Synthesis of phosphonoacetate analogues of the second messenger adenosine 5′-diphosphate ribose (ADPR)

Article abstract of DOI:10.1039/c9ra09284f

Adenosine 5′-diphosphate ribose (ADPR) is an intracellular signalling molecule generated from nicotinamide adenine dinucleotide (NAD⁺). Synthetic ADPR analogues can shed light on the mechanism of activation of ADPR targets and their downstream effects. Such chemical biology studies, however, are often challenging due to the negatively charged pyrophosphate that is also sensitive to cellular pyrophosphatases. Prior work on an initial ADPR target, the transient receptor potential cation channel TRPM2, showed complete pyrophosphate group replacement to be a step too far in maintaining biological activity. Thus, we designed ADPR analogues with just one of the negatively charged phosphate groups removed, by employing a phosphonoacetate linker. Synthesis of two novel phosphonoacetate ADPR analogues is described via tandem N,N′-dicyclohexylcarbodiimide coupling to phosphonoacetic acid. Neither analogue, however, showed significant agonist or antagonist activity towards TRPM2, underlining the importance of a complete pyrophosphate motif in activation of this particular receptor.

Full text of DOI:10.1039/c9ra09284f

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Synthesis of phosphonoacetate analogues of the
0
second messenger adenosine 5 -diphosphate
Cite this: RSC Adv., 2020, 10, 1776
ribose (ADPR)†
b
ab
c
c
Ondrej Baszczynski,‡ Joanna M. Watt,
Monika D. Rozewitz, Ralf Fliegert,
ˇ
ˇ
Andreas H. Guse and Barry V. L. Potter *ab
c
0
Adenosine 5 -diphosphate ribose (ADPR) is an intracellular signalling molecule generated from
+
nicotinamide adenine dinucleotide (NAD ). Synthetic ADPR analogues can shed light on the mechanism
of activation of ADPR targets and their downstream effects. Such chemical biology studies, however, are
often challenging due to the negatively charged pyrophosphate that is also sensitive to cellular
pyrophosphatases. Prior work on an initial ADPR target, the transient receptor potential cation channel
TRPM2, showed complete pyrophosphate group replacement to be a step too far in maintaining
biological activity. Thus, we designed ADPR analogues with just one of the negatively charged phosphate
groups removed, by employing a phosphonoacetate linker. Synthesis of two novel phosphonoacetate
Received 8th November 2019
Accepted 16th December 2019
0
ADPR analogues is described via tandem N,N -dicyclohexylcarbodiimide coupling to phosphonoacetic
DOI: 10.1039/c9ra09284f
acid. Neither analogue, however, showed significant agonist or antagonist activity towards TRPM2,
rsc.li/rsc-advances
underlining the importance of a complete pyrophosphate motif in activation of this particular receptor.
whole cells are oen hampered by ADPR's negative charges and
lack of membrane permeability. Studies of these processes
The linear diphosphate adenosine 5 -diphosphate ribose oen require technically demanding and time-consuming
Introduction
0
(
ADPR, Fig. 1a) is formed from nicotinamide adenine dinucle- patch clamp or microinjection experiments on single cells to
+
otide (NAD ) by two routes; either hydrolysis of the nicotin- deliver charged species physically through the cell membrane.
+
1
amide ribose by NAD glycohydrolases such as CD38 or via the Furthermore, ADPR is subject to hydrolysis of the pyrophos-
sequential action of poly-ADPR polymerase (PARP) and poly- phate by enzymes (intracellular enzymes, ectoenzymes in the
2
ADPR glycohydrolase (PARG). PARP inhibitors are currently plasma membrane and enzymes present in biological uids),
3
highly topical as drugs in oncology. ADPR's cyclic incarnation including hydrolases of the NUDIX family. This means that
as cyclic ADP-ribose (cADPR) is a second messenger in cellular ADPR itself is unlikely to be stable for any length of time in
4,5
calcium signalling. cADPR is also hydrolysed to ADPR as a biological setting, complicating the direct study of its effects.
a deactivation mechanism. ADPR targets include widespread Thus, synthetic ADPR analogues that are both membrane per-
6
macro domains and the non-selective cation channel, transient meant and stable in the biological environment would be
receptor potential cation channel, subfamily M, member 2 valuable tools to unravel the downstream mechanisms and
7,8
(
TRPM2). They are associated with a diverse range of physio- function of this ligand. Stable ADPR analogues may also be
logical processes that are still not yet fully understood. However, useful when clarication of the precise second messenger that
chemical biological studies of ADPR-mediated processes in is acting under physiological conditions has proved
9,10
challenging.
Small changes to the structure of ADPR have demonstrated
profound effects on activity and, as a result, synthetic analogues
closely based on the ADPR structure have been very informa-
a
Medicinal Chemistry and Drug Discovery, Department of Pharmacology, University of
Oxford, Manseld Road, Oxford, OX1 3QT, UK
b
Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and
11–13
11,13
tive.
terminal ribose
Analogues with modications in the adenosine
and
Pharmacology, University of Bath, Bath, BA2 7AY, UK. E-mail: barry.potter@pharm.
14–17
ox.ac.uk
c
motifs have been prepared via coupling of
0
0
The Calcium Signalling Group, Department of Biochemistry and Molecular Cell a modied 5 -O-phosphoryl adenosine or ribose to its 5 -O-
Biology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246
phosphoryl counterpart using morpholidate, diphenylphosph-
onate or imidazolide methods to activate one phosphate
towards nucleophilic attack and thus generate the pyrophos-
phate. Notwithstanding their utility for studying ADPR
processes using single cell techniques such as patch clamping,
Hamburg, Germany
1
13
†
3
Electronic supplementary information (ESI) available: H-NMR, C-NMR and
P-NMR spectra for compounds 1, 2 and 5–10. See DOI: 10.1039/c9ra09284f
Present address: Department of Organic Chemistry, Faculty of Science, Charles
1
‡
University, Hlavova 2030/8, 128 43 Prague 2, Czech Republic.
1776 | RSC Adv., 2020, 10, 1776–1785
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Fig. 1 (a) ADPR; (b–e) ADPR analogues modified in the pyrophosphate motif and (f) the targets 1 and 2 of the present work.
further studies even with many of these designed analogues are protected on some or all of the ADPR hydroxyl groups selectively
24
limited by the negative charge and potential biological insta- inhibited TRPM2.
These hydrolysis resistant analogues
bility of the pyrophosphate moiety. Such pyrophosphate- demonstrate that modication of the pyrophosphate can impart
containing ADPR analogues are thus not suitable for use in valuable attributes to the ADPR analogue. However, such meth-
whole cell studies or for in vivo applications. The cell permeable ylene bis-phosphonate analogues still carry two negative charges
peptide tat-M2NX has been shown to inhibit TRPM2 and reduce at physiological pH, and thus are not membrane permeable.
18
ischemic injury in male mice, suggesting that membrane
Our previous highly ambitious complete replacements of the
permeable analogues closely related to ADPR in structure would pyrophosphate group in ADPR (Fig. 1c and d), that are unlikely

nd similar utility.
to be charged at physiological pH, unfortunately did not show
11,13
The negative charge of ADPR may be reduced by masking the TRPM2 agonist or antagonist activity.
