Chemical synthesis of the novel Ca2+ messenger NAADP

Article abstract of DOI:10.1081/NCN-200061786

The first total chemical synthesis of nicotinamide adenine dinucleotide phosphate (β-NADP, 2) as a single isomer was achieved. This was subsequently converted into the important second messenger nicotinic acid adenine dinucleotide phosphate (β-NAADP) 1 an

Full text of DOI:10.1081/NCN-200061786

This article was downloaded by: [Dokuz Eylul University ]
On: 06 November 2014, At: 08:40
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic Acids
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/lncn20
CHEMICAL SYNTHESIS OF THE NOVEL CA2+ MESSENGER
NAADP
James Dowden a , Richard S. Brown a , Christelle Moreau a , Antony Galione b & Barry V. L.
Potter a
a Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology ,
University of Bath , Bath, UK
b Department of Pharmacology , University of Oxford , Oxford, UK
Published online: 15 Nov 2011.
To cite this article: James Dowden , Richard S. Brown , Christelle Moreau , Antony Galione & Barry V. L. Potter (2005)
CHEMICAL SYNTHESIS OF THE NOVEL CA2+ MESSENGER NAADP, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 513-518,
DOI: 10.1081/NCN-200061786
To link to this article: http://dx.doi.org/10.1081/NCN-200061786
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the
Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and
should be independently verified with primary sources of information. Taylor and Francis shall not be liable for
any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of
the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):513–518, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200061786
CHEMICAL SYNTHESIS OF THE NOVEL Ca2+ MESSENGER NAADP
5
James Dowden, Richard S. Brown, and Christelle Moreau
Wolfson Laboratory
of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath,
Bath, UK
5
Antony Galione
Department of Pharmacology, University of Oxford, Oxford, UK
5
Barry V. L. Potter
Wolfson Laboratory of Medicinal Chemistry, Department of
Pharmacy and Pharmacology, University of Bath, Bath, UK
5
The first total chemical synthesis of nicotinamide adenine dinucleotide phosphate (b-NADP, 2) as a
single isomer was achieved. This was subsequently converted into the important second messenger
nicotinic acid adenine dinucleotide phosphate (b-NAADP) 1 and the identity of this material confirmed
by biological evaluation. This flexible synthetic route offers new opportunities for the generation of
NAADP 1 analogues that cannot be generated directly from NADP 2 or mainly enzymatic methods.
RESULTS AND DISCUSSION
Nicotinic acid adenine dinucleotide phosphate (NAADP, Figure 1) 1, orig-
inally identified as a minor contaminant of commercial preparations of NADP 2,
potently induces calcium release in mammalian tissue, such as heart, T-cells, and
both pancreatic acinar and beta-cells at concentrations of 10–100 nM.[1,2] It is
interesting that NAADP 1 causes self-inactivation of this calcium release pathway in
certain cells. Glucose stimulation of pancreatic beta-cells leads to increased
endogenous levels of NAADP 1, suggesting its role as a second messenger[3] but
whether NAADP 1 addresses a discrete calcium pool to those of other messengers,
such as cyclic adenine dinucleotide phosphate ribose (cADPR), remains
contentious. The biological machinery that governs the intracellular chemistry of
NAADP 1 is poorly characterized. Meanwhile, the high potency of 1 means that
potential contamination of analogues prepared by direct modification of NADP 2
This research was funded by the Biotechnology and Biology Sciences Research Council (BBSRC) and an
Engineering and Physical Sciences Research Council (EPSRC) studentship. We acknowledge the use of the
EPSRC Chemical Database Service at Daresbury.
Address correspondence to James Dowden, Wolfson Laboratory of Medicinal Chemistry, Department of
Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK
513
Order reprints of this article at www.copyright.rightslink.com

