Full text of DOI:10.1002/cbic.201800133

Accepted Article
Title: Calcium mobilizing behaviors of neutral cyclic ADP-ribose mimics
which integrate the modifications of nucleobase, northern ribose
and pyrophosphate
Authors: Xuan Wang, Xiaoyan Zhang, Kehui Zhang, Jianxing Hu,
Zhenming Liu, Hongwei Jin, Liangren Zhang, and Lihe Zhang
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To be cited as: ChemBioChem 10.1002/cbic.201800133
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ChemBioChem
10.1002/cbic.201800133
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Calcium mobilizing behaviors of neutral cyclic ADP-ribose mimics
which integrate the modifications of nucleobase, northern ribose
and pyrophosphate
[
a]
Xuan Wang, Xiaoyan Zhang, Kehui Zhang, Jianxing Hu, Zhenming Liu, Hongwei Jin*, Liangren Zhang* ,
and Lihe Zhang
Abstract: Cyclic adenosine diphosphate ribose (cADPR) is an
phosphate dehydrogenase (GAPDH), etc., have been reported to
regulate the interaction of cADPR and RyRs.[4]
2+
endogenous Ca mobilizer involved in diverse cellular process.
Mimics of cADPR play crucial role in investigating the molecular
mechanism of cADPR mediated signaling pathway. Herein, a mimic
of cADPR (3) was firstly synthesized, in which neutral triazole moiety
and ether linkage were introduced to substitute pyrophosphate and
northern ribose, respectively. The pharmacological activities in Jurkat
cells indicated that this mimic is capable of permeating plasma
To explore the molecular mechanism inherent in the cADPR-
mediated signaling pathway, a series of cADPR analogues
including the modification on ribose, nucleobase, and
pyrophosphate have been synthesized. Northern ribose is the
most widely investigated moiety among the cADPR analogues,[
and the research results indicated that the northern ribose was
tolerant of chemical modification and relative to plasma
membrane permeability. For instance, cIDPRE (2) and its
derivatives, analogues of cyclic inosine diphosphate ribose
(cIDPR) in which the northern ribose is replaced by ether linkage,
5]
2+
membrane and inciting Ca release from endoplasmic reticulum (ER)
2+
via ryanodine receptors (RyRs) and trigger Ca influx. Furthermore,
uridine moiety was introduced and novel cADPR mimics (4, 5) were
synthesized. The results of biological investigation showed that these
2+
2+
[6]
mimics also targeted to RyRs and maintained moderate Ca
agonistic activities. The results indicated that the neutral cADPR
are membrane permeable Ca agonists.
2+
mimics had the same targets to motivate Ca signaling.
Introduction
Cyclic ADP-ribose (cADPR, 1), an endogenous nucleotide
+
synthesized from nicotinamide adenine dinucleotide (NAD ) by
ADP-ribosyl cyclases in cells, was firstly discovered in 1987 by
Lee and colleagues as
a
potent Ca2+ releasing second
[
1]
messenger. Recently, cADPR has drawn a great deal of
attention because of its potent calcium-mobilizing activities in
many cellular processes, such as fertilization, insulin secretion,
lymphocyte activation and proliferation.[2] CD38, the main
mammalian ADP-ribosyl cyclase, regulates both the formation
and hydrolysis of cADPR.[3] It has been shown that cADPR elicits
Ca2+ release from endoplasmic reticulum (ER) through the
ryanodine receptors (RyRs) and induces Ca2+ influx in many cell
types. Some accessory proteins, such as calmodulin (CaM),
FK506-binding protein 12.6 (FKBP12.6), and glyceraldehyde 3-
Scheme 1. Structures of cADPR (1), cIDPRE (2) and the novel cADPR mimics
3-5).
(
[
a]
X. Wang, X. Zhang, K. Zhang, J. Hu,Prof. Z. Liu, Dr. H. Jin, Prof. L.
Zhang, Prof. L. Zhang
Another main factor interrupting the permeability and activity of
State Key Laboratory of Natural and Biomimetic Drugs
School of Pharmaceutical Sciences
Peking University
Beijing 100191 (China)
E-mail: liangren@bjmu.edu.cn
cADPR is the strong negative charge at the pyrophosphate moiety.
