Total synthesis of cyclic ADP-carbocyclic-ribose, a stable mimic of Ca2+-mobilizing second messenger cyclic ADP-ribose

Article abstract of DOI:10.1021/ja010756d

The synthesis of cyclic ADP-carbocyclic-ribose (cADPcR, 4) designed as a stable mimic of cyclic ADP-ribose (cADPR, 1), a Ca²⁺-mobilizing second messenger, was achieved using as the key step a condensation reaction with the phenylthiophosphate-t

Full text of DOI:10.1021/ja010756d

8750
J. Am. Chem. Soc. 2001, 123, 8750-8759
Total Synthesis of Cyclic ADP-carbocyclic-ribose, a Stable Mimic of
Ca2+-Mobilizing Second Messenger Cyclic ADP-Ribose1
Satoshi Shuto,*,† Masayoshi Fukuoka,† Andrzej Manikowsky,† Yoshihito Ueno,†
Takashi Nakano,‡ Ritsu Kuroda,‡ Hideyo Kuroda,‡ and Akira Matsuda*,†
Contribution from the Graduate School of Pharmaceutical Sciences, Hokkaido UniVersity,
Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan, and Department of EnVironmental Biology and
Chemistry, Faculty of Science, Toyama UniVersity, 3190 Gofuku, Toyama, Toyama 930-8555, Japan
ReceiVed March 22, 2001
Abstract: The synthesis of cyclic ADP-carbocyclic-ribose (cADPcR, 4) designed as a stable mimic of cyclic
ADP-ribose (cADPR, 1), a Ca2+-mobilizing second messenger, was achieved using as the key step a
condensation reaction with the phenylthiophosphate-type substrate 14 to form an intramolecular pyrophosphate
linkage. The N-1-carbocyclic-ribosyladenosine derivative 16 was prepared via the condensation between the
imidazole nucleoside derivative 17, prepared from AICA-riboside (19), and the readily available optically
active carbocyclic amine 18. Compound 16 was then converted to the corresponding 5′′-phosphoryl-5′-
phenylthiophosphate derivatives 14. Treatment of 14 with AgNO3 in the presence of molecular sieves (3 Å)
in pyridine at room temperature gave the desired cyclization product 32 in 93% yield, and subsequent acidic
treatment provided the target cADPcR (4). This represents a general method for synthesizing biologically
1
important cyclic nucleotides of this type. H NMR analysis of cADPcR suggested that its conformation in
aqueous medium is similar to that of cADPR. cADPcR, unlike cADPR, was stable under neutral and acidic
conditions, where under basic conditions, it formed the Dimroth-rearranged N6-cyclized product 34. cADPcR
was also stable in rat brain membrane homogenate which has cADPR degradation activity. Furthermore, cADPcR
was resistant to the hydrolysis by CD38 cADPR hydrolase, while cADPR was rapidly hydrolyzed under the
same conditions. When cADPcR was injected into sea urchin eggs, it caused a significant release of Ca2+ in
the cells, an effect considerably stronger than that of cADPR. Thus, cADPcR was identified as a stable mimic
of cADPR.
Introduction
Cyclic ADP-ribose (cADPR, 1), a naturally occurring me-
tabolite of NAD+,2 has been shown to mobilize intracellular
Ca2+ in various cells, such as sea urchin eggs, pancreatic beta
cells, smooth muscle cells, cardiac myocytes, T-lymphocytes,
and cerebellar neurons, indicating that it is a general mediator
involved in Ca2+ signaling.3 The structure of cADPR had been
investigated4 and was recently confirmed by X-ray crystal-
lographic analysis as shown in Figure 1.4c
* Corresponding author. Satoshi Shuto. Telephone: 81-11-706-3229.
Fax: 81-11-706-4980. E-mail: shu@pharm.hokudai.ac.jp.
† Hokkaido University.
‡ Toyama University.
(1) This report constitutes Part 209 of Nucleosides and Nucleotides. Part
208, see: Ueno, Y.; Mikawa, M.; Hoshika, S.; Matsuda, A. Bioconjugate
Chem. 2001, 12, 635-642.
(2) Clapper, D. L.; Walseth, T. F.; Dargie, P. J.; Lee, H. C. J. Biol. Chem.
1987, 262, 9561-9568.
(3) (a) Galione, A. Science 1993, 259, 325-326. (b) Lee, H. C.; Galione,
A.; Walseth, T. F. Vitam. Horm. 1994, 48, 199-257. (c) Dousa, T. P.;
Chini, E. N.; Beers, K. W. Am J. Physol. 1996, 271, C1007-C1024. (d)
Lee, H. C. Physiol. Rev. 1997, 77, 1133-1164. (e) Lee, H. C. Cell. Biochem.
Biophys. 1998, 28, 1-17. (f) Galione, A.; Cui, Y.; Empson, R.; Iino, S.;
Wilson, H.; Terrar, D. Cell Biochem. Biophys. 1998, 28, 19-30. (g) Guse,
A. H. Cell Signal. 1999, 11, 309-316.
Figure 1. The structures of cADPR (1), cADPcR (4), and related
compounds.
(4) (a) Lee, C. H.; Walseth, T. F.; Bratt, G. T.; Hayes, R. N.; Clapper,
D. L. J. Biol. Chem. 1989, 264, 1608-1615. (b) Kim, H.; Jacobson, E. L.;
Jacobson, M. K. Biochem. Biophys. Res. Commun. 1993, 194, 1143-1147.
(c) Lee, H. C.; Aarhus, R.; Levitt, D. Nat. Struct. Biol. 1994, 1, 143-144.
(d) Gu, Q.-M.; Sih, C. J. J. Am. Chem. Soc. 1994, 116, 7481-7486. (e)
Wada, T.; Inageda, K.; Aritomo, K.; Tokita, K.; Nishina, H.; Takahashi,
K.; Katada, T.; Sekine, M. Nucleosides Nucleotides 1995, 14, 1301-1341.
In cells, cADPR is synthesized from NAD+ by ADP-
ribosylcyclase and acts as a second messenger; it is hydrolyzed
rapidly by cADPR hydrolase to give inactive ADP-ribose under
physiological conditions.3 cADPR is also known to be readily
hydrolyzed nonenzymatically at the unstable N-1-glycosidic
10.1021/ja010756d CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/18/2001

Cyclic ADP-Carbocyclic-Ribose
J. Am. Chem. Soc., Vol. 123, No. 36, 2001 8751
linkage of its adenine moiety to give ADP-ribose, even in neutral
aqueous solution.5 Although intensive studies of cADPR are
still needed because of its biological importance, the biological
as well as chemical instability of cADPR limits, to some extent,
further studies of its physiological role. Therefore, stable
analogues of cADPR exhibiting a Ca2+-mobilizing activity in
cells similar to that of cADPR are urgently required.
Scheme 1. Previous Synthesis of cIDPcR (8) and Its
8-Bromo Congener (9)
The synthesis of cADPR analogues and their biological effects
have been studied extensively.6,7 Potter and co-workers syn-
thesized cyclic aristeromycin-diphosphate-ribose (2) and showed
that it acted as a poorly hydrolyzable Ca2+-mobilizing mimic
of cADPR.6j However, 2 is hydrolyzed by enzymes, although
the rate is considerably slower than that of cADPR.6j More
recently, cyclic 3-deazaadenosine-diphosphate-ribose (3) was
synthesized by Walseth and co-workers as another analogue
resistant to hydrolysis.6p
We designed cyclic ADP-carbocyclic-ribose (cADPcR, 4) and
its inosine congener 5 (cIDP-carbocyclic-ribose, cIDPcR) as
stable mimics of cADPR,7 in which the oxygen atom in the
N-1-ribose ring of cADPR is replaced by a methylene group.
