an electrode in the pipette solution and a bath electrode in the bath solution surrounding the
cells. This voltage results in ions moving through channels in the membrane resulting in a current that
can be recorded.37,38 In HEK293 cells expressing TRPM2 addition of ADPR to the pipette solution
results in a current that is absent in wild type HEK293 cells. ADPR analogues can be tested for their
ability to activate the channel and they can also be tested for antagonist activity by adding them in
addition to ADPR. We found that adenosine 5ʹ-monophosphate (AMP) and adenosine 5ʹ-diphosphate
ADP) neither activated or inhibited the channel even when applied in large excess over ADPR.29
(
Interestingly, replacement of the terminal ribose of ADPR with small substituents led to analogues that
1
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
did not activate the channel, but inhibited actvation by ADPR indicating that they compete with ADPR
for the ligand binding site of TRPM2. Thus, activation of TRPM2 may be attributed to specific
interactions between the hydroxyl groups of the terminal ribose of ADPR and the ligand binding
NUDT9H domain of TRPM2. We evaluated the deoxy analogues 2 and 4 and and an ADPR analogue
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
where all hydroxyl groups of the terminal ribose are masked and could no longer act as hydrogen
bond donors (Figure 3). It was not possible to evaluate 3 biologically due to instability issues (see
above). During the experiments we included 8-Br-ADPR as a control. 8-Br-ADPR has previously been
shown to inhibit activation of TRPM2 by ADPR most likely by competing with the agonist.10
Figure 3. Effect of ADPR analogues on whole cell currents in TRPM2 expressing HEK293 cells
Conditions: a) Effect of ADPR analogues on whole cell currents in TRPM2 expressing HEK293 cells. Experiments were
done as outlined in the Experimental Section. ADPR or the indicated ADPR analogue were added to the pipette solution
at a concentration of 100 µmol/L. b) Effect of ADPR analogues on TRPM2 whole cell currents elicited by ADPR. In this
case the pipette solution contained either no nucleotide (buffer), 100 µM ADPR or a combination of 100 µM ADPR with
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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5
5
5
5
6
9
00 µM of the indicated ADPR analogue. 8-Br-ADPR was included as inhibitor control. Points indicate the maximum
current from individual cells. The number of cells for each condition is indicated at the bottom. Bars represent the mean of
the log-transformed currents. (ns = non-significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). Control conditions
on both panels include overlapping sets of data since on some days both agonist and antagonist experiments were
performed.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
As expected, the masked analogue 1″-O-Me-2″, 3″-iPr-ADPR 1 did not activate TRPM2 on its own
(Figure 3a). Since it is more space-filling than ADPR we were unsure whether it would compete with
ADPR for binding to the NUDT9H domain. When applied in excess over ADPR, however, it indeed
inhibited activation of TRPM2 (Figure 3b), showing that further steric constraints than those simply at
the 1''-position of the terminal ribose are tolerated and may potentially be exploitable for TRPM2
antagonist optimization.
Figure 4. The structures of masked ADPR 1, α-β-methylene ADPR (AMPCR) 32, 2ʺ-O-acetyl-ADPR
33, and 3ʺ-O-acetyl-ADPR 34.
NH2
NH2
N
N
1
N
1
N
9
N
9
N
R O
1
HO 1"
1"
N
N
O
O
O
O
2"
2'
2"
2'
R O
OH
R O
OH
2
O
X
O
1
O
O
O
3"
P
P
3"
P
P
R O
3
O OHO OH
OH
R O
2
O OHO OH
OH
X = O; R = Me (1''-OMe -anomer);
R = Ac; R = H; 33 (2´´-O-Acetyl-ADPR)
1
2
1
R , R = isopropylidene;
1
R = H; R = Ac; 34 (3´´-O-Acetyl-ADPR)
1 3
2
3
X = CH ; R = H; R , R = H;
AMPCPR 32
2
1
2
3
Interestingly, an analogue 32 (Figure 4) of the low-affinity partial TRPM2 agonist α-β-methylene
39
ADPR (AMPCPR) masked in the same way as 1 was recently described to be a high affinity TRPM2
4
0
antagonist that inhibits ADPR-elicited TRPM2 currents with an IC of 5.7 µM. Luo et al did not
5
0
explicitly mention whether they tested this compound for TRPM2 activation but, since it fully inhibits
the channel at 100 µM, it is unlikely to exhibit agonist activity. Methylene analogues may show
increased stability towards cellular pyrophosphatases like NUDT9 and NUDT5 which may be retained
1
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5
5
6
in the cell during whole cell patch-clamp experiments. Despite earlier reports that indicated an ADPR
1
pyrophosphatase activity of the NUDT9H domain of TRPM2, that might contribute to degradation of
3
agonists with pyrophosphate bridge, this has recently been disputed. The observed difference could
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2+
also result from different experimental conditions as we buffered the intracellular Ca concentration
to 200 nmol/L with 10 mmol/L of EGTA whereas Luo et al. left the Ca2+ concentration largely
unbuffered (50 µmol/L EGTA) which would result in a much steeper dependence of the current on the
agonist concentration and a more pronounced inhibition by the antagonist.
Contrary to our expectations both ADPR analogues 2, 4 lacking hydroxyl groups at the terminal ribose
did not induce significant whole cell currents in TRPM2-expressing HEK cells at 100 µmol/L (Figure
3a). This was unexpected as O-acetyl-ADPR, a product of NAD-dependent histone deacetylases of the
Sir2 family, is an effective activator of TRPM2 that binds to the NUDTH domain with similar affinity
as ADPR, as shown by UV crosslinking experiments and activates TRPM2 with a comparable
concentration dependence as ADPR.41 2″-O-acetyl-ADPR 33, the product of the deacetylases,
undergoes in neutral solution rapid intramolecular transesterification resulting in roughly equal
4
2
amounts of 2″-O-acetyl-ADPR 33 and 3″-O-acetyl-ADPR 34. Either the nucleotide binding site of
TRPM2 is indifferent to the position of the acetyl group, which would indicate that neither the 2″- nor
the 3″-hydroxyl group is essential as hydrogen bond donor, or there is a specificity for one of the
isomers which would mean that only half of the O-acetyl-ADPR molecules in solution are effective
agonists. Since the latter seems unlikely, this may suggest that the 2ʺ-OH and 3ʺ-OH groups are
hydrogen-bond acceptors, a feature that could be retained even when acetylated. We have previously
shown that α-O-methyl-ADPR and β-O-methyl-ADPR (Figure 1) do not activate TRPM2, but both
2
9
inhibit channel activation by ADPR. This indicates that the 1″-hydroxyl group in ADPR could not
be masked or that there is no space in this region to accommodate even a relatively small methyl group,
without losing agonist activity in contrast to the 2″- and 3″-hydroxyl groups. So why does 3″-deoxy-
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ADPR 4 not activate the channel? Possible explanations may be that the conformation of the furanose
ring or the ratio between the two anomeric forms of the ribose are affected by the absence of the 3″-
hydroxyl group in a way that prevents the proper hydrogen bonding necessary for channel activation,
or that this hydroxyl is required as a hydrogen bond acceptor.