Up to now, only a few structural analogues modified on the
pyrophosphate moiety have been synthesized, and most of these
are agonists. For example, cATPR, with a triphosphate bridge,
was proven to be considerably more potent than cADPR in
inducing Ca2+ release from rat brain microsomes.[7] And agonistic
activity was retained when the pyrophosphate linkage was shrunk
to a monophosphate.[8] Sulfur and selenium-substituted cyclic
jinhw@bjmu.edu.cn
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NaNO
THF, reflux, 16 h.
2
, AcOH, rt, 24 h; (vii) compound 9, DBU, DCM, -10 °C; (viii) CuI, DIPEA,
pyrophosphate cIDPRE derivatives also lowered the agonistic
activity.[6d, 9] Recently, Potter and colleagues have reported a
series of triazole analogues of cIDPR, albeit weak, retained the
ability to activate global cytosolic Ca2+ release in sea urchin egg
homogenate.[10] These findings raised a question that whether
cADPR mimics with the disappearance of the pivotal
pyrophosphate or even more structural diverse mimics still
possess similar mechanism to regulate the cADPR-involved Ca2+
signaling pathway.
To investigate the effect of pyrophosphate moiety on the
mechanism of Ca2+ mobilization, herein, a neutral cADPR mimic
Briefly, compound 6[12] was mixed with
P
O
5
and
2
dimethoxymethane in ice-water bath for 24 h to afford 7 (Scheme.
2 [
).
13]
Compound 7 was acetylated by acetic anhydride in the
presence of boron trifluoride−diethyl etherate to generate
compound 8, which was then reacted with TMSBr to produce 9. It
is worth mentioning that the reaction time of 8 should be handled
strictly to prevent byproducts. Compound 9 can be used directly
1
for next step without purification. The N -substitution product 12
(
3) was synthesized, in which the modifications of pyrophosphate
was carried out regioselectively by the reaction of 9 and 5’-deoxy-
and ribose were integrated, and the isopropylidene group at
southern ribose was maintained since our previous work showed
it did not affect activity to any great extent.[11] Furthermore, uridine
moiety was firstly introduced and novel cADPR mimics (4, 5) were
synthesized (Scheme.1). The biological studies were performed
in Jurkat cells. The findings of this research will help us to estabish
the structure-activity relationship of cADPR analogues in detail
and provide stable membrane-permeable probes to investigate
cADPR-regulated Ca2+ signaling pathway.
14]
5
’-azido-2’,3’-O-isopropylidene inosine (11)[ in the yield of 68%,
6
and the O -alkylation by-product was observed in 13% yield. After
the intramolecular click reaction catalyzed by CuI/DIPEA/THF,
neutral cADPR mimic 3 was afforded. The structure was
characterized by 1H, 13C-NMR and HRMS. In 1H-NMR
spectroscopy, the singlet at 7.19 ppm was observed, which
indicated the generation of a 1,4-disubstituted-1,2,3-triazole
(Figure. S1).
3
N -substitution of the uridine derivative was also performed
similarly by the reaction of 5’-deoxy-5’-azido-2’,3’-O-
isopropylidene uridine (14)[14] and compound 15[15] or 16 in the
presence of K CO , and uridine derivatives 17 or 18 were afforded,
2 3
Results
respectively (Scheme. 3). Subsequently, compound 17 or 18 was
converted to 4 or 5 using similar reaction condition as described
above. In addition, we tried to remove isopropylidene group,
however, treatment of 3-5 with aqueous HCOOH led to the rapid
decomposition of the starting material or get product in very low
yield.
Synthesis of neutral cADPR mimics (3-5).
The synthetic routes adopted for compounds 3-5 have two main
steps: (i) introduction of the northern chain onto N - or N -position,
which produces intermediates 12, 17 and 18; (ii) cyclization of
these intermediates to yield the target compounds 3-5 (Scheme.
1
3
2-3).
Scheme 3. Synthetic route of compounds 4 and 5. Reagents and conditions: (i)
DPPA, DBU, 1,4-dioxane, rt, 3 h, then NaN
5 or 16, K CO , DMF : acetone = 1:1, 55 C, 4 h; (iii) CuI, THF, DIPEA, reflux,
24 h.
3
, TBAI, 15-crown-5, reflux, 4 h; (ii)
1
2
3
Scheme 2. Synthetic route of compound 3. Reagents and conditions: (i)
propargyl bromide, KOH, H O, 0 °C - rt, 18 h; (ii) Methylal, P , DCM, 24 h,
9%; (iii) Ac O, BF ·OEt , 0°C, 2 h; (iv) TMSBr, DCM, 0 °C - reflux,18 h; (v)
DPPA, DBU, 1,4-dioxane, rt, 3 h, then NaN , TBAI, 15-crown-5, reflux, 4 h; (vi)
2
2 5
O
6
2
3
2
3
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Compounds 3-5 induce cytosolic Ca2+ increase in Jurkat
cells.