The mimics 4 and 5 should be resistant to both enzymatic and
chemical hydrolysis, since they have a chemically and biologi-
cally stable N-alkyl linkage instead of the unstable N-1-
glycosidic linkage of cADPR. These analogues preserve the
functional groups of cADPR except for this ring oxygen and
should have a conformation similar to that of cADPR. Therefore,
we expect that these analogues would effectively mobilize
intracellular Ca2+, as cADPR; therefore, they could be used as
pharmacological tools for further study of the mechanism of
cADPR-modulated Ca2+-signaling pathways.
cADPR and its analogues, including 2 and 3, have been
synthesized by enzymatic or chemo-enzymatic methods.6 ADP-
ribosyl cyclase from Aplysia californica mediates the intra-
molecular ribosylation of NAD+ and some modified NAD+
(prepared chemically or enzymatically) at the N-1-position of
the purine moiety, to yield cADPR or the corresponding
analogues.6 Although the specificity of ADP-ribosyl cyclase is
somewhat loose, the analogues obtained by this method are
limited by the substrate specificity of the enzyme.6o Furthermore,
even though ADP-ribosylcyclase catalyzes the cyclization of
the NAD+ analogues, in some cases the newly formed glycosidic
bond is attached to the N-7 nitrogen of the purine ring: e.g.,
the product of the enzymatic reaction of an inosine or guanosine
analogue of NAD+ is not the desired N-1-cyclized product, but
rather the N-7-cyclized product.6c,g
In the chemical synthesis of cADPR and its analogues, the
intramolecular condensation to form the pyrophosphate linkage
should be the key step; however forming such an intramolecular
pyrophosphate linkage has proved difficult for several groups
including ours,6o,7,9 thereby preventing completion of the
synthesis of target cADPR analogues. One of the first attempts
to prepare cADPR or its analogues by chemical intramolecular
condensation was reported by Gu and Sih.8 They tried to perform
a condensation between the two phosphate groups of N-1-
phosphoribosyl-AMP with EDC, but the yield of cADPR was
less than 1%.8 Later, Fortt and Potter attempted the synthesis
of an analogue of cIDPcR. However, the forming of the
intramolecular pyrophosphate linkage proved unsuccessful.9a
We investigated the intramolecular condensation of the N-1-
carbocyclic-ribosylinosine bisphosphate derivative 6 which
produced none of the desired cyclized product 8. However, we
found that cyclization of the bisphosphate 7 proceeded to give
the desired 9 when a bromo group was introduced at the
8-position probably by restricting the conformation of the
substrate to a product-like syn-form (Scheme 1a).7a,10 The
protecting groups were then removed to complete the synthesis
of cIDPcR (5).7a This was the first total synthesis of a cADPR-
related compound, but the over-all yield was low.7a More
recently, we have developed an efficient method for forming
the intramolecular pyrophosphate linkage by activation of
phenylthiophosphate type substrates, such as 10 or 11, with I2
or AgNO3 in the presence of molecular sieves (3 Å) in pyridine,
(5) Lee, H. C.; Aarhus, R. Biochim. Biophys. Acta 1993, 1164, 68-74.
(6) (a) Walseth, T. F.; Lee, H. C. Biochim. Biophys. Acta 1993, 1178,
235-242. (b) Lee, H. C.; Aarhus, R.; Walseth, T. F. Science 1993, 261,
352-355. (c) Graeff, R. M.; Walseth, T. J.; Fryxell, K.; Branton, W. D.;
Lee, H. C. J. Biol. Chem. 1994, 269, 30260-30267. (d) Zhang, F.-J.; Sih,
C. J. Bioorg. Med. Chem. Lett. 1995, 5, 1701-1706. (e) Zhang, F.-J.; Gu,
Q.-M.; Jing, P. C.; Sih, C. J. Bioorg. Med. Chem. Lett. 1995, 5, 2267-
2272. (f) Zhang, F.-J.; Yamada, S.; Gu, Q.-M.; Sih, C. J. Bioorg. Med.
Chem. Lett. 1996, 6, 1203-1208. (g) Zhang, F.-J.; Sih, C. J. Tetrahedron.
Lett. 1995, 63, 9289-9292. (h) Zhang, F.-J.; Sih, C. J. Bioorg. Med. Chem.
Lett. 1996, 6, 2311-2316. (i) Ashamu, G. A.; Galione, A.; Potter, B. V. L.
J. Chem. Soc. Chem. Commun. 1995, 1359-1356. (j) Bailey, B. C.; Fortt,
S. M.; Summerhill, R. J.; Galione, A.; Potter, B. V. L. FEBS Lett. 1996,
379, 227-230. (k) Bailey, V. C.; Sethi, J. K.; Fortt, S. M.; Galione, A.;
Potter, B. V. L. Chem. Biol. 1997, 4, 51-60. (l) Bailey, V. C.; Sethi, J. K.;
Galione, A.; Potter, B. V. L. J. Chem. Soc. Chem. Commun. 1997, 695-
696. (m) Sethi, J. K.; Empson, R. M.; Bailey, V. C.; Potter, B. V. L.;
Galione, A. J. Biol. Chem. 1997, 272, 16358-16363. (n) Ashamu, G. A.;
Sethi, J. K.; Galione, A.; Potter, B. V. L. Biochemistry 1997, 36, 9509-
9517. (o) Zhang, F.-J.; Gu, Q.-M.; Sih, C. J. Bioorg. Med. Chem. 1999, 7,
653-664. (p) Wong, L.; Aarhus, R.; Lee, H. C.; Walseth, T. F. Biochim.
Biophys. Acta 1999, 1472, 555-564.
(8) Gu, Q.-M.; Sih, C. J. J. Am. Chem. Soc. 1994, 116, 7481-7486.
(9) Synthetic approaches to carbocyclic analogues of cADP-ribose have
been published by other groups. Although the N-1-carbocyclic-inosine and
-adenosine structures have been constructed, formation of the intramolecular
pyrophosphate linkage has not been achieved. (a) Fortt, S.; Potter, B. V. L.
Tetrahedron Lett. 1997, 38, 5371-5374. (b) Hutchinson, E. J.; Taylor, B.
F.; Blackburn, G. M. J. Chem. Soc., Chem. Commun. 1997, 1859-1860.
(10) Although a predominance of anti- over syn-conformers is well-
known for natural nucleosides and their anologues, introducing a bulky
substituent, such as a bromo group, into the 8-position of purine nucleosides
restricts the conformation in a syn-form through steric repulsion with the
ribose moiety: Saenger, W. Principles of Nucleic Acid Structure; Springer-
Verlag: New York, 1983.
(7) (a) Shuto, S.; Shirato, M.; Sumita, Y.; Ueno, Y.; Matsuda, A. J. Org.
Chem. 1998, 63, 1986-1994. (b) Shuto, S.; Shirato, M.; Sumita, Y.; Ueno,
Y.; Matsuda, A. Tetrahedron Lett. 1998, 39, 7341-7344. (c) Fukuoka, M.;
Shuto, S.; Minakawa, N.; Ueno, Y.; Matsuda, A. Tetrahedron Lett. 1999,
40, 5361-5364. (d) Sumita, Y.; Shirato, M.; Ueno, Y.; Matsuda, A.; Shuto,
S. Nucleosides Nucleotides Nucleic Acids 2000, 19, 175-188. (e) Fukuoka,
M.; Shuto, S.; Minakawa, N.; Ueno, Y.; Matsuda, A. J. Org. Chem. 2000,
65, 5238-5248.