4
5
6
7
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9
1
1
1
1
1
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1
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The five-membered ribose sugar ring is puckered due to non-bonded interactions and the energetically
4
3
most stable conformation has all substituents as far apart as possible. Thus, the different substitution
patterns in the terminal ribose of ADPR, 1ʺ-deoxy-ADPR 2, 2ʺ-deoxy-ADPR 3 and 3ʺ-deoxy-ADPR
4
, might be expected to produce differing types of puckering. Extensive analysis of the ribose and 2ʹ-
deoxy-ribose rings in RNA and DNA nucleotides has shown that while ribose adopts a primarily C3ʹ-
44
endo configuration, 2ʹ-deoxy-ribose adopts a primarily C2ʹ-endo form. Such changes alter the
orientation of the ribose ring substituents, and this may affect the orientation of the substituents and
the way they are presented to the binding site, as well as the stability of a given ADPR analogue by
bringing nucleophilic hydroxyl groups into proximity for intramolecular attack on the pyrophosphate.
1
Unfortunately, the broad nature of the peaks in the H-NMR spectra of the ADPR analogues means
that it is not possible fully to determine the coupling constants, and hence to analyze fully each terminal
ribose ring conformation and suggests that rapid interconversion between configurations may be taking
place in solution.
ADPR is a mixture of terminal ribose α- and β-anomers and it is not known whether one or both
1
anomers activate TRPM2. Analysis of H-NMR spectra demonstrates that both 2ʺ-deoxy-ADPR 3 and
3ʺ-deoxy-ADPR 4 are also mixtures of the α and β-forms in the following ratios (α:β, 2ʺ-deoxy-ADPR
3
, 1:1.17;3ʺ-deoxy-ADPR 4; 1:4). Both analogues still adopt cyclic (as opposed to open chain form)
structures for the terminal ribose. Given that both anomers (α- and β-forms) are present in both cases,
it would seem unlikely that this could be the sole cause of their observed inactivity.
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6
Surprisingly, di-deoxy ADPR analogues 5 and 6 did not have significant antagonist activity (data not
shown). This was unexpected, since the potency of our previous antagonist 8-Ph-ADPR (IC = 11
5
0
uM) was improved by the 2'-deoxy modification (8-Ph-2'-deoxy-ADPR IC = 3 uM).
5
0
0
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The recent cryo-EM structures of human TRPM2 show that in the absence of ADPR interactions
between the NUDT9H domain and MHR1/2 domains of the same and adjacent subunits lock the
2
1
channel in a closed conformation. Binding of ADPR impacts the conformation of the NUDT9H
domain of TRPM2 in a way that leads to disengagement of the inter-subunit interactions, priming the
2
+
21
channel for full activation by binding of Ca ions to a cytosolic site near the pore region . The
NUDT9H domain of TRPM2 has a bi-lobed structure with the two lobes forming a cleft that is
supposed to bind ADPR. While comparison of the apo and the ADPR bound state shows a narrowing
2
1
of this cleft upon binding of ADPR , the low local resolution of the structure in the NUDT9H domain
does not currently allow for localization of ADPR in this binding pocket or for identification of the
interactions, that would trigger the conformational changes leading to the primed state. In the absence
of a high resolution structure, SAR data therefore remain essential for ligand based drug design. Our
current data further support the role of interactions involving the terminal ribose of ADPR in the gating
of TRPM2.
Recently, key differences between hTRPM2 and the invertebrate Nematostella vectensis variant
NvTRPM2 were identified. Thus, two reported synthetic ADPR analogue hTRPM2 antagonists were
4
5
NvTRPM2 agonists . Moreover, a regulatory function of NUDT9H in NvTRPM2 opposed to that in
hTRPM2 was uncovered through the action of another synthetic analogue, inosine 5'-diphosphate
ribose IDPR. Thus, ADPR analogues, such as those reported here, are at the cutting edge of progress
to unravel the mechanism of TRPM2 function.
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1
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1
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6
Building on our previous study that showed the essential role of the terminal ribose of ADPR in gating
0
1
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6
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9
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2+
of the Ca -permeable, non-selective cation channel TRPM2, we synthesized adenine, ribose and 2'-
deoxyribose analogues of ADPR 1-6 each lacking one of the hydroxyl groups of the terminal ribose.
The convergent synthesis of the targets 1-6 consisted of two separate steps: a) synthesis of particular
deoxyribose monophosphates 9-12 and b) coupling analogues 9-12 to the AMP and 2ʹ-deoxy-AMP
3
6
imidazolides 30-31, following the Dabrowski-Tumanski procedure . Such an approach seems to be
higly efficient in preparation of such complex molecules as modified ADPR analogues, offering
improved yields in our hands compared to pyrophosphate formation using AMP-morpholidates or
activation using CDI. While 2ʺ-deoxy-ADPR 3 proved to be unstable, analogues 1, 2, 4, 5, 6 showed
acceptable stability and were tested electrophysiologically in patch-clamp cell experiments. Neither
1ʺ-deoxy-ADPR 2 nor 3ʺ-deoxy-ADPR 4 was able to activate TRPM2 significantly; instead, both 2
and 4 were weak antagonists of ADPR-mediated TRPM2 activation in whole cell experiments, further
highlighting the sensitivity of channel activation to structural changes of the terminal ribose. 1″-O-Me-
2
″, 3″-iPr-ADPR, 1ʺ-deoxy-, and 3ʺ-deoxy-ADPR (1, 2 and 4) were all antagonists of ADPR-mediated
2
+
Ca -release in whole cell patch clamp experiments. Unlike in our previous observations, where
including an additional 2ʹ-deoxy-modification generated a more potent TRPM2 antagonist, the
corresponding 1ʺ- and 3ʺ, 2ʹ-dideoxy-analogues 5, 6 were less potent antagonists of TRPM2. Results
further highlight the significance of the ADPR terminal ribose for activation of TRPM2 and the use of
synthetic ADPR analogues in general. The synthesis of such ADPR analogues as chemical biology
4
5
tools using the methods outlined here is proving invaluable in the wider TRPM2 field. Further
analogues to interrogate individual hydroxyl group stereochemistry or individually mask each of the
three terminal hydroxyl groups to probe hydrogen bonding interactions at the TRPM2 binding site will
undoubtedly shed more light on the role of the termibal ribose.