The ability of the novel mimics to induce Ca2+ release in Jurkat
cells was investigated. The results showed that compounds 3-5
were all Ca2 agonists. Among them compound 3 produced
+
2+
strongest Ca elevations (Figure. 1a). Compound 4 triggered
2
+
impotent Ca increase, indicating the uridine base would mitigate
the ability of activating Ca2+ release, while compound 5 produced
much weaker Ca2+ increase even at high concentration. Therefore,
we chose compounds 3 and 4 for further investigation of biological
activity. Both of 3 and 4 were found to agonize Ca2+ signaling in a
dose-dependent manner under the presence or absence of
extracellular Ca2+ (Figure. 1b-1e). The cytosolic Ca2+ increase
observed in the presence of extracellular Ca2+ was more
pronounced than that in the absence of extracellular Ca2 (Figure.
+
1
b vs 1c; 1d vs 1e), suggesting that Ca2+ influx should also be
contributed.
Compounds 3 and 4 induce cytosolic Ca increase from ER
2
+
2
+
Ca pools via RyRs.
We determined whether compounds 3 and 4 had the same targets
as cADPR to trigger Ca2+ release. Ample evidence indicates that
cADPR targets RyRs on the ER membrane in many cell types.
Pretreating cells with thapsigargin (TG, 10 μM), a specific SERCA
2
+
inhibitor, rendered less pronounced Ca increase in the absence
2
+
2+
of external Ca , consistent with the fact that cADPR induce Ca
release from the ER pools (Figure. 2a). Pretreatment with a RyR
antagonist, ryanodine (20 μM), or cADPR antagonist, 8-Br-
cADPR (100 μM), or RyR2/RyR3 double knockdown[16]
2
+
significantly inhibited compound 3-induced Ca increase in
Jurkat cells (Figure. 2b, 2c, S4a). Similarly, compound 4-induced
2
+
Ca release was also inhibited by these antagonists or RyRs
knockdown (Figure. 2e, 2f, S4b), and even be abolished by TG in
Jurkat cells (Figure. 2d). In summary, these results demonstrate
that compounds 3 and 4 evokes Ca2+ release via RyRs.
Mitochondria participate in compound 3-induced Ca2+
release.
TG was not able to completely abolish compound 3-induced Ca2
release in the absence of extracellular Ca2+ (Figure. 2a), which
indicated that 3 might partially increase other Ca2+ pools in Jurkat
cells. For this reason, we explored whether the mitochondrial or
acidic Ca2+ stores participate in compound 3-induced Ca2+
release. Pretreatment with 2 µM FCCP[17], a mitochondrial
uncoupler, could lower compound 3-invoked Ca2+ increase
+
(
Figure. 3a). However, lysosomal inhibitors, 50 µM GPN[18] and 10
mM NH
4
Cl[19], failed to change the Ca2+ signaling (Figure. 3b, S5).
This evidence indicated that compound 3-induced Ca2+ signaling
was related to mitochondria, not lysosomes.
Compounds 3-5 have similar conformation as cADPR.
Three-dimensional conformation superimposition was performed
to assess the conformation similarity between cADPR and
compounds 3-5. All mimics were placed in similar positions with
cADPR after minimization, and showed pronounced overlap in the
southern ribose sugar and pyrophosphate backbone (Figure. 4).
These findings suggest that the triazole moiety can mimic the
pyrophosphate moiety to some extent.
Figure 1. Compounds 3-5 are cell permeable cADPR agonists. (a) Compound
3
4
(10 μM) induced strongest cytosolic Ca2+ increase among cIDPRE (100 μM),
(10 μM) and 5 (100 μM) in human Jurkat cells in the presence of extracellular
2+
2+
Ca . Concentration response relationship of 3 or 4-induced cytosolic Ca
increase in Jurkat cells in the presence (b, d) or absence (c, e) of extracellular
2+
Ca . Date are the mean ± S.D. (n=3, 20-40 cells in each independent
experiment).
