Chemo-enzymatic synthesis and biological evaluation of photolabile nicotinic acid adenine dinuclotide phosphate (NAADP+)

Article abstract of DOI:10.1039/b617344f

A chemo-enzymatic synthesis of novel caged NAADP⁺ without the formation of multiple cage compounds has been achieved. The biological activity of the caged NAADP⁺ was demonstrated by its fast uncaging in intact sea-urchin eggs. The Ro

Full text of DOI:10.1039/b617344f

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/obc | Organic & Biomolecular Chemistry
Chemo-enzymatic synthesis and biological evaluation of photolabile nicotinic
acid adenine dinuclotide phosphate (NAADP+)†
Raman Parkesh,*a Sridhar R. Vasudevan,a Alexandra Berry,a Antony Galione,a James Dowdenb and
Grant C. Churchilla
Received 28th November 2006, Accepted 28th November 2006
First published as an Advance Article on the web 11th December 2006
DOI: 10.1039/b617344f
A chemo-enzymatic synthesis of novel caged NAADP+ with-
out the formation of multiple cage compounds has been
achieved. The biological activity of the caged NAADP+ was
demonstrated by its fast uncaging in intact sea-urchin eggs.
(4,5-dimethoxy-2-nitroacetophenone
(DMNPE)–NAADP+,
Scheme 1). The presence of electron-donating methoxy groups on
the caging chromophore should lead to fast release of NAADP+.
The effect of structural variations on the photocleavage kinetics
of the nitrophenyl chromophore is well documented.8–10 Furuta
et al.11 and Ellis-Davies et al.12 have reported that DMNB-caged
compounds uncage faster than simple ortho-nitrobenzyl-caged
compounds in biological studies of photocaged molecules. Our
attempts to directly cage NAADP+ under a variety of conditions
all proved unsuccessful, yielding only the starting material and a
hydrolysis product, ADP-ribose phosphate. Initially, the highly
labile and photosensitive diazo caging reagent 1 was prepared
by the oxidation of 4,5-dimethoxy-2-nitroacetophenone with
MnO2 in CHCl3. The diazo compound was then added to NADP
under biphasic conditions (H2O–CHCl3) with vigorous stirring,
resulting in the formation of caged NADP+.13 Finally, ADP-
ribosyl cyclase catalyzed a base-exchange reaction between caged
NADP+ and excess nicotinic acid, resulting in the formation of
DMNPE-caged NAADP+ (Scheme 1). The negligible hydrolyzing
activity of this enzyme prevented formation of side-products,
resulting in the formation of caged NAADP+ in higher yield.14
The caging reaction was monitored by anion exchange high-
performance liquid chromatography (HPLC). Final purification
of DMNPE-caged NAADP+ was achieved by HPLC using AG
MP-1 columns (see ESI†).
The structure of DMNPE-caged NAADP+ was confirmed by
both 1H-NMR and 31P-NMR. The 1H-NMR of the purified
compound showed two additional resonance peaks in the aromatic
region, the H3 and H6 of the phenyl. Morever, the methoxy
groups at C4 and C5 are singlets, while the benzylic methyl is
a doublet. The 31P-NMR of DMNPE-caged NAADP+ showed
only two peaks at −2.08 ppm and −11.31 ppm (see ESI†), which
correspond to the 2ꢀ-phosphate and pyrophosphate groups. The
2ꢀ-phosphate is shifted upfield by about 2 ppm compared to
that of NAADP+, while the pyrophosphate signal is practically
unchanged. This dramatic shift is consistent with the caging
group being attached to the 2ꢀ-phosphate. As reported by Lee
et al.,7 we also observed peak broadening, which might be due
to intermolecular hydrogen bond formation. We believe that the
hydrophobic nature of the caging group will favour intermolecular
association, thus restricting the molecular motion of the caged
compound. The high concentration of the sample (5 mg ml−1)
needed to acquire the 31P-NMR spectra further promotes these
intermolecular and p–p stacking interactions.
NAADP+, first identified as a calcium-releasing messenger in sea-
urchin eggs,1 is a structural analogue of nicotinamide adenine
dinuclotide phosphate (NADP+) in which the amide group is
replaced with a carboxylic acid group. Interestingly, this minor
structural variation makes NAADP+ the most potent Ca2+-
mobilizing messenger.1,2 It is understood that the carboxylate,
amino and 2ꢀ-phosphate groups are crucial for NAADP+-induced
Ca2+ release, as several structural analogues of NAADP+ in
which these groups are modified show no effect on intracellular
Ca2+ release.3,4 The 2ꢀ-phosphate is particularly crucial for Ca2+
release, as NAAD+ which lacks this group has no Ca2+-mobilizing
property.3,4 Its mode of Ca2+ release is distinct from cyclic ADP-
ribose (cADPR) and inositol 1,4,5-trisphosphate.
NAADP+ operates on Ca2+ stores that have been identified
as acidic lysosome-related organelles,5 and recent studies have
demonstrated the calcium-mobilizing efficiency of NAADP+ in
a wide variety of cells, from plant to animal, including humans.6
However, NAADP+ signalling transduction, the receptors it acts
on and other regulatory functions are not clear. To fully un-
derstand the molecular basis underlying the NAADP+ signalling
process, it is necessary to deliver it intracellularly in a controlled
fashion. In this context, blocking the active functionalities in
NAADP+ and unblocking them when necessary is promising. Cell-
permeant photolabile caged NAADP+ is the ideal molecular tool
for investigating the localized signalling behavior of NAADP+. To
date there is only one report of caged NAADP+, based on the
caging reagent 1-(2-nitrophenyl)diazoethane (NPE), synthesized
by direct alkylation of NAADP+ at pH 1.3.7 These extremely
acidic conditions were used to avoid multiple caging of NAADP+,
but in spite of this precaution, significant caging of carboxylate
groups with NPE was observed. The unstable nature of most
caging reagents at low pH hindered the development of other
caged NAADP+ derivatives.
In the present study, we report the chemo-enzymatic synthesis
and biological investigation of
a
novel caged NAADP+
aDepartment of Pharmacology, University of Oxford, Mansfield Road,
Oxford, OX1 3QT, UK. E-mail: raman.parkesh@pharm.ox.ac.uk
bSchool of Chemistry, University of Nottingham, University Park, Notting-
ham, NG2 7RD, UK
The quantum yield (U) of the DMNPE-caged NAADP+
measured by comparison with a known standard, 4-methyl-7-
nitroindolinylglutamate, was found to be 0.20 at pH 7. Both the
† Electronic supplementary information (ESI) available: Synthetic detail
and biological experiments. See DOI: 10.1039/b617344f
This journal is
The Royal Society of Chemistry 2007
Org. Biomol. Chem., 2007, 5, 441–443 | 441
©