We therefore pursued
charged phosphate groups. Indeed, ADPR that was acetylated a more conservative approach in this study, designing two ADPR
and bioreversibly masked with a lipophilic group on the adeno- analogues 1 and 2 with only one phosphorus centre replaced
19
sine phosphate has been reported. However, pyrophosphates (Fig. 1f). We chose the isosteric phosphonoacetate group as
masked in this way can be more unstable towards hydrolysis by a pyrophosphate mimic between the adenosine and the
nucleophiles than their negatively charged counterparts, and this terminal ribose of ADPR, as this analogue would only carry
does not address the issue of degradation due to cellular pyro- a single negative charge at physiological pH. A phosphonoace-
phosphatases. The use of phosphate bioisosteres in medicinal tate motif should also be resistant to the intrinsic cellular
chemistry is an alternative method to improve cellular stability pyrophosphatase activity of some ADPR targets as the P–C bond
20
25
and potentially also improve membrane permeability. Previous is more chemically stable than the pyrophosphate P–O bond.
studies using pyrophosphate replacement in ADPR have Therefore, such analogues could potentially be used to probe
provided some new insights into ADPR binding. The methylene ADPR-mediated activities in isolation from pyrophosphatase
bis-phosphonate analogue of ADPR (a-b methylene ADPR, activity. The rst phosphonoacetate analogues of the 5-O-pyro-
AMPCPR, Fig. 1b) contains a non-hydrolysable P–C–P pyrophos- phosphate in diphosphoinositol polyphosphates (PP-InsPs)
phate substitution that is stable towards pyrophosphatases and were recognised and phosphorylated by the kinase domain of
has therefore generated a number of crystal structures of ADPR diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2),
21
targets bound to AMPCPR. T ´o th et al. showed that AMPCPR still showing that such substitution of a pyrophosphate did not
supports TRPM2 channel gating, albeit as a lower affinity partial prevent recognition of the analogue by its target enzyme and
22
agonist. AMPCPR has been used to uncouple the TRPM2 allowing downstream effects unrelated to dephosphorylation to
26
channel gating mechanism from its enzymatic activity by virtue be studied.
of its pyrophosphatase resistant methylene bis-phosphonate
We therefore report here the synthesis of phosphonoacetate
22,23
linker.
Two further ADPR analogues with similarly modied ADPR analogues 1 and 2, studies on the stability of both
pyrophosphates (one methylene bisphosphate and one analogues and their initial biological evaluation at TRPM2.
diuoromethylene bisphosphate, see Fig. 1e) that were also
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occurred at rt) plays a crucial role in the conversion of 6 to 7.
Synthesis of ADPR analogues
Finally, with the optimized conditions, the DCC mediated
0
0
0
0
Synthesis of adenosine-5 -O-(2-phosphoryl) acetate ribose coupling of 6 with 1 -O-methyl-2 , 3 -O-isopropylidene-b-D-ribo-
(
Fig. 1f, 1) started with commercially available triethylphos- furanoside 12 generated the desired ADPR analogue 7 (best
phonoacetate 3 (Scheme 1). Selective deprotection of the yield 57%). Deprotection of 7 led to target compound 1, which
27
carboxylic ester in the presence of the phosphate esters led to was obtained aer HPLC purication in 0.1 M TEAB buffer with
the corresponding carboxylic acid 4 (97%). Carboxylic acid 4 was a gradient of acetonitrile as the triethylammonium salt (36%).
0
esteried, by N,N -dicyclohexylcarbodiimide (DCC) mediated Until deprotection of analogue 7, all reactions are stereospe-
0
0
coupling, with 2 ,3 -O-isopropylidene adenosine 11 to obtain cic. The nal ADPR analogue 1 is a mixture of a and b anomers
compound 5 (82%). Diethylphosphonate ester 5 was then trans- at the terminal ribose (ratio a : b ¼ 2 : 3, as detailed in the
silylated by bromotrimethylsilane (TMSBr), followed by hydro- Experimental section).
0
lysis to obtain phosphonic acid 6 (best yield 46%). Selective
Synthesis of adenosine-5 -phosphonoacetyl ribose (Fig. 1f, 2)
removal of the phosphonate esters in the presence of the iso- started from diethylphosphonoacetic acid 4 (Scheme 2), that
0
0
0
propylidene group was achieved by carrying out the reaction in was coupled to 1 -O-methyl-2 ,3 -O-isopropylidene-b-D-ribofur-
pyridine, as the basic conditions buffer the HBr generated. anoside 12 using DCC, to give compound 8 (best yield 74%).
Carrying out the same conversion with DCM as a solvent Trans-silylation and hydrolysis of 8, followed by silica gel
generated a complex mixture due to concomitant deprotection chromatography gave phosphonic acid 9 (68%) contaminated
0
0
of the isopropylidene protecting group. Purication of the free with trace impurities. Compound 9 was directly coupled to 2 ,3 -
phosphate 6 was carried out using normal phase ash chro- O-isopropylidene adenosine 11 via previously optimized DCC
ꢀ
matography on silica gel. Impurities were eluted rst with an coupling at elevated temperature 120 C to give compound 10
isocratic solvent system of ethyl acetate, methanol and water (56%). Deprotection of 10 led to the nal compound 2, which
(
7 : 2 : 1 v/v/v) containing 0.1% triethylamine. The desired was puried several times using HPLC in 0.1 M TEAB buffer
product 6 was then eluted by switching to a second isocratic with a gradient of acetonitrile as the triethylammonium salt
solvent system of isopropyl alcohol, water and aqueous (best yield 67%). Until deprotection of analogue 10, all reactions
ammonia (7 : 2 : 1 v/v/v) containing 0.1% triethylamine. First are stereospecic. The nal ADPR analogue 2 is a mixture of
attempts to obtain compound 7 via DCC mediated coupling to a and b anomers at the terminal ribose (ratio a : b ¼ 2 : 3, as
0
0 0
-O-methyl-2 , 3 -O-isopropylidene-b-D-ribofuranoside 12 were detailed in the Experimental section).
1
unsuccessful. However, aer a short optimization, we found
ꢀ
that at elevated temperature (50–70 C) compound 6 forms
Stability of target phosphonoacetates 1
and 2
a mixture of anhydrides, including most likely adenosine
phosphonoacetate anhydride which only reacts with 12 at
temperatures above 100 C. Thus, the higher temperature of
20 C (compared to the DCC mediated reaction of 4 and 11 that
28
ꢀ
The stability of 1 and 2 in solution was monitored by analytical
HPLC, comparing freshly made samples from the solid
ꢀ
1
ꢀ
Scheme 1 Synthesis of phosphonoacetate ADPR analogue 1. Reagents and conditions: (a) NaOH, water, EtOH, 20 C (97%); (b) DCC, DMAP
ꢀ
ꢀ
ꢀ
ꢀ
(
cat.), THF, 20 C (82%); (c) TMSBr, pyridine, 20 C, 16 h (46%); (d) DCC, pyridine, 120 C, 16 h (57%); (e) aqueous TFA 75%, 2 h, 20 C (36%).
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ꢀ
Scheme 2 Synthesis of phosphonoacetate ADPR analogue 2. Reagents and conditions: (a) DCC, DMAP (cat.), THF, 20 C (74%); (b) TMSBr,
ꢀ
ꢀ
ꢀ
pyridine, 20 C, 16 h (68%); (c) DCC, pyridine, 120 C, 16 h (56%); (d) aqueous TFA 75%, 2 h, 20 C (60%).
ꢀ
compounds 1 and 2, stored at ꢁ20 C. Both nal analogues were nd that the corresponding sodium salt of 2 was even more
synthesised as the corresponding triethylammonium salts (1.0 unstable and degraded completely either at room temperature
ꢂ Et
3
N and 1.2 ꢂ Et
3
N equivalents respectively, calculated from or when stored in the fridge (Fig. 2).