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Figure 2. The requirement of RyRs for compounds 3 and 4-triggered Ca2+ release. Pretreatment with TG (1 μM) markedly reduced 3-induced Ca increase(a) or
2+
even blocked 4-induced Ca2+ increase(d) in the absence of extracellular Ca . Compound 3 (10 μM) (c) or 4 (1 mM) (f) induced cytosolic Ca increase was
2+
2+
2+
significantly inhibited by ryanodine (20 μM) or 8-Br-cADPR (100 μM) in the absence of extracellular Ca . RyR2/RyR3 double knockdown in Jurkat cells markedly
inhibited. Date are the mean ± S.D., n=3 (20-40 cells in each independent experiment). *, P < 0.05.
Discussion
the moiety that facilitates electrostatic interaction with binding
protein. Synthesis and study of the mechanism underlying
pyrophosphate-missed mimics have been challenging.
So far, no pyrimidine-based modification has been involved
since studies of major modifications have focused on the purine
scaffold. Purine-simplified moiety have been used to assess the
minimum structural requirement for activity, in which adenine was
substituted by triazole.[21] Three-dimensional conformation
analysis showed that pyrimidine-derivative and another neutral
[
20]
The cADPR-mediated Ca2+ signaling pathway is involved in a
wide variety of cellular processes.[2b] The molecular mechanism
underlying its action remains unclear even though many active
cADPR analogues have been used as probes. Chemical
modifications on pyrophosphate has been less investigated than
modifications on ribose and nucleobase. Pyrophosphate was
considered to participate in cADPR's calcium signaling and to be
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mimic (3) had positions similar to that of cADPR, and they all had
strong overlap in the southern ribose sugar and pyrophosphate
backbone (Figure. 4). In this way, our investigation of pyrimidine
derivative went the way we expected.
cADPR is readily hydrolyzed by CD38 NADase at the unstable
N1 link to produce ADPR, another Ca2+ second messenger.[22]
The biological activity of 4 and 5 showed that the uridine
substitution decreased agonistic activity (Figure. 1a), which
indicated the importance of purine of cADPR mimics. Moreover,
the oxygen atom of the northern ether chain is important since 5
activated less pronounced Ca2+ release than 4, as mentioned
above.[24]
Chemical modifications, such as cIDPR and cIDPRE, provided
relatively stable analogues.[23] Incubation of compounds 3-5 for 18
h with CD38 NADase did not result in any metabolism (Figure.
S2). Their stability suggested that the newly synthesized cADPR
mimics might be valuable probes to investigate cADPR mediated
Ca2+ signaling pathway.
cADPR regulates Ca2+ release directly or indirectly via RyRs.
Here we found that 3 and 4 also regulated similar Ca2+ signaling
pathway which involved Ca2+ release from ER via RyRs and
concomitant Ca2+ influx (Figure. 2, S4). The results indicated that
the major changing of structure, especially the substitution of
pyrophosphate moiety by triazole did not affect cADPR mimics to
motivate Ca2+ signaling via RyRs. Unexpectedly, 3 and 4 tended
to trigger Ca2+ influx, since the induced Ca2+ signaling still existed
in RyR2/RyR3 double knockdown Jurkat cells under the presence
of extracellular Ca2+ (Figure. 2c and 2f). Moreover, unlike cADPR
and other known cADPR agonists, the Ca2+ stimulation caused by
3 was inhibited by FCCP-induced mitochondrial depolarization
cIDPRE is a cell-permeable agonist, yet its activity to induce
Ca2+ release is still not strong enough to satisfy detailed biological
investigation. In present study, with the pyrophosphate linkage
changed to triazole moiety, 3 induced an immediate Ca2+ peak
and a subsequent plateau phase, indicating that triazole could
improve the potency of invoking Ca2+ release. Moreover, the Ca2+
inducing activity of 3 was much more pronounced than NPE-
cADPR (Figure. S3), a photolyzing caged cADPR analogue, or
cIDPRE (Figure. 1a). The result might be contributed by the
increased permeability of the neutral mimic. Unlike 3-induced
3
(Figure. 3a). Previous studies also proved that agonists of IP Rs
induced Ca2+ increase were inhibited by FCCP in
oligodendrocytes, HeLa and smooth muscle cells.[17, 25] Due to the
3
fact that RyRs and IP Rs are homogenous channels on ER, it
2
+
2+
Ca signaling, NPE-cADPR induced Ca signaling at 10 M was
found to decrease after a peak, and we reasoned that the small
quantity of cADPR released by NPE-cADPR after UV photolysis
was too slight to invoke the global Ca2+ increase and might be
immediately hydrolyzed in Jurkat cells.
follows that both might be related to mitochondrial activity.