View Article Online
Scheme 1 Chemical structure and synthesis of DMNPE-caged NAADP+. Reagents and conditions: (a) CHCl3–H2O, pH 4, 14 h, 60%; (b) ADP-ribosyl
cyclase, acetate buffer pH 4.4, 5 h, 80%.
DMNPE-caged NAADP+ and the standard were photolysed sep-
arately to check that there was no interference from simultaneous
photolysis. DMNPE-caged NAADP+ has an extinction coefficient
(e) of 18 000 and 8900 M−1 cm−1 at 250 nm and 350 nm respectively.
This value is comparable to the other DMNPE-caged compounds.
The photolysis of DMNPE-caged NAADP+ to NAADP+ was
confirmed by HPLC (see ESI†). The columns were packed
with AG MP-1 resin and eluted with non-linear gradient of
trifluoroacetic acid (150 mM). The HPLC trace of DMNPE-
caged NAADP+ showed a major peak with a retention time of
25.4 minutes. Upon photoirradiation, DMNPE-caged NAADP+
showed a new peak with a retention time of 21.2 minutes, identical
to that of NAADP+ (Fig. 1).
Fig. 1 HPLC chromatogram of the DMNPE-caged NAADP+ and
uncaged NAADP+. As can be seen, the uncaging reaction is quite facile,
and leads to a quantitative production of NAADP+ (left-hand trace).
The sea-urchin was used as a model system for studying the
Ca2+ response of caged NAADP+ because its large size (100
lm) facilitates microinjection. The sea-urchin eggs contain robust
Ca2+-release channels for the NAADP+ signalling system, making
it the most reliable biological system for directly studying Ca2+
release from intracellular stores. The ability of DMNPE-caged
NAADP+ to mobilize Ca2+ in intact sea-urchin eggs is depicted
in Fig. 2. It is well-known that even nanomolar concentrations of
NAADP+ are sufficient to bind the receptor and block the calcium
response.1,7,15 To avoid this desensitization of the receptor, caged
NAADP+ was pretreated with alkaline phosphate7 to remove any
trace amounts of free NAADP+.
in Ca2+ consistent with NAADP+ release. In another experiment,
eggs were injected with caged NAADP+ containing a small amount
of NAADP+ (10 nM) to inactivate the response. No Ca2+ was
observed after UV irradiation. This conforms that the calcium
response is indeed due to the NAADP+ released from the cage.
In conclusion, this report describes the novel chemo-enzymatic
synthesis of caged NAADP+ without the formation of multiple
cages. Biological evaluation in sea-urchin eggs demonstrated the
fast release of DMNPE-caged NAADP+. The mild enzymatic
route (pH > 4) will allow the caging of NAADP+ with novel caging
chromophores such as two-photon active reagents. Further studies
Calcium release was monitored by 488 BAPTA Dextran Oregon
Green, co-injected into the eggs along with DMNPE-caged
NAADP+. Exposure of the eggs to UV light resulted in an increase
442 | Org. Biomol. Chem., 2007, 5, 441–443
This journal is
The Royal Society of Chemistry 2007
©

