Synthesis of cyclic adenosine 5′-diphosphate ribose analogues: A C2′ endo/syn "southern" ribose conformation underlies activity at the sea urchin cADPR receptor

Article abstract of DOI:10.1039/c0ob00396d

Novel 8-substituted base and sugar-modified analogues of the Ca ²⁺ mobilizing second messenger cyclic adenosine 5′-diphosphate ribose (cADPR) were synthesized using a chemoenzymatic approach and evaluated for activity in sea urchin egg homogenate (SUH) and in Jurkat T-lymphocytes; conformational analysis investigated by ¹H NMR spectroscopy revealed that a C2′ endo/syn conformation of the "southern" ribose is crucial for agonist or antagonist activity at the SUH-, but not at the T cell-cADPR receptor.

Full text of DOI:10.1039/c0ob00396d

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PAPER
Synthesis of cyclic adenosine 5¢-diphosphate ribose analogues: a C2¢ endo/syn
“southern” ribose conformation underlies activity at the sea urchin cADPR
receptor†
Christelle Moreau,a Gloria A. Ashamu,a Victoria C. Bailey,a Antony Galione,b Andreas H. Gusec and
Barry V. L. Potter*a
Received 7th July 2010, Accepted 14th September 2010
DOI: 10.1039/c0ob00396d
Novel 8-substituted base and sugar-modified analogues of the Ca2+ mobilizing second messenger
cyclic adenosine 5¢-diphosphate ribose (cADPR) were synthesized using a chemoenzymatic approach
and evaluated for activity in sea urchin egg homogenate (SUH) and in Jurkat T-lymphocytes;
conformational analysis investigated by 1H NMR spectroscopy revealed that a C2¢ endo/syn
conformation of the “southern” ribose is crucial for agonist or antagonist activity at the SUH-,
but not at the T cell-cADPR receptor.
bonds betweenN9 and C1¢ of the ribose ring (or “southern” ribose)
and N1 and C1¢¢ of the second ribose moiety (or “northern”
ribose). The structure also revealed both N-glycosidic bonds
to be in the b-configuration and the “southern” ribose to be
predominantly in the C2¢-endo conformation.3 The cADPR/Ca2+
signalling system is active in diverse cellular systems such as animal
cells e.g. smooth, skeletal and cardiac muscle, acinar cells, protozoa
and plant cells.4 Pharmacological studies indicate that ryanodine
receptors are the intracellular Ca2+ channels involved in cADPR-
induced Ca2+ release.5–7
Many cADPR analogues have been synthesized since and their
Ca2+ mobilising activities examined in various systems, but mainly
in sea urchin egg homogenates (SUH) and Jurkat T cells (JTC).8–12
Despite these efforts and although many useful synthetic tools
have been developed, the structural features needed for both
agonist/antagonist activities at the receptor still remain somewhat
unclear. Early findings seemed to reveal that substitution at the 8-
position of the adenine ring of cADPR (2 and 3, Fig. 1) converts
a cADPR agonist into an antagonist in both SUH and JTC.13,14
However, it was later discovered that some 8-substituted cyclic
Introduction
Cyclic adenosine 5¢-diphosphoribose (cADPR, 1, Fig. 1), a
metabolite of nicotinamide adenine dinucleotide (NAD+), was
first discovered in 1987 by Lee and co-workers as a potent
Ca2+ releasing second messenger.1 Based on NMR and mass
spectroscopy this dinucleotide was suggested to possess a cyclic
structure with a glycosidic bond between N6 of the adenine ring
and the anomeric carbon C1¢¢ of the ribose linked to nicotinamide.2
Later, the structure of cADPR was finally revealed by X-ray
analysis to be a unique cyclic dinucleotide bearing two glycosidic
aWolfson Laboratory of Medicinal Chemistry, Department of Pharmacy
and Pharmacology, University of Bath, Bath, UK, BA2 7AY. E-mail: b.v.l.
potter@bath.ac.uk; Fax: +44 1225 386114; Tel: +44 1225 386639
bDepartment of Pharmacology, University of Oxford, Mansfield Road,
Oxford, UK, OX1 3QT
cInstitute of Biochemistry and Molecular Biology I: Cellular Signal Trans-
duction, University Medical Center Hamburg-Eppendorf, Germany
† This paper is part of an Organic & Biomolecular Chemistry web theme
issue on chemical biology.
Fig. 1 cADPR analogue structures and numbering system.
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adenosine diphosphocarbocyclic ribose (cADPcR, 4–7, Fig. 1)
analogues are agonists rather than antagonists in SUH, therefore
suggesting that the oxygen atom of the “northern” ribose could
be a crucial feature for antagonistic activity.15 A small number of
8-substituted cyclic inosine diphosphoribose (cIDPR, 8, Fig. 1)
analogues have been synthesised in our laboratory. Some of these
acted as agonists in T cells, suggesting the 6-amino group to be
an important structural feature for antagonistic activity.16–18 Use
of this template has lead to structural biology insight on the
cADPR hydrolase CD38.19 Modification of the base moiety of
cADPR has produced an agonist analogue in 3-deaza cADPR, 70
times more potent than cADPR in SUH.20 More radical structural
modifications of the “northern” ribose led to agonist analogues.21
Agonistic activity was also observed when the pyrophosphate
linkage was extended to a triphosphate.22 Further modifications
of the “southern” ribose revealed that the 2¢-OH has little
effect on agonist activity in SUH, but that 3¢-O-alkylation could
generate an antagonist.23 A recent NMR conformational study
of this compound related the antagonism observed to an altered
“backbone” conformation, but the evidence was not sufficient to
establish this idea.24
between analogues acting at both SUH and JTC cADPR receptors
it is becoming clear, not surprisingly, that the receptors in these
invertebrate and mammalian systems are different. In any case,
these results illustrate that the global structural features required
for cADPR agonism/antagonism are far from clear and are
also different from system to system. There is clearly a need to
rationalise the considerable range of activities now observed and
deduce trends to underpin future design strategies in this area for
chemical biological intervention.
Here, we report the synthesis of several new cADPR analogues
(9–18) modified at the “southern” ribose and in the purine ring,
as well as their biological evaluation in both SUH and JTC.
Also, we report, for the first time, how agonism/antagonism
in SUH may be linked to partial conformational preference in
cADPR. A preliminary account of some of the synthetic work has
appeared.14
Results and discussion
Chemistry
Recently, we successfully synthesized a series of 8-substituted
2¢-deoxy analogues of cADPR.25 Structure–activity relationship
studies revealed that deletion of the 2¢-OH group decreases
antagonistic activity but, more importantly, some classical an-
tagonist analogues unexpectedly showed agonistic activity at high
concentrations in SUH. While some parallel trends were observed
Synthesis of 8-modified cADPR analogues. All the cADPR
analogues (2, 3, 9–15) described herein were synthesized chemo-
enzymatically; the last step being the enzymatic cyclisation reac-
tion of the requisite NAD+ analogue catalyzed by ADP-ribosyl
cyclase from Aplysia californica. Syntheses of the 8-modified
cADPR analogues are summarised in Scheme 1.
Scheme 1 Synthesis of 8-modified cADPR analogues. Reagents and conditions: (a) PdCl2, PPh3, AlMe3, THF, reflux, 2.5 h. (b) POCl3, TEP, 0 ◦C. (c)
b-NMN+, DCC, pyridine:water (4 : 1), 7 days, rt. (d) Aplysia cyclase, 25 mM HEPES (pH 6.8), 20 min, rt.
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8-Bromo adenosine 5¢-monophosphate (19) was prepared using
established methodology developed by Yoshikawa et al.26 Crude
product was purified on an ion-exchange Q-Sepharose column
eluted with a gradient of 1 M TEAB followed by a second
column of activated charcoal to removed the inorganic phos-
phate contaminant. 8-Methyl adenosine 5¢-monophosphate (8-
Me-AMP, 23) was prepared in 2 steps by a palladium-catalysed
coupling reaction of 8-Br-adenosine with a methylating agent,
followed by phosphorylation. Other AMP derivatives (20–26)
were prepared by nucleophilic displacement on 8-bromo AMP
by the appropriate agent. 8-modified NAD+ analogues (31–39)
were synthesised by a method developed by Hughes et al. which
consists of the coupling of b-nicotinamide mononucleotide (b-
NMN+) with the corresponding monophosphate in the presence
of dicyclohexylcarbodiimide (DCC) as a coupling reagent.27 This
method, however, was low yielding (7–61%) and lately we have used
a better approach involving the coupling of a monophosphate with
a nucleotide phosphoromorpholidate in the presence of a Lewis
acid.16,25
Scheme 2 Synthesis of cAcycloDPR. Reagents and conditions: (a) POCl3,
TEP, 0 ◦C, 1.5 h then ice/water. (b) b-NMN+, DCC, 7 days, rt. (c) Aplysia
cyclase, 25 mM HEPES (pH 6.8), rt.
All of the final nucleotide pyrophosphates were carefully
separated from any monophosphates and purified to homogeneity
by ion-exchange chromatography. Subsequent incubation with
the Aplysia enzyme, followed by purification by ion-exchange
produced the desired 8-modified cADPR analogues. These
were: 8-bromo cyclic adenosine diphosphoribose (8-Br-cADPR,
2), 8-amino cyclic adenosine diphosphoribose (8-NH2-cADPR,
3), 8-methylamino cyclic adenosine diphosphoribose (8-NHMe-
cADPR, 9), 8-dimethylamino cyclic adenosine diphosphoribose
(8-NMe2-cADPR, 10), 8-methyl cyclic adenosine diphosphoribose
(8-Me-cADPR, 11), 8-methoxy cyclic adenosine diphosphoribose
(8-OMe-cADPR, 12), 8-piperidyl cyclic adenosine diphosphori-
bose (8-Pip-cADPR, 13), 8-oxy cyclic adenosine diphosphoribose
(8-Oxy-cADPR, 14) and 8-aza-9-deaza cyclic adenosine diphos-
phoribose (cFDPR, 15).
Synthesis of ribose-modified cADPR. To further investigate
the requirements of the adenosine ribose hydroxyls for Ca2+
mobilisation, three novel compounds were designed (i) cyclic
acycloadenosine diphosphate ribose (cAcycloDPR, 16) in which
the ribose ring was replaced with a flexible ether chain, (ii)
cyclic adenine 9-b-D-arabino ribofuranoside diphosphate ribose
(cAraDPR, 17) in which the stereochemistry of the 2¢-OH was
reversed and (iii) cyclic 2¢,3¢-O-isopropylidene adenosine diphos-
phate ribose (cAcetDPR, 18) in which the 2¢,3¢ diol was protected
with an isopropylidene group.
Scheme 3 Synthesis of cAraDPR. Reagents and conditions: (a) POCl3,
TEP, 0 ◦C, 1.5 h then ice/water. (b) b-NMN+, DCC, 7 days, rt. (c) Aplysia
cyclase, 25 mM HEPES (pH 6.8), rt.
