Synthesis and use of cell-permeant cyclic ADP-ribose

Article abstract of DOI:10.1016/j.bbrc.2012.01.025

Cyclic ADP-ribose (cADPR) is a second messenger that acts on ryanodine receptors to mobilize Ca²⁺. cADPR has a net negative charge at physiological pH making it not passively membrane permeant thereby requiring it to be injected, electroporated or loaded via liposomes. Such membrane impermeance of other charged intracellular messengers (including cyclic AMP, inositol 1,4,5-trisphosphate and nicotinic acid adenine dinucleotide phosphate) and fluorescent dyes (including fura-2 and fluorescein) has been overcome by synthesizing masked analogs (prodrugs), which are passively permeant and hydrolyzed to the parent compound inside cells. We now report the synthesis and biological activity of acetoxymethyl (AM) and butoxymethyl (BM) analogs of cADPR. Extracellular addition of cADPR-AM or cADPR-BM to neuronal cells in primary culture or PC12 neuroblastoma cells induced increases in cytosolic Ca²⁺. Pre-incubation of PC12 cells with thapsigargin, ryanodine or caffeine eliminated the response to cADPR-AM, whereas the response still occurred in the absence of extracellular Ca²⁺. Combined, these data demonstrate that masked cADPR analogs are cell-permeant and biologically active. We hope these cell-permeant tools will facilitate cADPR research and reveal its diverse physiological functions.

Full text of DOI:10.1016/j.bbrc.2012.01.025

Biochemical and Biophysical Research Communications 418 (2012) 353–358
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Synthesis and use of cell-permeant cyclic ADP-ribose
Daniel Rosen 1, Duncan Bloor-Young 1, James Squires 1, Raman Parkesh, Gareth Waters,
⇑
Sridhar R. Vasudevan, Alexander M. Lewis, Grant C. Churchill
University of Oxford, Department of Pharmacology, Mansfield Road, Oxford OX1 3QT, UK
a r t i c l e i n f o
a b s t r a c t
Cyclic ADP-ribose (cADPR) is a second messenger that acts on ryanodine receptors to mobilize Ca2+. cAD-
PR has a net negative charge at physiological pH making it not passively membrane permeant thereby
requiring it to be injected, electroporated or loaded via liposomes. Such membrane impermeance of other
charged intracellular messengers (including cyclic AMP, inositol 1,4,5-trisphosphate and nicotinic acid
adenine dinucleotide phosphate) and fluorescent dyes (including fura-2 and fluorescein) has been over-
come by synthesizing masked analogs (prodrugs), which are passively permeant and hydrolyzed to the
parent compound inside cells. We now report the synthesis and biological activity of acetoxymethyl
(AM) and butoxymethyl (BM) analogs of cADPR. Extracellular addition of cADPR-AM or cADPR-BM to
Article history:
Received 3 January 2012
Available online 17 January 2012
Keywords:
Cyclic ADP-ribose
Cell-permeant
Calcium
PC12
Ryanodine receptor
Chemical probe
neuronal cells in primary culture or PC12 neuroblastoma cells induced increases in cytosolic Ca2+
.
Pre-incubation of PC12 cells with thapsigargin, ryanodine or caffeine eliminated the response to
cADPR-AM, whereas the response still occurred in the absence of extracellular Ca2+. Combined, these data
demonstrate that masked cADPR analogs are cell-permeant and biologically active. We hope these
cell-permeant tools will facilitate cADPR research and reveal its diverse physiological functions.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
area (279 square Å) and net negative charge at physiological pH
[28]. Of these parameters that dictate membrane permeability,
Intracellular Ca2+ mobilization is mediated not only by inositol
1,4,5-trisphosphate (IP3) [1–3] but also by nictoinic acid adenine
dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR)
[4–6]. Although NAADP and cADPR are bona fide messengers
[5,7,8], their role in cell physiology and the identity of their molec-
ular targets remain hotly contested [9–11].