8752 J. Am. Chem. Soc., Vol. 123, No. 36, 2001
Scheme 2. Retrosynthetic Analysis of cADPcR (4)
Shuto et al.
Scheme 3
and have successfully synthesized the cIDPcR and its 8-bromo
congener (Scheme 1b).7c,e This method is very effective for
producing the desired cyclization products, even without the
conformational restriction of the substrate to a product-like syn-
form by the introduction of the 8-bromo substituent. The
8-unsubstituted cyclization product 12 and the 8-bromo product
13 were obtained in 81% and quantitative yields from the
corresponding substrates 10 and 11, respectively.
With these encouraging results in mind, we next tried to
synthesize cADPcR (4). In this report, we describe the synthesis
and chemical and biological properties of 4, a stable mimic of
cADPR.
Results and Discussion
Synthetic Plan. As described above, we first tried to
synthesize the inosine congener cIDPcR (5), since its synthesis
was thought to be easier than that of cADPcR (4). The charged
N-1-substituted moiety (pKa of cADPR is 8.34b), which was
expected to be unstable, especially under basic and nucleophilic
conditions, might prove to be troublesome. In the synthesis of
the target cADPcR, construction of the N-1-carbocyclic structure
and the condensation between the two phosphate moieties
forming an intramolecular pyrophosphate linkage should be the
two most important steps. Our plan for the synthesis is shown
in Scheme 2. Formation of the intramolecular pyrophosphate
linkage was planned to investigate by treating 5′-phenylthio-
phosphate 14 or its regioisomer 5′′- phenylthiophosphate 15 as
the substrate with AgNO3 or I2/molecular sieves (3 Å) as a
promoter. The phenylthiophosphates 14 and 15 would be
obtained by functional group transformations of N-1-carbocyclic-
ribosyladenosine derivative 16. The N-1-carbocyclic-ribosyl-
adenosine structure of 16 could be constructed from the imid-
azole nucleoside derivative 17 and the optically active carbo-
cyclic amine 18. Compounds 17 and 18 would be readily pre-
pared from 5-aminoimidazole-4-carboxamide riboside (AICAR,
19) and commercially available (1R)-(-)-2-azabicyclo[2.2.1]-
hept-5-en-3-one (20).9b
used it effectively for the synthesis of cIDPcR as described
above.7c,e
Construction of the N-1-Carbocyclic-Ribosyl-Adenosine
Structure. Development of an efficient method for constructing
the N-1-carbocyclic-ribosyladenosine structure was essential for
completing the synthesis of cADPcR (4). Recently, Blackburn
and co-workers reported that treatment of imidazole nucleoside
21 and carbocyclic amine 18 under basic conditions provided
N-1-carbocyclic-ribosyladenosine derivative 16 (Scheme 3).9b
Although their method was likely to be the most efficient one
for the construction of N-1-carbocyclic-ribosyladenosine struc-
ture known so far, the yield was insufficient (40%).
We attempted to improve their method using the N-benzyl-
imidazole derivative 22 as a model substrate, and the results
are shown in Scheme 4 and Table 1. In the condensation reaction
of Blackburn and co-workers,9b it may be that the Dimroth
rearrangement12 of the desired N-1-carbocyclic product 16
(11) (a) Nakagawa, I.; Konya, S.; Ohtani, S.; Hata, T. Synthesis 1980,
556-557. (b) Sekine, M.; Kamimura, T.; Hata, T. J. Chem. Soc., Perkin
Trans. 1 1985, 997-1000. (c) Sekine, M.; Nishiyama, S.; Kamimura, T.;
Osaki, Y.; Hata, T. Bull. Chem. Soc. Jpn. 1985, 58, 850-860. (d) Fukuoka,
K.; Suda, F.; Suzuki, R.; Ishikawa, M.; Takaku, H.; Hata, T. Nucleosides
Nucleotides 1994, 13, 1557-1567.
(12) (a) Brookers, P.; Lawley, P. D. J. Chem. Soc. 1960, 539-545. (b)
Ames, B. N.; Martin, R. G.; Garry, B. J. J. Biol. Chem. 1961, 236, 2019-
2026.
The key condensation reaction between a phenylthiophosphate
and a phosphate forming the pyrophosphate linkage promoted
by I2 or AgNO3 was originally developed by Hata and
co-workers.11 We recently improved the Hata’s reaction and

Cyclic ADP-Carbocyclic-Ribose
J. Am. Chem. Soc., Vol. 123, No. 36, 2001 8753
Scheme 4
Scheme 5a
a Reagents and conditions: a) HC(OMe)3, cat. CF3CO2H, reflux,
quant; b) 18, K2CO3, MeOH, rt, 83%; c) PSS, TPSCI, py, rt.
Table 1. Condensation Reactions between 18 and 22,a and Basic
Treatments of 23 and 24.
sine structure was efficiently constructed from the imidazole
nucleoside derivative 17 and the readily available optically active
carbocyclic amine 18. This should be a general method for
preparing N-1-carbon-substituted adenosine derivatives without
the occurrence of the Dimroth rearrangement.
yield (%)b
entry substrate
conditions
23
24
25
1
2
3
4
5
6
7
22 + 18 CF3CO2H/MeCN, reflux 73
trace
12
-
-
-
-
-
-
-
22 + 18 MeOH, rt
22 + 18 EtOH, rt
80
92
-
Introduction of a bis(phenylthio)phosphoryl group at the 5′′-
position of 16 to produce 27 was attempted (Scheme 5). When
16 was treated with a cyclohexylammonium S,S-diphenylphos-
phorodithioate (PSS)/2,4,6-triisopropylbenzenesulfonyl chloride
(TPSCl)/pyridine system,11 progress of the reaction was ob-
served by TLC. However the product 27 was too unstable to
be isolated; the spot likely to be for 27 disappeared during the
workup after the reaction. Therefore, we decided to forego
preparation of the 5′′-phenylthiophosphate-type substrate 15 via
27 and subsequently investigated the introduction of a bis-
(phenylthio)phosphoryl group at the 5′-positon of the ribose
moiety, as shown in Scheme 6. After protection of the
5′′-hydroxyl of 16 with a monomethoxytrityl (MMTr) group,
the 5′-O-TBS group of the product 28 was removed with TBAF
to give 29. Treatment of 29 with PSS/TPSCl in pyridine
successfully gave the 5′-bis(phenylthio)phosphate 30 in 66%
yield. Removal of the 5′′-O-MMTr group of 30 with aqueous
AcOH gave 31 in 77% yield. An unprotected phosphoryl group
was introduced at the resulting 5′′-primary hydroxyl of 31 by
Yoshikawa’s method with POCl3/(EtO)3PO,13 followed by
treating the product with H3PO2 and Et3N in pyridine,14
affording 5′-phenylthiophosphate 14, the substrate for the
intramolecular condensation reaction, in 75% yield as a triethyl-
ammonium salt after purification by a C18 column chromatog-
raphy.