The synthesis of these three analogues 16, 17 and 18 is sum-
marised in Schemes 2, 3 and 4 respectively. Acyclic adenosine28,29
(43), arabinofuranoside (44) and isopropylidene protected adeno-
sine (45) were selectively phosphorylated at the 5¢-hydroxyl using a
POCl3, triethylphosphate and water mixture to give their respective
monophosphates 28, 29 and 30. Subsequent pyrophosphate bond
formation followed by cyclase incubation under the general
conditions described for the 8-modified analogues, generated the
cAcycloDPR (16), cAraDPR (17), cAcetDPR (18) analogues
respectively.
Biology
Scheme 4 Synthesis of cAcetDPR. Reagents and conditions: (a) POCl3,
TEP, 0 ◦C, 1.5 h then ice/water. (b) b-NMN+, DCC, 7 days, rt. (c) Aplysia
cyclase, 25 mM HEPES (pH 6.8), rt.
Biological evaluation of 8-modified analogues of cADPR on
Ca2+-release in sea urchin egg homogenate. The first indication
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Table 1 Antagonistic activitiesa of cADPR analogues 2, 3, 9–14
that an exocyclic substitution in position 8 of the adenine
ring of cADPR might be important in designing an antagonist
of cADPR-induced Ca2+ release was demonstrated by Walseth &
Lee.13 They found that substitution of HA8 with an amino group
converted the cADPR from an agonist into an antagonist.25
Two other analogues, 8-bromo and 8-azido-cADPR were also
found to be antagonists, but with lesser potency. The potency of
8-azido-cADPR was in between that of 8-amino-cADPR and
8-bromo-cADPR, although IC50 values were not reported. It was
suggested that the size of the substituent (expressed in atomic
units) may be responsible for the difference in potency of the three
8-substituent analogues synthesised as antagonists. The larger the
size of the substituent at this position the lower the potency, as
an increase in size from 16 (-NH2) to 42 (-N3) to 79 (-Br) resulted
in a decrease in potency as an antagonist in this order. We have
therefore synthesized various 8-substituted analogues of cADPR
in order to further investigate this hypothesis.
8-Amino-cADPR (3) was also synthesized as a control to
enable us to carry out comparative studies. 8-Amino-cADPR was
confirmed as a potent antagonist in SUH with an IC50 value
of 0.01 mM. Replacement of the amino group with a methyl
group as in 8-methyl-cADPR (11) gave an antagonist with an
IC50 value of 0.53 mM. The –CH3 group is similar in atomic
mass to –NH2 and yet 8-Me-cADPR was 53 times less potent
as an antagonist compared to 8-NH2-cADPR (3). Substitution
with an oxy group (with an atomic mass of 17) was attempted.
The analogue obtained (14) showed still weaker activity as an
antagonist with an approximate IC50 of 2 mM. 8-“Hydroxy” AMP
(26a) is known to exist predominantly in the keto form (26b) at
physiological range 5 < pH < 9,30,31 hence the nitrogen N7 is
protonated (Fig. 2). It is reasonable to assume that 8-oxy cADPR
is also predominantly in the keto form at the same pH range.
The reduction in activity could be due to the protonation at N7
which may affect receptor binding. Substitution with groups of
varying size such as -NHMe, -NMe2 and -piperidyl produced novel
compounds (9, 10 and 13 respectively) with perturbations to the 8-
amino motif, which were investigated for antagonist activity (Table
1). 8-NHMe-cADPR (9) was a much weaker antagonist compared
to 8-NH2-cADPR (3) with an IC50 of ~40 mM, 8-NMe2 cADPR
(10) was weaker than 8-NHMe-cADPR (9) as an antagonist, but
8-piperidyl (13) was not active at all as an antagonist up to 50
mM. Other analogues such as 8-OCH3-cADPR (12) and 8-Br-
cADPR (2) were also synthesised for comparative study. These
analogues were antagonists with IC50 values of 4.8 and 0.97 mM
respectively. It appears, therefore, from this study, that the ability
of the molecule to hydrogen bond at the 8-position with its target
protein in SUH is not critical for activity as an antagonist as 8-
CH3-cADPR (11) is a better antagonist compared to those that
can form hydrogen bonds such as 8-OCH3-cADPR (12) and 8-
NHMe-cADPR (9).
8-X-cADPR
IC50 (mM) in SUH
IC50 (mM) in JTC
8-NH2-cADPR (3)
8-Me-cADPR (11)
8-Br-cADPR (2)
8-Oxy-cADPR (14)
8-OMe-cADPR (12)
8-NHMe-cADPR (9)
8-NMe2-cADPR (10)
8-Pip-cADPR (13)
0.01
0.53
0.97
2
1
n.d
b
n.d
0.2
n.d
n.d
n.d
4.8
<40
>40
No effect
a Ca2+ mobilisation from sea urchin egg microsomes was evaluated
fluorimetrically using 2.5% egg homogenates containing fluo-3 (3 mM)
as previously described.25,33 Ca2+ release assay in Jurkat T cells was carried
out as reported previously.34,35 b IC50 was not reached (partial antagonist).
nd = active compound, IC50 was not determined.
Modification in the purine ring as in 8-aza-9-deaza cADPR
(15), resulted in an analogue that is 10 times less potent as an
agonist compared to cADPR (EC50 = 0.31 mM). The C8 carbon
in the purine ring was replaced by a nitrogen atom and the
N9 nitrogen was replaced by a carbon atom. Modification in
the purine ring hence did not produce an antagonist compared
to exocyclic modification as discussed above. The reduction in
activity could be due to protonation of the N7 nitrogen which
may affect receptor binding. A small modification at this position
as in 7-deaza cADPR (51), has been reported to result in partial
agonist activity 32
Biological evaluation of sugar-modified analogues of cADPR on
Ca2+ release in sea urchin egg homogenate. Preliminary work
has already been published. The importance of the 2¢-OH was
investigated. It was found that 2¢-deoxy-cADPR is a potent agonist
in SUH but not in T cells.23 Other groups reported that 2¢-
cADPRP is an agonist in T cells but is inactive in SUH.8,36
Moreover, 3¢-deoxy cADPR was found to be a poor agonist in
SUH,23 therefore indicating the importance of the 3¢-OH group for
agonistic activity in SUH, whilst the 2¢-hydroxyl group appeared
to be less important.
In order to further study these interactions three compounds
(16, 17 and 18) were designed, synthesised and evaluated for their
Ca2+ mobilising activities in SUH. Both cAcycloDPR (16) and
cAraDPR (17) were inactive whilst cAcetDPR (18) was a poor
agonist (EC50 = 12 mM). A careful study of the biological data
obtained for these compounds suggested that instead of there
being a relationship between the groups attached to the ribose ring
and activity, the conformation of the ribose ring is of importance
and will be discussed later.
Biological evaluation of 8-modified analogues of cADPR on
Ca2+-release in permeabilised Jurkat T-cells. In order to design
antagonists for cADPR-induced mechanisms in Jurkat T-cells
(JTC), we decided to investigate whether 8-NH2-cADPR (3) and
8-Br-cADPR (2) would have a similar effect to that seen in the
SUH system. We found that these two analogues inhibited Ca2+
release mediated by cADPR. 8-Br-cADPR was less effective as an
antagonist compared to 8-NH2-cADPR. We decided to investigate
whether there is a link between the size of the 8-substituent and the
potency of the antagonist as an aid to designing potent inhibitors
of the cADPR-induced mechanism. The extension to mammalian
cells is essential for the aim of defining a wider applicability for the
Fig. 2 Tautomeric form of 8-“hydroxyl” AMP (26a) vs. 8-oxy AMP (26b).
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cADPR signalling system. Substitution of any group other than
hydrogen into position 8 of the adenine ring resulted in analogues
with antagonist activity in this system. Hence, all the 8-substituted
analogues synthesized inhibited cADPR-induced Ca2+-release. 8-
NH2-, 8-CH3- and 8-NMe2-cADPR (3, 11 and 10) are potent
inhibitors. The activity of 8-NMe2-cADPR (10), although weaker
than its parent (3), suggests that hydrogen bond donation via the
8 position to the receptor is not a requirement for antagonism. 8-
CH3-cADPR (11) completely abolished cADPR induced release,
suggesting further that hydrogen bond interaction between the
substituent and the receptor site is unlikely to be responsible for
antagonist activity, as the -CH3 group in 8-Me-cADPR cannot
form a hydrogen bond. There is, however, a possibility of a
hydrophobic interaction taking place, supported by the fact that
8-NHMe-cADPR (9) antagonized at higher concentrations and
shows less potent inhibition as compared to 8-NMe2-cADPR
(10), the -NMe2 substituent being more hydrophobic than the -
NHMegroup. Moreover, andinstrongcontrasttothelackofeffect
in SUH, 8-piperidyl-cADPR antagonized at low concentrations,
broadly comparable to 8-NH2-cADPR (3). The piperidyl group,
though large, is also hydrophobic and flexible. Hence, it can change
its conformation to fit a receptor site. 8-Br-cADPR (2) showed
relatively poor inhibition, possibly due to the bulk of the bromo
substituent and, unlike the piperidyl group, the bromo atom is
a large rigid structure and hence is potentially hindered from
fitting tightly into the receptor site. 8-Oxo-cADPR (14) showed
poor inhibition even though the substituent is similar in size
to a -NH2 and -CH3 substituent. There are potential multiple
alterations in this analogue compared to cADPR as the 8-oxo
form can tautomerise to the 8-hydroxy form. This change, though
small, may be sufficient to affect the interaction of this analogue
with the receptor. 8-OCH3-cADPR (12) showed good inhibition
comparable to 8-CH3-cADPR (11). This compound has found
application in a seminal study on the role of cADPR in T cells.37
Overall, it appears that the presence of a hydrophobic group
in position 8 enhances antagonist activity of the analogue in
permeabilised JTC.
Fig. 3 Schematic representation of the ribofuranose ring in both C2¢ endo
and C3¢ endo conformation.
developed by Altona and Sundaralingam have established that the
C2¢-endo/C3¢-endo ratio can be mathematically calculated from a
1H NMR spectrum by adaptation of the equation C2¢-endo =
[J1¢,2¢/(J1¢,2¢ + J3¢,4¢)] ¥ 100. Moreover, they have also shown that the
sum J1¢,2¢ + J3¢,4¢ is close to 10 and therefore the ratio C2¢-endo/C3¢-
39,40
endo can be estimated from the equation 10J1¢,2¢
.
In cADPR, the adenine base is oriented in the syn conformation
about the glycosidic bond both in the crystal structure3 and
in solution.24,38 Information on the conformation about the
glycosidic bond has been well documented, and has revealed
that the chemical shift of the H-2¢ proton could be used very
effectively as a good indicator for the syn/anti equilibrium in
nucleosides and nucleotides.41,42 Typically, purine nucleosides and
nucleotides with a bulky substituent at C8 display a characteristic
downfield shift of H-2¢ upon 8-substitution. The chemical shift
values for the protons common to some AMP, NAD+ and cADPR
analogues prepared during the course of this study are listed
in Table 2 and indeed, in agreement with early reports, we
have found that the NMR resonance for H-2¢ can be used very
effectively to assign the favoured glycosyl bond conformation.