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6
Experimental Section
General
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All reagents and solvents were of commercial quality and were used without further purification,
unless described otherwise. Triethylamine was dried over potassium hydroxide, distilled and then
stored over potassium hydroxide pellets. H O was of MilliQ quality. Unless otherwise stated, all
2
1
13
31
reactions were carried out under an inert atmosphere of nitrogen. All H, C, and P NMR spectra of
the final compounds were collected, either on a Varian Mercury 400 MHz or Bruker Avance III 500
MHz Spectrometer. Chemical shifts () are reported in parts per million (ppm) and all H- and 13C-
NMR assignments are based on COSY, HSQC, HMBC, and DEPT experiments.
Abbreviations for splitting patterns are as follows: br, broad; s, singlet; d, doublet; t, triplet; m,
multiplet etc. High resolution time-of-flight mass spectra were obtained
on a Bruker Daltonics micrOTOF mass spectrometer using electrospray
ionisation (ESI). Analytical HPLC analyses were carried out on a Waters 2695 Alliance
module equipped with a Waters 2996 Photodiode Array Detector (210 – 350 nm). The
chromatographic system consisted of a Hichrom Guard Column for HPLC and a Phenomenex Synergi
1
4
u MAX-RP 80A column (150 × 4.60 mm), eluted at 1 mL/min with a gradient of MeCN in 0.05 M
TEAB. Semi-preparative HPLC was carried out on a Waters 2525 pump with manual FlexInject. The
chromatographic system consisted of a Phenomenex Gemini, 5u, C18, 110A column (250 x 10.00
mm), eluted at 5 mL/min.
1
-O-Methyl-2, 3-O-isopropylidene-D-ribofuranose (7).30
Compound
7
was prepared
1
according to the literature from D-ribose. Yield (4.28 g, 63%). NMR and HRMS confirmed. HNMR
(400MHz, CDCl ) δ 4.93 (s, 1H, H-1), 4.78 (d, J = 6.0 Hz, 1H, H-2), 4.54 (d, J = 6.0 Hz, 1H, H-3),
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.38 (bs, 1H, H-4), 3.66-3.55 (m, 2H, H-5 ), 3.38 (s, 3H, OCH ), 3.19 (bs, 1H, OH), 1.44 (s, 3H,
a, b 3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CH ), 1.27 (s, 3H, CH ).
3
3
Dibenzyl 1-O-methyl-2,3-O-isopropylidene-β-D-ribofuranose-5-phosphate (8). 1-O-methyl-2,3-
O-isopropylidene-β-D-ribofuranose 7 (100 mg, 0.49 mmol) and 5-phenyl-1H-tetrazole (143 mg, 0.98
mmol) were co-evaporated with dry toluene (2×). Then the solid mixture was suspended in
dichloromethane (2 mL) and dibenzyl N, N-diisopropylphosphoramidite (250 µL, 0.669 mmol) was
added dropwise. The mixture was stirred at 20 °C for 2 hours. TLC analysis (petroleum ether/EtOAc,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
:1 v/v) showed complete phosphitylation. The reaction was cooled to 0 °C and triethylamine (407 µL,
.92 mmol) was added followed by aqueous hydrogen peroxide (35%, 109 µL, 1.24 mmol). The
resulting mixture was stirred at 20 °C for 1 h. The mixture was diluted with EtOAc (20 mL) and
extracted with aqueous Na SO (10%, w/v). The organic phase was dried (Na SO ), solids were
2
3
2
4
removed by filtration and the solvent was evaporated in vacuo. The crude product was purified by
Isco-Flash chromatography using petroleum ether/EtOAc (1:0 → 0:1, v/v). This procedure afforded
1
the title compound 8 as a colorless liquid (180 mg, 79%). HNMR (400MHz, CDCl ) δ 7.40-7.31 (m,
3
1
0H, Bn-2, 3, 4), 5.10-5.0 (m, 4H, Bn-CH ), 4.93 (s, 1H, H-1), 4.60 (dd, 1H, J = 6.0 Hz, J = 0.4 Hz,
2
H-2), 4.50 (d, 1H, J = 6.0 Hz, H-3), 4.28 (dt, 1H, J = 6.0 Hz, J = 0.4 Hz, H-4), 4.0-3.88 (m, 2H, H-5),
3
1
3.26 (s, 3H, OMe), 1.46 and 1.28 (2  s, 2  3H, CH iPr). PNMR (160MHz, CDCl ) δ -1.23 (s, P).
3
3
13
1
C{ H}NMR (100MHz, CDCl ) δ 135.7 and 135.6 (2 x C-ipso-Bn), 128.5 and 127.9 (10 x C-Bn-o,
3
m, p), 112.5 (C(CH )), 109.3 (C-1), 84.9 (C-3), 84.6 (d, J = 9.0 Hz, C-4), 81.4 (C-2), 69.4 and 69.4
3
+
(CH -Bn), 67.1 (d, J = 6.0 Hz, C-5), 55.0 (OMe), 26.3 and 24.9 (CH iPr). HRMS (ES ) calcd for
2
3
+
C H NaO P 487.1492 [M+Na] , found 487.1501.