23
K2CO3/EtOH, rt
98
22 + 18 K2CO3/MeCN, rt
22 + 18 K2CO3/MeOH, rt
trace 73
trace 87
24
1 M NaOH, reflux
-
-
quant
a
The reactions were performed using a slightly excess (1.1 equiv)
b
of 18 to 22. Isolated yield.
(Scheme 3) takes place, which produces N6-alkyladenine
products from N-1-alkyladenines under basic conditions, thereby
decreasing the yield. Consequently, we first treated a mixture
of 18 and 22 under acidic (entry 1) or neutral (entries 2 and 3)
conditions, and these reactions gave the coupled but not cyclized
product 23 in excellent yield. Since compound 23 could be an
intermediate for forming the desired N-1-carbocyclic product
24, we examined its pyrimidine ring-closure reaction and found
that treatment of 23 under mild basic conditions, namely with
K2CO3/EtOH at room temperature, quantitatively produced the
desired compound 24 (entry 4). The direct treatment of a mixture
of 18 and 22 with K2CO3 in MeCN or MeOH was next
performed to give 24 in 73 and 87% yield (entries 5 and 6).
We confirmed that the Dimroth rearrangement of 24, described
above, actually occurred to form the N6-carbocyclic product 25
quantitatively (Scheme 4), when 24 was treated under basic
conditions, such as heating it in 1 M NaOH (entry 7).
Synthesis of cADPcR (4). On the basis of the above results,
we next investigated the synthesis of cADPcR (4), which is
summarized in Schemes 5 and 6. A sugar-protected imidazole
nucleoside 26,9b prepared from AICAR (19), was heated in
HC(OMe)3 under reflux in the presence of a catalytic amount
of TFA to give the methoxymethylene derivative 17 quantita-
tively. Compound 17 was next subjected to the pyrimidine ring-
closure reaction with the carbocyclic amine 18 under the
conditions described above. When a mixture of 17 and 18 (1.2
equiv) was treated with K2CO3 in MeOH at room temperature,
the desired ring-closure product 16 was obtained in 83% yield
(Scheme 5). The N-1-carbocyclic-ribosyladenine structure of 16
was confirmed by its HMBC spectrum; a correlation between
the H-2 of the adenine and the C-1′′ of the cyclopentane ring
was observed. Thus, the desired N-1-carbocyclic-ribosyladeno-
The intramolecular condensation reaction of 14 under high
dilution conditions was next investigated (Scheme 6). When a
solution of 14 in pyridine was added slowly, using a syringe
pump, to a mixture of a large excess of AgNO3 and Et3N in the
presence of molecular sieves (3 Å) in pyridine at room
temperature,7c,e the desired cyclization product 32 was obtained
in 93% yield as a triethylammonium salt, after purification by
C18 column chromatography. Although a similar reaction with
I2 instead of AgNO3 as a promoter also produced the cyclization
product 32 as the major product, the yield lowered (50%) and
several byproducts were observed by HPLC analysis. Nucleo-
(13) Yoshikawa, M.; Kato, T.; Takenishi. T. Bull. Chem. Soc. Jpn. 1969,
42, 3505-3508.
(14) Hata, T.; Kamimura, T.; Urakami, K.; Kohno, K.; Sekine, M.;
Kumagai, I.; Shinozaki, K.; Miura, K. Chem. Lett. 1987, 117-120.

8754 J. Am. Chem. Soc., Vol. 123, No. 36, 2001
Shuto et al.
Scheme 6a
a Reagents and conditions: a) MMTrCI, pyridine, rt, 71%; b) TBAF, THF, AcOH, rt, quant; c) PSS, TPSCI, py, rt, 66%; d) aq 80% AcOH, rt,
77% e) 1) POCI3, (MeO)3PO, rt, 2) H3PO2, Et3N, pyridine, rt, 75% (2 steps); f) AgNO3, MS 3 Å, Et3N, py, rt, 93%; g) 60% HCO2H, rt, 88%.
philic attack by iodide ion, formed in the reaction, to the
positively charged electron-deficient purine moiety of 32 might
occur in this case to form the byproducts. The desired cyclic
pyrophosphate structure of 32 was confirmed by the following
data: (1) the molecular-ion peak corresponding to 32 was
observed at m/z 618 in the FAB mass spectrum; (2) its 31P NMR
spectrum showed two signals at -10.39 and -10.80 ppm, which
are typical chemical shifts for a pyrophosphate moiety, with a
coupling constant (J ) 15.3 Hz) similar to that of cADPR
(-9.92 and -10.67 ppm, J ) 14.6 Hz).4e Finally, the cyclic
Figure 2. NOE data of cADPcR (4) in D2O.
pyrophosphate 32 was treated with aqueous HCO2H at room
temperature to give the target cADP-carbocyclic-ribose (4) in
88% yield.
The results of this study as well as the previous synthesis of
the corresponding N-1-ribose moiety of cADPR, due to the
presence of the protons at the 6′′-position in cADPcR. Thus, a
very strong NOE was observed at the H-2 (14.5%), when the
H-6′′b was irradiated. Irradiation of the H-2 of cADPcR
produced a strong NOE at H-6′′b (7.2%) together with moderate
ones at both the H-2′′ (4.8%) and the H-3′′ (3.9%), revealing a
C6′′-endo envelop-like conformation of the carbocyclic moiety,
in which the C1′′-C2′′-C3′′-C4′′ unit is almost flat. This C6′′-
endo envelop-like conformation is also supported by a large
J1′′,6′′a value (10.2 Hz) and a rather small J1′′,6′′b value (2.9 Hz).
It has been proposed that the N-1-ribose moiety of cADPR
adopts a C1′′-C2′′-C3′′-C4′′ flat conformation in aqueous
solution, since the H-2-H-2′′ and the H-2-H-3′′ correlations with
a similar strength were observed in its ROESY spectrum.4e On
the other hand, the X-ray crystallographic analysis of cADPR
showed the C3′′-exo envelop conformation of the N-1-ribose
moiety.4c The J1′′,2′′, J2′′,3′′, and J3′′,4′′ values of cADPcR obtained
in this study are similar to the corresponding J values of
cADPR.4e Thus, it is likely that both the N-1-carbocyclic moiety
in cADPcR and the N-1-ribose moiety in cADPR adopt an
analogous C1′′-C2′′-C3′′-C4′′ flat/C6′′(O1′′)-endo envelop
conformation in aqueous solution.
cIDPcR (5)7c,e clearly demonstrate that the strategy using a
phenylthiophosphate-type substrate in the key intramolecular
condensation reaction forming the pyrophosphate linkage is very
efficient for the total syntheses of cADPR related compounds.
The Structure of cADPcR. The structure of cADPcR (4)
1
was fully confirmed by H, 13C, and 31P NMR, HMBC, NOE,
HR-FAB, and UV spectra. We investigated the conformation
of cADPcR and compared it with that of cADPR reported
previously.4c,e 1H NMR data of cADPcR in D2O, together with
those of cADPR,4e are summarized in S-4 (see Supporting
Information). It has been demonstrated that the ribose moiety
of the adenosine residue in cADPR adopts a C2′-endo confor-
1
mation by H NMR4e and X-ray crystallographic4c analyses.