Therefore, AMP, 8-amino- and 8-aminomethyl AMP appear to be
predominantly anti as previously reported.43,44 The H-2¢ resonance
of 7-deaza AMP suggests that this nucleotide has significant
anti glycosyl bond character, a result that is not surprising due
to its resemblance to AMP. Our data show a similar trend for
the H-2¢ resonance values in the NAD+ series as in the AMP
series. We had previously observed in the hypoxanthine series that
the glycosyl bond conformation is unaffected by pyrophosphate
bond formation.16 However, during the cyclization reaction, the
NAD+ analogues in the anti conformation e.g NAD+, 8-amino-,
8-methylamino- and 7-deaza NAD+ have their cyclic counterpart
predominantly in the syn configuration.
Modification in the purine ring in analogue (15) did not affect
the Ca2+ release property of the analogue. This analogue showed
a similar Ca2+ release profile compared to cADPR in JTC even
though the 8-position is altered (although not outside the ring).
These alterations appear to be unimportant for Ca2+ releasing
activity in this system.
We have therefore extracted information on the preferred
conformation about the glycosidic bond based on NMR results,
and have determined the conformation of the “southern” ribose
using Altona’s approach for all the cADPR analogues synthesized
in our laboratory as well as those from other groups. The results
of our analysis are summarized in Table 3. Structures of analogues
46–62 are shown in Fig. 4 and 5.
At first glance, it can be seen that most cADPR analogues
adopt a syn conformation about the N9-glycosyl linkage except
cAcetDPR (18), cAraDPR (17) and 2¢¢,3¢¢-dideoxydidehydro
cADPcR (62). These same compounds also adopt a C3¢ endo form
in the N9-ribose moiety.
Conformational analysis
Despite significant past work by several groups, the structural
features required for cADPR-mediated agonism/antagonism
remain unclear. Recently, Shuto et al. reported that 2¢¢,3¢¢-
dideoxydidehydro cADPcR (62), an inactive compound, adopted
a major C3¢ endo and a high anti conformation in aqueous
solution,38 therefore indicating that both the N9-ribose moiety
and the N9-glycosidic bond conformations may be of crucial
importance for the Ca2+ release activities of cADPR analogues.
In solution, nucleosides and nucleotides exist in conformational
equilibrium between C2¢-endo and C3¢-endo forms (Fig. 3). In
addition, the nucleobase can be oriented towards (syn) or away
(anti) from the ribose ring. These local changes will affect the
overall conformation of the cyclic dinucleotides. Extensive studies
Additionally, 3¢-deoxy cADPR, 2¢-cADPRP and 3¢-cADPRP
also display a C3¢ endo form, but with a predominantly syn
glycosidic bond. It appears that these compounds are either
inactive or are really poor agonists in SUH.23 Conversely, all
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Table 2 1H NMR chemical shifts (d) and conformational analysis of 8-X-AMP, 8-X-NAD+ and 8-X-cADPR in D2O
8-X-AMP
8-X-NAD+
8-X-cADPR
a
X
H-1¢
H-2¢
D1¢-2¢
Confb
H-1¢
H-2¢
D1¢-2¢
Conf
H-1¢
H-2¢
D1¢-2¢
Conf
H
Br
Me
NH2
NHMe
NMe2
Pip
OMe
Oxy
7-Deaza
7-Deaza-8-Br
6.1
5.8
5.8
5.8
5.8
5.6
5.6
5.7
5.6
6.1
6.0
4.8
5.1
4.8
4.6
4.6
5.1
5.1
4.8
4.9
4.5
5.1
1.3
0.7
1.0
1.2
1.2
0.5
0.5
0.9
0.7
1.6
0.9
anti
syn
syn
anti
anti
syn
syn
syn
syn
anti
syn
6.0
5.7
5.7
5.9
5.7
5.9
5.5
5.7
5.5
6.1
6.0
4.7
5.0
4.7
4.6
4.5
5.4
5.0
4.8
4.9
4.4
5.1
1.3
0.7
1.0
1.2
1.2
0.5
0.5
0.9
0.6
1.7
0.9
anti
syn
syn
anti
anti
syn
syn
syn
syn
anti
syn
5.8
6.3
5.8
5.8
5.7
6.2
5.9
5.9
5.8
5.7
6.0
5.2
5.6
5.3
5.4
5.6
5.6
5.6
5.6
5.6
5.3
5.4
0.6
0.7
0.5
0.4
0.1
0.6
0.3
0.3
0.2
0.4
0.6
syn
syn
syn
syn
syn
syn
syn
syn
syn
syn
syn
a Difference in chemical shifts between H-1¢ and H-2¢. b Favoured glycosidic bond conformation.
Table 3 N9 ribosyl moiety and N9 glycosidic bond conformation of cADPR analogues and their Ca2+ release activities in sea urchin egg homogenate
Compounds
J1¢,2¢
J3¢,4¢
C2¢ endo
H-1¢
H-2¢
D1¢-2¢
Syn/Anti
Activity in SUH
Compounds synthesized by our group14,23,25,32,45–50
cADPR (1)
5.6
3.2
n.da
n.d
n.d
n.d
n.d
n.d
n.d
—
8.1
3.1
n.d*
n.d
n.d
n.d
2.7
n.d
3.5
n.d
64%
53%
56%
55%
64%
53%
59%
53%
—
47%
22%
70%
30%
40%
37%
67%
64%
63%
66-69%
5.8
6.3
5.8
5.7
6.2
5.8
5.9
5.9
—
5.2
5.6
5.3
5.6
5.6
5.3
5.6
5.0
—
0.6
0.7
0.5
0.1
0.6
0.5
0.3
0.9
—
Syn
Syn
Syn
Syn
Syn
Syn
Syn
Syn
—
Anti
Anti
Syn
Syn
Syn
Syn
Syn
Syn
Syn
Syn
Agonist EC50 = 32 nM
Antagonist IC50 = 0.97 mM
Antagonist IC50 = 0.01 mM
Weak antagonist IC50 = 40 mM
Weak antagonist IC50>40 mM
Antagonist IC50 = 0.53 mM
Antagonist IC50 = 4.8 mM
Agonist EC50 = 0.3 mM
Inactive
8-Bromo-cADPR (2)
8-NH2 cADPR (3)
8-NHMe cADPR (9)
8-NMe2 cADPR (10)
8-Me cADPR (11)
8-OMe cADPR (12)
8-Aza-9-deaza cADPR (15)
cAcycloDPR (16)
5.3
5.6
5.5
6.4
5.3
5.9
5.3
—
7.3
2.0
7.0
3.0
4.0
3.7
5.6
6.4
5.9
6.9-
6.6
cAraDPR (17)
6.2
6.2
5.1
5.1
1.1
1.1
Inactive
cAcetDPR (18)
Poor agonist EC50 = 12 mM
Agonist EC50 = 58 nM
Poor agonist EC50 = 5 mM
Inactive
2¢-Deoxy cADPR (46)
3¢-Deoxy cADPR (47)
2¢-cADPRP (48)
5.9
6.2
6.0
5.9
5.7
6.0
6.06
5.2
5.7
5.3
5.3
5.3
5.4
—
0.7
0.5
0.7
0.6
0.4
0.6
—
3¢-cADPRP (49)
Inactive
3¢-OMe cADPR (50)
7-Deaza cADPR (51)
7-Deaza-8-Br-cADPR (52)
8-Amino-2¢-deoxy cADPR (53)
Antagonist IC50 = 4.8 mM
Agonist EC50 = 90 nM
Antagonist IC50 = 0.73 mM
Antagonist IC50 = 0.22 mM
Compounds synthesized by Shuto’s group15,38,51,52
cADPcR (4)
8-Cl-cADPcR (5)
6.1
6.3
6.3
6.2
6.1
6.2
6.3
6.2
5.9
5.4
6.3
5.3
1.5
2.6
2.4
n.d
n.d
2.6
n.d
2.3
n.d
3.0
2.4
2.4
2.2
6.3
70%
72%
63%
62%
70%
62%
73%
62%
66%
69%
72%
70%
19%
6.0
6.1
5.9
5.9
6.1
6.1
5.9
5.9
6.1
6.1
6.0
6.1
6.1
5.1
5.2
5.2
5.2
5.2
5.2
5.3
5.2
5.2
5.2
5.1
5.2
5.0
0.9
0.9
0.7
0.7
0.9
0.9
0.6
0.7
0.9
0.9
0.9
0.9
1.1
Syn
Syn
Syn
Syn
Syn
Syn
Syn
Syn
Syn
Syn
Syn
Syn
Anti
Agonist EC50 = 79 nM
Agonist EC50 = 19 mM
8-NH2-cADPcR (6)
8-N3-cADPcR (7)
Agonist EC50 = 80 nM
Agonist EC50 = 3.9 mM
Agonist EC50 = 14 nM
3¢¢-Deoxy cADPcR (54)
8-Cl-3¢¢-deoxy cADPcR (55)
8-NH2-3¢¢-deoxy cADPcR (56)
8-N3-3¢¢-deoxy cADPcR (57)
2¢¢-Deoxy cADPcR (58)
2¢¢,3¢¢-Dideoxy cADPcR (59)
2¢¢-OMOM-3¢¢-OMe cADPcR (60)
2¢¢-OMOM-3¢¢-deoxy cADPcR (61)
2¢¢,3¢¢-Dideoxy- didehydro cADPcR (62)
Partial agonist EC50 = 0.19 mM
Partial agonist EC50 = 17 nM
Partial agonist EC50 = 0.49 mM
Agonist EC50 = 0.61 mM
Agonist EC50 = 0.73 mM
Agonist EC50 = 0.88 mM
Agonist EC50 = 0.88 mM
Poor agonist EC50 > 20 mM
a Not determined. The compounds in italics do not display a C2¢ endo/syn conformation.
the other analogues in this table displaying a C2¢ endo/syn
conformation are either agonists or antagonists in SUH.
2¢-deoxy cADPR (46) is inactive in T cells, but is a potent agonist in
SUH, whereas 2¢-cADPRP (48) is active in T cells but not in SUH
(Table 4). 3¢-OMe cADPR (50) was designed to investigate further
the role of the hydroxyl group. 3¢-Deoxy cADPR (47) can neither
donate or accept a hydrogen bond at the 3¢ position, whilst 3¢-OMe
cADPR can only act as a hydrogen bond acceptor. This compound
is an antagonist in SUH, thus showing that the oxygen atom must
interact with the receptor in order to inhibit Ca2+ release. However,
Whilst this analysis does not provide structural clues about
relative agonistic/antagonistic activity or potency in either case,
it does seem that the C2¢ endo/syn conformation may be a key
requirement for activity in SUH only. Indeed, it has been demon-
strated earlier that there are differences in ligand recognition by the
protein between the sea urchin and T cell receptor. For example,
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Table 4 Ca2+ release activities of cADPR analogues in SUH and JTC
Compounds
Activity in SUH Activity in JTC Ref.
cADPR (1)
Agonist
Agonist
1,34
14,25
13,34
50
8-Bromo cADPR (2)
8-NH2 cADPR (3)
8-NHMe cADPR (9)
Antagonist
Antagonist
Weak
Antagonist
Antagonist
Antagonist
antagonist
Weak
8-NMe2 cADPR (10)
Antagonist
50
antagonist
Antagonist
Antagonist
Inactive
8-Me cADPR (11)
8-OMe cADPR (12)
8-Piperidyl cADPR (13)
Antagonist
Antagonist
Antagonist
Active
Not tested
Not tested
Not tested
Inactive
Inactive
Agonist
Inactive
Not tested
Antagonist
46
37
14
50
49
49
49
23
8-Aza-9-deaza cADPR (15) Active
cAcycloDPR (16)
cAraDPR (17)
cAcetDPR (18)
2¢-Deoxy cADPR (46)
3¢-Deoxy cADPR (47)
2¢-cADPRP (48)
3¢-cADPRP (49)
3¢-OMe cADPR (50)
Inactive
Inactive
Poor agonist
Agonist
Poor agonist
Inactive
23
23,47
23,47
23
Fig. 4 Structures of the cADPR analogues mentioned herein – our group.