2
3
29
8
1
-O-Methyl-2,3-O-isopropylidene-β-D-ribofuranose-5-phosphate (9). Dibenzyl 1-O-methyl-2,3-
O-isopropylidene-β-D-ribofuranose-5-phosphate 8 (180 mg, 0.39 mmol) was dissolved in MeOH-
water (17:3 v/v, 8 mL). After vacuum/argon deoxygenation of the reaction mixture the catalytic
amount of palladium on charcoal (10%, 20 mg) was added. The reaction was stirred under positive
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6
pressure of hydrogen atmosphere (balloon) at 20 °C for 5 hours. Then the reaction was flushed with
argon and neutralized by addition of triethylammonium bicarbonate buffer (TEAB, 1 mL, 1M). The
solvent was evaporated and the crude product was co-evaporated (2 ) with dry toluene and dried
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
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6
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9
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4
5
6
7
8
9
0
under high-vacuum to give a colourless solid film, compound 9 (1.17  Et N salt, 122 mg, 82%).
3
1
HNMR (400MHz,CD OD) δ 4.83 (s, 1H, H-1), 4.77 (m, 1H, H-2), 4.53 (d, 1H, J = 8.0 Hz, H-3),
3
4
.24-4.18 (m, 1H, H-4), 3.83-3.72 (m, 2H, H-5), 3.24 (s, 3H, OMe), 3.08 (q, 7H, J = 8.0 Hz,
3
1
NCH CH ), 1.37 (t, 3H, CH iPr), 1.27-1.21 (m, 13.5 H, NCH CH , CH iPr). PNMR (160MHz, d -
2
3
3
2
3
3
4
1
3
1
MeOD) δ 0.87 (s, P). C{ H}NMR (100MHz, CD OD) δ 113.3 (C(CH )), 110.7 (C-1), 86.9 (d, J =
3
3
9.0 Hz, C-4), 86.4 (C-3), 83.4 (C-2), 66.3 (d, J = 5.0 Hz, C-5), 55.1 (OMe), 47.5 (NCH CH ), 26.7
2
3
-
-
and 25.0 (CH iPr), 9.2 (NCH CH ). HRMS (ES ) calcd for C H O P 283.0588 [M-H] , found
3
2
3
9
16
8
2
83.0575. (The product was transformed into its tributylammonium salt before the pyrophosphate
coupling). Transformation of 9 to mono-tributylammonium salt: The triethylammonium salt of
compound 9 (89 mg, 0.23 mmol) was dissolved in water (2 mL) and treated with Dowex® D50 (H+).
Then the resin was removed by filteration and the solution of 9 (free acid) was titrated with
tributylamine (54 µL, 0.23 mmol, 1 equivalent, pH 7). The solution of 9 was freeze-dried overnight to
1
obtain a colorless film, compound 9 (mono-tributylammonium salt, quant.). H NMR (400MHz, H O,
2
water suppression) δ 5.09 (s, 1H, H-1), 4.90 (d, 1H, J = 5.6 Hz, H-2), 4.65 (d, 1H, suppressed by H O,
2
H-3), 4.43-4.35 (m, 1H, H-4), 3.89-3.82 (m, 2H, H-5), 3.37 (s, 1H, OMe), 3.15-3.07 (m, 6H, NCH2),
1.69-1.59 (m, 6H, NCH CH ), 1.49 (s, 3H, CH iPr), 1.40-1.30 (m, 9H, N(CH ) CH , CH iPr), 0.95-
2
2
3
2 2
3
3
31
13
1
0
1
.87 (m, 9H, N(CH ) CH ). PNMR (160MHz, D O) δ 0.14 (s, P). C{ H}NMR (100MHz, D O) δ
2
3
3
2
2
13.2 (C(CH )), 108.5 (C-1), 85.0 (d, J = 9.0 Hz, C-4, HSQC), 86.4 (C-3), 81.0 (C-2), 65.2 (d, J = 7.0
3
Hz, C-5, HSQC), 54.8 (OMe), 52.8 (NCH ), 25.3 (CH iPr), 25.3 (NCH CH ), 23.7 (CH iPr), 19.3
2
3
2
2
3
(
N(CH ) CH ), 12.8 (N(CH ) CH ).
2 2 2 2 3 3
2
, 3-O-Isopropylidene-5-O-trityl-D-ribofuranose (14). 2,3-O-Isopropylideneribose 13 (4 g, 21.1
mmol) was dissolved in pyridine (10 mL). Trityl chloride (7.05 g, 25.3 mmol) was added to the stirred
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solution and the mixture was stirred at 20 °C for 16 hours. Water (30 mL) was added and the mixture
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
3
3
3
3
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3
4
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4
4
4
4
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4
4
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5
5
5
5
5
5
5
5
5
5
6
was stirred additional 10 min. The aqueous phase was extracted with dichloromethane (3). The
combined organic phase was dried with MgSO , solids were removed by filtration, and the solvent was
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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5
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7
8
9
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
evaporated in vacuo. The crude product was purified by silica gel chromatography petroleum
ether/EtOAc (1:0 → 0:1, v/v). This procedure afforded the title compound 14 as a white amorphous
1
25
solid (5.9 g, 65%). HNMR (400MHz, d -DMSO) δ 7.42-7.32 and 7.30-7.24 (2  m, 12 + 3H, o, m,
6
p-Tr), 6.41 (d, 1H, J = 4.0 Hz, OH), 5.15 (d, 1H, J = 4.0 Hz, H-1), 4.56 (dd, 1H, J = 6.0 Hz, J = 1.0
Hz, H-2), 4.36 (d, 1H, J = 6.0 Hz, H-3), 4.11-4.05 (m, 1H, H-4), 3.16-3.05 (m, 2H, H-5), 1.37 and 1.23
+
+
(2 × s, 2 × 3H, CH iPr). HRMS (ES ) calcd for C H NaO 455.1829 [M+Na] , found 455.1842.