View Article Online
Notes and references
1 H. C. Lee and R. J. Aarhus, J. Biol. Chem., 1995, 270, 2152–2157;
H. C. Lee, Recent Prog. Hormone Res., 1996, 52, 357–299; H. C. Lee,
Physiol. Rev., 1997, 77, 1133–1164; A. Galione and O. H. Petersen,
Mol. Intervention, 2005, 5, 73–79.
2 J. M. Cancela, G. C. Churchill and A. Galione, Nature, 1999, 398,
74–76.
3 E. N. Chini, K. W. Beers and T. P. Dousa, J. Biol. Chem., 1995, 270,
3216–3223.
4 H. C. Lee, Annu. Rev. Pharmacol. Toxicol., 2001, 41, 317–345.
5 G. C. Churchill, Y. Okada, J. M. Thomas, A. A. Genazzani, S. Patel
and A. Galione, Cell, 2002, 111, 703–708.
6 J. Bak, J. R. A. Billington, G. Timar, A. C. Dutton and A. A. Genazzani,
Curr. Biol., 2001, 11, 987–990; S. Patel, G. C. Churchill, T. Sharp and
A. Galione, J. Biol. Chem., 2000, 275, 36495–36497.
7 H. C. Lee, R. Aarhus, K. R. Gee and T. J. Kestner, J. Biol. Chem., 1997,
272, 4172–4178; K. R. Gee and H. C. Lee, Methods Enzymol., 1998,
291, 403–415.
8 C. P. J. Holmes, J. Org. Chem., 1997, 62, 2370–2380.
9 S. Bu¨hler, I. Lagoja, H. Giegrich, K.-P. Stengele and W. Pfleiderer, Helv.
Chim. Acta, 2004, 87, 620–659.
10 M. Schwrer and J. Wirz, Helv. Chim. Acta, 2001, 84, 1441.
11 Y. Hirayama, M. Iwamura and T. Furuta, Bioorg. Med. Chem. Lett.,
2003, 13, 905–908.
12 S. Kantevari, C. J. Hoang, J. Ogrodnik, M. Egger, E. Niggli and G. C.
Ellis-Davies, ChemBioChem, 2006, 7, 174–180.
Fig. 2 (A) Effect of photoreleasing DMNPE-caged NAADP+ on Ca2+
signalling in intact sea-urchin eggs. (B) Injecting sub-threshold amounts
of NAADP+ (10 nM), prior to photoreleasing DMNPE-caged NAADP+,
failed to elicit a response, suggesting the inactivation of NAADP+
receptors. Images are self-ratio, with colours representing the ratio
according to the calibration scale. Traces are of average ratio over times.
Eggs contained Oregon Green 488 BAPTA Dextran (10 lM) and
DMNPE-caged NAADP+ (0.5 lM) as labelled. Caged NAADP+ was
photoreleased as indicated by the arrows.
13 B. E. Cohen, B. L. Stoddard and D. E. Koshland, Jr., Biochemistry,
1997, 39, 9035–9044; M. Wilcox, R. W. Viola, K. W. Johnson, A. P.
Billington, B. K. Carpenter, J. A. McCray, A. P. Guzikowski and G. P.
Hess, J. Org. Chem., 1990, 55, 1585–1589.
14 F.-J. Zhang, Q.-M. Gu, P. Jing and C. J. Sih, Bioorg. Med. Chem. Lett.,
1995, 19, 2267–227.
to measure the two-photon absorption cross-sectional area and its
biological application are currently underway.
This study was supported by a grant from Wellcome Trust,
UK. We thank Prof. Hon-Cheung Lee for a generous gift of ADP-
ribosyl cyclase. We thank Dr Rethi Madathil and Dr Chunnu for
helpful discussions.
15 A. A. Genazzani, R. M. Empson and A. Galione, J. Biol. Chem., 1996,
271, 11599–11602.
This journal is
The Royal Society of Chemistry 2007
Org. Biomol. Chem., 2007, 5, 441–443 | 443
©