Inactive
Antagonist
7-Deaza-8-bromo cADPR Antagonist
37,48,49
(52)
8-Bromo cIDPR
Inactive
Agonist
17
variety of strategies.53–56 Taking our observations reported here
into account it could be productive to apply such approaches also
to the cADPR field and these could hopefully extend the work
reported here to encompass receptor bound conformations.
Conclusion
A series of novel cADPR derivatives has been synthesized in order
to investigate the determinants for both agonist and antagonist
activity. These compounds were tested for their Ca2+ releasing
activity in both SUH and JTC. A careful analysis of all the cADPR
analogues synthesized over the past decade reveals that a C2¢
endo/syn conformation (Fig. 6) is crucial for activity (agonistic or
antagonistic), whereas compounds in their C3¢ endo conformation
Fig. 5 Structures of the cADPR analogues mentioned herein – Shuto’s
group.
the same compound is an agonist in JTC, thus showing that the
OMe group has little effect on the activity of cADPR. These results
further highlight that there may be differences in the cADPR-Ca2+
release mechanism for the ryanodine receptor of SUH and JTC.
This trend appears to be valid only with cADPR analogues and
not with the cIDPR series. Indeed, some cIDPR derivatives (e.g
8-Br cIDPR) show agonist activity in T cells but are apparently
inactive in SUH.
We naturally need to invoke the caveat for all work of this nature
that, while our study focuses upon cADPR analogue conformation
in solution, we can draw no firm conclusions regarding actual
ligand conformation as bound to the cADPR receptor. A recent
trend in nucleoside/nucleotide work in general has been to employ
the use of conformationally locked rigid ribose motifs using a
Fig. 6 Model of 8-Br cADPR (2) with its “southern” ribose in the C2¢
endo/syn conformation. Hydrogen atoms are not shown for clarity.
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are either inactive or are poor agonists. This trend appears to be
valid in sea urchin egg homogenate only (not in T cells) and with
cADPR analogues only (not cIDPR).
slightly. Fura 2/free acid (1 mM final concentration) was added and
the free Ca2+ concentration was monitored at 340 and 380 nm
as alternating excitation wavelengths and 495 nm as emission
wavelength. Experiments were started by addition of creatine
kinase (20 units/mL) and creatine phosphate (20 mM), followed
by addition of 1 mM ATP to allow loading of the intracellular Ca2+
pools by sarcoplasmic/endoplasmic Ca2+ ATPases. Chelex resin
was added generally to solutions of compounds to be tested for
Ca2+ release to avoid any Ca2+ contamination. The quality of the
permeabilized cell suspension was checked by its responsiveness
to cADPR and D-myo-inositol 1,4,5-trisphosphate.
Experimental
General Procedures
All reagents and solvents were of commercial quality and were
used directly unless otherwise described. Pyridine was dried
overnight with calcium hydride, distilled and stored over potas-
sium hydroxide pellets. Aplysia californica ovotestis homogenate
containing ADP-ribosyl cyclase was prepared as previously
described.57 The protein concentration of the Aplysia cyclase
used in all cases was estimated as 10 mg ml-1 using a Bio-
rad protein estimation assay. Ion-exchange chromatography was
performed on an LKB-Pharmacia medium pressure ion-exchange
chromatograph using a Sepharose Q fast flow column with
gradients of triethylammonium bicarbonate buffer (TEAB) pH
7.6 as eluent. 1 M TEAB was prepared by bubbling carbon dioxide
gas into 1 M triethylamine solution. HPLC was performed on a
Shimadzu LC-6A chromatograph with the UV detector operating
at 259 nm using a combination of a Partisil 10 m SAX guard column
(10 ¥ 0.46 cm) and a Technicol (10 ¥ 0.46 cm) 10 m SAX HPLC
column, or using a Spherisorb 10 m SAX (25 ¥ 0.46 cm) column
with an isocratic elution using phosphate buffer (KH2PO4), pH 3.0
at a flow rate of 1 ml min-1. 1H NMR and 31P NMR spectra were
recorded on either Jeol JNM GX-270 FT NMR or Jeol EX-400
FT NMR spectrometers. Chemical shifts were measured in ppm
Chemistry
Synthesis of nucleoside analogues
Synthesis of 8-bromo-adenosine 5¢-monophosphate (8-Br-AMP,
19). 8-Bromoadenosine (0.212 g, 0.61 mmol) was dissolved in
triethylphosphate (4 mL) by heating with a heat gun. The flask was
fitted with a CaCl2 drying tube and the solution cooled to 0 ◦C in an
ice bath. Phosphorus oxychloride (200 mL, 2.16 mmol) was added
dropwise and the flask was left stirring for 3 h at rt. HPLC analysis
of the reaction mixture dissolved in water showed the presence of
a phosphorylated material with the same retention time as an
authentic sample of 8-Br-AMP. Rt 8-Br-adenosine (1.2 min) 8-Br-
AMP (1.8 min). The reaction mixture was quenched with 8 mL of
pyridine:water (1 : 3, v/v) and left stirring for a further 30 min. The
sample was dried in vacuo and 31P NMR spectroscopy showed the
presence of inorganic phosphate and 8-Br-AMP at d -0.1 and 0.4
ppm respectively. Inorganic phosphate was removed by passing
the nucleotide mixture dissolved in 50 mL of water through a
charcoal column. This column (25 ¥ 4 cm) was prepared by using
ca. 1 cm of celite as a bed on top of which 7 cm of activated
charcoal (NoritB) were added. The nucleotide solution in water
was poured unto the column and the eluent was collected. Water
was used to flush inorganic phosphate off the column and a total
of 75 mL of eluent was collected. The inorganic phosphate-free
nucleotide was eluted off the column using ethanol:water:conc.
ammonia (25 : 24 : 1, v/v, 2 ¥ 500 mL). dH (D2O, 400 MHz) 3.8–
3.9 (2H, m, H-5¢a, H-5¢b), 4.25 (1H, m, H-4¢), 4.56 (1H, dd, J3¢,2¢
5.8, J3¢,4¢ 4.2, H-3¢), 5.19 (1H, dd, J2¢,3¢ 5.8, J2¢,1¢ 5.7, H-2¢), 6.07 (1H,
d, J1¢,2¢ 5.8, H-1¢), 8.14 (1H, s, H-2). dC (D2O, 100 MHz) 65.3 (d,
JCP 3.7, C-5¢), 70.5 (C-3¢), 71.8 (C-2¢), 84.4 (d, JCP 9.2, C-4¢), 89.2
(C-1¢), 118.3 (C-5), 139.9 (C-8), 150.7 (C-4), 153.6 (C-2), 154.9
(C-6). dP (D2O, 162 MHz, 1H-decoupled) 3.4 (s, 1P) and dP (D2O,
1
relative to deuterated water (D2O) for H NMR and to external
85% H3PO4 for 31P NMR. 31P NMR spectra were measured at
162 MHz or 109 MHz in D2O. J values are given in Hertz (Hz).
Mass spectra were recorded at the EPSRC Mass Spectrometry
Service Center at the University of Swansea and at the University
of Bath. Ultraviolet (UV) absorbance was measured with a Perkin-
Elmer Lambda 3 UV/VIS spectrophotometer. Melting points
were determined with a Reichert-Jung Thermo Galen Kofler block
and are uncorrected.
Biology
Biological testing of cADPR analogues in SUH. Sea urchin
egg homogenates were prepared as described.1 Before use the
homogenate was defrosted and diluted to give a 2.5% egg
suspension using a buffer containing an ATP regenerating sys-
tem, mitochondrial inhibitors and protease inhibitors. Finally,
Fluo-3 fluorescent dy◦e (3 mM) was added and the homogenate
was incubated at 17 C. Extra-microsomal Ca2+ was measured
by monitoring Fluo-3 fluorescence in a Perkin Elmer LS-50B
fluorimeter.25,33
1
162 MHz, H-coupled) 3.7 (s, J 5.7, 1P). lmax 264 nm (e 15100,
pH 7.0).
8-Amino-adenosine 5¢-monophosphate (8-NH2-AMP, 20). To a
solution of 8-azido AMP (25 mg, 59.2 mmol) in 50 mM TEAB
(7.5 mL, pH 8.0) was added dithiothreitol (14 mg, 90.6 mmol) and
the mixture was left stirring for 16 h at room temperature.58 The
reaction was judged to be complete by a change in UV absorption
maximum from 282 to 274 nm. The solvent was removed in vacuo
and the residue purified by ion-exchange chromatography using
a gradient of 50–1000 mM TEAB pH 7.6. Pure 8-NH2-AMP
eluted as the triethylammonium salt between 310–390 mM TEAB
(38.7 mmol, 65%).
Calcium release measurements in JTC. Permeabilized cells
were prepared as follows: cells were transferred into an intracel-
lular medium (nominal Ca2+ free, pH 7.2), permeabilized with
55 mg ml-1 saponin for 20 min, and then washed 3 times to remove
all saponin. Then the cells were left on ice for approximately
2 h to allow for re-sealing of intracellular Ca2+ stores. Finally,
approximately 1.5 ¥ 107 permeabilized cells were transferred into
a quartz cuvette and placed into an F2000 fluorimeter (Hitachi
Instruments). The cell suspension was warmed to 37 ◦C and stirred
d
H (D2O, 270 MHz) 3.8-3.9 (2H, m, H-5¢a, H-5¢b), 4.2-4.1 (1H,
m, H-4¢), 4.3-4.4 (m, 1H, H-3¢), 4.7 (1H, app.t, J2¢,1¢ = J2¢,3¢ 7.6,
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H-2¢), 5.9 (1H, d, J1¢,2¢ 7.6, H-1¢), 7.8 (1H, s, H-2). dP (D2O,
162 MHz, H-decoupled) 0.35 (s, 1P). lmax 274 nm (e 16000, pH
8.3).
