Novel nucleobase-simplified cyclic ADP-ribose analogue: A concise synthesis and Ca2+-mobilizing activity in T-lymphocytes

Article abstract of DOI:10.1039/b925295a

A purine nucleobase-simplified cyclic ADP ribose (cADPR) analogue 6b was synthesized, in which a 1,2,3-triazole-4-amide was constructed, instead of a purine moiety, and the northern ribose was replaced by an ether strand. Compound 6b exhibits calcium rele

Full text of DOI:10.1039/b925295a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Novel nucleobase-simplified cyclic ADP-ribose analogue: A concise synthesis
and Ca2+-mobilizing activity in T-lymphocytes†
Lingjun Li,‡a,b Cornelia C. Siebrands,‡c Zhenjun Yang,a Liangren Zhang,a Andreas H. Gusec and Lihe Zhang*a
Received 2nd December 2009, Accepted 22nd January 2010
First published as an Advance Article on the web 18th February 2010
DOI: 10.1039/b925295a
A purine nucleobase-simplified cyclic ADP ribose (cADPR) analogue 6b was synthesized, in which a
1,2,3-triazole-4-amide was constructed, instead of a purine moiety, and the northern ribose was
replaced by an ether strand. Compound 6b exhibits calcium release activity in intact T-lymphocytes and
indicates that it is a membrane-permeable cADPR mimic. Thus, the cADPR analogue containing
1,2,3-triazole-4-amide provides a novel template for further designing cADPR analogues and
elucidating their structure–activity relationships.
cADPR, which are applied well in different types of cells,16 and
the analogues with the 6-NH2 replaced by an oxygen atom, e.g.
Introduction
Since cyclic ADP-ribose (cADPR) (Fig. 1), a metabolite of NAD+,
was discovered by Lee and co-workers in 1987,1 numerous cell
systems have been described to utilize the cADPR/ryanodine
receptor (RyR)/Ca2+ signaling system to control Ca2+-dependent
cellular responses, such as fertilization, secretion, contraction,
proliferation and many more.2,3 A large number of key proteins are
involved in specifically shaping Ca2+ signals. However, it is unclear
whether cADPR elicits calcium release by direct binding to RyR,
or via an additional binding protein. A main approach to explore
the molecular mechanism of calcium release is to investigate the
relationship of the structure of cADPR and cADPR analogues
with their respective biological activities. Much effort has been
focused on the syntheses of structural derivatives to elucidate the
structure–activity relationship and supply tools for investigating
cellular Ca2+ signaling.4–6
cIDPR, are fully agonist resistant to enzymatic hydrolysis.20 It was
reported that 7-deaza- and 3-deaza-cADPR are agonists of the
cADPR/RyR signaling system, and 3-deaza-cADPR is a 70 times
more potent agonist than cADPR itself.18,21 Therefore, the purine
moiety in cADPR plays a very important role for the recognition
between cADPR and its receptor. However, as these reported
modifications of the nucleobase in cADPR are always based on
the purine structure, it would be interesting to synthesize the
purine-simplified cADPR analogues and investigate whether the
whole purine structure in cADPR is essential for the recognition
of cADPR and its receptor. Two cADPR analogues containing
4-amide-1,2,3-triazole have been successfully prepared previously
in our group,22 in which a click reaction was used to construct
the 4-amide-1,2,3-triazole nucleobase and simultaneously
connect the two building blocks (Scheme 1, Fig. 2). In the
current paper, we report a microwave-assisted intramolecular
pyrophosphorylation method for the scale-up preparation of 6b,
and a biological study of compound 6b on the calcium release
activity in intact T-lymphocytes, exhibiting that compound 6b is
a membrane-permeable cADPR mimic.