514
J. Dowden et al.
FIGURE 1
can never be ruled out, further, important analogues such as C-glycosides cannot be
prepared in this way. We therefore sought to develop a flexible synthetic route
toward a range of chemical probes to this end. While NAD 3 has been prepared on
a kilogram scale,[4] the synthesis and isolation of NADP 2, or NAADP 1 as a single
isomer has not been published. We report the first chemical synthesis of NADP 2,
its conversion to NAADP 1, and confirmation of its identity by biological
evaluation as an important milestone toward the preparation of chemical probes.
A convergent route culminating in penultimate pyrophosphate bond formation
was expected to deliver sufficient flexibility for later production of analogues.
b-Nicotinamide mononucleotide (b-NMN, 6) was therefore generated with
trimethylsilyl trifluoromethanesulfonate activation of 1,2,3,5-tetra-O-acetyl-D-ribofu-
ranose 4 facilitating installation of nicotinamide. Subsequent in situ methanolysis
provided nicotinamide riboside, 5, in good yield, which was converted to b-NMN,
6, upon reaction with POCl3 in trimethyl phosphate[4,5] (Scheme 1).
The 2’-phosphate adenosyl ribose motif is important for the activity of NAADP
1 but makes the synthesis somewhat more complex than that of NAD analogues
because the phosphate must be installed regioselectively and maintained without
migration. The 1,1,3,3,-tetraisopropyl disiloxane protected adenosine 7 allowed
simultaneous protection of 3’-and 5’-hydroxyls and unambiguous, robust
installation of the 2’-phosphate. 1,3-Dichloro-1,1,3,3-tetraisopropyl disiloxane was
reacted with adenosine in pyridine providing good yields of the protected
precursor, 7. Imidazolium triflate catalysed reaction with phosphoramidites, such
as 8, occurred exclusively at the hydroxyl group in high yields (98% for
bis(benzyloxy)phosphate 10) after one pot oxidation using m-chloroperoxybenzoic
acid (mCPBA). Imidazolium triflate is less reactive than more commonly used
1H-tetrazole such that protection of the purinyl amine is not required. Exclusive
protodesilylation at the 5’-silyl ether only unveiled the 5’-alcohol 11, using
SCHEME 1 Reagents & conditions: a) Nicotinamide, CH3CN, then TMSOTf, RT, 1.5 h; then MeOH, RT; 1 h;
b) POCI3, PO(OMe)3, 0°C, 4 h.

J. Dowden et al.
515
carefully controlled acidic conditions.[6] Phosphoramidite reagents were again
explored for installation of orthogonally protected 5’-phosphates. Reaction times
were generally much slower, presumably a result of the more crowded steric
environment. That a number of reagents unexpectedly proved to be incompatible
with this route was a source of significant frustration. Bis(prop-2-enyloxy)-N,N-
diisopropylaminophopshane 9, reacted only sluggishly when imidazolium triflate
was used as catalyst. 1H-tetrazole appeared to provide smooth reaction after
appropriate precautions were taken to protect the purinyl amine, but led to
1
material of higher mass than expected. Examination of the H NMR spectrum
showed that the allylic protons integrated to a total of four, rather than the
expected two, possibly due to the formation of an unexpected silanol phosphate
17. This functional group proved to be relatively robust and we were not able to
convert this product into the desired precursor (Scheme 2).
Instead, tris-imidazolylphosphine was generated in situ from PCl3 and
imidazole and reacted with the 5’-hydroxyl, rapidly furnishing the H-phosphonate
12, providing a convenient alternative route that proceeded in high yield. Sub-
sequent oxidation was carried out using conditions originally reported by Sekine
and coworkers[7] with monitoring by 31P NMR. The H-phosphonate triethylam-
monium salt (d(31P) = 5.5 ppm) was converted to the bis(trimethylsilylphosphonate)
(d(31P) = 116 ppm) after treatment with N,O-bis(trimethylsilyl)acetamide for approx-
imately 1 h. Addition of (1S)-(+)-(10-camphorsulfonyl)oxaziridine (CSO) resulted in
oxidation to the bis(trimethylsilyl)phosphate (d(31P) = À16 ppm) after approxi-
mately 20 min, which was then slowly hydrolized to the phosphate (d(31P) = 1 ppm)
upon addition of D2O. Using this route we were able to obtain the phosphate
precursor 13, from adenosine in 72% yield (69% from 9) (Figure 2).
Our initial attempts to generate the phosphomorpholidate from protected
adenosine phosphate were low yielding and did not encourage further exploration
SCHEME 2 Reagents & conditions: c) 6, CH2Cl2, RT; 3 h, then mCPBA, À78°C, 0.5 h, 98%; d) TFA/H2O/THF
1:1:4, 0°C, 3 h, 98%; e) PCI3, imidazole, Et3N, THF, 0°C, 15 mins, then 1 m TEAB aq. pH 7, RT, 15 mins, quant.