Q-sepharose column eluted with 1 M TEAB. The product eluted
off between 110–160 mM TEAB, and the inorganic phosphate
impurity was removed by passing the solution through a charcoal
column as previously described to afford the nucleotide as a white
solid (0.38 mmol, 60.3%). dH (D2O, 400 MHz) dH (D2O, 400 MHz)
2.46 (3H, s, Me), 3.8-3.9 (2H, m, H-5¢a, H-5¢b), 4.1-4.21 (1H, m,
H-4¢), 4.3 (app.t, J3¢,2¢ = J3¢,4¢ = 6.2 Hz, 1H, H-3¢), 4.8 (1H, app.t,
1
8-Methylaminoadenosine 5¢-monophosphate (8-NHMe-AMP,
21). A solution of 8-Br-AMP (270.8 mmol) in anhydrous 2 M
methylamine in methanol (10 mL) was heated to 60–70 ◦C for
2 h. The crude sample was dried down in vacuo, the residue
dissolved in milliQ water and the product purified by ion-exchange
chromatography using a gradient of 0–350 mM TEAB. The
product eluted between 200–300 mM TEAB to yield the title
compound as a white solid (176 mmol, 65%). dH (D2O, 400 MHz)
2.8 (3H, s, NMe), 3.8-3.9 (2H, m, H-5¢a, H-5¢b), 4.1 (1H, m, H-4¢),
4.26 (1H, app.t, J3¢,2¢ = J3¢,4¢ 5.5, H-3¢), 4.6 (1H, app.t, J2¢,1¢ = J2¢,3¢
5.8, H-2¢), 5.8 (1H, d, J1¢,2¢ 8, H-1¢), 7.8 (1H, s, H-2). dP (D2O,
162 MHz, 1H-decoupled) 3.2 (s, 1P). m/z (ES-) 375.4 [100%, (M
- H)-]. lmax 278 nm (e 17700, pH 8.3). HPLC Rt = 2.3 mins.
J
2¢,1¢ = J2¢,3¢ 6.2, H-2¢), 5.8 (1H, d, J1¢,2¢ 6.8, H-1¢), 7.9 (1H, s, H-2). dP
(D2O, 162 MHz, 1H-decoupled) 4.0 (s, 1P) and dP (D2O, 162 MHz,
1H-coupled) 3.7 (s, J 5.6, 1P). lmax 260 nm (e 15300, pH 8.3).
8-Methoxy-adenosine 5¢-monophosphate (8-OCH3-AMP, 24).
8-Br-AMP (0.229 mmol was treated with 0.5 M sodium methoxide
in methanol (20 mL) under reflux overnight. The pH of the
solution was adjusted to 7 with glacial acetic acid and the solvent
was removed in vacuo. The residue was redissolved in milliQ water
and purified by ion-exchange chromatography using a TEAB
gradient from 50–350 mM. The pure sample eluted off between
210–250 mM TEAB and was obtained in 79% yield. dH (D2O,
400 MHz) 3.7-3.0 (2H, m, H-5¢), 4.0 (3H, s, OMe), 4.2 (1H, m,
H-4¢), 4.25 (1H, dd, J3¢,2¢ 5.8, J3¢,4¢ 4.2, H-3¢), 4.8 (1H, dd, J2¢,3¢ 5.8,
J2¢,1¢ 5.5, H-2¢), 5.7 (1H, d, J1¢,2¢ 5.5, H-1¢), 7.8 (1H, s, H-2). dP
(D2O, 162 MHz, 1H-decoupled) 3.7 (s, 1P). lmax 260 nm (e 13500,
pH 8.3).
8-Dimethylamino-adenosine 5¢-monophosphate (8-NMe2-AMP,
22). 8-Br-AMP (0.147 mmol) was added to an anhydrous
solution of 2 M dimethylamine in methanol (2 M, 10 mL) and
the solution was stirred under reflux overnight, after which the
solvent was removed in vacuo. The crude sample was purified on
an ion-exchange column using a TEAB gradient of 0–400 mM.
Pure sample eluted off between 140–210 mM TEAB to afford
the desired product (99.9 mmol, 68%). dH (D2O, 400 MHz) 2.8
(6H, s, NMe2), 3.8-3.9 (2H, m, H-5¢), 4.1 (1H, m, H-4¢), 4.3 (1H,
app.t, J3¢,2¢ = J3¢,4¢ 5.0, H-3¢), 4.6 (1H, app.t, J2¢,1¢ = J2¢,3¢ 6.4, H-2¢),
5.6 (1H, d, J1¢,2¢ 6.7, H-1¢), 7.9 (1H, s, H-2). dP (D2O, 162 MHz,
1H-decoupled) 2.2 (s, 1P). m/z (FAB-) 389 [90%, (M - H)-]. lmax
274 nm (e 10900, pH 8.3). HPLC Rt = 4.9 mins.
8-Piperidyl-adenosine 5¢-monophosphate (8-pip-AMP, 25).
A
solution of 8-Br-AMP (111.3 mmol) in dry piperidine (0.4 mL)
◦
was stirred at 50 C for 40 h after which HPLC analysis showed
the presence of a new peak at 5.1 min. Piperidine was removed in
vacuo, the residue dissolved in milliQ water and product purified
by ion-exchange chromatography using a gradient of 0–300 mM
TEAB. Pure 8-pip-AMP eluted between 30–65 mM TEAB (65%
yield). dH (D2O, 400 MHz) 1.6 (6H, m, 3 ¥ CH2), 3.2 (4H, m,
2 ¥ CH2), 3.8-3.9 (2H, m, H-5¢), 4.0 (1H, m, H-4¢), 4.34 (1H,
app.t, J3¢,2¢ = J3¢,4¢ 6.0, H-3¢), 5.1 (1H, app.t, J2¢,1¢ = J2¢,3¢ 6.0, H-2¢),
5.6 (1H, d, J1¢,2¢ 6.4, H-1¢), 7.9 (1H, s, H-2). dP (D2O, 162 MHz,
1H-decoupled) 2.0 (s, 1P). m/z (FAB-) 429 [85%, (M - H)-]. lmax
275 nm (e 11660, pH 8.3). HPLC Rt = 5.1 mins.
8-Methyladenosine 5¢-monophosphate (8-Me-AMP, 23). 8-
Bromoadenosine (1 g, 2.89 mmol) was dissolved in hexamethyl-
disilazane (20 mL) and dry dioxane (20 mL) in a three-necked
flask. A catalytic amount of ammonium sulfate was added to
the suspension and the mixture was left under reflux at 130 ◦C
for 2–3 h. Triphenylphosphine (76 mg, 2.89 mmol), palladium
dichloride (26 mg, 0.144 mmol) and trimethylaluminium (2.89 mL,
5.78 mmol) were added to the solution in dry THF under N2. The
reaction was left under reflux and a gentle stream of N2 for 2.5 h
(Rf 0.36, DCM–MeOH 9 : 1). The crude mixture was dried in vacuo
to give a green residue which was dissolved in methanol (50 mL)
and refluxed for 4 h with a small amount of ammonium chloride
(Rf 0.05, DCM–MeOH 9 : 1). The crude sample was purified on
a short silica gel column eluted with CHCl3–EtOH (3 : 2) to give
a white solid which was further recrystallised from water (0.39 g,
1.4 mmol, 48%). dH (D2O, 270 MHz) 3.5–3.7 (2H, m, H-5¢a, H-
5¢b), 4.0 (m, 1H, H-4¢), 4.1 (m, 1H, H-3¢), 4.8 (dd, J2¢,1¢ = 7.3 and
J2¢,3¢ = 5.1 Hz, 1H, H-2¢), 5.8 (1H, d, J1¢,2¢ 7.3, H-1¢), 8.0 (1H, s,
H-2). m/z (FAB+) 282 [100%, (M + H)+]. HRMS (FAB+) calcd for
C11H16N4O4 282.1207 (M + H)+ found 282.1202. m.p. 208 ◦C.
Phosphorus oxychloride (100 mL, 1.08 mmol) was added
dropwise at 0 ◦C to a solution of 8-methyl adenosine (170 mg,
0.6 mmol) in triethylphosphate (4 mL) under N2. The reaction
was left stirring for 3 h at rt after which HPLC analysis showed
the presence of a new product at 2 min. The reaction mixture
was quenched by stirring in 8 mL of pyridine:water (1 : 3) for
30 min and the solvents were removed in vacuo. The residue was
dissolved in milliQ water (350 mL) and applied to an ion-exchange
8-Oxy-adenosine 5¢-monophosphate (8-oxy-AMP, 26). 8-Br-
AMP (128 mg, 301.9 mmol) was co-evaporated with pyridine (3 ¥
5 mL) and anhydrous sodium acetate (50 mg) and acetic anhydride
(4.4 mL) were added. The solution was refluxed at 150–165 ◦C for
2 h and then stirred for 24 h at room temperature after which 2 mL
of methanol were added. The solvents were removed, the residue
was dissolved in 1 M NaOH (11 mL) and stirred for 24 h at rt after
which it was neutralised with 1 M HCl. The sample was purified
on an ion-exchange column using a gradient between 0–400 mM
TEAB. Pure product eluted off between 300–360 mM TEAB in
51% yield (0.155 mmol). dH (D2O, 400 MHz) 3.7–3.9 (2H, m, H-
5¢), 3.9 (1H, m, H-4¢), 4.2 (1H, dd, J3¢,2¢ 5.0, J3¢,4¢ 4.6, H-3¢), 4.9 (1H,
dd, J2¢,1¢ 5.5, J2¢,3¢ 5.0, H-2¢), 5.6 (1H, d, J1¢,2¢ 5.5, H-1¢), 8.0 (1H, s,
H-2). dP (D2O, 162 MHz, 1H-decoupled) -0.4 (s, 1P). lmax 270 nm
(e 10545, pH 8.3). HPLC Rt = 3.7 mins.
Acycloadenosine monophosphate (AcycloAMP, 28). 9-[(2-
Hydroxyethoxy)methyl]adenine29 43 (50 mg, 0.24 mmol) was
dissolved in triethylphosphate (2 mL), cooled to 0 ◦C and POCl3
(75 mL, 0.53 mmol) was added slowly. The reaction was warmed
to rt and stirred for 1.5 h after which it was quenched by the
addition of iced water (5 g) and the product was purified by
286 | Org. Biomol. Chem., 2011, 9, 278–290
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ion-exchange chromatography eluting with a gradient of 1 M
TEAB. The appropriate fractions were combined and the solvent
removed in vacuo to give the title monophosphate in 55% yield.
The inorganic phosphate was removed on a charcoal column as
previously described with 95% recovery followed by precipitation
using MeOH:acetone 1 : 10 (2 mL) to give the pure product 21.
dH (400 MHz, D2O) 3.57–3.60 (2H, m, POCH2CH2), 3.74–3.78
(2H, m, POCH2), 5.66 (2H, s, OCH2base), 8.16 (1H, s, H-2), 8.26
(1H, s, H-8), and. dC (D2O, 100 MHz) 64.5 (d, JCP 5.5, POCH2),
69.8 (d, JCP 9.2, POCH2), 73.9 (OCH2base), 119.2 (C-5), 143.4
(C-8), 149.7 (C-4), 153.6 (C-2), 156.3 (C-6). dP (D2O, 161 MHz)
1.48. m/z (FAB+) 290.1 [100%, (M + H)+]. lmax 259 nm (e 15300).