2
+
Mitochondria are important Ca -storing organelles closely
2
+ [26]
associated with ER and can rapidly sequester Ca , yet little is
known about their effect on cADPR/RyRs/Ca2+ pathway. Further
research on cADPR mimics, RyRs and mitochondria is still
underway.
Figure 4. Superimposition of minimized conformations of cADPR and
compounds 3-5. cADPR is shown in light blue, compound 3 in green, compound
4
in purple and compound 5 in yellow.
Conclusions
In present study, we synthesized three novel neutral cADPR
mimics (3-5) and then explored their pharmacological activities in
Jurkat cells. The agonistic activities of cADPR mimics might result
from the consistence of conformations even if the structures were
simplified both in the nucleobase and pyrophosphate regions
(Figure. 4). Besides, the nucleobase mimics 4 and 5 showed that
the uridine modification of adenine decrease agonistic activity and
the purine ring is important for maintaining high agonistic activity.
Moreover, it showed that 3 and 4 invoked Ca2+ release via RyRs.
Highly polar phosphate groups of adenosine nucleotide-based
Figure 3. The requirement of mitochondria for compound 3-triggered Ca2+
releases. Compound 3 (10 μM) induced Ca2+ increase was inhibited by FCCP
(
2 μM) pretreatment in the absence of extracellular Ca2+ (a). Pretreatment of
2+
cells with GPN (50 μM) did not affect compound 3 (10 μM) induced Ca
increase in the absence of extracellular Ca2+ (b). Date are the mean ± S.D.,
n=3-5 (20-40 cells in each independent experiment). *, P < 0.05.
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1
second messengers - such as cADPR, NAADP and ADPR -
probably correspond to positively-charged or electron-poor
residues in their binding counterpart, an interaction often
presumed to be critical for their activity. These findings proved
that the cADPR mimics with the neutral modification to
pyrophosphate keep most of the same targets as cADPR and
cIDPRE in regulating the Ca2+ pathway. These findings provided
a new perspective for understanding the cADPR signaling
Synthesis
of
N -cyclic-ethoxylmethyl-inosine-5’-deoxy-5’-(4’”-
methyl-1’”,2’”,3’”-triazole)-2’,3’-O-isopropylidene ribose (3): To 12 (95
mg, 0.21 mmol) in THF (95 mL) was added DIPEA (79 μL, 0.460mmol).
The solution was degassed with argon (30 min), CuI (52.5 mg, 0.315
mmol) added and then stirred at 65C for 24 h. All solvents were
evaporated, and the residue was purified by silica gel column
chromatography eluting with PE: acetone (5:1 v/v) to afford compound 3
[20]
1
3
in 21% (20 mg)as a while amorphous solid. H NMR (400 MHz, CDCl ): δ
7.89 (s, 1H), 7.79 (s, 1H), 7.20 (s, 1H), 6.11 (s, 1H), 5.93 (s, 1H), 5.23 (s,
1H), 5.09 (s, 1H), 4.97 (s, 1H), 4.88 (s, 1H), 4.55 (s, 2H), 4.39 (s, 2H), 3.94
(s, 1H), 3.73 (s, 1H), 3.64 (s, 1H), 3.53 (s, 1H), 1.65 (s, 3H), 1.37 (s, 3H);
13C NMR (101 MHz, CDCl
25.2, 115.2, 88.8, 83.5, 83.0, 78.2, 74.8, 69.7, 69.3, 64.7, 48.9, 27.3, 25.7.
pathway. Considering the cell selectivity of cADPR mimics in
mobilizing calcium signaling,[27] the biological behaviors of these
neutral mimics in other cell systems are underway.
3
): δ 156.9, 148.6, 146.2, 144.3, 139.4, 129.5,
1
+
24 7 6
HRMS (m/z): [(M+H)] calcd for C19H N O , 446.1788; found, 446.1791.