Each coupling constant between the ribose protons of the
adenosine residue in cADPcR is similar to the corresponding
one in cADPR. These similar J values between cADPcR and
cADPR suggest that the ribose moiety of cADPcR also adopts
a C2′-endo conformation as cADPR does.4e
The NOE experiments on cADPcR demonstrated that the
adenosine residue is restricted in a syn-form around the
glycosidic linkage due to the cyclic structure of the molecule:
irradiation of the H-8 produced a strong NOE (14.4%) at the
H-1′ as shown in Figure 2. The similar syn-conformation of
the adenosine moiety of cADPR has been confirmed by X-ray
On the basis of these results, we concluded that the three-
dimensional structure of cADPcR in aqueous solution is
analogous to that of cADPR, as we had expected.
Chemical Stability of cADPcR. It has been suggested that
cADPcR is stable in aqueous media, since the final acidic
removal of the isopropylidene groups of 32 was successful in
furnishing cADPcR (4) in high yield as described above
1
1
crystallographic4c and H NMR4e studies. When the H NMR
technique is used, conformation of the carbocyclic moiety of
cADPcR should be analyzed more easily and more precise than

Cyclic ADP-Carbocyclic-Ribose
J. Am. Chem. Soc., Vol. 123, No. 36, 2001 8755
analogous to that of N6-methyladenosine derivatives,15 (5) a
correlation of the H-1′′ was not observed at the C-2 but at the
C-6 in the HMBC spectrum, supporting its N6-carbocyclic-
ribosyl structure.
The structure of cADPR was originally presented as the N6-
cyclic ribosyl compound 35 (Figure 3) by Lee and co-workers,4a
and later revised as N-1-cyclic regioisomer 1 by its X-ray
1
crystallographic,4c UV,4b,d and H NMR4e analyses. The N6-
cyclized regioisomer 35 of cADPR has not been synthesized;
Jacobson and co-workers reported that the alkaline treatment
of cADPR does not result in a Dimroth rearrangement but
instead in hydrolysis at the N-1-position to afford ADP-ribose
because of its unstable N-1-glycosidic linkage.4b Accordingly,
the Dimroth rearranged N6-carbocyclic product 34, which
corresponds to the carbocyclic congener of 35, may be of
biological importance.
Stability of cADPcR in Rat Brain Extract. The degradation
enzymes of cADPR (1) are known to be widely distributed in
brain extracts of mammals as well as in invertebrates.16 We
investigated whether cADPcR (4) is stable in brain extract
having cADPR degradation enzymes. Brain extract from rats
was prepared according to the previous method.16 cADPR was
first treated with the extract at 37 °C to be rapidly degraded, as
shown in S-2 (see Supporting Information). As expected, under
the same conditions, cADPcR was almost completely resistant
to degradation by the extract. After 120 min of treatment, about
90% of cADPR was degraded, whereas most of cADPcR
remained. Thus, it was suggested that cADPcR would be stable
under physiological conditions.
Resistance of cADPcR to the CD38 cADPR Hydrolase.
The cell-surface antigen CD38, a transmembrane glycoprotein,
is known to synthesize cADPR (1) from NAD+.17 It is also
known CD38 catalyzes not only the formation of cADPR but
also the hydrolysis of cyclic ADPR.17 Therefore, we planned
to investigate whether cADPcR (4) was resistant to the hy-
drolysis by CD38. The recombinant CD38 fused with thio-
redoxin (ThioHis-CD38), which involves the extracellular
domain of CD38, was expressed in Escherichia coli BL21(DE3)
and purified according to the previously reported method by
Katada and co-workers.18
The prepared extracellular domain of CD38 rapidly hydro-
lyzed cADPR, and ADP-ribose was the sole product observed
on HPLC (S-3, see Supporting Information), as previously
reported.18 However, when cADPcR was treated under the same
hydrolysis conditions, it was, not surprisingly, completely
resistant to the CD38 hydrolase as shown in S-3 (see Supporting
Information).
Ca2+-Mobilizing Induced by cADPcR in Sea Urchin Eggs.
Since the discovery of cADPR (1), sea urchin eggs have been
used for bioassay of cADPR and its analogues. We examined
whether cADPcR (4) could induce the [Ca2+]i increase when it
was injected into the unfertilized eggs of A. crassispina. Figure
4 shows representative time courses of the [Ca2+]i changes
induced by cADPR (a) or cADPcR (b). Injection of cADPR at
a final concentrations of 30 to 500 nM induced a small increase
in [Ca2+]i. Injection of cADPcR at a final concentration of 30
Figure 3. Structures of Dimroth rearrangement product 34 and related
compounds.
(Scheme 6). Stability of cADPcR (4) and cADPR (1) in aqueous
solution was examined in detail. The two compounds were
treated in acidic (pH 2.0), neutral (pH 7.0), and alkaline (pH
12.0) buffers at 37 °C, and the time courses were analyzed by
HPLC. The results are shown in S-1 (see Supporting Informa-
tion). As we hypothesized, cADPcR is very stable and com-
pletely resistant to hydrolysis under acidic and neutral condi-
tions, whereas cADPR rapidly decomposed with a t1/2 of 33.8
h at pH 2.0 and 60.5 h at pH 7.0. The product derived from
cADPR in neutral and acidic solutions was identified as ADP-
ribose by HPLC.
On the other hand, when cADPR and cADPcR were treated
under high alkaline conditions (pH12.0), both the compounds
decomposed with a t1/2 of 36.0 h (cADPR) and 37.1 h
(cADPcR). However, HPLC analyses suggested that the reaction
course of cADPcR in alkaline solution was different from that
of cADPR: only one product was formed from cADPcR by
alkaline treatment while the formation of at least three products
was observed by HPLC analysis during treatment of cADPR
under the same conditions.
The Dimroth Rearrangement of cADPcR: Formation of
N6-cyclic ADP-carbocyclic-Ribose. As described above, when
the N-1-carbocyclic-ribosyladenine derivative 24 was treated
with aqueous NaOH, the Dimroth rearrangement occurred to
give the corresponding N6-carbocyclic product 25 (Scheme 4).
Ames and co-workers reported that N-1-(phosphoribosyl)-
adenosine 5′-triphosphate underwent the Dimroth rearrangement
to form the corresponding N6-phosphoribosyl-product 33 (Figure
3).12b Therefore, it would be reasonable to assume that the
product of the alkaline treatment of cADPcR (4) should be the
Dimroth-rearranged N6-cyclic ADP-carbocyclic-ribose (34, Fig-
ure 3). We treated cADPcR with aqueous NaOH/KCl buffer
(pH 12) at 37 °C for 4 days on a preparative scale. After
purification by ion-exchange column chromatography, the N6-
product 34 was obtained in 70% yield as a triethylammonium
salt. The structure of 34 was confirmed by the following data:
(1) the molecular-ion peak was observed at m/z 538 in the FAB
mass spectrum; (2) two signals around -10 ppm in the 31P NMR
spectrum supported the cyclic pyrophosphate structure;4e (3) a
(15) Chang. C.-J.; Ashworth, D. J.; Chern, L-.J.; Gomes, D.; Lee, C.-
G.; Mou, P. W.; Narayan, R. Org. Magn. Reson. 1984, 22, 671-675.
(16) Lee, H. C.; Aarhus, R. Biochim. Biophys. Acta 1993, 1164, 68-74.
(17) (a) Howard, M.; Girimaldi, J. C.; Bazan, J. F.; Lund, F. E.; Santos-
Argumedo, L.; Parkhouse, R. M. E.; Walseth, T. F.; Lee, H. C. Science
1994, 262, 1056-1059. (b) Takasawa, S.; Tohgo, A.; Noguchi, N.; Kogura,
T.; Nata, K.; Sugimoto, T.; Yonekura, H.; Okamoto, H. J. Biol. Chem. 1994,
268, 26052-26054.