HPLC Rt = 2.5 mins.
room temperature for 7 days. The resulting mixture was poured
into 100 mL of cold distilled water and left at 4 ◦C for 2 h to
precipitate the DCU formed in the reaction. DCU was filtered off
and the filtrate was extracted with 3 ¥ 50 mL aliquots of chloroform
to remove other water insoluble organic impurities. The aqueous
layer was collected and dried in vacuo. The residue was dissolved
in milliQ water and purified by ion-exchange chromatography on
Q-sepharose eluted with a gradient of 1 M TEAB.
Nicotinamide 8-bromoadenine dinucleotide (8-Br-NAD+, 31).
b-NAD+ (200 mg, 0.3 mmol) free acid was dissolved in acetate
buffer (5 mL, pH 3.9) and bromine (0.2 mL) was added dropwise.
The reaction was left for 30 min after which a 1.25 M solution
of NaHSO3 was added dropwise to discharge the bromine colour
and the unreacted bromine was extracted with CHCl3. The sample
was dried in vacuo, the residue dissolved in milliQ water and
the product purified by ion-exchange chromatography using a
gradient of 0–350 mM TEAB. Pure 8-Br-NAD+ eluted between
45–70 mM TEAB and was obtained in 61% yield (184.1 mmol). dH
(D2O, 270 MHz) 4.0–4.4 (9H, m, ribose-H), 5.0 (1H, t, J2¢,1¢ = J2¢,3¢
5.8, H-2¢), 5.7 (1H, d, J1¢,2¢ 5.5, H-1¢), 5.8 (1H, d, J1¢¢,2¢¢ 4.8, H-1¢¢),
7.8 (1H, s, HA2), 8.0 (1H, t, J 7, HN5), 8.6 (1H, d, J4,5 8.2, HN4),
8.9 (1H, d, J6,5 6.2, HN6), 9.1 (1H, s, HN2). dP (D2O, 109 MHz,
1H-decoupled) -11.54 and -11.92 (AB system, JAB 21.2, 2P). m/z
(ES-) 741.9 [50% (M)-]. lmax 264 nm (e 15500, pH 8.3). HPLC Rt =
2.4 mins.
Adenine 9-b-D-arabinofuranoside 5¢-monophosphate (AraAMP,
29). Adenine 9-b-D-arabino-ribofuranoside 44 (177 mg,
0.66 mmol) was dissolved in triethylphosphate (2 mL), cooled
to 0 ◦C and POCl3 was added (140 mL, 0.70 mmol). The reaction
mixture was stirred overnight at 4 ◦C after which it was quenched
with ice (10 g) and the material was purified by ion exchange
chromatography eluting with a gradient of 1 M TEAB. The
product obtained was passed through a charcoal column to
remove the inorganic phosphate. The desired monophosphate was
obtained in 58% yield. dH (270 MHz, D2O) 4.3-4.0 (3H, m, H-4¢
and H-5¢), 4.38 (1H, dd, J3¢,2¢ 6.6, J3¢,4¢ 6.4, H-3¢), 4.59 (1H, dd, J2¢,3¢
6.6, J2¢,1¢ 5.5, H-2¢), 6.42 (1H, d, J1¢,2¢ 5.5, H-1¢), 8.38 (1H, s, H-8),
8.53 (1H, s, H-2). dC (D2O, 100 MHz) 63.1 (C-5¢), 68.9 (C-3¢), 80.8
(C-2¢), 86.4 (d, JCP 7.4, C-4¢), 89.5 (C-1¢), 123.3 (C-5), 149.0 (C-4
or C-8), 150.1 (C-4 or C-8), 153.5 (C-2), 155.1 (C-6). dP (D2O,
161 MHz) 3.10. m/z (FAB+) 346 [100%, (M + H)+]. lmax 259 nm
(e 15300). HPLC Rt = 2.21 mins.
Nicotinamide 8-aminoadenine dinucleotide (8-NH2-NAD+, 32).
8-NH2-AMP (14 mg, 38.7 mmol) was coupled to b-NMN+
(8.08 mg, 24.2 mmol) with 0.32 g of DCC in 6.5 mL of pyri-
dine:water (4 : 1) as described. The pure dinucleotide was obtained
in 16% yield. dH (D2O, 400 MHz) 4.2-4.6 (9H, m, ribose-H), 4.7-
4.8 (1H, m, H-2¢), 5.86 (1H, d, J1¢,2¢ 7, H-1¢), 6.1 (1H, d, J1¢¢,2¢¢ 4.7,
H-1¢¢), 8.0 (1H, s, HA2), 8.2 (1H, t, J5,6 = J5,4 6.6, HN5), 8.7 (1H,
d, J4,5 6.6, HN4), 9.1 (1H, d, J6,5 6.6, HN6), 9.3 (1H, s, HN2). dP
2¢,3¢-Isopropylidene adenosine monophosphate (AcetAMP, 30).
To a cooled solution of dry 2¢,3¢-O-isopropylidene adenosine
45 (200 mg, 0.65 mmol) suspended in triethylphosphate (2 mL)
was added POCl3 (140 mL, 0.70 mmol) and the reaction stirred
1
◦
(D2O, 162 MHz, H-decoupled) -11.83 and -11.97 (AB system,
overnight at 4 C. It was quenched by the addition of ice (10 g)
J
AB 19.4, 2P). m/z (ES-) 677 [10% (M - H)-]. HPLC Rt = 4.1 mins.
and the mixture was purified by ion exchange chromatography
eluted with a gradient of 1 M TEAB. The product was purified
further on a charcoal column to remove the inorganic phosphate
and the title nucleotide was obtained in 47% yield. dH (400 MHz,
D2O) 1.43 (3H, s, CH3), 1.66 (3H, s, CH3), 3.98 (2H, m, H-5¢), 4.60
(1H, m, H-4¢), 5.04 (1H, dd, J3¢,2¢ 5.9, J3¢,4¢ 2.0, H-3¢), 5.35 (1H, dd,
Nicotinamide 8-methylaminoadenine dinucleotide (8-NHMe-
NAD+, 33). 8-Methylamino-AMP (74.03 mmol) was coupled to
b-NMN+ as described above using 2 g of DCC. dH (D2O, 400 MHz)
2.8 (3H, s, NHMe), 4.0–4.5 (10H, m, ribose-H), 5.7 (1H, d, J1¢,2¢
7.0, H-1¢), 5.9 (1H, d, J1¢¢,2¢¢ 4.6, H-1¢¢), 7.9 (1H, s, HA2), 8.0 (1H,
t, J 7, HN5), 8.6 (1H, d, J 7.9, HN4), 9.0 (1H, d, J 5.8, HN6), 9.1
J
2¢,3¢ 5.9, J2¢,1¢ 3.4, H-2¢), 6.19 (1H, d, J1¢,2¢ 3.4, H-1¢), 84.3 (C-3¢), 8.12
(1H, s, H-8), 8.42 (1H, s, H-8). dC (D2O, 100 MHz) 27.2, 29.0 (both
CH3), 67.3 (d, JCP 3.7, C-5¢), 86.7 (C-2¢), 87.8 (d, JCP 9.2, C-4¢),
92.7 (C-1¢), 117.6 (C), 121.1 (C-5), 143.0 (C-8), 151.4 (C-4), 158.5
(C-2),158.1 (C-6). dP (D2O, 161 MHz) 3.51. m/z (FAB+) 388.1
[100%, (M + H)+]. lmax 259 nm (e 15200). HPLC Rt = 2.98 mins.
1
(1H, s, HN2). dP (D2O, 109 MHz, H-decoupled) -11.7 (s, 2P).
m/z (FAB+) 693 [60%, [M + H)+]. lmax 273 nm (e 13000, pH 8.3).
HPLC Rt = 4.7 mins.
Nicotinamide 8-dimethylaminoadenine dinucleotide (8-Me2NH-
NAD+, 34). 8-Dimethylamino-AMP (161 mmol) was coupled
to b-NMN+ (149.6 mmol) with 2 g of DCC in 12.5 mL of
pyridine:water (4 : 1) as described. The pure product was obtained
in 11% (17.1 mmol) yield. dH (D2O, 400 MHz) 3.1 (3H, s, NMe2),
4.4-5.0 (9H, m, ribose-H), 5.0 (1H, app.t, J2¢,1¢ = J2¢,3¢ 6.3, H-2¢),
5.9 (1H, d, J1¢,2¢ 6.3, H-1¢), 6.3 (1H, d, J1¢¢,2¢¢ 5.4, H-1¢¢), 8.2 (1H, s,
HA2), 8.4 (1H, t, J 7, HN5), 8.9 (1H, d, J 8.0, HN4), 9.3 (1H, d,
J 6.3, HN6), 9.5 (1H, s, HN2). dP (D2O, 109 MHz, 1H-decoupled)
-11.42 and -11.82 (AB system, JAB 21, 2P). m/z (FAB+) 707 [70%
(M + H)+]. lmax 273 nm (e 16100, pH 8.3). HPLC Rt = 5.8 mins.
Synthesis of NAD+ analogues
General method. All NAD+ analogues with the exception of
8-Br-NAD+ were prepared essentially by an adaptation of a
literature method described by Hughes et al.27 using dicyclohexyl-
carbodiimide as the coupling agent.
b-NMN+ and the appropriate AMP analogue were dissolved in
water in a round bottom flask, dry pyridine was added to make
a 4 : 1 pyridine:water mixture and excess DCC was subsequently
added to the nucleotide mixture. The solution was left stirring at
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Nicotinamide 8-methyladenine dinucleotide (8-Me-NAD+, 35).
8-Me-AMP (139.2 mmol) was coupled to b-NMN+ (149.6 mmol)
with 2 g of DCC in 12.5 mL of pyridine:water (4 : 1) as described.
The pure dinucleotide was obtained in 12% yield after 2 purifi-
cations. dH (D2O, 400 MHz) 2.5 (3H, s, Me), 4.2–4.6 (9H, m,
ribose-H), 4.7 (1H, d, J2¢,3¢ = J2¢,1¢ 6.4, H-2¢), 5.7 (1H, d, J1¢,2¢ 6.4,
H-1¢), 5.8 (1H, d, J1¢¢,2¢¢ 5.2, H-1¢¢), 7.9 (1H, s, HA2), 8.1 (1H, t,
J 7.0, HN5), 8.6 (1H, d, J4,5 6.3, HN4), 8.9 (1H, d, J6,5 6.1, HN6),
Nicotinamide 9-[(2-hydroxyethoxy)methyl] adenine dinucleotide
(NAcycloD+, 40). AcycloAMP (14 mmol) was coupled to b-
NMN+ (15 mg, 45 mmol) with 0.25 g of DCC in 1.5 mL of
pyridine:water (4 : 1) as described. The pure dinucleotide was
obtained in 12% yield. dH (D2O, 400 MHz) 3.81 (2H, m,
POCH2CH2), 4.07 (2H, m, POCH2), 4.21-4.75 (5H, m, H-2¢¢, H-
3¢¢, H-4¢¢ and H-5¢¢), 5.66 (2H, s, OCH2base), 6.11 (1H, d, J1¢¢,2¢¢
5.5, H-1¢¢), 8.18-8.26 (3H, m, HN5, HA2 and HA8), 8.89 (1H, d, J4,5
6.9, HN4), 9.20 (1H, d, J6,5 6.2, HN6), 9.36 (1H, s, HN2). dP (D2O,
1
9.3 (1H, s, HN2). dP (D2O, 162 MHz, H-decoupled) -10.96 and
-11.34 (AB system, JAB 20.5, 2P). m/z (ES-) 676 [100% (M - H)-].
lmax 259 nm (e 13755, pH 8.3). HPLC Rt = 2.46 mins.