It was rather unexpected that ADPR analogue 1 is stable
1
H-NMR spectra). While phosphonoacetate 1 is stable in this
form in MilliQ water, TEAB (0.1 M) or MeCN solutions, we were under various aqueous conditions whereas ADPR analogue 2 is
surprised to observe that phosphonoacetate 2, degrades slowly not. This led us to consider which feature of 2 might be
in TEAB (pH 8–9, 0.1 M TEAB, ESI, Fig. S1†). HPLC of 2 (R ¼ 3.9 responsible for the observed degradation. Our observations
t
minutes) aer 16 h in 0.1 M TEAB buffer showed an additional suggest that 2 is sensitive to basic hydrolysis of the phospho-
peak at R
t
¼ 2.7 minutes. This decomposition product showed noacetate ester in TEAB buffer but that, under the same
a characteristic adenosine UV absorption (lmax ¼ 259) and was conditions, the phosphonoacetate ester of 1 is stable. This
+
ꢁ
00
suggests that the terminal ribose 5 -O-ester may inuence the
analysed using mass spectrometry (ESI found [M ꢁ H]
388.0676, C12
H
15
N
5
O
8
P requires 388.0664) suggesting loss of stability in a way that is not observed with the adenosine ribose
0
the terminal ribose to generate adenosine 5 -phosphonoacetate
(ESI, Fig. S1†). We suspect it is most likely that 2 undergoes
basic hydrolysis of the terminal ribose from the phosphonoa-
cetate ester mediated by triethylamine from the TEAB buffer.
We then measured the stability of phosphonoacetate 2 under
various aqueous conditions (Fig. 2). According to these ndings,
analogue 2 was stable in pure MilliQ water (pH 7) as the trie-
thylammonium salt when stored in the fridge. Importantly, this
should be sufficient for biological assays using the pure phos-
phonoacetate 2 when used immediately from a frozen sample.
Increased pH 8–9 (in 0.1 M TEAB buffer) resulted in slow
decomposition of 2 either at room temperature or when stored
in the fridge. We prepared the sodium salt of analogue 2 in an
attempt to overcome the possibility of triethylammonium
counter ions generating a basic solution and thus causing the
decomposition of 2. The triethylammonium salt of 2 was stirred
+
with Chelex (Na form) resin, which was removed by ltration to
generate the sodium salt of 2. However, we were surprised to
Fig. 2 Stability of compound 2 under various aqueous conditions.
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0
5
-O-ester. The triethylamine base present in TEAB buffer may
catalyse hydrolysis of the ester bond between the phospho-
0
0
noacetate ester and the 5 -O-terminal ribose. Indeed, we have
previously observed that modications to the terminal ribose
can generate ADPR analogues that are particularly prone to
14
instability. Analogue 1 is not susceptible to hydrolysis because
the more stable phosphonate monoester is adjacent to the
terminal ribose. Phosphate monoesters are stable under basic
conditions due to the negative charge on the phosphonate
0
oxygen preventing nucleophilic attack. Presumably, the 5 -O-
ester in analogue 1 is more sterically hindered, due to its
proximity to the less exible adenosine, and thus is less
susceptible to hydrolysis under mildly basic conditions. In
future, synthesis of the attractive corresponding phosphona-
mide ADPR analogues in which the ester is replaced by an
amide linker, may be one method to ensure stable analogues
are generated, as exemplied in a recent synthesis of an inositol
2
9
polyphosphate pyrophosphate analogue.
Fig. 3 Effect of 1 and 2 on whole cell currents in hTRPM2 expressing
HEK293 cells. Electrophysiological analysis of the impact of ADPR
analogues on human TRPM2. (a) During whole cell patch clamp
experiments using HEK293 cells with stable expression of the ion
channel, the analogues were included in the pipette solution at 100
Biological evaluation of 1 and 2
Due to our stability studies, we were aware of the possibility of
ꢁ1
ꢁ1
mmol L or 1000 mmol L . The maximum outward current at +15 mV
degradation of the unstable ester bond of compound 2. This over a time of 450 s from individual cells is plotted at a logarithmic
would have been undesirable in our assay to determine the scale with a horizontal bar indicating the mean value. Neither
compound did result in a significantly higher current than the buffer
agonist and antagonist effects of 2. In order to prevent degra-
control. (b) To check for potential inhibition of TRPM2 currents from
dation, the compounds were reconstituted using MilliQ water
pH 7), which was shown to generate a stable solution of both
ꢁ1
activation by ADPR, the compounds were added at 300 mmol L or
(
ꢁ1
ꢁ1
9
00 mmol L to a pipette solution already containing 100 mmol L
compounds (Fig. 2). The assay was carried out at pH 7.2. ADPR. Neither compound reduced the TRPM2 current significantly
Immediately before the assay for agonist or antagonist effects, when compared to the control (pipette solution containing only
ADPR). The numbers at the bottom indicate the number of cells
analysed.
the solution of the analogue to be tested was veried by HPLC.
No degradation was detected during the entire sequence of
testing, or even when the pH 7 solution of the analogue was
ꢀ
stored at ꢁ80 C for 1 year.
molecule may lead to the complete loss of activity. The single
atom substitution in the methylene bis-phosphonate analogue
ADPR analogues 1 and 2 were tested for their agonist and
antagonist activity on human TRPM2 (hTRPM2) using patch
AMPCPR corresponded to a roughly 40 fold reduction in
clamp experiments in HEK293 clones with stable expression of
potency at hTRPM2 and AMPCPR is also only a partial hTRPM2
13
hTRPM2. Because the compound is infused into the cyto-
plasm of the cell via the patch-pipette, this method allows
evaluation of the activity of the compounds independently of
their membrane permeability. Following the results of our
stability testing, phosphonoacetate 2 was stored as a lyophilized
22
agonist. Alternatively the substitution of the pyrophosphate
with the phosphonoacetate bioisostere may alter the presenta-
tion of other critical binding partners in the hTRPM2 binding
pocket. Our molecular modelling suggests the phosphonoace-
tate linker theoretically seems to be a very good spatial mimic of
the pyrophosphate unit, with sufficient exibility to allow the
adenosine and terminal ribose binding partners to position
themselves in the same orientation as ADPR (ESI, Fig. S2†), but
this may not reect the reality once solvent effects and binding
interactions are taken into account.
ꢀ
dry solid at ꢁ20 C and made up in fresh MilliQ water imme-
diately before testing to ensure that decomposition did not
occur (vide supra). When applied at 100 mM or 1000 mM, neither
nor 2 had signicant agonist effects at hTRPM2 (Fig. 3a).
1
When applied with ADPR in the patch pipette (100 mM of ADPR
with 300 mM or 900 mM 1 or 2), neither analogue showed
signicant antagonist effects on the ADPR-induced TRPM2
current (Fig. 3b). If activity was observed, we would have
determined the membrane permeability of 1 and 2 by applying
them extracellularly to whole cells, however this was not
possible.
These results highlight the sensitivity of hTRPM2 channel
activation to modications of the ADPR molecular structure. It
is possible that both phosphate motifs of the pyrophosphate
bridge are required for binding and activation or inhibition of
the channel, so that even a subtle change in this part of the
Cryo-EM structures of hTRPM2 have recently improved
understanding of the zebrash hTRPM2 apo and ADPR-bound
30
states importantly for hTRPM2 also in the presence of
31,32
bound ligands.
However, only recently has their resolution
become detailed enough to place ADPR in the binding pocket
and has revealed, for the rst time, ADPR binding sites in
32
hTRPM2 that may act synergistically. The requirement for an
ADPR mimic to bind to both sites, which possess very distinct
30
shapes, may explain why very few synthetic ADPR analogues
11,15
have activated the channel.