λ
max in the UV spectrum was observed at 271 nm, which is the
typical absorption for N6-alkyladenosine derivatives;4b (4) the
(18) Kukimoto, I.; Hoshino, S.; Kontani, K.; Inageda, K.; Nishina, H.;
Takahashi, K.; Katada, T. Eur. J. Biochem. 1996, 239, 177-182.
13C NMR chemical shift pattern of the base moiety was

8756 J. Am. Chem. Soc., Vol. 123, No. 36, 2001
Shuto et al.
suggest that cADPcR is a stable mimic of cADPR, as we
hypothesized.
Experimental Section
Chemical shifts are reported in ppm downfield from TMS (1H and
1
13C) or H3PO4 (31P). All of the H NMR assignments described were
in agreement with COSY spectra. Thin-layer chromatography was done
on Merck coated plate 60F254. Silica gel chromatography was done on
Merck silica gel 5715. Reactions were carried out under an argon
atmosphere.
1-Benzyl-4-cyano-5-[(methoxymethylene)amino]imidazole (22). A
mixture of 5-amino-1-benzyl-4-cyanoimidazole21 (1.03 g 5.22 mmol),
TFA (20 µL, 0.26 mmol), and trimethyl orthoformate (2.86 mL, 26.1
mmol) in CH3CN (30 mL) was stirred at 105 °C for 1 h. The mixture
was evaporated, and the residue was purified by column chromatog-
raphy (SiO2, 25% acetone in CHCl3) to give 22 (920 mg, 74%) as a
pale yellow oil: 1H NMR (DMSO-d6, 400 MHz) δ 8.42 (s, 1 H), 7.88
(s, 1 H), 7.25-7.38 (m, 5 H), 5.12 (s, 2 H), 3.88 (s, 3 H).
1-Benzyl-4-cyano-5-[[(1R,2S,3R,4R)-2,3-(isopropylidenedioxy)-4-
hydroxymethyl)cyclopentyl]aminomethyleneamino]imidazole (23;
Table 1, entry 3). A mixture of the optically active carbocyclic amine
18 (356 mg, 1.90 mmol) and 22 (415 mg, 1.73 mmol) in EtOH (9 mL)
was stirred at room temperature for 23 h. The precipitated solid was
filtered, washed by cold EtOH and Et2O and dried to give 23 (524 mg,
76%) as a white solid. The filtrate was evaporated, and the residue
was purified by column chromatography (SiO2, 5% MeOH in CHCl3)
to give further 23 (107 mg, 16%) as a white solid: mp (MeOH) 107-
109 °C: 1H NMR (DMSO-d6, 400 MHz) δ 8.14 (m, 1 H, NH,
exchangeable with D2O), 8.05 (d, 1 H, NdCH, J ) 3.3 Hz), 7.66 (s,
1 H, H-2), 7.24-7.30 (m, 5 H, C6H5), 5.04 (s, 2 H, N-CH2), 4.76 (t,
1 H, 5′-OH, J ) 5.0 Hz, exchangeable with D2O), 4,41 (m, 2 H, H-2′,
H-3′), 4.17 (m, 1 H, H-1′), 3.39 (t, 2 H, H-5′, J ) 5.8 Hz), 2.19-2.26
(m, 1 H, H-6′b), 2.09 (m, 1 H, H-4′), 1.44-1.51 (m, 1 H, H-6′a), 1.19,
1.37 (each s, each 3 H, isopropyl CH3); 13C NMR (DMSO-d6, 100
MHz) δ 152.6, 150.0, 137.1, 135.0, 128.5, 127.8, 127.6, 117.9, 110.9,
94.2, 84.8, 81.6, 62.0, 56.8, 46.4, 46.3, 32.5, 27.2, 24.8; HRMS (FAB,
positive) calcd for C21H26N5O3 (MH+) 396.2035, found 396.2030; UV
(MeOH) λmax 289 nm. Anal. Calcd for C21H25N5O3: C, 63.78; H, 6.37;
N, 17.71. Found: C, 63.79; H, 6.44; N, 17.80.
9-Benzyl-6-imino-1-[(1R,2S,3R,4R)-2,3-(isopropylidenedioxy)-4-
hydroxymethyl)cyclopentyl]purine (24). From 23 (Table 1, entry 4):
A mixture of 23 (500 mg, 1.26 mmol) and K2CO3 (9.0 mg, 76 µmol)
in EtOH (70 mL) was stirred at room temperature for 3 h. The mixture
was evaporated, and the residual yellow oil was purified by column
chromatography (SiO2, 14% MeOH in CHCl3) to give 24 (487 mg,
98%) as a white solid. From 18 and 22 (Table 1, entry 6): A mixture
of 22 (110 mg, 0.46 mmol), 18 (94 mg, 0.51 mmol), and K2CO3 (3.0
mg, 23 µmol) in MeOH (3 mL) was stirred at room temperature for 18
h. The resulting solution was evaporated, and the residue was purified
by column chromatography (SiO2, 12% MeOH in CH2Cl2) to give 24
(157 mg, 87%) as a white solid: 1H NMR (DMSO-d6, 400 MHz) δ
8.13 (s, 1, H-2), 8.04 (s, 1 H, H-8), 7.35-7.26 (m, 5 H, C6H5), 7.05
(br s, 1 H, NH, exchangeable with D2O), 5.28 (s, 2 H, N-CH2), 5.14
(m, 1 H, H-1′), 5.06 (t, 1 H, H-2′, J ) 6.1 Hz), 4.73 (br s, 1 H, 5′-OH,
exchangeable with D2O), 4.47 (m, 1 H, H-3′), 3.50 (m, 1 H, H-5′b),
3.45 (m, 1 H, H-5′a), 2.03-2.17 (m, 3 H, H-4′, H-6′), 1.20, 1.44 (each
s, each 3 H, isopropyl CH3); 13C NMR (DMSO-d6, 100 MHz) δ 153.9,
146.8, 140.8, 139.1, 136.8, 128.6, 127.6, 127.3, 122.1, 111.9, 82.2,
81.0, 62.3, 61.6, 46.3, 46.0, 33.1, 27.6, 25.3; HRMS (FAB, positive)
calcd for C21H26N5O3 (MH+) 396.2035, found 396.2039; UV (MeOH)
Figure 4. Time courses of the [Ca2+]i changes induced by cADPR (1,
a) and cADPcR (4, b) injected into eggs of A. crassispina: (b) 30
nM, (9) 200 nM, (2) 500 nM.
nM induced a [Ca2+]i increase comparable to the peak value of
the 500 nM cADPR-induced Ca2+ transient. Injection of
cADPcR at a final concentration of 200 nM induced the peak
value of [Ca2+]i at about 3.3 µM, which was more than 10 times
higher that induced by cADPR. These results clearly show a
potent Ca2+-mobilizing activity of cADPcR in comparison to
that of cADPR.19 This difference seems to be caused by the
fact that cADPcR is much more biologically stable than cADPR,
as described above.20
Conclusions
We have successfully synthesized cADPcR (4) designed as
a stable mimic of cADPR (1). The N-1-carbocyclic adenosine
structure was efficiently constructed via condensation between
an imidazole nucleoside derivative 17, prepared from AICA-
riboside (19), and a readily available optically active carbocyclic
amine 18. The key intramolecular condensation reaction of the
phenylthiophosphate-type substrates 14 proceeded with AgNO3/
molecular sieves (3 Å) to give the cyclization product 32 in
high yield. This method would be applicable to the synthesis
of other cADPR analogues and may be an entry to a general
method for synthesizing biologically important cyclic nucleo-
tides of this type. cADPcR was shown to be resistant to chemical
and biochemical hydrolysis. It released Ca2+ in sea urchin eggs,
and the activity was stronger than that of cADPR. These results
λ
max 263 nm, sh 270. Anal. Calcd for C21H25N5O3‚0.3 H2O: C, 62.92;
H, 6.44; N, 17.47. Found: C, 62.92; H, 6.54; N, 17.45.