3
27 28
5
2, 3-O-Isopropylidene-5-O-trityl-D-ribitol (15). 2, 3-O-Isopropylidene-5-O-trityl-D-ribofuranose
4 (5.2 g, 11.98 mmol) was dissolved in EtOH and cooled to 0 °C. Sodium borohydride (460 mg, 12.1
1
mmol) was added to the solution in three portions over the period of 30 min. The mixture was stirred
at 20 °C for 2 h. Water (80 mL) was added and the solution was carefully acidified with acetic acid to
pH 6. Then the aqueous phase was extracted with dichloromethane (3). The combined organic phase
was dried (Na SO ), solids were removed by filtration and the solvent was evaporated in vacuo. The
2
4
crude product was purified by Isco-Flash chromatography using CH Cl /Acetone (1:0 →1:1, v/v). This
2
2
1
procedure afforded the title compound 15 as an amorphous white solid (4.35 g, 83%). HNMR
400MHz, d -DMSO) δ 7.48-7.41 (m, 6H, o-Tr), 7.35-7.28 (m, 6H, m-Tr), 7.26-7.20 (m, 3H, p-Tr),
(
6
5.15 (d, J = 5.6 Hz, OH-4), 4.80 (t, J = 5.6 Hz, OH-1), 4.17-4.10 (m, 1H, H-2), 4.08-4.01 (m, 1H, H-
3
1
), 3.78-3.66 (m, 2H, H-4 and H-1 ), 3.51-3.43 (m, 1H, H-1 ), 3.15-3.11 (m, 1H, H-5 ), 3.06-3.01 (m,
a b a
1
3
1
H, H-5 ), 1.20 (s, 6H, CH iPr). C{ H}NMR (100MHz, d -DMSO) δ 144.0 (3 x C-ipso-Tr), 128.4
b
3
6
(
6 x C-o-Tr), 127.7 (6 x C-m-Tr), 126.8 (3 x C-p-Tr), 107.4 (C(CH )), 85.7 (C(Tr)), 78.2 (C-2), 76.4
3
+
(C-3), 68.2 (C-4), 66.1 (C-1), 60.0 (C-5), 27.8 and 25.4 (CH iPr). HRMS (ES ) calcd for C H NaO
3
27 30
5
+
4
57.1985 [M+Na] , found 457.2004.
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-Deoxy-2, 3-O-isopropylidene-5-O-trityl-D-ribofuranose (16).2,3-O-Isopropylidene-5-O-trityl-
D-ribitol 15 (1.89 g, 4.35 mmol) was dissolved in pyridine (15 mL) and tosyl chloride (2.49 g, 13.06
mmol) was added. The mixture was stirred at 60 °C for 16 h. TLC analysis showed the reaction was
complete. The tosyl chloride surplus was decomposed by addition of water (50 mL) and the aqueous
phase was extracted with dichloromethane (3). The combined organic phase was dried (Na SO ),
0
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2
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4
5
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7
8
9
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solids were removed by filtration and the solvent was evaporated in vacuo. The crude product was
purified by Isco-Flash chromatography using petroleum ether/EtOAc (1:0 → 1:1, v/v), affording the
1
title compound 16 as an amorphous white solid (1.44 g, 80%). HNMR (400MHz, CDCl ) δ 7.44-7.39
3
(m, 6H, o-Tr), 7.33-7.27 (m, 6H, m-Tr), 7.26-7.21 (m, 3H, p-Tr), 4.91-4.86 (m, 1H, H-2), 4.65 (dd,
1
H, J = 6.4 Hz, J = 1.2 Hz, H-3), 4.24-4.19 (m, 1H, H-4), 4.15 (dd, 1H, J = 10.4 Hz, J = 4.4 Hz, H-
1a), 4.05 (dd, 1H, J = 10.0 Hz, J = 1.0 Hz, H-1 ), 3.27 (dd, 1H, J = 10.0 Hz, J = 4.0 Hz, H-5 ), 3.11
b
a
1
3
1
(dd, 1H, J = 10.0 Hz, J = 4.4 Hz, H-5 ), 1.51 and 1.34 (2  s, 6H, CH iPr). C{ H}NMR (100MHz,
b
3
CDCl ) δ 143.6 (3  C-ipso-Tr), 128.6 (6  C-o-Tr), 127.8 (6  C-m-Tr), 127.2 (3  C-p-Tr), 112.3
3
(C(CH )), 87.1 (C(Tr)), 84.0 (C-4), 83.0 (C-3), 81.6 (C-2), 74.3 (C-1), 64.5 (C-5), 26.6 and 25.1
3
+
+
(CH iPr). HRMS (ES ) calcd for C H NaO 439.1880 [M+Na] , found 439.1868.
3
27 28
4
1-Deoxy-2,3-O-isopropylidene-D-ribofuranose (17). 1-Deoxy-2,3-O-isopropylidene-5-O-trityl-D-
ribofuranose 16 (100 mg, 0.24 mmol) was dissolved in diethylether-HCOOH (2 mL, 1:1, v/v) and
stirred at 20 °C for 16 h. TLC analysis (petroleum ether/EtOAc, 1:1, v/v) indicated the complete
conversion of reaction and the mixture was evaporated to dryness. The dry residue was suspended in
water and the suspension was filtered through a cotton pad. Evaporation of water afforded the title
1
compound 17 as a colorless viscous liquid (14.5 mg, 32%). HNMR (400MHz, CDCl ) δ 4.83-4.78
3
(m, 1H, H-2), 4.59 (dd, 1H, J = 6.4 Hz, J = 2.0 Hz, H-3), 4.13-4.09 (m, 1H, H-4), 3.98-3.95 (m, 2H,
H-1 ), 3.65 (dd, 1H, J = 11.6 Hz, J = 4.0 Hz, H-5 ), 3.58 (dd, 1H, J = 11.6 Hz, J = 6.4 Hz, H-5 ), 1.51
a, b
a
b
1
3
1
and 1.33 (2  s, 6H, CH iPr). C{ H}NMR (100MHz, CDCl ) δ 113.0 (C(CH )), 84.8 (C-4), 81.8 (C-
3
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), 81.0 (C-2), 72.8 (C-1), 61.8 (C-5), 26.7 and 25.0 (CH iPr). HRMS (ES ) calcd for C H NaO
3 8 14 4
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
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2
2
3
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+
1
97.0784 [M+Na] , found 197.0791.