516
J. Dowden et al.
of this route. Instead, contrary to other reports,[4] we found that b-NMN imidazolide
(14, d(31P) = À10.6 ppm) reacted with good conversion when monitored by 31P
NMR spectroscopy. Unfortunately, the pyrophosphate product 15 is amphiphilic
and purification by HPLC or ion exchange chromatography was sufficiently
difficult to force further manipulation of the crude. Treatment of intermediate 15
with an acetic acid buffered solution of 1M TBAF in THF effected simultaneous
cleavage of the silanol ether and one of the phosphate benzyl protecting groups,
allowing ion exchange purification of the resulting monobenzyl NADP 16. This
was the only compound detectable using this purification method, suggesting that
no migration of the 2’-phosphate had occurred. The best yield for this reaction was
22% over these two steps, and although this yield is modest, it is comparable with
many literature reports for pyrophosphate bond formation (Scheme 3).
Ultimately, transfer hydrogenation effected clean removal of the remaining
benzyl-protecting group, providing clean NADP 2 in a reasonable 77% yield after
purification using ion exchange chromatography. In seeking a route to NAADP 1,
we sought to confirm the identity of the synthetic NADP, by subjecting it to base
exchange in the presence of nicotinic acid and crude Aplysia ADP ribosyl cyclase
[E.C.3.2.2.5] to provide NAADP 1, in 63% isolated yield.
Vesicles derived from intracellular stores remain intact and sequester calcium
when supplemented with and ATP-regenerating system. Using a calcium reported
dye fluo-3 and cuvette-based fluorimetry, concentration-dependent calcium release
can be observed when the homogenate is challenged with appropriate second
messengers. After careful quantification by total phosphate assay, the identity
of the synthetic NAADP 1 was checked by comparing its ability to induce Ca2+
release from aliquots of cell-free sea urchin egg homogenate with that of authen-
tic material.
FIGURE 2

J. Dowden et al.
517
SCHEME 3 Reagents & conditions: g) DMF, RT, 16 h; h) 1M TBAF/THF, AcOH, 0°C, 1.5 h, 22% i) 10% Pd/C,
cyclohexadiene, MeOH/H2O, RT, 2 h, 77%; j) Nicotinic acid, Aplysia ADP-ribosyl cyclase, 1M NaOAc aq. pH 4,
RT, 5 h, 63%.
The synthetic material 1 behaved in an identical manner to authentic NAADP,
inducing calcium release at 100 nM concentrations and potently causing time-
dependent inactivation of calcium release at subthreshold concentrations.
In summary, we have completed the first synthesis of NADP 2 and converted
this material into the second messenger NAADP.[1] This route provides robust and
high yielding synthesis of adenosine analogues that furnishes and maintains the
important 2’-phosphate functional group, without apparent migration. Synthesis of
the b-NMN, 6 portion of this molecule is more challenging, however, and we are
currently engaged in developing efficient synthesis of analogous nicotinic acid
structures toward total chemical synthesis of NADP. The latter pyrophosphate bond
formation and penultimate protecting group removal provides sufficient material
for biological evaluation.
Much remains to be learned about the proteins that govern the intracellular
chemistry of this interesting second messenger and we recognize a need for
NAADP analogues that can be used as chemical probes to interrogate its chemical
biology. This first total synthesis of NADP represents a significant milestone toward
this wider objective.
REFERENCES
1. Dowden, J.; Moreau, C.; Brown, R.S.; Berridge, G.; Galione, A.; Potter, B.V.L. First chemical synthesis of the
novel second messenger nicotinic acid adenine dinucleotide monophosphate NAADP via total synthesis of
nicotinamide adenine dinucleotide monophosphate NADP. Angew. Chem. Int. Ed. 2004, 43, 4637–4640.

518
J. Dowden et al.
2. Lee, H.C. Calcium signaling: NAADP ascends as a new messenger. Curr. Biol. 2003, 13, R186–R188.
3. Masgrau, R.; Churchill, G.C.; Morgan, A.J.; Ashcroft, S.J.H.; Galione, A. NAADP: a new second messenger
for glucose-induced Ca2+ responses in clonal pancreatic beta cells. Curr. Biol. 2003, 13, 247–251.
4. Lee, J.; Churchil, H.; Choi, W.B.; Lynch, J.E.; Roberts, F.E.; Volante, R.P.; Reider, P.J. A Chemical synthesis of
nicotinamide adenine dinucleotide NAD(+). Chem. Commun. 1999, 729–730.
5. Tanimori, S.; Ohta, T.; Kirihata, M. An efficient chemical synthesis of nicotinamide riboside (NAR) and
analogues. Bioorg. Med. Chem. Lett. 2002, 12, 1135–1137.
6. Zhu, X.F.; Williams, H.J.; Scott, A.I. Aqueous trifluoroacetic acid—an efficient reagent for exclusively cleaving
the 5’-end of 3’,5’-TIPDS protected ribonucleosides. Tetrahedron Lett. 2000, 41, 9541–9545.
7. Wada, T.; Mochizuki, A.; Sato, Y.; Sekine, M. A convenient method for phosphorylation involving a facile
oxidation of H-phosphonate monoesters via bis(trimethylsilyl) phosphates. Tetrahedron Lett. 1998, 39, 7123–
7126.
8. Brown, R.S.; Moreau, C.; Dowden, J.; Potter, B.V.L. A concise synthesis of Tiazofurin. Tetrahedron Lett.
2002, 43, 6561–6562.