Despite this recent advance, the
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structure activity relationship for hTRPM2 is not fully under- spectrometer with electrospray ionisation (ESI). Analytical
stood, and ADPR analogues remain essential for ligand-based HPLC was performed on a Waters 2695 Alliance module
drug design.
coupled to a Waters 2996 PDA Detector (210–350 nm) equipped
The crystal structure of the macro domain of thermophilic with Hichrom Guard Column for HPLC and a Phenomenex
˚
protein Af1521 bound to ADPR was solved in high resolution Synergi column (4u, MAX-RP, 80 A, 150 ꢂ 4.60 mm), eluted at 1
6
ꢁ1
˚
(
1.5 A, PDB 2BFQ). With the exception of the adenosine ribose, mL min with a gradient of MeCN in 0.05 M TEAB. Semi-
each ADPR structural feature interacts closely with the macro preparative HPLC was performed on a Waters 2525 pump
0
domain structure. The adenosine 5 -O-phosphate has two with manual FlexInject using a Phenomenex Gemini column
ꢁ1
˚
hydrogen bonds to the backbone N–H of valine 43 and glycine (5u, C18, 110 A, 250 ꢂ 10.00 mm), eluted at 5 mL min
.
0
1
43. The ribose 5 -O-phosphate has hydrogen bonds to the
1-O-Methyl-2,3-O-isopropylidene-b-D-ribofuranoside
(12).
backbone N–H of serine 141 and tyrosine 145. Furthermore, the Compound 12 was prepared according to the literature from D-
32
1
pyrophosphate is stabilized by the helix dipole. Thus, the ribose. Yield 63%. H NMR (400 MHz, CDCl ) d 4.93 (s, 1H, H-
3
demonstrated highly specic binding interactions of macro 1), 4.78 (d, J ¼ 6.0 Hz, 1H, H-2), 4.54 (d, J ¼ 6.0 Hz, 1H, H-3), 4.38
3
domains and apparent similar selectivity of hTRPM2 binding (bs, 1H, H-4), 3.66–3.55 (m, 2H, H-5a,b), 3.38 (s, 3H, OCH ), 3.19
require very specic ADPR binding partners. Our initial results (bs, 1H, OH), 1.44 (s, 3H, CH
with hTRPM2 suggest that phosphonoacetate analogues 1 and 2 Diethylphosphonoacetic acid (4). Compound 4 was prepared
may not be able to full all these demands, but 1 and 2 may from commercially available triethyl phosphonoacetate
3 3
), 1.27 (s, 3H, CH ).
27
potentially nd applicability in other systems that bind ADPR. following the described procedure. Yield 97% of compound 4.
1
H NMR (400 MHz, d -DMSO) d 12.6 (bs, 1H, OH), 4.09 (q, 4H, J
6
¼
7.2 Hz, CH CH ), 3.02 (d, 2H, J
¼ 21.2, CH P), 1.29 (t, 6H,
2
3
CH
2
–P
2
Conclusion
3
1
J ¼ 7.2 Hz, CH
2
3 6
CH ). P NMR (400 MHz, d -DMSO) d 20.93 (s).
0
0
0
We have synthesised two phosphonoacetate analogues of ADPR,
5 -O-[(Diethoxyphosphoryl)acetate]-2 ,3 -O-isopropylidene
in which the pyrophosphate is substituted by a phosphonoace- adenosine (5). Diethylphosphonoacetic acid 4 (0.9 g, 4.59
0
0
tate linker. These analogues retain all other ADPR features but mmol) and 2 ,3 -O-isopropylidene adenosine (1 g, 3.25 mmol)
0
would be predicted to carry only a single negative charge at were dissolved in THF (50 mL). N,N -Dicyclohexylcarbodiimide
physiological pH. Suitably protected ribose and adenosine (DCC, 1 g, 4.8 mmol) was added to the stirred solution in one
building blocks were sequentially coupled to the phosphonoa- portion, followed by catalytic amount of DMAP (20 mg, 0.16
cetate core by carbodiimide-mediated coupling, followed by mmol). The resulting mixture was stirred at room temperature
0
a global deprotection to give phosphonoacetates 1 and 2. overnight under argon atmosphere. The precipitate (N,N -dicy-
Analogue 2, in which the phosphonoacetate ester is attached to clohexylurea) was ltered off and the solution was evaporated.
0
0
ꢀ
the terminal ribose 5 -hydroxyl, has unexpected instability in The residue was dissolved in EtOAc, cooled to 5 C, and ltered
TEAB buffer. Future efforts would seek to avoid using ester links through a sintered glass lter. The solution was evaporated and
in such analogues, but likely employ amide linkages. Neither the crude product was puried by Isco-Flash chromatography
analogue showed any agonist activity at hTRPM2, nor antago- eluted with petroleum ether–EtOAc (6 : 4 v/v) and a gradient of
nist effects on the ADPR-induced current at TRPM2, suggesting MeOH (0–10%). This procedure afforded the title compound 5
1
the full pyrophosphate motif is required for activity and adding as colourless glass (0.98 g, 62%). H NMR (400 MHz, d -DMSO)
6
to the growing picture of a highly specic binding interaction d 8.34 (s, H-8), 8.16 (s, H-2), 7.34 (s, 2H, NH
2
), 6.19 (d, 1H, J ¼
0
0
with many requisite ADPR features.
2.68 Hz, H-1 ), 5.47 (dd, 1H, J ¼ 6.0 Hz, J ¼ 2.96 Hz, H-2 ), 5.04
0
0
(
dd, 1H, J ¼ 6.4 Hz, J ¼ 3.09 Hz, H-3 ), 4.39–4.35 (m, 1H, H-4 ),
0
0
4
.31–4.27 ðm; 1H; H ꢁ 5 Þ; 4.23–4.18 ðm; 1H; H-5 Þ; 4.04–3.95
Experimental section
General
a
b
(m, 4H, CH
2
CH
3
), 3.11 (d, 2H, JH–P ¼ 21.36 Hz, CH
2
P), 1.54 (s,
3
H, C(CH ) ), 1.33 (s, 3H, C(CH ) ), 1.18 (m, 6H, J ¼ 3.16 Hz,
3
3
2
1
3 2
13
Reagents and solvents were purchased from commercial sour- CH
ces and used without further purication, unless described (100 MHz, d
otherwise. Triethylamine was dried with potassium hydroxide, 148.81 (C-4), 139.83 (C-8), 119.08 (C-5), 113.49 (C(CH
CH
). P NMR (161 MHz, d
-DMSO) d 19.64 (s). C NMR
2
3
6
-DMSO) d 165.44 (COO), 156.14 (C-6), 152.75 (C-2),
6
3
)
2
0
), 89.00
0
0
0
0
puried by distillation, and stored over potassium hydroxide. (C-1 ), 83.31 (C-4 ), 83.08 (C-2 ), 81.02 (C-3 ), 64.56 (C-5 ), 62.05
MilliQ quality water was used for all aqueous experiments and and 61.99 (CH CH
purications. All reactions were performed under an inert 25.19 (C(CH ), 16.06 and 16.01 (CH
29 5 8
atmosphere of argon unless otherwise stated. For NMR experi- for C19H N O P 486.1748, found [M + H] 486.1789.
2
3
), 33.22 (d, JC–P ¼ 130.57 Hz, C–P), 26.99 and
+
3
)
2
2 3
CH ). HRMS (ES ) calcd
+
1
13
31
0 0 0
5 -O-Phosphonic acid-2 ,3 -O-isopropylidene adenosine (6).