1
-Deoxy-2,3-O-isopropylidene-D-ribofuranose-5-O-di-tert-butylphosphate (18). 1-deoxy-2,3-O-
0
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0
isopropylidene-D-ribofuranose 17 (94 mg, 0.49 mmol) and 5-phenyl-1H-tetrazole (145 mg, 0.99
mmol) were co-evaporated with dry toluene (2). Then, the dry mixture was dissolved in
dichloromethane (2 mL) and di-tert-butyl N, N-diisopropylphosphoramidite (234 µL, 0.74 mmol) was
added dropwise. The mixture was stirred at 20 °C for 2 hours after which TLC analysis (petroleum
ether/EtOAc, 2:1, v/v) showed the reaction was complete. It was cooled to 0 °C and triethylamine (411
µL, 2.97 mmol) added, followed by hydrogen peroxide (35% aq., 109 µL, 1.24 mmol). The resulting
mixture was stirred at 20 °C for 1 hour. The mixture was diluted with EtOAc (20 mL) and extracted
with aqueous Na SO (10%, w/v). The organic phase was dried (Na SO ), solids were filtered off and
2
3
2
4
the solvent was evaporated in vacuo. The crude product was purified by Isco-Flash chromatography
using petroleum ether/EtOAc (1:0 → 0:1, v/v), to afford the title compound 18 as a colorless liquid
1
(74 mg, 41%). HNMR (400MHz, CDCl ) δ 4.85-4.81 (m, 1H, H-2), 4.76 (dd, 1H, J = 6.4 Hz, J = 1.2
3
Hz, H-3), 4.23-4.18 (m, 1H, H-4), 4.2-3.94 (m, 4H, H-1, H-5), 1.51 (s, 3H, CH iPr), 1.48 (s, 18H, CH
3
3-
3
1
13
1
tBu), 1.33 (s, 3H, CH iPr). PNMR (160MHz, CDCl ) δ -10.1 (s, P). C{ H}NMR (100MHz, CDCl )
3
3
3
δ 112.6 (C(CH )), 83.1 (d, J = 8.0 Hz, C(tBu)), 82.6 (d, J = 7.0 Hz, C-4), 82.3 (C-3), 81.2 (C-2), 73.9
3
+
(
C-1), 66.6 (d, J = 7.0 Hz, C-5), 29.8-29.7 (m, CH tBu), 26.5 and 24.9 (CH iPr)). HRMS (ES ) calcd
3
3
+
for C H NaO P 389.1700 [M+Na] , found 389.1699.
1
6
31
7
Tributylammonium 1-deoxy-2,3-O-isopropylidene-D-ribofuranose-5-phosphate (10). Di-tert-
butyl 1-deoxy-2,3-O-isopropylidene-D-ribofuranose-5-phosphate 18 (36 mg, 0.098 mmol) was
dissolved in aqueous methanol (1:1, v/v, 2 mL) and the solution was cooled to 0 °C. Trifluoroacetic
acid (2 mL) was added dropwise and the solution was allowed to warm up to 20 °C and stirred for an
additional 4 h. The solution was evaporated to dryness and the residue co-evaporated with water (3)
and with methanol (3). The procedure afforded the pure phosphate derivative as a colorless gum (21
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mg, quantitative) which was directly dissolved in water (2 mL), neutralized with tributylamine (33 µL,
0
.14mmol), the solvent evaporated and the residue co-evaporated with EtOH (2). The title compound
1
10 was obtained as the mono-tributylammonium salt (18.7 mg, 49%). HNMR (400MHz, MeOH) δ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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0
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.21-4.16 (m, 1H, H-2), 4.12-4.07 (m, 1H, H-3), 4.03-3.87 (m, 4H, H-1 , 4, 5 ), 3.69 (dd, 1H, J = 9.6
a a,b
Hz, J = 4.0 Hz, H-1 ), 3.10-3.03 (m, 6H, NCH ), 1.73-1.64 (m, 6H, NCH CH ), 1.41 (m, 6H,
b
2
2
2
N(CH ) CH ), 1.00 (t, 9H, J = 7.6 Hz, CH Bu). 31PNMR (160MHz, MeOH) δ 1.09 (s, P).
2
2
2
3
13
1
C{ H}NMR (100MHz, MeOH) δ 83.3 (d, J = 8.0 Hz, C-4), 73.7 (C-1), 73.6 (C-3), 72.5(C-2), 66.3
-
(
d, J = 5.0 Hz, C-5), 54.0 (NCH ), 27.0 (NCH CH ), 21.0 (N(CH ) CH ), 14.0 (CH Bu). HRMS (ES )
2
2
2
2 2
2
3
-
calcd for C H O P 213.0170 [M-H] , found 213.0162.
5
11
7
2-Deoxy-D-ribofuranose-5-phosphate sodium salt (19). Compound 19 (sodium salt) was purchased
from Sigma Aldrich (UK) and found to be a mixture of α and β anomers in the ratio 1/1.17 α/β).
1
HNMR (500MHz, D O) δ 5.52-5.47 (m, 1.6H, H-1α, β, deuterium exchange), 4.51-4.46 (m, 1H, H-
2
3
β), 4.38-4.34 (m, 1H, H-3α), 4.25-4.20 (m, 1H, 4α), 4.04-3.99 (m, 1H, H-4β), 3.85-3.81 (m, 2H, H-
5β), 3.81-3.75 (m, 2H, H-5α), 2.51-2.42 (m, 1H, H-2α), 2.22-2.14 (m, 2H, H-2β), 1.91-1.83 (m, 1H,
31
13
1
H-2α). PNMR (202MHz, D O) δ 3.65 (P-β) and 3.68 (P-α). C{ H}NMR (126MHz, D O) δ 98.4
2
2
(C-1β), 98.0 (C-1α), 84.9 (d, J = 10.08 Hz, C-4β), 84.6 (d, J = 8.82 Hz, C-4α), 71.6 (C-3β), 71.1 (C-
3α), 64.6 (d, J = 5.0 Hz, C-5β), 63.7 (d, J = 3.8 Hz, C-5α), 40.8 (C-2β), 40.6 (C-2α). Compound 19
was transformed to its tributylammonium salt.