The
structural
modifications
of
cADPR
include
pyrophosphate,7–9 ribose10–15 and adenine moieties.16–21 The drastic
reduction in calcium release activity of cADPR(CH2) (Fig. 1)
indicates that the bridging oxygen in the pyrophosphate moiety is
important in cADPR action.7 A series of N1-glycosyl-subsitituted
(cADPcR, cIDPRE, cADPRE) or N9-glycosyl-subsitituted
cADPR analogues with carbon riboses10–13 (cyclic aristeromycin
diphosphoribose), or simplified structures, e.g. ether stands14
(cIDPDE), show good agonist activities, indicating that the
northern and southern ribose moieties are alterable, to some
extent, in cADPR action. Modifications on adenine moieties,
such as 8-NH2-cADPR and 8-Br-cADPR, give antagonists of
Results and discussion
1. Microwave-assisted intramolecular pyrophosphorylation for
the preparation of cTDPRE 6b
The synthesis of cADPR analogues has been extensively
investigated.23–29 Analogues of cADPR can be prepared from
NAD+ derivatives by enzymatic and chemo-enzymatic methods,
using ADP-ribose cyclase to catalyze the cyclization reaction.24,27,28
However, the analogues obtained by these methods are limited
because of the substrate specificity of ADP-ribose cyclase.19,20
Shuto et al. developed a chemical method for synthesis of cADPR
analogues through intramolecular cyclization for the formation
of the pyrophosphate linkage.11,26,29 The total chemical synthesis
allowed extensive modifications of both the ribose and nucleobase
moieties.11–14,25 However, as a disadvantage, this method includes
many steps for the protection and deprotection of building blocks
in constructing the substituted nucleoside and pyrophosphate
aState Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University, Beijing 100083, P.R. China.
E-mail: zdszlh@bjmu.edu.cn; Fax: 86-10-82802724; Tel: 86-10-82801700;
86-10-82802567
bCollege of Chemistry and Environmental Science, Henan Normal University,
Xinxiang 453007, P.R. China
cThe Calcium Signaling Group, University Medical Center Hamburg-
Eppendorf, Center of Experimental Medicine, Institute of Biochemistry
and Molecular Biology I: Cellular Signal Transduction, 20246 Hamburg,
Germany
† Electronic supplementary information (ESI) available: Further experi-
mental details and NMR spectra. See DOI: 10.1039/b925295a
‡ These authors contributed equally.
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Fig. 1 cADPR and its analogues.
Scheme 1 Synthesis of cTDPRE derivatives. Reagents and conditions: (a) CuI, DIPEA, CH3CN; (b) 1. POCl3, DIPEA, CH3CN, 0 ◦C, 12 h 2. 0.1 M
˚
TEAB; (c) I2, 3 A MS, pyridine; (d) 50% HCOOH.
linkage. Furthermore, in most of the cases of total chemical
synthesis of cADPR analogues, super-dilute solutions of interme-
diates are needed for the key intramolecular pyrophosphorylation.
Under these super-dilute solution conditions, the reaction is
usually completed by using a syringe-pump over 15 h, even for
10 mg of precursor. In addition, the reaction is very sensitive to
traces of water.26 This strict dry environment and the long reaction
time in a super-dilute solution render the experimental work-up
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Table 1 Microwave-assisted intramolecular cyclopyrophosphorylation of compound 4b under different conditions
Entrya
Temperature/K
Molecular sieves/g ml-1 of py.
Time/min
Concentration of 4b
Yieldb of 5b (ratio of 7 : 5b : 8)c
1
2
3
4
5
6
7
8
r.t.
r.t.
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
1200
150
5
Super-dilute
2.27 mM
2.27 mM
2.27 mM
2.27 mM
2.27 mM
2.27 mM
2.27 mM
2.27 mM
2.27 mM
4.54 mM
6.81 mM
82.5 (9 : 83 : 8)
68 (0 : 70 : 30)
40 (57 : 40 : 3)
45 (51 : 45 : 3)
51 (46 : 51 : 3)
63 (35 : 63 : 3)
83 (15 : 83 : 2)
88 (10 : 88 : 2)
95 (0 : 98 : 2)
95 (0 : 98 : 2)
95 (0 : 98 : 2)
95 (0 : 98 : 2)
50 ◦C (MW)
50 ◦C (MW)
50 ◦C (MW)
60 ◦C (MW)
70 ◦C (MW)
80 ◦C (MW)
90 ◦C (MW)
90 ◦C (MW)
90 ◦C (MW)
90 ◦C (MW)
10
15
15
15
15
15
15
15
15
9
0.1
10
11
12
none
none
none
a 100 mg I2 as the catalyst of the reaction was added for 10 mg compound 1. b Isolated yield. c The ratio of 7 : 5b : 8 was based on analysis by HPLC.
Fig. 2 Structural design of target compounds cTDPRC 6a and cTDPRE 6b.
tedious, and thus, the large-scale preparation of cADPR analogues
is limited.