ments; H, C, and P NMR spectra were collected using either
a Varian Mercury 400 MHz or Bruker Avance III 500 MHz Bromotrimethylsilane (1.2 mL, 9.12 mmol) was added drop-
1
13
0
0
0
Spectrometer. All H and C NMR assignments are based on wise to a solution of 5 -O-[(diethoxyphosphoryl) acetate]-2 ,3 -
COSY, HSQC, HMBC, and DEPT experiments. Chemical shis O-isopropylidene adenosine 5 (0.553 g, 1.14 mmol) in dry
ꢀ
(
d) are reported in parts per million (ppm) and splitting patterns pyridine (6 mL) at 0 C. The mixture was stirred at room
are abbreviated as follows: br, broad; s, singlet; d, doublet; t, temperature for 5 h and then another portion of bromo-
triplet; m, multiplet etc. High resolution mass spectra (HRMS) trimethylsilane (0.5 mL) was added dropwise. The mixture
were obtained on
a Bruker Daltonics micrOTOF mass was stirred at room temperature and monitored via
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 1776–1785 | 1781

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RSC Advances
Paper
3
1
00 00
6
-DMSO) d 8.14. Aer 2 ,3 -O-isopropylidene-b-D-ribofuranoside 7 (10.7 mg, 0.0165
phosphorus NMR, P NMR (161 MHz, d
6 h, the solvent was evaporated and the residue was co- mmol) was dissolved in 75% aqueous CF COOH (8 mL) and the
1
3
evaporated with aqueous ammonia. The crude material was mixture was stirred at room temperature for 2 h. The reaction
ꢀ
puried by silica gel chromatography. Impurities were eluted mixture was quickly evaporated at 30 C to dryness and the
with EtOAc–MeOH–H
product was eluted using iPrOH–H
v/v/v + 0.1% Et N). This procedure afforded the title (TEAB, 2 mL, 0.1 M) and puried by semi-preparative, reverse-
compound 6 as a white foam, as the mono-triethylammo- phase HPLC using a gradient of 0.1 M TEAB-acetonitrile
2
O (7 : 2 : 1 v/v/v + 0.1% Et
3
N) and the residue was co-evaporated (3ꢂ) with Milli-Q water. The crude
2
O–aq. ammonia (7 : 2 : 1 product was dissolved in triethylammonium bicarbonate buffer
3
1
nium salt (179 mg, 30%). H NMR (400 MHz, d
6
-DMSO) (95 : 5 / 35/65 v/v). The title compound 1 was afforded as
d 8.59 (s, 1H, H-8), 8.16 (s, 1H, H-2), 7.33 (s, 2H, NH ), 6.13 colourless glass, as a mixture of a and b anomers in the ratio
2
0
1
1
(
d, 1H, J ¼ 3.16 Hz, H-1 ), 5.54 (dd, 1H, J ¼ 6.4 Hz, J ¼ 3.2 Hz, a : b ¼ 2 : 3, (3.1 mg, 36%, Et N salt). DOSY H NMR (water
3
3
0
0
0
H-2 ), 5.10 (dd, 1H, J ¼ 6.0 Hz, J ¼ 3.0 Hz, H-3 ), 4.39 (m, 1H, suppression, 500 MHz, D O) d 8.32 (s, 1H, H-8), 8.18 (s, 1H, H-2),
2
0
0
00
H-4 ), 4.22 ðdd; 1H; J ¼ 12:4 Hz; J ¼ 4:8 Hz; H-5 Þ; 4.06 6.03 (d, 1H, J ¼ 4.5 Hz, H-1 ), 5.16 (d, 0.4H, J ¼ 4.0 Hz, H-1 a),
a
0
00
ðdd; 1H; J ¼ 12:4 Hz; J ¼ 4:0 Hz; H-5 Þ; 2.89 (q, 6H, J ¼ 5.10 (d, 0.6H, J ¼ 2.5 Hz, H-1 b), 4.77 (t, 1H, obscured by D
2
O
b
0
0
7
.25, NCH
CH P), 1.53 (s, 3H, C(CH
H, J ¼ 7.26 Hz, NCH CH
2
CH
3
), 2.53 (dq, 2H, JH–P ¼ 22.4 Hz, Jgem ¼ 8.0 Hz, peak, J ¼ 2.5 Hz, H-2 ), 4.45 (t, 1H, J ¼ 5.0 Hz, H-3 ), 4.41–4.32
0
0
00
2
3
)
2
), 1.18 (s, 3H, C(CH
). P NMR (161 MHz, d
3
)
2
), 1.11 (t, (m, 3H, H-4 , 5 ), 4.12 (t, 0.6H, J ¼ 5.0 Hz, H-3 b), 4.07–4.04 (m,
3
1
00
00
00
00
00
a;b
6
2
3
6
-DMSO) 0.4H, H-4 a), 4.01–3.75 ðm; 4H; H-2 a=b; 3 a; 4 b; 5 Þ; 3.12 (q,
1
3
d 8.14 (s). C NMR (100 MHz, d -DMSO) d 176.66 (COO) 2H, J ¼ 7.0 Hz, NCH CH ), 2.81 and 2.79 (2 ꢂ dd, 2H, J
¼ 15.5
6
2
3
H–P
HMBC, 156.06 (C-6), 152.76 (C-2), 149.13 (C-4), 139.84 (C-8), and 16 Hz, J
¼ 5.0 and 4.5 Hz, CH P a, b), 1.20 (t, 3H, J ¼
gem
2
0
0
31
1
8
3
18.79 (C-5), 113.27 (C(CH ) ), 88.69 (C-1 ), 83.40 (C-4 ), 7.0 Hz, NCH CH ). P NMR (203 MHz, D O) d 14.40 (s), 14.30
3
2
2
3
2
0
0
0
13
3.16 (C-2 ), 81.01 (C-3 ), 63.52 (C-5 ), 45.05 (NCH
8.52 and 38.02, very low intensity, CH
2
CH
3
), (d, (s). C NMR (126 MHz, D
2
O) d 169.70 (COO) HMBC, 155.53 (C-
2
P signal partially 6), 152.73 (C-2), 149.02 (C-4), 139.99 (C-8), 118.87 (C-5), 101.05
hidden by the DMSO signal, estimated from HSQC), 27.02 (C-1 b), 96.28 (C-1 a), 87.58 (C-1 ), 81.85 and 81.16 (C-4 , 4 a
and 25.20 (C(CH ) ), 8.70 (NCH CH ). HRMS (ES ) calcd for and b), 75.06 (C-2 b), 73.44 (C-2 ), 70.65, 70.44, 70.00, 69.85 (C-
0
0
00
0
0
00
ꢁ
00 0
3 2 2 3
ꢁ
00
0
00
0
00
C
15
H
2
19
0
N
5
O
8
P 428.0977, found [M ꢁ H] 428.0985.
2 a, 3 a/b, 3 ), 65.31 (d, J ¼ 4.5 Hz, C-5 ), 64.00 (d, J ¼ 5.5 Hz, C-
0
0
,3 -O-Isopropylidene
adenosine-5 -O-(2-phosphoryl) 5 ), 46.65 (NCH CH ), 35.03 and 34.34 (d, J
acetate-1 -O-methyl-2 ,3 -O-isopropylidene-b-D-ribofuranoside
7). 5 -O-Phosphonic acid-2 ,3 -O-isopropylidene adenosine 6 (NCH CH ). HRMS (ES ) calcd for C H N O P 520.1086,
¼ 121.4 Hz,
2
3
C–P
00
00 00
CH P), 35.27 and 34.29 (d, J
¼ 122.1 Hz, CH P), 8.19
2
C–P
2
0
0
0
ꢁ
(
(
2
3
17 23 5 12
ꢁ
28 mg, 0.053 mmol) and 1-O-methyl-2,3-O-isopropylidene-b-D- found [M ꢁ H] 520.1097. Analytical HPLC: R
t
¼ 4.14 min.
ribofuranoside 12 (20 mg, 0.098 mmol) were dissolved in
5-O-[(Diethoxyphosphoryl)acetate]-1-O-methyl-2, 3-O-isopro
0
anhydrous pyridine (1 mL). N,N -dicyclohexylcarbodiimide pylidene-b-D-ribofuranoside (8). 1-O-Methyl-2,3-O-isopropylidene
(
20 mg, 0.097 mmol) was added in one portion to the stirred -b-D-ribofuranoside 12 (100 mg, 0.49 mmol) and dieth-
ꢀ
mixture and the mixture was heated at 120 C for 16 h. The ylphosphonoacetic acid 4 (96 mg, 0.49 mmol) were dissolved in
reaction mixture was evaporated to dryness and the compound dry THF (5 mL). N,N -Dicyclohexylcarbodiimide (142 mg, 0.68
0
was puried by silica-gel chromatography CH Cl –MeOH-0.1% mmol) was added to the stirred solution in one portion followed
2
2
Et N (10–40%) to afford the title compound 7 as a white solid by catalytic amount of DMAP (2 mg, 0.016 mmol). The resulting
3
1
8
1
(
(
3
6
5
18.6 mg, 54%, Et
3
N salt). H NMR (400 MHz, d
6
-DMSO) d 8.69 mixture was stirred at room temperature for 16 h under argon.