2
-Deoxyribose-5-phosphate mono-tributylammonium salt (11). Commercially available 2-
deoxyribose monophosphate sodium salt 19 (Sigma Aldrich) (8 mg, 0.029 mmol) was dissolved in
+
water (2 mL) and acidified to pH 1-2 by using the ion-exchange resin Dowex® (D50 H ). The resin
was filtered off and the free phosphate was neutralized by addition of one equivalent of tributylamine
(6.9 µL, 0.029 mmol). The aqueous solution was freeze-dried (quant.). (NMR analysis showed partial
decomposition (approx. 20-30%), of 2ʹ-deoxyribose). The compound itself is sensitive to basic
1
conditions (pH 9)). HNMR (500MHz, D O) δ 5.50-5.47 (m, 0.54H, H-1β), 5.43-5.41 (dd, 0.46H, J =
2
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.5 Hz, J = 2.5 Hz, H-1α), 4.43-4.39 (m, 0.53H, H-3β), 4.29-4.25 (m, 0.47H, H-3α), 4.15-4.1 (m,
.46H, H-4α), 4.02-3.97 (m, 0.7H, H-5β), 3.94-3.85 (m, 1.32H, H-4β, 5α, 5β), 3.84-3.77 (m, 0.52H,
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H-5α), 3.09-3.01 (m, 10H, NCH (CH ) CH ), 2.38-2.30 (m, 0.5H, H-2α), 2.12-1.99 (m, 1H, H-2β),
2
2 2
3
0
1
2
3
4
5
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1
.86-1.79 (m, 0.5H, H-2α), 1.72-1.60 (m, 10H, NCH CH CH CH ), 1.47-1.35 (m, 10H,
2 2 2 3
3
1
NCH CH CH CH ), 1.04-0.94 (m, 15H, NCH (CH ) CH ). PNMR (202MHz, D O) δ 1.10 (P-β)
2
2
2
3
2
2 2
3
2
1
3
1
and 1.03 (P-α). C{ H}NMR (126MHz, D O) δ 100.0 (C-1β), 99.5 (C-1α), 86.3 (d, J = 8.4 Hz, C-4β),
2
8
5.6 (d, J = 8.7 Hz, C-4α), 73.5 (C-3β), 73.0 (C-3α), 67.3 (d, J = 5.4 Hz, C-5β), 66.1 (d, J = 5.3 Hz,
C-5α), 53.8 (NCH (CH ) CH ), 43.0 (C-2β), 42.6 (C-2α), 26.9 (NCH CH CH CH ), 21.1
2
2 2
3
2
2
2
3
(
NCH CH CH CH ), 14.0 (NCH (CH ) CH ).
2 2 2 3 2 2 2 3
1
, 2-O-Isopropylidene-D-xylofuranose (20).34 Finely powdered D-xylose (10 g, 67 mmol) was
suspended in acetone (260 mL) containing sulphuric acid (10 mL) and stirred for 30 min until
dissolved. The solution was cooled to 0 °C and a solution of Na CO (13 g in 112 mL H O) was added.
2
3
2
The mixture was stirred at 20 °C for 1 hour and then solid Na CO (7 g) was added. The slurry was
2
3
stirred at 20 °C for an further 30 min. Solids were removed by filteration and washed with acetone.
Acetone was evaporated and the water phase was extracted with EtOAc (3) and dried with Na SO .
2
4
Isco-Flash chromatography using petroleum ether/EtOAc (1:0 → 1:1, v/v) afforded the title compound
20 as a colorless viscous oil (4.35 g, 34%) and doubly protected 1, 2-O-isopropylidene-3,5-O-
isopropylidene-D-xylofuranose (3.29 g, 21%).34 1HNMR (400MHz, CDCl ) δ 5.98 (d, 1H, J = 3.6 Hz,
3
H-1), 4.52 (d, 1H, J = 3.6 Hz, H-2), 4.32 (d, 1H, J = 2.4 Hz, H-4), 4.19-4.11 (m, 2H, H-3, 5 ), 4.05
a
1
3
1
(dd, 1H, J = 12.4 Hz, J = 2.4 Hz, H-5 ), 1.48 and 1.32 (CH iPr). C{ H}NMR (100MHz, CDCl ) δ
b
3
3
1
12.0 (C(CH ) ), 105.1 (C-1), 85.9 (C-2), 78.7 (C-3), 77.3 (C-4), 61.5 (C-5), 26.9 and 26.3 (CH iPr).
3 2 3
+
+
HRMS (ES ) calcd for C H NaO 213.0733 [M+Na] , found 213.0787.
8
14
5
5
-O-tert-Butyl(diphenyl)silyl-1, 2-O-isopropylidene-D-xylofuranose (21). 1, 2-O-Isopropylidene-
D-xylofuranose 20 (3.63 g, 19.08 mmol) and DMAP (5 mg) were dissolved in pyridine (15 mL). tert-
Butyl(diphenyl)silyl chloride (4.89 mL, 19.08 mmol) was added dropwise and the solution was stirred
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at 20 °C for 16 hours. Solvent was evaporated and the crude product was purified by Isco-Flash
chromatography using Petroleum ether/EtOAc (1:0 → 1:1, v/v). This procedure afforded the title
1
compound 21 as a white solid (6.58 g, 83%). HNMR (400MHz, CDCl ) δ 7.74-7.65 (m, 4H, o-Ph),
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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2
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4
5
6
7
8
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1
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4
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0
7
1
1
1
.48-7.38 (m, 6H, m, p-Ph), 6.00 (d, 1H, J = 3.6 Hz, H-1), 4.54 (d, 1H, J = 3.6 Hz, H-2), 4.39-4.35 (m,
H, H-4), 4.16-4.12 (m, 1H, H-3), 4.12-4.09 (m, 2H, H-5), 3.99 (d, 1H, J = 2.4 Hz, 3-OH), 1.47 and
1
3
1
.33 (CH iPr), 1.06 (CH tBu). C{ H}NMR (100MHz, CDCl ) δ 135.8 and 135.7 (o-Ph), 132.6 and
3
3
3
32.1 (C-ipso-Ph), 130.2 and 128.1 (2  d, J = 2.2 Hz, J = 1.7 Hz, m, p-Ph), 111.8 (C(CH ) ), 105.2
3
2
(
C-1), 85.6 (C-2), 78.6 (C-3), 77.0 (C-4, obstructed by CDCl signal), 62.9 (C-5), 27.0 and 26.3
3
+
+
(CH iPr), 26.9 (CH tBu), 19.2 (C(CH ) ). HRMS (ES ) calcd for C H NaO Si 451.1911 [M+H] ,
3
3
3 3
24 32
5
found 451.1929.