In general, intramolecular reactions are favored in dilute reac-
tions, and intermolecular reactions are favored in concentrated
reactions. We first tried the intramolecular pyrophosphorylation
of compound 4b in pyridine at 2.27 mM concentration. Com-
pound 4b was prepared from 1b and 2 by the published procedure
(Scheme 1).22 Thus, 5 mg of compound 4b was reacted in a 2.5 ml
dry pyridine solution containing 50 mg I2, at ambient temperature
for 3 h; water was added into the reaction mixture for another
1 h to hydrolyze the non-reacted precursor 4b into compound
7 (Scheme 2). After separation and purification, compound 5b
was obtained as a triethylammonium salt with 69% yield and a
dimer 8 (Fig. 3) was separated in 30% yield (entry 2, Table 1). In
Fig. 3 Structure of compound 8.
comparison with the yield of 5b obtained from the dilute solution
(entry 1, Table 1), this is a reasonable yield.
Microwave-assisted organic chemistry has been recently devel-
oped for the fast synthesis of functional compounds and libraries
in many areas.30,31 With the assistance of microwave, the reaction
rates and yields were increased greatly in some cases. We applied
this technology to the intramolecular pyrophosphorylation of
compound 4b for the preparation of cTDPRE 6b. Interestingly,
the intramolecular pyrophosphorylation of compound 4b was
carried out in a highly concentrated solution, and the amount
of dimer 8 was reduced considerably (Table 1). Optimization of
these reaction conditions was done by changing temperature and
reaction time. Increases of both the temperature and the reaction
time under microwave irradiation dramatically increased the yield
of compound 5b (entry 3–9, Table 1). Compound 5b was obtained
in 95% yield when the reaction was carried out at 90 ◦C for
15 min (entry 9, Table 1). In all of the cases with microwave
irradiation, the yield of the intermolecular pyrophosphorylation
product 8 was less than 3%. Thus, contamination with by-product
8 was less than in the reaction using the super-dilute solution
(entry 1, Table 1). Since water obviously increases the amount of
open chain products in the intramolecular pyrophosphorylation
reaction in super-dilute solution, the removal of trace amounts
of water using the molecular sieves is important.26 However,
in the microwave-assisted intramolecular pyrophosphorylation,
molecular sieves are not necessary, as shown in Table 1 (entry
10). Even further increases in the concentration of precursor 4b
up to 4.54 or 6.81 mM resulted in a 95% yield of 5b (Table 1).
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Scheme 2 Microwave-assisted intramolecular cyclopyrophosphorylation for the preparation of cTDPRE 6b.
After deprotection of the 2¢,3¢-O-isopropyl group, compound 6b
was obtained from 5b in quantitative yield. Taken together, an
efficient microwave-assisted intramolecular pyrophosphorylation
approach was developed and successfully applied to the key
step for the preparation of cTDPRE 6b. The microwave-assisted
reaction allows for an efficient cyclopyrophosphorylation that is
completed within 15 min at high precursor concentrations, with
95% yield, without requiring super-dilute precursor solutions and
molecular sieves. Compound 6b prepared by this new method was
linear analogue TDPRE 7 was applied in a concentration of
1 mM, but this compound did not increase [Ca2+]i significantly
(Fig. 4C).