0
s, 1H, H-8), 8.16 (s, 1H, H-2), 7.34 (bs, 2H, NH
2
), 6.13 (d, 1H, J ¼ The precipitate (N,N -dicyclohexylurea) was ltered off and the
0
0
.0 Hz, H-1 ), 5.56 (q, 1H, J ¼ 2.99 Hz, H-2 ), 5.07 (dd, 1H, J ¼ solution was evaporated to dryness. The residue was dissolved in
0
00
ꢀ
.4 Hz, J ¼ 2.93 Hz, H-3 ), 4.86 (s, 1H, H-1 ), 4.75 (d, 1H, J ¼ EtOAc, cooled to 5 C, and ltered through a sintered glass lter.
00
00
.96 Hz, H-2 ), 4.52 (d, 1H, J ¼ 5.96 Hz, H-3 ), 4.42–4.39 (m, 1H, The solution was evaporated to dryness and the crude product was
0
0
00
H-4 ), 4.26–4.22 ðm; 1H; H-5 Þ; 4.15–4.11 (m, 1H, H-4 ), 4.06– puried by Isco-Flash chromatography eluting with petroleum
a
0
00
4
6
.02 ðm; 1H; H-5 Þ; 3.66–3.57 ðm; 2H; H-5 Þ; 3.53 (q, 0.7H, J ¼ ether–EtOAc (1 : 0 / 0 : 1 v/v) to afford the title compound 8 as
b
a;b
1
.32 Hz, NCH CH ), 3.19 (s, 3H, CH O), 2.60–2.47 (m, 2H, a colourless glass (112 mg, 60%). H NMR (400 MHz, CDCl ) d 4.96
2
3
3
3
CH
2
P), 1.53 and 1.33 (2 ꢂ s, 2 ꢂ 3H, C(CH
3
)
2
-Adn), 1.36 and 1.23 (s, 1H, H-1), 4.67 (d, 1H, J ¼ 6.0 Hz, H-2), 4.59 (d, 1H, J ¼ 6.0 Hz, H-
3
1
(
2 ꢂ s, 2 ꢂ 3H, C(CH
3
)
2
-Rib). P NMR (161 MHz, d
6
-DMSO) 3), 4.35 (t, 1H, J ¼ 7.2 Hz, H-4), 4.21–4.13 (m, 6H, H-5, CH
2
CH
P), 1.46 (s, 3H,
), 1.30 (s, 3H, C(CH ).
3
) d 19.22. C NMR (100 MHz, CDCl )
3
),
1
3
d 7.60 (s). C NMR (100 MHz, d
6
-DMSO) d 169.60 (COO) HMBC, 3.31 (s, 3H, OCH
3
), 2.98 (d, 2H, J ¼ 21.6, CH
), 1.34 (t, 6H, J ¼ 7.2 Hz, CH CH
2
1
5
8
8
3
56.04 (C-6), 152.76 (C-2), 149.13 (C-4), 140.15 (C-8), 118.75 (C- C(CH
), 113.23 (Adn C(CH
8.75 (C-1 ), 85.18 (C-4 ), 84.42 (C-3 ), 83.52 (C-4 ), 83.30 (C-2 ), d 165.72 (COO), 112.90 (C-(CH ) ), 109.79 (C-1), 85.50 (C-3), 84.29
)
3 2
2
13
3
3 2
)
0
0
0
31
3
00
)
2
), 111.22 (Rib C(CH
3
)
2
), 108.45 (C-1 ),
P NMR (161 MHz, CDCl
3
0
00
0
3
2
00
0
00
0
1.46 (C-2 ), 81.04 (C-3 ), 64.00 (C-5 ), 63.50 (C-5 ), 54.15 (CH O), (C-4), 82.15 (C-2), 65.88 (C-5), 63.19 (d, JC–P ¼ 6.06 Hz, CH CH ),
3
2
3
6.32 (d, J ¼ 112.0 Hz, CH P a and b, HMBC), 27.03 and 25.21 55.34 (OCH ), 34.67 (d, J ¼ 133.79 Hz, CH P), 26.73 (CH ), 25.29
2
3
C–P
2
3
ꢁ
+
(C(CH
3
)
2
-Adn), 26.22 and 24.61 (C(CH
3
)
2
-Rib). HRMS (ES ) (CH
3
), 16.70 (CH
2
CH
3
). HRMS (ES ) calcd for C15
H O
28 9
P 383.1465,
ꢁ
+
calcd for C24
Analytical HPLC, R
H
33
N
5
O
12P 614.1869, found [M ꢁ H] 614.1885. found [M + H] 383.1474.
t
¼ 8.62 min.
5-O-Phosphonic acid-1-O-methyl-2,3-O-isopropylidene-b-D-
0
0
0
Adenosine-5 -O-(2-phosphoryl)acetate ribose (1). 2 ,3 -O-Iso- ribofuranoside (9). Bromotrimethylsilane (1.33 mL, 10.1 mmol)
propylidene adenosine-5 -O-(2-phosphoryl) acetate-1 -O-methyl- was
0
00
added
dropwise
to
the
solution
of
5-O-
1782 | RSC Adv., 2020, 10, 1776–1785
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Paper
(diethoxyphosphoryl)
RSC Advances
[
acetate]-1-O-methyl-2,3-O-iso- co-evaporated with water (3ꢂ). The crude product was dissolved
propylidene-b-D-ribofuranoside 8 (0.642 g, 1.68 mmol) and in triethylammonium bicarbonate buffer (TEAB, 2 mL, 0.1 M)
triethylamine (4.18 mL, 30.25 mmol) in dry dichloromethane and puried by semi-preparative, reverse-phase HPLC eluting
ꢀ
(
1
d
20 mL) at 0 C. The mixture was stirred at room temperature for with a gradient of 0.1 M TEAB-acetonitrile (95 : 5 / 35 : 65 v/v)
3
1
6 h and monitored via phosphorus NMR, P NMR (161 MHz, to afford the title compound 2 as a colourless glass, as a mixture
1
6
-DMSO) d 8.36. The solution was evaporated to dryness and of a and b anomers a/b ¼ 2/3 (7.6 mg, 60%, Et
3
N salt). DOSY
3
1
the residue was co-evaporated with aqueous ammonia. The
H NMR (water suppression, 500 MHz, D
2
O) d 8.37 (s, 1H, H-8),
0
crude material was puried by silica gel chromatography. 8.15 (s, 1H, H-2), 6.04 (d, 1H, J ¼ 5.5 Hz, H-1 ), 5.21–5.19 (m,
0
0
00
Impurities were eluted with EtOAc–MeOH–H
2
O (7 : 2 : 1 v/v/v + 0.4H, H-1 a), 5.11 (d, 0.6H, J ¼ 1.5 Hz, H-1 b), 4.67 (t, 1H, J ¼
0
0
0
0
.1% Et N) and the product was eluted using iPrOH–H O–aq. 5.0 Hz, H-2 ), 4.44–4.40 (m, 1H, H-3 ), 4.31–4.27 (m, 1H, H-4 ),
ammonia (7 : 2 : 1 v/v/v + 0.1% Et N) to afford phosphonic acid 4.24
3
2
0
ðdd; 1H; J ¼ 12:0 Hz; J ¼ 3:5 Hz; H-5 Þ;
4.71–3.96
3
a;b
0
0
00
00
0
00
9
(603 mg, 68%, mono-triethylammonium salt) which was used ðm; 5:4H; H-2 a; 3 a=b; 4 a=b; 5 ; 5 a=bÞ; 3.90–3.88 (m, 0.6H,
a;b
1
00
for the next reaction without any further purication. H NMR H-2 b), 3.11 (q, 2H, J ¼ 7.0 Hz, NCH
2
3
CH
3
), 2.87–2.79 (m, 2H,
). P NMR (203 MHz, D O)
O) d 169.72 (COO),
1
(
400 MHz, d
6
-DMSO) d 4.91 (s, 1H, H-1), 4.75 (d, 1H, J ¼ 7.5 Hz, CH
2
P), 1.19 (t, 3H, J ¼ 7.5, NCH
2
CH