162 MHz, H-decoupled) -10.9 and -10.6 (AB system, JAB 18.5,
2P). lmax 259 nm (e 17300, pH 8.3). HPLC Rt = 3.1 mins.
1
Nicotinamide 8-methoxyadenine dinucleotide (8-OCH3-NAD+,
36). 8-OMe-AMP (166.6 mmol) was coupled to b-NMN+ (50 mg,
149.6 mmol) with 2 g of DCC in 12.5 mL of pyridine:water (4 : 1)
as described. The pure dinucleotide was obtained in 16% yield.
dH (D2O, 400 MHz) 4.0 (3H, s, OMe), 4.6-4.0 (9H, m, ribose-H),
4.8 (1H, dd, J2¢,3¢ 5.8, J2¢,1¢ 5.2, H-2¢), 5.7 (1H, d, J1¢,2¢ 5.2, H-1¢),
5.8 (1H, d, J1¢¢,2¢¢ 4.9, H-1¢¢), 7.9 (1H, s, HA2), 8.1 (1H, dd, J5,4 7.9,
J5,6 6.1, HN5), 8.6 (1H, d, J4,5 7.9, HN4), 9.0 (1H, d, J6,5 6.1, HN6),
Nicotinamide adenine 9-b-D-arabinofuranoside dinucleotide
(NAraD+, 41). AraAMP (240 mmol) was coupled to b-NMN+
(50 mg, 150 mmol) with 2 g of DCC in 12.5 mL of pyridine:water
(4 : 1) as described. The pure dinucleotide was obtained in 10%
yield. dH (D2O, 400 MHz) 4.57-4.13 (10H, m, ribose-H), 6.05 (1H,
d, J1¢,2¢ 4.2, H-1¢), 6.27 (1H, d, J1¢¢,2¢¢ 5.7, H-1¢¢), 8.13 (1H, s, HA2),
8.19 (1H, m, HN5), 8.34 (s1H,, HA8), 8.83 (1H, d, J4,5 8.1, HN4),
9.17 (1H, d, J6,5 5.7, HN6), 9.31 (1H, s, HN2). dP (D2O, 162 MHz,
1H-decoupled) -11.44 and -11.82 (AB system, JAB 20.8, 2P). m/z
(FAB-) 663 [100%, (M - H)-]. lmax 259 nm (e 17300, pH 8.3).
HPLC Rt = 2.32 mins.
1
9.1 (1H, s, HN2). dP (D2O, 162 MHz, H-decoupled) -11.38 and
-11.85 (AB system, JAB = 20.9 Hz, 2P). m/z (ES+) 694 [100%,
M+H)+]. lmax 259 nm (e 13000, pH 8.3). HPLC Rt = 2.4 mins.
Nicotinamide 2¢,3¢-isopropylidene adenosine dinucleotide
(NAcetD+, 42). AcetAMP (240 mmol) was coupled to b-NMN+
(50 mg, 150 mmol) with 2 g of DCC in 12.5 mL of pyridine:water
(4 : 1) as described. The pure dinucleotide was obtained in 10%
yield. dH (D2O, 400 MHz) 1.31(3H, s, CH3), 1.52 (3H, s, CH3),
4.53-4.03 (8H, m, ribose-H), 5.04 (1H, dd, J3¢,2¢ 5.8, J3¢,4¢ 2.6,
H-3¢), 5.32 (1H, dd, J2¢,3¢ 5.8, J2¢,1¢ 2.8, H-2¢), 6.04 (1H, d, J1¢¢,2¢¢
5.8, H-1¢¢), 6.12 (1H, d, J1¢,2¢ 2.8, H-1¢), 8.07 (1H, s, HA2), 8.15
(1H, dd, J5,4 7.9, J5,6 5.8, HN5), 8.20 (1H, s, HA8), 8.83 (1H, d,
J4,5 7.9, HN4), 9.16 (1H, d, J6,5 5.8, HN6). 9.38 (1H, s, HN2), dP
Nicotinamide 8-piperidyladenine dinucleotide (8-pip-NAD+, 37).
8-Pip-AMP (107.8 mmol) was coupled to b-NMN+ (45 mg,
134 mmol) with 1.8 g of DCC in 12.5 mL of pyridine:water (4 : 1)
as described. The pure dinucleotide was obtained in 7% yield after
2 purifications. dH (D2O, 400 MHz) 1.6 (6H, m, 3 ¥ CH2), 3.2
(4H, m, 2 ¥ CH2), 4.0–4.5 (9H, m, ribose-H), 5.0 (1H, d, J2¢,3¢ 6.4,
J2¢,1¢ 6.1, 1H, H-2¢), 5.5 (1H, d, J1¢,2¢ 6.1, H-1¢), 5.8 (1H, d, J1¢¢,2¢¢
5.5, H-1¢¢), 7.9 (1H, s, HA2), 8.1 (1H, dd, J5,4 7.0, J5,4 6.1, HN5),
8.6 (1H, d, J4,5 7.0, HN4), 8.9 (1H, d, J6,5 6.1, HN6), 9.1 (1H, s,
HN2). dP (D2O, 162 MHz, 1H-decoupled) -11.44 and -11.84 (AB
system, JAB 20.8, 2P). m/z (ES-) 746 [50%, (M - H)-]. lmax 274 nm
(e 12800, pH 8.3). HPLC Rt = 6.3 mins.
1
(D2O, 162 MHz, H-decoupled) -11.8 (m, 2P). m/z (FAB-) 580
[M+ - nicotinamide]. lmax 259 nm (e 17300, pH 8.3). HPLC Rt =
4.18 mins.
Nicotinamide 8-oxyadenine dinucleotide (8-oxy-NAD+, 38). 8-
Oxy-AMP (155.3 mmol) was coupled to b-NMN+ (50 mg,
149.6 mmol) with 2 g of DCC in 12.5 mL of pyridine:water (4 : 1) as
described. The pure dinucleotide was obtained in 9% yield after 2
purifications. dH (D2O, 400 MHz) 4.4-4.0 (9H, m, ribose), 4.9 (1H,
app.t, J2¢,3¢ = J2¢,1¢ 5.6, H-2¢), 5.5 (1H, d, J1¢,2¢ 5.6, H-1¢), 5.85 (1H, d,
J1¢¢,2¢¢ 4.5, H-1¢¢), 7.7 (1H, s, HA2), 8.1 (1H, t, J 7.0, HN5), 8.5 (1H,
d, J4,5 6.5, HN4), 8.9 (1H, d, J6,5 6.0, HN6), 9.1 (1H, s, HN2). dP
Synthesis of analogues of cyclic-adenosine diphosphate ribose
General method. All cyclic dinucleotides were obtained by
incubating the NAD+ analogues in HEPES buffer (25 mM, pH
6.8) with the crude Aplysia cyclase. The mixtures were left for
20 min after which they were purified on Q-sepharose eluted with
a gradient of 1 M TEAB.
1
(D2O, 162 MHz, H-decoupled) -11.44 and -11.92 (AB system,
Cyclic adenosine 5¢-diphosphate ribose (cADPR, 1). A solution
of NAD+ (2.5 mg) in HEPES (2.5 mL) was incubated with the
cyclase (10 mL) as described. cADPR was obtained in 60% yield.
dH (D2O, 400 MHz) 3.8 (1H, m, H-5¢b), 4.0 (1H, m, H-5¢¢b), 4.2
(1H, m, H-4¢), 4.3 (1H, m, H-5¢¢a), 4.4 (2H, m, H-3¢¢ and H-5¢a),
4.6 (2H, m, H-2¢¢ and H-4¢¢), 4.7 (1H, m, H-3¢), 4.9 (1H, app.t,
J2¢,3¢ = J2¢,1¢ 7.0, H-2¢), 5.85 (1H, d, J1¢,2¢ 7.0, H-1¢), 6.0 (1H, d, J1¢¢,2¢¢
3.8, H-1¢¢), 8.2 (1H, s, HA8), 8.9 (1H, s, HA2). dP (D2O, 162 MHz,
1H-decoupled) -11.36 and -11.84 (AB system, JAB 14.9, 2P). m/z
(FAB-) 540 [100%, (M - H)-]. lmax 254 nm (e 14300, pH 8.3).
JAB 21.0, 2P). m/z (ES-) 678 [100%, (M - H)-]. lmax 266 nm (e
13100, pH 8.3). HPLC Rt = 3.4 mins.
8-Aza-9-deaza nicotinamide adenine dinucleotide (NFD, 39).
Formycin 5¢-monophosphate monoammonium salt (205 mmol)
was coupled to b-NMN+ (50 mg, 149.6 mmol) with 2 g of
DCC in 12.5 mL of pyridine:water (4 : 1) as described. The pure
dinucleotide was obtained in 9% yield. dH (D2O, 400 MHz) 4.0-5.0
(10H, m, ribose-H), 5.2 (1H, d, J1¢,2¢ 7.1, H-1¢), 6.0 (1H, d, J1¢¢,2¢¢
5.3, H-1¢¢), 8.0 (1H, s, HA2), 8.1 (1H, m, HN5), 8.6 (1H, d, J4,5
8.0, HN4), 9.05 (1H, d, J6,5 5.3, HN6), 9.2 (1H, s, HN2). dP (D2O,
8-Bromo cyclic adenosine 5¢-diphosphate ribose (8-Br-cADPR,
2). A solution of 8-Br-NAD+ (2.25 mg) in HEPES (2.5 mL) was
incubated with the cyclase (10 mL) as described. 8-Br-cADPR was
obtained in 60% yield. dH (D2O, 400 MHz) 4.2-5.0 (9H, m, ribose),
1
162 MHz, H-decoupled) -11.88 and -11.78 (AB system, JAB
=
21.6 Hz, 2P). m/z (ES-) 662 [30%, (M - H)-]. lmax 275 nm (e 4600,
pH 8.3). HPLC Rt = 2.2 mins.
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5.6 (1H, app.t, J2¢,3¢ = J2¢,1¢ 5.0, H-2¢), 6.0 (1H, d, J1¢¢,2¢¢ 2.0, H-1¢¢),
6.3 (1H, d, J1¢,2¢ 6.1, H-1¢), 8.8 (1H, s, HA2). dP (D2O, 162 MHz,
1H-decoupled) -10.83 and -11.05 (AB system, JAB 13.2, 2P). m/z
(ES-) 619.9 [100%, (M - H)-]. lmax 264 nm (e 15730, pH 8.3).