One impediment to the widespread investigation of cADPR is its
lack of cell permeability, which has made research into the effect of
cADPR itself in intact cells the preserve of labs with specialist
equipment and techniques. Techniques to introduce cADPR into
cells include microinjection [12] and delivery through cADPR-
loaded liposomes [13]. An alternative to using cADPR itself is to
use cell-permeant cADPR analogs [14–20]; however, their very
hydrophilic nature makes passive permeability extremely low
[21] and uptake likely depends on transporters [22–26]. Moreover,
if the transporters do not exist on all cell types, as with the carrier-
mediated uptake of NAADP [27], hydrophilic analogs are not a uni-
versally applicable solution.
charge dominates [21]. This has been recognized for numerous
charged molecules in the past and several chemical-protecting
techniques can be used to mask the charge to make the molecule
cell permeant [29]. Likewise, the hydrophilic contribution of hy-
droxyl groups can be reduced with cleavable protecting groups
[30]. Once the molecule penetrates the cell, the ester is hydrolyzed
by nonselective esterases regenerating the parent molecule
[29,31]. A particularly successful approach is to make the methox-
yester method (first reported for penicillin [32]), which has been
used to great success with Ca2+ chelators [31], fluorescent probes
[33] and second messengers including IP3 [34–36], cyclic AMP
and cyclic GMP (Li1997) and NAADP [37].
2. Materials and methods
2.1. Materials
All reagents were obtained from Sigma–Aldrich, unless other-
wise stated. All water used was double deionized by a reverse
osmosis filter (Purite Ltd., UK).
It should be possible to make cADPR cell-permeant through
chemical modification. Like other second messengers, cADPR is
passively impermeant due to its size (541 Da), large polar surface
2.2. High-performance liquid chromatography (HPLC)
⇑
Corresponding author.
E-mail address: grant.churchill@pharm.ox.ac.uk (G.C. Churchill).
These authors contributed equally to this work.
Anion-exchange HPLC was used to separate nucleotides, moni-
tor progress of reactions and isolate synthesized compounds.
1
0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2012.01.025

354
D. Rosen et al. / Biochemical and Biophysical Research Communications 418 (2012) 353–358
Samples were run over a strong anion-exchange resin, AG MP-1,
packed into a 3 ꢀ 150 mm borosilicate glass column (Omnifit,
New Jersey, USA). Samples were injected via a Waters 600E Multi-
solvent Delivery System and analyzed by a Waters 2487 Dual
Wavelength Absorbance Detector set at 254 nm (Waters Corpora-
tion). Elution was achieved with trifluoroacetic acid, run as an
exponential gradient over 40 min [38].
Br or BM-Br was made after 24 h. After stirring the reaction mix-
ture at room temperature for 2 days, the product was vacuum
dried and tested for purity by HPLC. Esterified cADPR was stored
under argon at ꢁ20 °C. The solid orange–brown product was taken
up in chloroform and analyzed by HPLC, phosphorous nuclear mag-
netic resonance, proton nuclear magnetic resonance and mass
spectroscopy. The cADPR–acetoxymethyl ester (resin) was stored
under argon at ꢁ80 °C. The ability to hydrolyze the cADPR back
to cADPR was evaluated with alkaline hydrolysis (100 mM NaOH,
1 h, 23 °C) and incubation with a guinea pig heart homogenate
(10% weight/volume).
Due to the low yields and instability of cADPR esters, as re-
ported for the direct esterification of cAMP [29,42], we were unable
to obtain enough material for NMR for absolute structural assign-
ment. However, mass spectroscopy revealed a family of peaks sep-
arated by 73 atomic mass units indicating multiple species with
varying numbers of AM groups.
2.3. Synthesis of cADPR
cADPR was synthesized from NAD as described previously
[12,39]. Briefly, we incubated b-NAD 2 mM with purified ADP-ribo-
syl cyclase (0.1 U/mL) from Aplysia (provided by H.C. Lee; [39–41])
in Hepes–NaOH 50 mM, pH 7 for 3 h at room temperature. The
reaction was monitored and reactants and products separated by
HPLC. The cADPR peak was collected, dried with rotary evaporation
under vacuum, washed three times with methanol, then three
times with acetone and stored as the free acid or sodium salt at
ꢁ80 °C and was routinely >95% pure by HPLC.