Experimental Section
3
Synthesis of N -[(2”-propynyl)ethoxylethyl]-5’-azido-5’-deoxy-2’,3’-O-
isopropylidene uridine (17): 15[15] (111 mg, 0.544 mmol) was added
2
Agents and materials: NPE-cADPR[28], cIDPRE[29] and 8-Br-cADPR[30]
were synthesized as detailed previously. Other chemicals purchased from
Beijing J&K Chemical Ltd. and Shanghai Sigma Trading Co Ltd.. All
anhydrous reagents and solvents were obtained by dehydration according
dropwise to the solution of 14 (140mg, 0.454 mmol) and anhydrous K CO3
(141 mg, 1.02 mmol) in DMF (6 mL) and acetone (6 mL), and the resulting
mixture was refluxed at 55C for 4 h. The mixture was evaporated to
dryness, and the residue was applied to
chromatography eluting with PE: acetone (8:1 v/v) to yield 17 in 58% (114
mg) as a pale yellow oil. H NMR (400 MHz, CDCl ): δ 7.23 (d, J = 8.1 Hz,
1H), 5.76 (d, J = 8.1 Hz, 1H), 5.63 (d, J = 1.6 Hz, 1H), 4.97 (dd, J = 6.3,
1.6 Hz, 1H), 4.80 (dd, J = 6.3, 4.2 Hz, 1H), 4.23 (dd, J = 9.4, 4.9 Hz, 1H),
4.15 (dd, J = 10.7, 4.1 Hz, 4H), 3.71 (t, J = 5.9 Hz, 2H), 3.65 (s, 4H), 3.60
a silica gel column
2
to the standard methods before use. DCM was distilled from CaH . THF
was freshly distilled from sodium. Other reagents and solvents were
obtained locally and of analytical grade. Recombinant soluble mouse
CD38 and ADP-ribosyl cyclase from Aplysia californica were supplied by
Zhao, Y. J. (Shenzhen Graduate School of Peking University, China).
1
3
(
d, J = 5.2 Hz, 2H), 2.41 (t, J = 2.3 Hz, 1H), 1.55 (s, 3H), 1.34 (s, 3H); 13
NMR (101 MHz, CDCl ): δ 162.3, 150.5, 140.3, 114.8, 102.4, 95.6, 85.8,
84.4, 81.2, 79.6, 74.4, 69.8, 69.1, 67.6, 58.3, 52.2, 39.7, 27.1, 25.4.
C
Synthesis of 3-[2-(methoxymethoxy)ethoxy]-1-propyne (7): To the
solution of 6[12] (2 g, 20 mmol) and dimethoxymethane (4 mL, 45 mmol) in
3
2 5
DCM (4 mL) was added P O (1 g, 7 mmol) at 0C. After 24 h, DCM and
NaHCO
3
(sat. aq.) were added, and the organic layer dried with anhydrous
Synthesis of N -cyclic-ethoxylethyl-uridine-5’-deoxy-5’-(4’”-methyl-
3
Na SO
2
4
and evaporated. Then the residue was applied to a column of
1’”,2’”,3’”-triazole)-2’,3’-O-isopropylidene ribose (4): To 17 (170 mg,
0.391mmol) in THF (170 mL) was added DIPEA (150 μL, 0.870mmol). The
solution was degassed with argon (30 min), CuI (100 mg, 0.591 mmol)
added and then stirred at 65C for 24 h. All solvents were evaporated, and
the residue was purified by silica gel column chromatography eluting with
silica gel with PE: EtOAc (8:1 v/v) to yield 7 in 69% (1.98 g) as a colourless
oil. 1H NMR (400 MHz, CDCl
): δ 4.59 (s, 2H), 4.15 (d, J = 2.4 Hz, 2H),
.64 (s, 4H), 3.30 (s, 3H), 2.40 (t, J = 2.4 Hz, 1H); 13C NMR (101 MHz,
3
3
3
CDCl ): δ 96.5, 79.5, 74.6, 68.9, 66.5, 58.3, 55.1.