A couple of cADPR analogues modified in the nucleobase
have been shown to retain agonist activity. Examples for such
biologically active compounds are cIDPR derivatives,19 3-deaza-
cADPR,20 and 7-deaza-cADPR.21 These examples suggest that
the nucleobase may be modified at several sites without a major
loss of agonist activity. The triazole-based cADPR derivative
6b presented here represents a much more radical approach to
simplify the structure of cADPR analogues, as compared to any
other cADPR derivatives. The imidazole moiety of the natural
nucleobase adenine is replaced by the similar triazole ring, and
the amino-pyrimidine moiety of adenine is replaced by an amide
bond partially mimicking C6 and N1 of adenine. Interestingly, we
demonstrate here that regardless of this dramatic simplification
of the purine nucleobase, the principal ability to mobilize Ca2+
is retained in cTDPRE 6b. Although the peak Ca2+ obtained
at 1 mM extracellular concentration was below about 200 nM,
a sustained Ca2+ signal was observed indicating coupling of
Ca2+ release to capacitative Ca2+ entry. Importantly, the relative
amplitude (defined as the difference between basal [Ca2+]i and
[Ca2+]i at a certain time point upon addition of the compound)
of sustained Ca2+ signaling upon cTDPRE addition was almost
100 nM (Fig. 4C), and thus, is clearly comparable to the sustained
relative Ca2+ signal slightly above 100 nM observed with the
benchmark cADPR analogue, cIDPRE.12
1
identified by H and 31P NMR, and HRMS, and compound 6b
is consistent with the compound reported by us previously by
HPLC.22
2. Evaluation of Ca2+ mobilizing activity of cTDPRE 6b in
Jurkat T cells
The biological activity of cTDPRE 6b was assessed in intact
human Jurkat T-lymphocytes as described previously.12–14 Appli-
cation of cTDPRE 6b (1 mM final extracellular concentration)
resulted in an increase in the free cytosolic Ca2+ concentration
([Ca2+]i). Lag periods between approx. 50 and a few hundred
seconds were sufficient to reach a threshold concentration of
cTDPRE 6b inside the cell to promote Ca2+ release (Fig. 4A and
B). Some cells responded with oscillatory Ca2+ signaling patterns,
while other cells showed a sustained increase in [Ca2+]i (Fig. 4).
Similar to the application of the anti-CD3 monoclonal antibody
OKT3, cTDPRE 6b resulted in a fast increase in [Ca2+]i (peak),
followed by a sustained level of elevated [Ca2+]i (plateau). This
effect was concentration-dependent, resulting in smaller signals
with 300 and 100 mM final concentrations of 6b (Fig. 4C). The
In conclusion, here we report an efficient microwave-assisted
intramolecular cyclopyrophosphorylation approach that was suc-
cessfully applied to the key step for the preparation of cTDPRE
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Fig. 4 Quantitative analysis of the Ca2+ mobilizing effects of cTDPRE 6b and TDPRE 7. A representative field of six cells was imaged for [Ca2+]i at
different time points. cTDPRE (1 mM final concentration) was added as indicated by the arrow (90 s). Changes in [Ca2+]i are shown as pseudocolor
images (A; scale bar 10 mm) and by quantification of the mean [Ca2+]i in each single cell. (B) Color encoding of localization of individual cells in the
microscopy field is as indicated in the inset in (A). (C) Changes in mean [Ca2+]i after the application of compound or buffer control were averaged for at
least three independent experiments (number of cells are indicated in the bars) for each condition. The peak was analyzed as the maximum within 200 s
after application, the plateau was analyzed 500 s after application. Data are presented as mean SD. * Indicates a significant difference compared to the
respective negative control (p < 0.01). The response to the application of the anti-CD3 monoclonal antibody OKT3 served as a positive control while
addition of buffer was used as a negative control.
6b. With this microwave-assisted reaction, an efficient cyclopy-
rophosphorylation can be completed in 15 min at high precursor
concentrations, resulting in 95% yield. To our best knowledge, this
is the first report about a high-yield intramolecular cyclization
in highly concentrated precursor solutions for the preparation
of cADPR analogues. Together with its Ca2+ release activity,
cell membrane-permeant property, and the concise and efficient
synthesis, cTDPRE 6b is a promising valuable tool for the
investigation of cADPR-mediated Ca2+ signals. The 1,2,3-triazole-
modified cADPR mimics may open a completely new direction for
the medicinal chemistry and pharmacology of cADPR.
Compound 4b was prepared based on the method that we
reported previously.22 The mixture of 10 mg of compound 4b
(12.4 mmol) and 100 mg of iodine in pyridine (5 ml) was stirred
at 90 ◦C, assisted by microwave irradiation, for 15 min. Then
the pyridine was evaporated, and the residue was partitioned
between CHCl3 and H2O. The aqueous layer was evaporated and
the residue was dissolved in 0.05 M TEAB buffer (1.0 ml), which
was applied to a C18 reversed-phase column (2.2 ¥ 5 cm). The
column was eluted using a linear gradient of 0–50% CH3CN in
TEAB buffer (0.05 M, pH 7.5) within 50 min to give 5b at 34.1 min
(8.2 mg, 95%) as a triethylammonium salt. 1H NMR (300 MHz,
D2O), d 8.48 (s, 1H, H-5), 5.96-5.98 (d, 1H, J = 8 Hz, H-1¢), 4.81-
4.89 (m, 2H, H-2¢, 3¢), 4.57-4.60 (m, 1H, H-4¢), 4.21-4.26 (d, J =
13 Hz, 1H, H-5¢a), 3.83-3.88 (d, J = 13 Hz, 1H, H-5¢b), 3.39-3.60
(m, 8H, H-1¢¢,2¢¢,4¢¢,5¢¢), 1.30, 1.45 (each s, each 3H, -C(CH3)2).