2.6. Neuronal cell culture and Ca2+ imaging
1H NMR (D2O, 400 MHz): d 3.94 (m, 1H), 4.02 (m, 1H), 4.27 (m,
2H), 4.37 (m, 2H), 4.65 (m, 3H), 5.25 (t, 1H, J = 5.4 Hz), 5.97 (d, 1H,
J = 5.8 Hz), 6.05 (d, 1H, J = 4.0 Hz), 8.30 (s, 1H), 8.91 (s,1H); MS (ESI)
m/z 540 (M–H)ꢁ.
Primary neuronal cells were cultured from p1 Wistar rat pups
(Harlan UK Ltd.) as described previously [37]. Primary rat neuronal
cells were loaded with fluo-3 by incubation in medium with 5 lM
fluo-3-acetoxymethyl ester (Invitrogen) for 1 h at room tempera-
ture. A coverslip was mounted in a perfusion chamber and placed
onto the stage of a Leica LS2000 confocal microscope supported on
a vibration-isolated table. Cells were viewed through 63ꢀ water
objective lens. Excitation was at 488 nm (Argon laser) and fluores-
cence was detected after a 515-nm long-pass filter and captured at
512 ꢀ 512 pixels.
2.4. Synthesis of butyryloxymethyl bromide
Butyric acid (5.27 g, 5.5 mL, 59.8 mmol) was added to 2 M NaOH
(60 mL) in a round bottom flask and the mixture was stirred for
30 min. To this was added tetrabutylammonium hydrogen sulfate
(20.4 g, 60.1 mmol) and the reaction mixture was further stirred
for 30 min at room temperature. After this, the aqueous layer was
extracted with dichloromethane (4 ꢀ 100 mL) and the organic layer
dried over magnesium sulfate. The solution was evaporated on a
rotavap and the crude product was then purified by vacuum distil-
lation at 140 °C for 2 h to afford 7.20 g of methylene dibutyrate.
1H NMR (CDCl3, 200 MHz) d 0.93 (t, 6H, J = 7.43), 1.65 (m, 4H,
J = 7.43), 2.32 (t, 4H, J = 7.43), 5.73 (s, 2H).
PC12 cells (American Type Culture Collection) were cultured in
DMEM containing 5% fetal bovine serum, 10% horse serum, 1% pen-
icillin/streptomycin and 1%
L-glutamine and maintained in a
humidified incubator (95% O2, 5% CO2). Before use, PC12 cells were
trypsinized and plated onto glass coverslips (25-mm diameter)
coated with poly-D-lysine (0.1 mg/mL) and were differentiated
with 50 ng/mL nerve growth factor. Ca2+ imaging experiments
were performed 5 days after plating. PC12 cells were loaded with
fura-2 by incubation with 4 M of its acetoxymethylester (Invitro-
gen) for 20 min at room temperature. Coverslips were mounted
into a perfusion chamber and maintained in HBSS and imaged with
a Zeiss Axiovert 2000 microscope. Fluoresence was detected with a
CCD camera during alteranting 340 and 380 nm excitation after
510 long-pass filtering controlled with Metafluor 7.0 software
(Molecular Devices).
Methylene dibutyrate (7.20 g, 48.49 mmol), trimethylsilylbro-
mide (7.42 g, 6.4 mL, 48.49 mmol) and zinc bromide (0.43 g,
19.10 mmol) were charged in 50 mL of round bottom flask and
the reaction mixture stirred for 24 h at room temperate. Then a
further portion of trimethylsilylbromide (6.4 mL, 48.49 mmol)
was added and the mixture left to stir for another 24 h at room
temperature. After the completion of the reaction as judged by
TLC, diethyl ether (20 mL) and 1 M HCl (10 mL) were added and
the reaction stirred for 15 min after which 1 M Na2CO3 (20 mL)
was added. The reaction mixture was further stirred for 30 min
and the aqueous layer extracted with ether (3 ꢀ 50 mL). The organ-
ic layer was dried over magnesium sulfate and solvent evaporated
on rotavap. The crude product was purified by vacuum distillation
at 120 °C to yield 4.5 g of butyryloxymethyl bromide (BM-Br) as
pure product.