5
-O-tert-Butyl(diphenyl)silyl-1,
2-O-isopropylidene-3-thiocarbonylimidazolo-D-xylofuranose
(22). A mixture of 5-O-tert-butyl(diphenyl)silyl-1, 2-O-isopropylidene-D-xylofuranose 21 (3.0 g, 7.01
mmol) and 1, 1ʹ-thiocarbonyldiimidazole (2.37 g, 13.32 mmol) was gently refluxed in dichloroethane
at 85 °C for 2 hours. The solvent was evaporated and the crude product was purified by Isco-Flash
chromatography using petroleum ether/EtOAc (1:0 → 0:1, v/v) to afford the title compound 22 as a
1
white solid (2.4 g, 63%). HNMR (400MHz, CDCl ) δ 8.24-8.21 (m, 1H, Imidazole), 7.64-7.60 (m,
3
2H, Ph), 7.55-7.51 (m, 2H, Ph), 7.47 (t, 1H, J = 1.6 Hz, Imidazole), 7.46-7.33 (m, 4H, Ph), 7.29-7.23
(m, 2H, Ph), 7.04 (q, 1H, J = 0.8 Hz, Imidazole), 5.98 (d, 1H, J = 2.8 Hz, H-3), 5.94 (d, 1H, J = 4.0
Hz, H-1), 4.72 (d, 1H, J = 4.0 Hz, H-2), 4.61 (dq, 1H, J = 8.8 Hz, J = 2.8 Hz, H-4), 4.02 (dd, 1H, J =
10.0 Hz, J = 4.4 Hz, H-5 ), 3.82 (dd, 1H, J = 10.0Hz, J = 8.4 Hz, H-5 ), 1.60 and 1.36 (2  s, 2  3H,
a
b
13
1
CH iPr), 1.02 (s, 9H, CH tBu). C{ H}NMR (100MHz, CDCl ) δ 182.6 (C=S), 136.9 (Imidazole),
3
3
3
1
35.6 and 135.4 (Ph), 132.8 and 132.7 (C-ipso-Ph), 131.2 (Imidazole), 130.1 (Ph), 127.9 and 127.8
(Ph), 117.8 (Imz), 112.8 (C(CH ) ), 105.0 (C-1), 84.2 (C-3), 82.7 (C-2), 78.9 (C-4), 60.3 (C-5), 26.9
3
2
+
(
CH tBu), 26.7 and 26.4 (CH iPr), 19.2 (C(CH ) ). HRMS (ES ) calcd for C H N NaO SSi
3 3 3 3 28 34 2 5
+
5
61.1850 [M+Na] , found 561.1837.
2
8
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Page 29 of 47
The Journal of Organic Chemistry
1
2
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5
-O-tert-Butyl(diphenyl)silyl-1, 2-O-isopropylidene-3-deoxy-D-ribofuranose (23). Tributyltin
hydride (1.07 mL, 3.99 mmol) was added into boiling toluene (90 mL) under argon followed by AIBN
(13 mg, 0.08 mmol). Then 5-O-tert-butyl(diphenyl)silyl-1, 2-O-isopropylidene-3-
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9
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thiocarbonylimidazolo-D-xylofuranose 22 (1.26 g, 2.35 mmol) was added dropwise in toluene (20
mL). The mixture was refluxed for 3 hours. Solvent was evaporated and the crude product was purified
by Isco-Flash chromatography using petroleum ether/EtOAc (1:0 → 0:1, v/v) to afford the title
1
compound 23 as a white solid (731 mg, 76%). HNMR (400MHz, CDCl ) δ 7.71 (m, 4H, o-Ph), 7.45-
3
7
.35 (m, 6H, m, p-Ph), 5.83 (d, 1H, J = 3.6 Hz, H-1), 4.75 (t, 1H, J = 4.0 Hz, H-2), 4.33 (dq, 1H, J =
0.4 Hz, J = 4.0 Hz, H-4), 3.82 (dd, 1H, J = 11.2 Hz, J = 4.4 Hz, H-5 ), 3.76 (dd, 1H, J = 11.2 Hz, J =
1
a
4.0 Hz, H-5 ), 3.08 (dd, 1H, J = 13.6 Hz, J = 4.8 Hz, H-3 ), 1.88 (ddd, 1H, J = 13.6 Hz, J = 10.4 Hz, J
b
a
1
3
1
=
4.8 Hz, H-3 ), 1.52 and 1.34 (2  s, 2  3H, CH iPr), 1.06 (s, 9H, CH tBu). C{ H}NMR (100MHz,
b
3
3
CDCl ) δ 135.8 and 135.7 (o-Ph), 133.7 and 133.5 (C-ipso-Ph), 129.8 and 129.8 (p-Ph), 127.8 and
3
127.8 (m-Ph), 111.3 (C(CH ) ), 105.9 (C-1), 80.6 (C-2), 78.7 (C-4), 64.9 (C-5), 35.1 (C-3), 27.1
3
2
+
(CH iPr), 27.0 (CH tBu), 26.5 (CH iPr), 19.4 (C(CH ) ). HRMS (ES ) calcd for C H NaO Si
3 3 3 3 3 24 32 4
+
4
35.1962 [M+Na] , found 435.1995.
1
, 2-O-Isopropylidene-3-deoxy-D-ribofuranose (24). A mixture of TBAF.3H O (1.55 g, 5.0 mmol)
2
and acetic acid (300 µL, 5.25 mmol) in DMF (10 mL) was stirred at room temperature for 30 min. The
mixture was cooled to 0 °C and 5-O-tert-butyl(diphenyl)silyl-1, 2-O-isopropylidene-3-deoxy-D-
ribofuranose 23 (687 mg, 1.67 mmol) was added dropwise in DMF (5 mL). The resulting mixture was
stirred at 20 °C for 3 hours. Solvent was evaporated and the crude product was purified by Isco-Flash
chromatography using petroleum ether/EtOAc (1:0 → 0:1, v/v). This procedure afforded the title
1
compound 24 as white solid (260 mg, 82%). HNMR (400MHz, CDCl ) δ 5.79 (d, 1H, J = 3.6Hz, H-
3
1
α), 4.73 (t, 1H, J = 4.4 Hz, H-2), 4.31 (dtd, 1H, J = 10.8 Hz, J = 4.4 Hz, J = 3.2 Hz, H-4), 3.84 (dd,
1H, J = 12.0 Hz, J = 2.8 Hz, H-5 ), 3.54 (dd, 1H, J = 12.0 Hz, J = 4.4 Hz, H-5 ), 1.97 (dd, 1H, J = 13.6
a
b
1
3
1
Hz, J = 4.8 Hz, H-3 ), 1.85-1.76 (m, 1H, H-3 ), 1.48 and 1.29 (2  s, 2  3H, CH iPr). C{ H}NMR
a
b
3
2
9
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