13C NMR (100 MHz, D2O), d 127.9, 124.3, 123.6, 113.3, 94.9,
85.4, 84.1, 80.8, 68.9, 67.0, 64.8, 63.8, 45.6, 38.5, 24.9, 23.2. 31P
NMR (D2O, 81 MHz), d -9.11 (br, s), -11.4 (br, s). HRMS (ESI)
calculated for (M + H+) C15H25N4O12P2 515.0939, found 515.0925,
(M + Na+) C15H24N4O12P2 537.0758, found 537.0746.
Experimental
(i) Preparation of cTDPRE 6b by MW-assisted intramolecular
pryophosphorylation reaction
Mass spectra were obtained on either VG-ZAB-HS or Bruker
APEX. HR-ESI-MS (electrospray ionization) were performed
with a Bruker BIFLEXTM III. 1H NMR was recorded with a
JEOL AL300 spectrometer using D2O as solvent. Chemical shifts
were reported in parts per million downfield from TMS. 31P NMR
spectra were recorded at room temperature by use of a Bruker
Avance 200 spectrometer (81 MHz); orthophosphoric acid (85%)
was used as an external standard. Compound 6b was purified twice
on an Alltech preparative C18 reversed phase column (2.2 ¥ 25 cm)
using a Gilson HPLC by buffer system: MeCN–TEAB (pH 7.5)
and MeCN–TEAA (pH 7.0) before the structural identification
and biological study.
A solution of 5b (5 mg, 7.1 mmol) in 50% HCOOH (1.5 ml) was
stirred for 2 h and then evaporated under reduced pressure. The
purification of the residue was performed with the same procedure
as for compound 5b by HPLC on a C18 reversed-phase column,
eluted with a linear gradient of 0–65% CH3CN in TEAB buffer
(0.05 M, pH 7.5) to give the target molecule 6b in quantitative
1
yield. H NMR (300 MHz, D2O), d 8.67 (s, 1H, H-5), 6.03-6.05
(d, J = 9 Hz, H-1¢), 4.54-4.56 (m, 2H, H-2¢¢, 3¢¢), 4.14-4.18 (m, 1H,
H-4¢¢), 3.93-4.02 (m, 2H, H-5¢a, 5¢b), 3.80-3.66 (m, 8H, H-1¢¢,2¢¢,
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4¢¢,5¢¢). 31P NMR (D2O, 81 MHz), d -8.91 (br, s), -11.39 (br, s).
HRMS (ESI) (M - H+) calculated for C12H20N4O12P2 473.0480,
found: 473.0477.
TEAB
TMS
triethylammonium bicarbonate
tetramethylsilane
Acknowledgements
HPLC analysis of the microwave-assisted reactions. The re-
action mixture was evaporated and the residue was partitioned
between CHCl3 and H2O. The aqueous layer was evaporated and
the residue was dissolved in 0.05 M TEAB buffer (1.0 ml), which
was applied to a C18 reversed-phase column (2.2 ¥ 5 cm). The
column was eluted using a linear gradient of 0–50% CH3CN in
TEAB buffer (0.05 M, pH 7.5) within 50 min to give 7 at 24.2 min,
5b at 34.1 min, and 8 at 38.0 min; the molecular weights of 7,
5b and 8 were given by ESI-TOF- with 531.0, 513.0, and 1027.1,
respectively.
This study was supported by the National Natural Sciences
Foundation of China (grant no. 90713005, 20802017), Ministry of
Education of China (grant no. 200800010078), and the Deutsche
Forschungsgemeinschaft (grant no. GU 360/13-1).
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cIDPR
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cIDPRE
cIDP-DE
DCC
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31P NMR
HR-ESI-MS
cyclic adenosine 5¢-diphosphoribose
cyclic inosine 5¢-diphosphoribose
cyclic adenosine 5¢-diphospho- carboribose
cyclic inosine diphosphoribose-ether
cyclic inosine diphospho-diether
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