3. Results and discussion
3.1. Synthesis cADPR-AM
1H NMR (CDCl3, 200 MHz) d 0.94 (t, 3H, J = 7.43), 1.68 (m, 2H),
2.35(t, 2H, J = 7.43), 5.80 (s, 2H).
To synthesize cADPR we incubated NAD with Aplysia ADP-ribo-
syl cyclase (Fig. 1A). The reaction is reversible [39] and reached an
equilibrium of about 40% cADPR and 60% NAD at 4 h as demon-
strated by anion-exchange HPLC (Fig. 1D). The cADPR peak was
collected, dried down in a rotary evaporator and dissolved in dou-
ble-deionized water and found to be >90% pure (Fig. 1E, left trace)
and was confirmed with NMR and mass spectroscopy (Fig. 1C; see
Section 2). When cADPR was reacted with the reagent acetoxy-
methyl bromine (AM-Br) in anhydrous acetonitrile for 48 h, it
was entirely converted into products that did not bind to the anion
exchange column (Fig. 1E, right trace). Analysis of this isolated
peak with mass spectroscopy was consistent with cADPR with
multiple AM groups (see Section 2). Further evidence for this peak
being cADPR-AM was that cADPR could be obtained upon
2.5. Synthesis of acetoxymethyl (AM) and butyryloxymethyl (BM)
cADPR analogs
Addition of AM and BM groups to cADPR was adapted from a
protocol developed by Schultz et al. [42] to esterify cAMP. In a
round bottom flask, 40 mg cADPR (free acid form produced by
passing through a Dowex 50 Columns, acid form), 50 lL diisopro-
pylethylamine (DIEA) and 2.5 mL of dry acetonitrile were stirred
together for 2 h under a dry argon atmosphere. The esterification
reaction was initiated by addition of 100
l
L acetoxymethyl bro-
L addition of AM-
mide (AM-Br) or 100 L BM-Br. A further 100
l
l

D. Rosen et al. / Biochemical and Biophysical Research Communications 418 (2012) 353–358
355
O
O
A
B
C
OHOH
O
OHOH
O
NH2
NH2
O
N+
N
O
O
O
O
O
O
Nicotinamide
R
O
Br
N
N
-O
-O
P
O
P
O
O
+
O
O
O
O
P
O
P
O
O
+
R
R
N
NH2
N
NH2
OH OH
N
-O
-O
P
O
O
NH2
N
N
O
N
N
O
N
O
ADP-ribosyl cyclase
CH3CN, DIEA
N
O
N
P
O
O
OHOH
OHOH
O
R = AM = CH -
OH OH
BM = CH33CH2CH2CH2-
NAD
cADPR
D
E
F
NAD
Nicotinamide + cADPR
0 h 3 h
cADPR
0 h
cADPR-AM
48 h
cADPR-AM
cADPR
Homogenate
1 h
NaOH
1 h
5 min
Fig. 1. Synthesis of cADPR and its acetyoxymethyl ester (cADPR-AM). (A) Chemical structures and synthetic schemes. cADPR was synthesized enzymatically with Aplysia
ADP-ribosyl cyclase. cADPR-AM was synthesized by dissolving its disopropylethylamine (DIEA) salt in acetonitrile and esterifying with acetyoxymethylbromine. (B)
Chromatography traces showing cADPR synthesis from NAD+ catalyzed by ADP-ribosyl cyclase. (C) Chromatography traces showing cADPR-AM synthesis from the purified
cADPR. (D) Chromatography traces showing cADPR-AM de-esterified by either alkaline hydrolysis (1 M NaOH) or by incubation with rat heart homogenate (10% weight/
volume).