9-Benzyl-N6-[[(1R,2S,3R,4R)-2,3-(isopropylidenedioxy)-4-hydroxy-
methyl]-cyclopentyl]adenine (25; Table 1, entry 7). A suspension
of 24 (150 mg, 0.38 mmol) in 1 M NaOH (2 mL) was heated under
reflux for 1 h, and then MeOH (1 mL) was added. The resulting solution
was extracted with CHCl3 (three times), and the organic layer was
washed with brine, dried (Na2SO4), and evaporated. The residue was
(19) Preliminary experiments showed that cADPcR (4) also induced
[Ca2+]i increases in patch-clamped NG108-15 neuronal cells: Hashii, M.;
Higashida, H. unpublished results.
(20) Although cADPR synthesized from NAD+ by ADP-ribosylcyclase
might be very rapidly hydrolyzed in cells, e.g., within 1 min, because of its
role as a second messenger (see ref 3), the reaction conditions of cADPR
hydrolysis by the brain extract or CD38 in this study were selected to be
suitable for the HPLC assay (t1/2 ) 15-40 min).
(21) Hosmane, R. S.; Lim, B. B.; Burnett, F. M. J. Org. Chem. 1988,
53, 382-386.

Cyclic ADP-Carbocyclic-Ribose
J. Am. Chem. Soc., Vol. 123, No. 36, 2001 8757
purified by column chromatography (SiO2, 5% MeOH in CHCl3) to
give 25 (148 mg, 98%) as a white solid: 1H NMR (DMSO-d6, 400
MHz) δ 8.26 (s, 1 H, H-8), 8.23 (s, 1 H, H-2), 7.92 (br s, 1 H, NH,
exchangeable with D2O), 7.34-7.24 (m, 5 H, C6H5), 5.37 (s, 2 H,
N-CH2), 5.00 (br s, 1 H, 5′-OH, exchangeable with D2O), 4.60 (m, 1
H, H-1′), 4.54 (dd, 1 H, H-2′, J ) 3.2, 5.9 Hz), 4.44 (m, 1 H, H-3′),
3.47 (m, 2 H, H-5′), 2.28 (m, 1 H, H-6′a), 2.13 (m, 1 H, H-4′), 1.53
(m, 1 H, H-6′b), 1.39, 1.19 (each s, each 3 H, isopropyl CH3); 13C
NMR 153.9, 152.5, 148.9, 140.7, 137.1, 128.6, 127.4, 118.9, 110.6,
85.1, 82.0, 62.5, 55.9, 46.4, 46.1, 33.5, 27.2, 24.8; FAB-MS calcd for
C21H26N5O3 (MH+) 396.2035, found 396.2053; UV (MeOH) λmax 270
nm. Anal. Calcd for C21H25N5O3: C, 63.78; H, 6.37; N, 17.71. Found:
C, 63.81; H, 6.58; N, 17.50.
H, H-1′′), 4.89 (dd, 1 H, H-3′, J3′,2′ ) 6.1, J3′,4′ ) 2.3 Hz), 4.56 (m, 1
H, H-3′′), 4.37 (m, 1 H, H-4′), 3.84 (dd, 1 H, H-5′a, J5′a,4′ ) 4.0, J5′a,5′b
) 11.2 Hz), 3.79 (s, 3 H, OCH3), 3.76 (m, 1 H, H-5′b), 3.35 (dd, 1 H,
H-5′′a, J5′′a,4′′ ) 3.2, J5′′a,5′′b ) 8.5 Hz), 3.18 (dd, 1 H, H-5′′b, J5′′b,4′′
)
5.4, J5′′b,5′′a ) 8.5 Hz), 2.46-2.39 (m, 3 H, H-4′′, H-6′′), 1.62, 1.53,
1.39, 1.28 (each s, each 3 H, isopropyl CH3), 0.88 (s, 9 H, tert-butyl),
0.06, 0.05 (each s, each 3 H, dimethyl); 13C NMR (CDCl3, 67.8 MHz)
δ 158.5, 154.2, 146.3, 144.6, 144.5, 140.4, 136.6, 135.7, 130.3, 128.4,
127.7, 126.8, 124.0, 114.1, 113.0, 91.0, 87.0, 86.1, 85.2, 82.8, 81.6,
81.3, 64.2, 63.6, 63.4, 55.2, 45.0, 33.6, 27.7, 27.2, 25.9, 25.3, 18.3,
-5.4, -5.5; HRMS (FAB, positive) calcd for C48H62N5O8Si 864.4367
(MH+), found 864.4384; UV (MeOH) λmax 261 nm, sh 297 nm. Anal.
Calcd for C48H61N5O8Si: C, 66.72; H, 7.12; N, 8.10. Found C, 66.65;
H, 7.13; N, 7.97.
5-[(Methoxymethylene)amino]-1-[5-O-(tert-butyldimethylsilyl)-
2,3-O-(isopropylidene)-â-D-ribofuranosyl]imidazole-4-nitrile (17).
Compound 17 (1.35 g, quant) was obtained from 5-amino-1-[5′-O-(tert-
butyldimethylsilyl)-2,3-O-(isopropylidene)-â-D-ribofuranosyl]imidazole-
4-nitrile (26,9b 1.18 g, 3.0 mmol) as described for the synthesis of 22
after purification by column chromatography (SiO2, 25% EtOAc in
hexane) as a yellow oil: 1H NMR (CDCl3, 500 MHz) δ 8.39 (s, 1 H,
NdCH), 7.64 (s, 1 H, H-2), 5.84 (d, 1 H, H-1′, J1′,2′ ) 2.9 Hz), 4.78
N-1-[(1R,2S,3R,4R)-2,3-(Isopropylidenedioxy)-4-[(5-monometh-
oxytrityl)oxymethyl]cyclopentyl]-2′,3′-O-isopropylideneadenosine (29).