PE: acetone (5:1 v/v) to afford compound 4 in 16% (27 mg) as a while
1
Synthesis of 3-[2-(acetoxymethoxy)ethoxy]-1-propyne (8): To 7 (670
mg, 4.65 mmol) in acetic anhydride (1.1mL) was added boron
trifluoride−diethyl etherate (0.30 mL) at 0C, then the solution was stirred
amorphous solid. H NMR (400 MHz, CDCl
3
): δ 7.71 (s, 1H), 7.09 (d, J =
8.0 Hz, 1H), 5.75 (d, J = 8.0 Hz, 1H), 5.32 (dd, J = 6.3, 3.0 Hz, 1H), 5.24
(d, J = 3.0 Hz, 1H), 4.97 (dd, J = 14.3, 11.3 Hz, 1H), 4.85 – 4.73 (m, 2H),
4.76 – 4.56 (m, 4H), 3.92 – 3.89 (m, 2H), 3.72 – 3.51 (m, 5H), 1.56 (s, 3H),
for 2 h. The reaction mixture was diluted with DCM, washed with NaHCO
3
1.37 (s, 3H); 13C NMR (101 MHz, CDCl
(
sat. aq.) and dried with anhydrous Na SO . After evaporation the residue
2
4
3
) δ 162.5, 150.7, 146.2, 142.6,
was applied to a column of silica gel with PE: EtOAc (7:1 v/v) to yield 8 in
124.5, 114.5, 102.3, 101.8, 84.2, 82.6, 82.2, 71.3, 69.4, 67.8, 65.0, 50.7,
1
+
3
4
3
2
8% (2.76 g) as a colourless oil. H NMR (400 MHz, CDCl
.16 (s, 2H), 3.79-3.77 (m, 2H), 3.67-3.65 (s, 2H), 2.43 (s, 1H), 2.05 (s,
3
): δ 5.26 (s, 2H),
26 5 7
40.9, 27.2, 25.1. HRMS (m/z): [(M+H)] calcd for C19H N O , 436.1832;
found, 436.1835.
H);13C NMR (101 MHz, CDCl
0.9.
3
): δ 170.5, 89.2, 79.4, 74.7, 69.3, 68.7, 58.4,
3
Synthesis of N
isopropylidene uridine (18): 16 (109 mg, 0.54 mmol) was added
dropwise to the solution of 14 (140mg, 0.45 mmol) and anhydrous K CO
-[(2”-propynyl) pentyloxy]-5’-azido-5’-deoxy-2’,3’-O-
1
Synthesis of N -[(2”-propynyl) ethoxylmethyl]-5’-azido-5’-deoxy-2’,3’-
O-isopropylidene inosine (12): To 8 (145 mg, 0.841 mmol) in DCM (2mL)
was added TMSBr (0.25 mL) at 0C, then the solution was refluxed for 18
h. The residue was concentrated under reduced pressure to afford 3-[2-
2
3
(141 mg, 1.02 mmol) in DMF (6 mL) and acetone (6 mL), and the resulting
mixture was refluxed at 55C for 4 h. The mixture was evaporated to
dryness, and the residue was applied to silica gel column chromatography
(
Bromomethoxy)ethoxy]-1-propyne (9). Subsequently, 9 (140 mg, 0.730
mmol) was added to the solution of 11[14] (60 mg, 0.180 mmol) and DBU
0.2 mL, 1.51 mmol) in DCM (2 mL) at 0C. After being stirred for 4 h, the
eluting with PE: acetone (8:1 v/v) to yield 18 in 60% (117 mg) as a yellow
1
oil. H NMR (400 MHz, CDCl
3
): δ 7.21 (d, J = 8.0 Hz, 1H), 5.74 (d, J = 8.0
(
Hz, 1H) 5.61 (s, 1H), 4.95 (d, J = 6.4 Hz, 1H), 4.77 – 4.75 (m, 1H), 4.21 (d,
J = 4.4 Hz, 1H), 4.09 (s, 2H), 3.86 (t, J = 7.4 Hz, 2H), 3.58 (d, J = 4.9 Hz,
2H), 3.48 (t, J = 6.4 Hz, 2H), 2.39 (s, 1H), 1.59 (dd, J = 14.7, 7.1 Hz, 4H), ,
1.53 (s, 3H), 1.39 (dd, J = 14.7, 7.7 Hz, 2H), 1.32 (s, 3H); 13C NMR (101
solvent was evaporated and the residue was purified by silica gel column
chromatography with PE: acetone (5:1 v/v) to afford compound 12 in 68%
(
1
54 mg). H NMR (400 MHz, CDCl
3
): δ 8.13 (s, 1H), 7.91 (s, 1H), 6.05 (d,
J = 2.7 Hz, 1H), 5.55 (s, 2H), 5.24 (dd, J = 6.4, 2.7 Hz, 1H), 4.94 (dd, J =
.4, 3.4 Hz, 1H), 4.36 – 4.32 (m, 1H), 4.14 (d, J = 2.4 Hz, 2H), 3.86 – 3.81
m, 2H), 3.66 (dd, J = 5.4, 3.4 Hz, 2H), 3.58 (dd, J = 8.3, 5.4 Hz, 2H), 2.40
s, 1H), 1.60 (s, 3H), 1.37 (s, 3H); 13C NMR (101 MHz, CDCl
): δ 156.6,
47.9, 147.0, 139.0, 125.1, 115.1, 90.2, 84.9, 84.3, 81.6, 79.0, 75.0, 74.6,
9.3, 68.6, 58.1, 52.1, 27.4, 25.4.