3
2
1
3
H-2), 4.57 (d, 1H, J ¼ 7.5 Hz, H-3), 4.22 (t, 1H, J ¼ 9.0 Hz, H-4), d 14.61 (s), 14.50 (s). C NMR (126 MHz, D
2
3
CH
.98–3.86 (m, 2H, H-5), 3.22 (s, 3H, MeO), 2.78 (q, 2H, J ¼ 9.0 Hz, 155.52 (C-6), 152.77 (C-2), 149.1 (C-4), 139.74 (C-8), 118.69 (C-5),
31
00
00
00
0
0
2
P), 1.37 (s, 3H, C(CH
3
)
2
), 1.25 (s, 3H, C(CH
3
)
2
). P NMR (161 101.18 (C-1 b), 96.34 (C-1 a), 87.02 (C-1 ), 83.76 (C-4 ), 79.83 and
1
3
00
00
0
MHz, d -DMSO) d 8.36 (s). C NMR (400 MHz, d -DMSO) 79.44 (C-3 a/b, 4 a/b), 74.93 (C-2 b), 74.20 (C-2 ), 70.50 and
6
6
0
0
00
0
0
d 168.51 (d, J ¼ 6.5 Hz, COO), 111.44 (C(CH ) ), 108.73 (C-1), 70.28 and 70.10 (C-3 a/b, 2 a, 3 ), 65.30 ðC-5 Þ; 64.58 and
3
2
a;b
0
0
8
4
4.44 (C-3), 83.37 (C-4), 81.23 (C-2), 64.03 (C-5), 54.23 (MeO), 63.84 (C-5 a/b), 46.63 (NCH CH ), 35.14 and 34.18 (d, J
C–P
¼
2
3
ꢁ
ꢁ
4.82 (CH
2
P), 26.20 and 24.63 (2 ꢂ C(CH
3
ꢁ
)
2
). HRMS (ES ) calcd 121.1 Hz, CH
2
P a and b), 8.18 (NCH
2
CH
3
). HRMS (ES ) calcd for
ꢁ
for C11
H
0
18
O
9
P 325.0694, found [M ꢁ H] 325.0702.
C
17
H
23
N
5
O
12P 520.1086, found [M ꢁ H] 520.1086. Analytical
0
0
00
2
,3 -O-Isopropylidene adenosine-5 -O-phosphonoacetyl-1 - HPLC: RT ¼ 3.68 min.
00
00
O-methyl-2 ,3 -O-isopropylidene-b-D-ribofuranoside (10). 5-O-
Phosphonic acid-1-O-methyl-2,3-O-isopropylidene-b-D-ribofur-
Pharmacology
0
0
anoside 9 (65 mg, 0.123 mmol) and 2 ,3 -O-isopropylidene
adenosine 11 (57 mg, 0.185 mmol) were dissolved in anhydrous
pyridine (2 mL). N,N -Dicyclohexylcarbodiimide (51 mg, 0.246 cillin (100 U mL ) and streptomycin (100 mg mL ) at 37 C and
Cell culture. HEK293 cells were kept in DMEM medium (4.5 g
ꢁ1
L
glucose, Glutamax-I) supplemented with 10% FBS and peni-
0
ꢁ1
ꢁ1
ꢀ
mmol) was added in one portion to the stirred mixture and the 5% CO . The medium for the stable cell lines additionally con-
2
ꢀ
ꢁ1
mixture heated to 120 C for 16 h. The reaction mixture was tained 400 mg mL G418 sulfate. The parental HEK293 cells used
evaporated to dryness and the compound was puried by silica- to generate the cell line with stable expression of human TRPM2
gel chromatography eluted with CH
0.1% Et N) to afford the title compound 10 as a white solid Biochemistry and Signal Transduction, University Medical Centre
2 2
Cl
–MeOH (9 : 1 / 6 : 4 v/v were provided by Prof. Dr Manfred J u¨ cker (Institute of
+
3
1
(52 mg, 56%). H NMR (500 MHz, D O) d 8.41 (s, 1H, H-8), 8.24 Hamburg-Eppendorf). The cells were authenticated by short
2
0
(
s, 1H, H-2), 6.27 (d, 1H, J ¼ 3.0 Hz, H-1 ), 5.43–5.41 (m, 1H, H- tandem repeat proling (DSMZ service for the authentication of
0
0
00
2
4
6
5
), 5.20 (dd, 1H, J ¼ 5.0 Hz, J ¼ 2.0 Hz, H-3 ), 4.93 (s, 1H, H-1 ), cell lines, https://www.dsmz.de/services/human-and-animal-cell-
0
00
.71–4.66 (m, 1H, H-4 ), 4.58 (d, J ¼ 6.0 Hz, H-2 ), 4.43 (d, 1H, J ¼ lines/authentication-of-human-cell-lines) and are being tested
00
00
0
.0 Hz, H-3 ), 4.18–4.12 (m, 3H, H-4 , 5 ), 4.00–32.96 (m, 1H, H- regularly regarding mycoplasma contamination using an enzy-
0
0
00
3
a), 3.86–3.81 (m, 1H, H-5 b), 3.23 (s, 3H, CH O), 2.84 (dq, 2H, matic assay (MycoAlert mycoplasma detection kit; Lonza).
J
H–P ¼ 20.5 Hz, J ¼ 13.5 Hz, Jgem ¼ 5.0 Hz, CH
2
P), 1.68 (s, 3H,
Generation of HEK293 cells with stable expression of
C(CH -Adn), 1.40 (s, 3H, C(CH -Adn), 1.46 (s, 3H, C(CH
Rib), 1.27 (s, 3H, C(CH ) -Rib). P NMR (161 MHz, D O) d 14.11 and EGFP were generated as described before. In brief the
3
)
2
3
)
2
3
)
2
-
TRPM2. HEK293 cells with stable expression of human TRPM2
3
1
13
3
2
2
1
3
(
s). C NMR (126 MHz, D O) d 169.17 (d, J ¼ 6.8 Hz, COO), cells were transfected with pIRES2-EGFP-TRPM2 (containing
2
1
5
9
8
5
55.56 (C-6), 152.79 (C-2), 148.78 (C-4), 139.86 (C-8), 118.55 (C- the coding sequence for the full-length of human TRPM2 under
0
0
), 114.76 (C(CH
3
)
2
-Adn), 112.96 (C(CH
3
)
2
-Rib), 108.69 (C-1 ), a CMV promoter followed by an internal ribosome entry site
0
0
00
0
0.16 (C-1 ), 84.90 (d, J ¼ 8.78 Hz, C-4 ), 84.06 (C-3 ), 83.96 (C-2 ), (IRES) and the coding sequence for EGFP). Cells that remained
00
0
00
00
3.40 (C-4 ), 81.46 (C-3 ), 80.94 (C-2 ), 65.17 (C-5 ), 64.80 (d, J ¼ untransfected were killed by addition of G418 sulfate to the
0
.41 Hz, C-5 ), 54.69 (CH
3
O), 34.89 and 33.93 (d, JC–P ¼ 120.5 Hz, medium. Surviving cells were subjected to limiting dilution
CH
2
P), 26.14 and 24.35 (2 ꢂ C(CH
3
)
2
-Adn), 25.06 and 23.41 (2 ꢂ cloning. The derived clones were then tested for TRPM2
ꢁ
2+
C(CH ) -Rib); HRMS (ES ) calcd for C H N O P 614.1869, expression by Ca measurement and patch-clamp analysis.
found [M ꢁ H] 614.1889.