HPLC Rt = 3.9 mins.
and -11.76 (AB system, JAB 15.9, 2P). m/z (ES-) 623 [100%, (M -
H)-]. lmax 282 nm (e 12000, pH 8.3). HPLC Rt = 16.2 mins.
8-Oxy cyclic adenosine 5¢-diphosphate ribose (8-oxy-cADPR,
14). A solution of 8-Oxy-NAD+ (3 mmol) in HEPES (2.5 mL)
was incubated with the cyclase (20 mL) as described. 8-Oxy-
cADPR was obtained in 49% yield. dH (D2O, 400 MHz) 4.6-3.9
(9H, m, ribose), 5.6 (1H, app.t, J2¢,3¢ = J2¢,1¢ 5.6, H-2¢), 5.8 (1H, d,
J1¢,2¢ 5.6, H-1¢), 6.0 (1H, d, J1¢¢,2¢¢ 4.0, H-1¢¢), 8.8 (1H, s, HA2). dP
(D2O, 162 MHz, 1H-decoupled) -10.85 and -11.3 (AB system, JAB
16.1, 2P). m/z (ES-) 556 [20%, (M - H)-]. lmax 279 nm (e 9800, pH
8.3). HPLC Rt = 5.95 mins.
8-Amino cyclic adenosine 5¢-diphosphate ribose (8-NH2-cADPR,
3). A solution of 8-NH2-NAD+ (2.25 mg) in HEPES (1 mL) was
incubated with the cyclase (10 mL) as described. 8-NH2-cADPR
was obtained in 48% yield. dH (D2O, 400 MHz) 4.2-5.0 (10H, m,
ribose), 6.1 (1H, d, J1¢,2¢ 7.6, H-1¢), 6.4 (1H, d, J1¢¢,2¢¢ 5.0, H-1¢¢),
1
9.0 (1H, s, HA2). dP (D2O, 162 MHz, H-decoupled) -13.35 and
-14.05 (AB system, JAB 17.5, 2P). m/z (ES-) 555.2 [100%, (M -
H)-]. lmax 274 nm (e 15730, pH 8.3). HPLC Rt = 6.4 mins.
8-Aza-9-deaza cyclic adenosine 5¢-diphosphate ribose (cFDPR,
15). A solution of NFD+ (6 mmol) in HEPES (4 mL) was
incubated with the cyclase (20 mL) as described. cFDPR was
obtained in 40% yield. dH (D2O, 400 MHz) 4.0–5.0 (9H, m, ribose-
H), 5.2 (1H, d, J1¢,2¢ 7.1, H-1¢), 6.0 (1H, d, J1¢¢,2¢¢ 5.3, H-1¢¢) and 8.2
(1H, s, HA2). dP (D2O, 162 MHz, 1H-decoupled) -11.65 and -11.95
(AB system, JAB 15.7, 2P). m/z (ES-) 540 [70%, (M - H)-]. lmax
275 nm (e 10300, pH 8.3). HPLC Rt = 5.9 mins.
8-Methylamino cyclic adenosine 5¢-diphosphate ribose (8-NHMe-
cADPR, 9). A solution of 8-NHMe-NAD+ (2.5 mg) in HEPES
(2.5 mL) was incubated with the cyclase (15 mL) as described. 8-
NHMe-cADPR was obtained in 42% yield. dH (D2O, 400 MHz)
2.8 (3H, s, NHMe), 4.4-3.8 (9H, m, ribose), 5.6 (1H, app.t, J2¢,3¢
=
J2¢,1¢ 5.5, H-2¢), 5.7 (1H, d, J1¢,2¢ 5.5, H-1¢), 5.9 (1H, d, J1¢¢,2¢¢ 4.0,
H-1¢¢), 8.6 (1H, s, HA2). dP (D2O, 162 MHz, 1H-decoupled) -10.87
and -11.58 (AB system, JAB = 14.9 Hz, 2P). m/z (ES-) 569 [100%,
(M - H)-]. lmax 279 nm (e 11050, pH 8.3). HPLC Rt = 7.8 mins.
Cyclic acycloadenosine 5¢-diphosphate ribose (cAcycloDPR, 16).
A solution of NAcycloD+ (1 mmol) in HEPES (2.5 mL) was
incubated with the cyclase (10 mL) as described. cAcycloDPR
was obtained in 21% yield. m/z (FAB+) 493 [100%, (M + H)+].
lmax 257 nm (e 14300, pH 8.3). HPLC Rt = 5.1 mins.
8-Dimethylamino cyclic adenosine 5¢-diphosphate ribose (8-
NMe2-cADPR, 10). A solution of 8-NMe2-NAD+ (2.98 mg)
in HEPES (2.5 mL) was incubated with the cyclase (15 mL) as
described. 8-NMe2-cADPR was obtained in 62% yield. dH (D2O,
400 MHz) 3.2 (6H, s, NMe2), 4.2-5.0 (9H, m, ribose), 5.6 (1H, dd,
Cyclic adenine 9-b-D-arabino ribofuranoside 5¢-diphosphate ri-
bose (cAraDPR, 17). A solution of NAraD+ (7.5 mmol) in
HEPES (17.5 mL) was incubated with the cyclase (75 mL) as
described. cAcycloDPR was obtained in 54% yield. dH (D2O,
400 MHz) 4.59-3.90 (8H, m, ribose), 4.65 (1H, dd, J3¢,2¢ 8.2, J3¢,4¢
8.1, H-3¢), 5.17 (1H, dd, J2¢,3¢ 8.2, J2¢,1¢ 7.3, H-2¢), 6.03 (1H, d, J1¢¢,2¢¢
4.0, H-1¢¢), 6.24 (1H, d, J1¢,2¢ 7.3, H-1¢), 8.20 (1H, s, HA8), 8.91 (1H,
s, HA2). dP (D2O, 162 MHz, 1H-decoupled) -10.23 (br s, 1P) and
-11.76 (br s, 1P). m/z (ES-) 540 [100%, (M - H)-]. lmax 257 nm (e
14300, pH 8.3). HPLC Rt = 4.34 mins.
J
2¢,1¢ 6.4, J2¢,3¢ 5.0, H-2¢), 6.2 (1H, d, J1¢,2¢ 6.4, H-1¢), 6.3 (1H, d, J1¢¢,2¢¢
1
4.0, H-1¢¢), 9.1 (1H, s, HA2). dP (D2O, 162 MHz, H-decoupled)
-5.66 and -6.42 (AB system, JAB 15.4, 2P). m/z (ES-) 583 [100%,
(M - H)-]. lmax 282 nm (e 11800, pH 8.3). HPLC Rt = 9.7 mins.
8-Methyl cyclic adenosine 5¢-diphosphate ribose (8-Me-cADPR,
11). A solution of 8-Me-NAD+ (3 mmol) in HEPES (2 mL) was
incubated with the cyclase (20 mL) as described. 8-Me-cADPR
was obtained in 84% yield. dH (D2O, 400 MHz) 2.5 (3H, s, Me),
4.0-4.6 (9H, m, ribose), 5.3 (1H, app.t, J2¢,3¢ = J2¢,1¢ 5.5, H-2¢), 5.85
(1H, d, J1¢,2¢ 5.5, H-1¢), 6.0 (1H, d, J1¢¢,2¢¢ 4.0, H-1¢¢), 8.8 (1H, s, HA2).
dP (D2O, 162 MHz, 1H-decoupled) -8.3 and -9.1 (AB system, JAB
16.1, 2P). m/z (ES-) 554 [100%, (M - H)-]. lmax 260 nm (e 10000,
pH 8.3). HPLC Rt = 4.2 mins.
Cyclic 2¢,3¢-O-isopropylidene adenosine 5¢-diphosphate ribose
(cAcetDPR, 18). A solution of NAcetD+ (7.5 mmol) in HEPES
(17.5 mL) was incubated with the cyclase (75 mL) as described.
cAcetDPR was obtained in 46% yield. dH (D2O, 400 MHz) 1.30
(3H, s, CH3), 1.49 (3H, s, CH3), 3.80-4.43 (8H, m, ribose), 4.65
(1H, dd, J3¢,2¢ 6.1, J3¢,4¢ 3.1, H-3¢), 5.17 (1H, dd, J2¢,3¢ 6.1, J2¢,1¢ 2.0,
H-2¢), 6.03 (1H, d, J1¢¢,2¢¢ 3.7, H-1¢¢), 6.25 (1H, d, J1¢,2¢ 2.0, H-1¢), 8.26
(1H, s, HA8), 8.91 (1H, s, HA2). dP (D2O, 162 MHz, 1H-decoupled)
-10.14 and -11.52 (AB system, JAB 13.9, 2P). m/z (ES-) 580 [100%,
(M - H)-]. lmax 257 nm (e 14300, pH 8.3). HPLC Rt = 7.1 mins.
8-Methoxy cyclic adenosine 5¢-diphosphate ribose (8-OCH3-
cADPR, 12). A solution of 8-OMe-NAD+ (4 mmol) in HEPES
(2 mL) was incubated with the cyclase (20 mL) as described. 8-
OMe-cADPR was obtained in 53% yield. dH (D2O, 400 MHz) 4.1
(3H, s, OMe), 4.2–4.62 (9H, m, ribose), 5.6 (1H, app.t, J2¢,3¢ = J2¢,1¢
5.4, H-2¢), 5.9 (1H, d, J1¢,2¢ 5.9, H-1¢), 6.0 (1H, d, J1¢¢,2¢¢ 4.0, H-1¢¢),
8.8 (1H, s, HA2). dP (D2O, 162 MHz, 1H-decoupled) -10.9 (s, 2P).
m/z (ES-) 570 [100%, (M - H)-]. lmax 259 nm (e 10300, pH 8.3).
HPLC Rt = 4.0 mins.
Acknowledgements
8-Piperidyl cyclic adenosine 5¢diphosphate ribose (8-pip-cADPR,
13). A solution of 8-Pip-NAD+ (3.75 mg) in HEPES (3.3 mL)
was incubated with the cyclase (10 mL) as described. 8-Pip-cADPR
was obtained in 49% yield. dH (D2O, 400 MHz) 1.7 (6H, m, 3 ¥
CH2), 3.2 (4H, m, 2 ¥ CH2), 4.0-4.5 (9H, m, ribose), 5.6 (1H, app.t,
We thank the Wellcome Trust for Project Grant no. 084068 [to
BVLP and AHG] and MRC for a studentship (VCB). Work in
the Guse lab has been supported by the Deutsche Forschungsge-
meinschaft, the Gemeinnu¨tzige Hertie-Stiftung and the Deutsche
Akademische Austauschdienst. We thank Dr M Thomas for
providing Fig. 6.
J
2¢,3¢ = J2¢,1¢ 4.2, H-2¢), 5.9 (1H, d, J1¢,2¢ 5.8, H-1¢), 6.1 (1H, d, J1¢¢,2¢¢ 4.0,
H-1¢¢), 8.9 (1H, s, HA2). dP (D2O, 162 MHz, 1H-decoupled) -10.92
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