A
B
C
NAADP 0.5 µM
cADPR 0.5 µM
IP3 10 µM
150 cADPR 0.5 µM
100
200
150
100
50
cADPR-AM 100 µM
50
10 min
5 min
Fig. 2. Esterified cADPR does not release Ca2+ in the sea urchin egg homogenate bioassay. (A) Sea urchin egg homogenate releases Ca2+ in response to NAADP, cADPR and IP3.
(B) cADPR releases Ca2+; whereas (C) cADPR-AM does not.
hydrolysis by either NaOH (Fig. 1F, left trace) or heart homogenate
(Fig. 1F, right trace), a rich source of esterases [12,31].
cADPR eliminates its Ca2+-releasing activity. This is consistent with
what is known about the importance of the phosphates and
hydroxyls in cADPR for its biological activity [46,47].
3.2. Esterification of cADPR eliminates biological activity in sea urchin
egg homogenate bioassay
3.3. cADPR-AM induces Ca2+ increases in neuronal cells
The sea urchin egg homogenate is a sensitive and robust bioas-
say for all three Ca2+-releasing messengers, IP3, NAADP and cADPR
(Fig. 3A) [5,12,28,43]. Indeed, two of these three messengers, cAD-
PR [6] and NAADP [43] were discovered using this bioassay. We
used this sea urchin egg homogenate bioassay to determine
whether cADPR-AM had any Ca2+-releasing activity because it is
sensitive to sensitive to all three Ca2+-releasing messengers
(Fig. 2A) [5,6,12] and Lytechinus pictus sea urchin eggs contain very
limited esterase activity [12,44,45] thereby minimizing the con-
founding effect of cADPR-AM hydrolysis. In the same batch of sea
urchin egg homogenate, a supramaximal concentration of cADPR
To determine whether AM addition made cADPR cell-permeant,
we tested cADPR-AM on both primary rat neurons and PC12 cells,
each of which has advantages and disadvantages. While primary
neurons are arguably closer to the physiological situation, PC12
cells are better characterized in regard to cADPR signaling
[48,49]. PC12 cells are a pheochromocytoma stable line and widely
used as a nerve cell model [50]. Importantly, for testing cADPR-AM,
PC12 cells have been reported to utilize cADPR-mediated Ca2+ sig-
naling [48,49], possess functional ryanodine receptors [51], the
putative cADRP receptor [5,9,11], and use cADPR as an endogenous
messenger in the nitric oxide [48] and agonist-mediated [49] sig-
naling pathways.
(0.5 l lM) re-
M) released Ca2+ (Fig. 3B), whereas cADPR-AM (100
leased minimal Ca2+ (Fig. 3C). These results demonstrate that addi-
tion of AM groups to the phosphates and likely the hydroxyls on
The addition of extracellular cADPR (100
lM) failed to elicit an
increase in intracellular Ca2+ in primary neurons, even after a

356
D. Rosen et al. / Biochemical and Biophysical Research Communications 418 (2012) 353–358
A
D
B
cADPR-AM
cADPR
C
20
15
10
5
cADPR-AM
cADPR-AM
cADPR-BM
0
10 min
10 min
-7 -6 -5 -4 -3
Log [cADPR-ester] (M)
E
F
cADPR-AM
1.8
1.4
1.0
cADPR
-7 -6 -5 -4 -3
Log [cADPR-AM] (M)
10 min
10 min
Fig. 3. Extracellular cADPR esters induce Ca2+ responses in neuronal cells. (A–C) Effects of extracellular cADPR and cADPR-AM on intracellular Ca2+ in primary cultures of rat
neurons. (A) Addition of extracellular cADPR-AM (100 M) but not cADPR (100
M) increases intracellular Ca2+. (B) Reducing the concentration of extracellular cADPR-AM
(0.1
M) results in a latency in the increase in intracellular Ca2+. (C) Plot showing the relationship between cADPR ester concentration and latency to the Ca2+ increase. (D–F)
l
l
l
Effects of extracellular cADPR and cADPR-AM on intracellular Ca2+ in cultured PC-12 cells. (D) Addition of cADPR-AM (100 M) induces an intracellular Ca2+ increase. (G)
l
Addition of extracellular cADPR (100
l
M) does not affect intracellular Ca2+. (H) Concentration–response relationship for cADPR-AM and Ca2+ increase (mean standard error
of the mean, n = 3–6).