3
MHz, CDCl ): δ 162.5, 150.6, 139.9, 114.6, 102.4, 95.8, 85.6, 84.4, 81.7,
80.0, 74.1, 69.9, 57.8, 52.3, 41.1, 29.1, 27.3, 27.1, 25.3, 23.5.
6
(
(
1
6
3
3
Synthesis of N -cyclic-pentyloxyl-uridine-5’-deoxy-5’-(4’”-methyl-
1’”,2’”,3’”-triazole)-2’,3’-O-isopropylidene ribose (5): To 18 (173 mg,
0.40 mmol) in THF (173 mL) was added DIPEA (150 μL, 0.87 mmol). The
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solution was degassed with argon (30 min), CuI (100 mg, 0.60 mmol)
added and then stirred at 65C for 24 h. All solvents were evaporated, and
the residue was purified by silica gel column chromatography eluting with
Statistics: All data were averaged from at least three independent
experiment. Moreover, the data represent mean ± S.D. and the Graph Pad
Prism 5.01 was used for statistical analyzing. Results comparison
analyzing was including two-tailed Student’s t-test for two groups.
Statistical significance was defined as p<0.05.
PE: acetone (5:1 v/v) to afford compound 5 in 15% (26 mg) as a while
1
amorphous solid. H NMR (400 MHz, DMSO-d
6
): δ 7.90 (d, J = 8.0 Hz, 1H),
7
4
1
1
2
.69 (s, 1H), 5.71 (d, J = 8.0 Hz, 1H), 5.61 (s, 1H), 5.22 (d, J = 6.0 Hz, 1H),
.81 – 4.72 (m, 2H), 4.64 (dd, J = 14.8, 6.0 Hz, 1H), 4.55 (d, J = 13.2 Hz,
H), 4.42 (d, J = 13.2 Hz, 1H), 4.26 (d, J = 6.8 Hz, 1H), 3.84 – 3.77 (m,
H), 3.64 – 3.58 (m, 1H), 3.45 – 3.40 (m, 2H), 1.52 (s, 3H), 1.45 – 1.40 (m,
H), 1.35– 1.31 (m, 2H), 1.30 (s, 3H), 1.22 – 1.20 (m, 2H); 13C NMR (101
Acknowledgements
MHz, DMSO-d
2.6, 82.4, 79.7, 68.6, 63.9, 49.4, 27.8, 27.5, 25.9, 25.8, 23.5; HRMS(m/z):
6
): δ 162.5, 150.4, 145.1, 144.0, 125.1, 113.8, 100.9, 96.1,
This work was supported by National Natural Science Foundation
of China (NSFC) 1673279 (to L.Z.) and 81573273 (to L-H.Z.).
8
+
[
28 5 6
(M+H)] calcd for C20H N O , 434.2040; found, 434.2038.
Keywords: Nucleotides • Signal transduction •cADPR mimics •
Metabolic stability: Compounds 3-5 (50 μM) were incubated with
recombinant soluble mouse CD38 (0.75 mg/mL) in buffer (20 mM HEPES,
RyRs
1
4 2 2 4
40 mM NaCl, 5 mM KCl, 1 mM MgSO , 1 mM CaCl , 1 mM NaH PO , 5
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Entry for the Table of Contents
Layout 2:
FULL PAPER
Xuan Wang, Xiaoyan Zhang, Kehui
Zhang, Jianxing Hu, Zhenming Liu,
Hongwei Jin*, Liangren Zhang*, and
Lihe Zhang
Page No. – Page No.
Calcium mobilizing behaviors of
neutral cyclic ADP-ribose mimics
which integrate the modifications of
nucleobase, northern ribose and
pyrophosphate
Mimics of cADPR play crucial role in investigating the molecular mechanism of
cADPR-mediated calcium signaling pathway. In this study, mimics of cADPR were
synthesized, in these, neutral triazole moiety, ether linkage and uridine were
introduced to substitute pyrophosphate, northern ribose and nucleobase,
respectively. The pharmacological activities in Jurkat cells indicated that these
neutral mimics motivated the similar Ca2+ signaling pathway as cADPR.
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