3
2
24 33
5
12
ꢁ
Whole cell patch-clamp recordings. Whole cell patch-clamp
0
0
0
13
Adenosine-5 -phosphonoacetyl-ribose (2). 2 ,3 -O-Isopropyl recordings were done as described before. In brief, HEK cells
0
00
00 00
idene adenosine-5 -O-phosphonoacetyl-1 -O-methyl-2 ,3 -O-iso- with stable expression of human TRPM2 were seeded to 35 mm
propylidene-b-D-ribofuranoside 10 (15.0 mg, 0.024 mmol) was dishes and kept as described above. 24 h aer seeding the
3
dissolved in 75% aqueous CF COOH (8 mL) and the mixture culture medium was removed and replaced by an extracellular
ꢁ
1
was stirred at room temperature for 2 h. The reaction mixture solution (in mmol L : 140 NMDG, 5 KCl, 3.3 MgCl
2
, 1 CaCl
2
, 5
ꢀ
was quickly evaporated at 30 C to dryness and the residue was D-glucose, 10 HEPES, pH 7.4 with HCl). All recordings were done
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at room temperature. Patch pipettes were prepared from boro-
2 B. Buelow, Y. Song and A. M. Scharenberg, The Poly(ADP-
ribose) polymerase PARP-1 is required for oxidative stress-
induced TRPM2 activation in lymphocytes, J. Biol. Chem.,
2008, 283, 24571–24583.
3 C. J. Lord and A. Ashworth, PARP inhibitors: synthetic
lethality in the clinic, Science, 2017, 355, 1152–1158.
4 H. C. Lee, Cyclic ADP-ribose and nicotinic acid adenine
dinucleotide phosphate (NAADP) as messengers for
calcium mobilization, J. Biol. Chem., 2012, 287, 31633–31640.
5 J. M. Swarbrick, R. Graeff, C. Garnham, M. P. Thomas,
A. Galione and B. V. L. Potter, Click cyclic ADP-ribose:
a neutral second messenger mimic, Chem. Commun., 2014,
50, 2458–2461.
silicate glass tubing 1.05 ꢂ 1.50 ꢂ 80 mm, Science Products
(Hoeim, Germany) using a Sutter P-97 horizontal puller
resulting in a pipette resistance of 1.5 MU to 3.5 MU. An EPC-10
amplier was used in conjunction with PatchMaster (v2x32,
HEKA Elektronik, Lambrecht/Germany) to record the data. Aer
break-in series resistance was compensated to 70% and the
holding potential set to ꢁ50 mV. A total of 90 voltage ramps
from ꢁ85 mV to +20 mV over 140 ms were applied every 5 s to
follow activation of TRPM2 activation by ADPR or ADPR
ꢁ
1
analogues added to the pipette solution (in mmol L : 120 KCl,
NaCl, 1 MgCl , 10 HEPES, 10 EGTA, 5.6 CaCl , pH 7.2 with
8
2
2
ꢁ
1
2+
KOH, 200 nmol L free Ca as calculated with MaxChelator
https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/
(
6 G. I. Karras, G. Kustatscher, H. R. Buhecha, M. D. Allen,
C. Pugieux, F. Sait, M. Bycro and A. G. Ladurner, The
macro domain is an ADP-ribose binding module, EMBO J.,
2005, 24, 1911–1920.
maxchelator/CaEGTA-NIST.htm)). The peak current at +15 mV
during the course of the recording was taken for further quan-
titative analysis of TRPM2 activity.
Statistics. For statistical evaluation of patch-clamp data
GraphPad Prism (version 7.04, GraphPad Soware Inc.) was used.
Currents from the patch-clamp recordings showed a positively
skewed distribution and were normalized by log-transformation.
The normalized data were tested using one-way-ANOVA with post-
hoc testing against the appropriate control using Bonferroni
correction for multiple testing (a ¼ 0.05).
7 A. L. Perraud, A. Fleig, C. A. Dunn, L. A. Bagley, P. Launay,
C. Schmitz, A. J. Stokes, Q. Q. Zhu, M. J. Bessman,
R. Penner, J. P. Kinet and A. M. Scharenberg, ADP-ribose
gating of the calcium-permeable LTRPC2 channel revealed
by Nudix motif homology, Nature, 2001, 411, 595–599.
8 J. C. Belrose and M. F. Jackson, TRPM2: a candidate
therapeutic target for treating neurological diseases, Acta
Pharmacol. Sin., 2018, 39, 722–732.
9
B. T ´o th, I. Iordanov and L. Csan ´a dy, Ruling out pyridine
dinucleotides as true TRPM2 channel activators reveals
novel direct agonist ADP-ribose-2 -phosphate, J. Gen.
Author contributions
0
J. M. W. and B. V. L. P. designed the study. O. B. and J. M. W.
equally contributed and O. B. synthesized the ADPR analogues
Physiol., 2015, 145, 419–430.
supervised by J. M. W.; M. D. R. carried out patch-clamp 10 T. Rosenbaum, Activators of TRPM2: getting it right, J. Gen.
experiments with R. F. and A. H. G.; O. B., J. M. W. and
B. V. L. P. wrote the manuscript with input from all authors.
Physiol., 2015, 145, 485–487.
11 R. Fliegert, A. Bauche, A. M. W. Perez, J. M. Watt,
M. D. Rozewitz, R. Winzer, M. Janus, F. Gu, A. Rosche,
A. Harneit, M. Flato, C. Moreau, T. Kirchberger, V. Wolters,
Conflicts of interest
0
0
B. V. L. Potter and A. H. Guse, 2 -Deoxyadenosine 5 -
diphosphoribose is an endogenous TRPM2 superagonist,
Nat. Chem. Biol., 2017, 13, 1036–1044.
There are no conicts to declare.
1
2 F. J. P. K u¨ hn, J. M. Watt, B. V. L. Potter and A. L u¨ ckhoff,
Different substrate specicities of the two ADPR binding
sites in TRPM2 channels of Nematostella vectensis and the
role of IDPR, Sci. Rep., 2019, 9, 12.
Acknowledgements
This work was supported by the Deutsche For-
schungsgemeinscha (DFG) (Project number 335447717;
SFB1328, project A01 to A. H. G, SFB1328, project A05 to R. F) 13 C. Moreau, T. Kirchberger, J. M. Swarbrick, S. J. Bartlett,
and the Wellcome Trust. BVLP is a Wellcome Trust Senior
Investigator (Grant 101010). Research in the Guse/Fliegert labs
is also supported by the Joachim-Herz-Foundation, Infecto-
physics consortium, project 4; and EU project INTEGRATA -
DLV-813284.
R. Fliegert, T. Yorgan, A. Bauche, A. Harneit, A. H. Guse
and B. V. L. Potter, Structure-Activity Relationship of
Adenosine 5 -diphosphoribose at the Transient Receptor
Potential Melastatin 2 (TRPM2) Channel: Rational Design
of Antagonists, J. Med. Chem., 2013, 56, 10079–10102.
0
1
4 O. Baszczy nˇ ski, J. M. Watt, M. D. Rozewitz, A. H. Guse,
R. Fliegert and B. V. L. Potter, Synthesis of Terminal
Ribose Analogues of Adenosine 5 -Diphosphate Ribose as
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