lar Ca2+ increase but with a longer latency between the time of
addition and the time of the response (Fig. 3B). This relationship
was concentration dependent and could be fit with a single-site
A
B
Ca2+ free
Thapsigargin
cADPR-AM
cADPR-AM
model with an EC50 of 1 lM (Fig. 3C). In contrast to the concentra-
tion dependence observed with latency, there was no relationship
with the amplitude of the response and the concentration of cAD-
PR-AM and was typically all or none (Fig. 3A versus B). This may
reflect that the target of cADPR is likely ryanodine receptors
[5,9,11] that show an all-or-none response [1,52].
10 min
C
10 min
E
We next determined whether cADPR-AM also was effective in
Caffeine
1.3
1.2
1.1
1.0
PC12 neurons. cADPR-AM (100 lM) resulted in an increase in
intracellular Ca2+ (Fig. 3D) whereas cADPR itself did not (Fig. 3E).
In contrast to primary neurons, in PC12 cells exhibited a well-de-
fined relationship between cADPR-AM concentration and the
cADPR-AM
***
**
***
amplitude of the Ca2+ response with an EC50 of 22
lM (Fig. 4F).
***
10 min
3.4. cADPR-BM induces Ca2+ increases in neuronal cells
D
When Li et al. [34] synthesized cell-permeant esters of IP3, they
tested AM, propionyloxymethyl (PM) and butoxymethyl (BM)
groups and found that the AM group was less effective than the
PM or BM groups likely due to the balance between the rate of
hydrolysis (fewer methylenes was faster) versus hydrophobicity
that dictates passive permeability [34]. Therefore, we also synthe-
sized and tested cADPR protected with BM groups. Unlike IP3-BM
[34], cADPR-BM did not show a dramatically greater potency based
on a concentration–response relationship based on latency, but the
BM group did shift the curve slightly to the left (Fig. 3C). As the
advantages of cADPR-BM were slight and the BM precursor had
to be synthesized in-house rather than simply purchased, all fur-
ther experiments were conducted with cADPR-AM.
Ryanodine
cADPR-AM
cADPR-AM 30 µM
10 min
Fig. 4. Pharmacological characterization of the Ca2+ response to cADPR-AM in PC-
12 cells. (A) Reducing extracellular Ca2+ with EGTA did not reduce the Ca2+ response
to cADPR-AM (100
stores with the Ca2+ pump inhibitor thapsigargin (1
response to cADPR-AM (100
M). Depleting intracellular Ca2+ stores containing
ryanodine receptors with (C) 100 M ryanodine or (D) 20 mM caffeine eliminated
the Ca2+ response to cADPR-AM (100
M). (E) Summary of the responses to cADPR
l
M). (B) Depleting intracellular endoplasmic reticulum Ca2+
l
M) eliminated the Ca2+
l
l
l
and cADPR-AM in response to the various pharmacological treatments. Bars
represent the mean standard error of the mean for n = 3–6. Statistical significance
was evaluated with a one-way analysis of variance followed by Dunnett’s multiple
comparison test against the control, ⁄p 6 0.05, ⁄⁄p 6 0.01, and ⁄⁄⁄p 6 0.001.
3.5. cADPR-AM responses require intracellular and not extracellular
Ca2+
cADPR can activate not only ryanodine receptors but also cer-
tain plasma membrane Ca2+ channels such as TRPM2 [53]. There-
fore, we tested the involvement of extracellular Ca2+ influx and
mobilization from intracellular stores. Lowering extracellular Ca2+
did not significantly affect the amplitude of the response to
10-min incubation (Fig. 3A). In contrast, in the same cells, the addi-
tion of cADPR-AM (100
Ca2+ within 10 s (Fig. 3A). The addition of extracellular cADPR-
AM at the lower concentration of 0.1 M resulted in an intracellu-
lM) elicited an increase in intracellular
l

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