A mixture of 28 (1.74 g, 2.0 mmol), TBAF (1.0 M in THF, 22.6 mL,
22.6 mmol), and AcOH (657 µL, 10.4 mmol) in THF (7 mL) was stirred
at room temperature for 1 h. The mixture was evaporated, and the
residue was purified by column chromatography (SiO2, 3.5% MeOH
in CHCl3) to give 29 (1.52 g, quant) as a white form: 1H NMR (CDCl3,
500 MHz) δ 7.71 (s, 1 H, H-8), 7.62 (s, 1 H, H-2), 7.44-6.81 (m, 14
H, Ar-H), 5.76 (d, 1 H, H-1′, J1′,2′ ) 3.6 Hz), 5.49 (br s, 1 H, 5′-OH),
5.10 (dd, 1 H, H-2′′, J2′′,1′′ ) 5.1, J2′′,3′′ ) 6.8 Hz), 5.04 (m, 2 H, H-2′,
(dd, 1 H, H-3′, J3′,2′ ) 6.0, J3′,4′ ) 1.9 Hz), 4.74 (dd, 1 H, H-2′, J2′,1′
)
2.9, J2′,3′ ) 6.0 Hz), 4.38 (ddd, 1 H, H-4′, J4′,3′ ) 1.9, J4′,5′a ) 2.5, J4′,5′b
) 2.9 Hz), 3.95 (s, 3 H, OCH3), 3.86 (dd, 1 H, H-5′a, J5′a,4′ ) 2.5,
J5′a,5′b ) 11.5 Hz), 3.77 (dd, 1 H, H-5′b, J5′b,4′ ) 2.9, J5′b,5′a ) 11.5 Hz),
1.56, 1.34 (each s, each 3 H, isopropyl CH3), 0.86 (s, 9H, tert-butyl),
0.06, 0.06 (each s, each 3H, dimethyl); 13C NMR (CDCl3, 67.8 MHz)
δ 159.9, 143.4, 133.5, 115.7, 113.8, 99.2, 91.4, 86.5, 86.1, 81.0, 63.5,
54.6, 27.2, 25.8, 25.3, 18.3, -5.5, -5.7; HRMS (FAB, positive) calcd
for C20H33N4O5Si 437.2220 (MH+), found 437.2195; UV (MeOH) λmax
273 nm. Anal. Calcd for C20H32N4O5Si: C, 55.02; H, 7.39; N, 12.83.
Found C, 54.86; H, 7.43; N, 12.70.
H-3′), 4.97 (m, 1 H, H-1′′), 4.53 (dd, 1 H, H-3′′, J3′′,2′′ ) 5.8, J3′′,4′′
)
2.7 Hz), 4.37 (m, 1 H, H-4′), 3.92 (dd, 1 H, H-5′a, J5′a,4′ ) 1.0, J5′a,5′b
) 12.5 Hz), 3.80 (s, 3 H, OCH3), 3.77 (m, 1 H, H-5′b), 3.34 (dd, 1 H,
H-5′′a, J5′′a,4′′ ) 3.5, J5′′a,5′′b ) 8.8 Hz), 3.17 (dd, 1 H, H-5′′b, J5′′b,4′′
)
5.8, J5′′b,5′′a ) 8.8 Hz), 2.42 (m, 3 H, H-4′′, H-6′′), 1.63, 1.53, 1.37,
1.27 (each s, each 3 H, isopropyl CH3); 13C NMR (CDCl3, 67.8 MHz)
δ 158.5, 153.8, 146.4, 144.5, 144.5, 139.3, 138.0, 135.7, 130.3, 128.4,
127.7, 126.8, 125.4, 114.2, 113.2, 113.0, 93.8, 86.1, 85.8, 83.6, 82.4,
81.5, 81.3, 64.2, 63.2, 55.2, 44.7, 33.6, 30.8, 27.7, 27.5, 25.3, 25.2;
HRMS (FAB, positive) calcd for C42H48N5O8 750.3503 (MH+), found
750.3509; UV (MeOH) λmax 261 nm, sh 300 nm. Anal. Calcd for
C42H47N5O8‚0.5H2O: C, 66.48; H, 6.38; N, 9.23. Found C, 66.50; H,
6.44; N, 8.98.
N-1-[(1R,2S,3R,4R)-2,3-(Isopropylidenedioxy)-4-(hydroxymeth-
yl)cyclopentyl]-5′-O-(tert-butyldimethylsilyl)-2′,3′-O-isopropylidene-
adenosine (16). A mixture of 17 (2.6 g, 6.0 mmol), 18 (1.35 g, 7.2
mmol), and K2CO3 (41 mg, 0.3 mmol) in MeOH (60 mL) was stirred
at room temperature for 4 h. The mixture was evaporated, and the
residue was partitioned between EtOAc and H2O. The organic layer
was washed with brine, dried (Na2SO4), and evaporated. The residue
was purified by column chromatography (SiO2, 6% MeOH in EtOAc)
to give 16 (2.94 g, 83%) as a white form: 1H NMR (CDCl3, 500 MHz)
δ 7.79 (s, 1 H, H-8), 7.66 (s, 1 H, H-2), 7.19 (br s, 1 H, NH), 5.99 (d,
N-1-[(1R,2S,3R,4R)-2,3-(Isopropylidenedioxy)-4-[(5-monometh-
oxytrityl)oxymethyl]cyclopentyl]-5′-O-[bis(phenylthio)phosphoryl]-
2′,3′-O-isopropylideneadenosine (30). After stirring a mixture of PSS
(2.19 g, 5.7 mmol) and TPSCl (1.74 g, 5.7 mmol) in pyridine (15 mL)
at room temperature for 1.0 h, 29 (1.44 g, 1.9 mmol) was added, and
the resulting mixture was stirred at room temperature for 1.0 h. The
mixture was evaporated, and the residue was partitioned between EtOAc
and H2O. The organic layer was washed with brine, dried (Na2SO4),
and evaporated. The residue was purified by column chromatography
(SiO2, 2% MeOH in CHCl3) to give 30 (1.27 g, 66%) as a white form:
1H NMR (CDCl3, 500 MHz) δ 7.66 (s, 1 H, H-8), 7.64 (s, 1 H, H-2),
7.51-6.81 (m, 24 H, Ar-H), 5.97 (d, 1 H, H-1′, J1′,2′ ) 2.4 Hz), 5.28
(br s, 1 H, NH), 5.13 (dd, 1 H, H-2′, J2′,1′ ) 2.4, J2′,3′ ) 6.3 Hz), 5.11
(m, 1 H, H-2′′), 4.93 (m, 1 H, H-1′′), 4.92 (dd, 1 H, H-3′, J3′,2′ ) 6.3,
J3′,4′ ) 2.8 Hz), 4.53 (m, 1 H, H-3′′), 4.43-4.36 (m, 3 H, H-4′, H-5′),
3.79 (s, 3 H, OCH3), 3.79 (m, 1 H, H-5′′a), 3.73 (m, 1 H, H-5′′b), 2.41
(m, 3 H, H-4′′, H-6′′), 1.60, 1.52 1.36, 1.25 (each s, each 3 H, isopropyl
CH3); 13C NMR (CDCl3, 67.8 MHz) δ 158.5, 154.1, 146.6, 144.6, 144.5,
137.3, 135.7, 135.3, 135.2, 135.1, 135.1, 130.3, 129.6, 129.4, 128.4,
127.7, 126.7, 125.9, 124.4, 114.6, 113.0, 90.7, 86.1, 84.9, 84.8, 84.4,
82.6, 81.6, 81.1, 66.4, 66.3, 64.3, 64.1, 55.2, 44.9, 33.5, 27.7, 27.1,
1 H, H-1′, J1′,2′ ) 2.7 Hz), 5.31 (dd, 1 H, H-2′′, J2′′,1′′ ) 5.2, J2′′,3′′
)
5.8 Hz), 5.06 (dd, 1 H, H-2′, J2′,1′ ) 2.7, J2′,3′ ) 6.2 Hz), 4.86 (dd, 1 H,
H-3′, J3′,2′ ) 6.2, J3′,4′ ) 2.6 Hz), 4.74 (dd, 1 H, H-3′′, J3′′,2′′ ) 5.8,
J3′′,4′′ ) 2.7 Hz), 4.57 (ddd, 1 H, H-1′′, J1′′,2′′ ) 5.2, J1′′,6′′a ) 9.7, J1′′,6′′b
) 9.6 Hz), 4.35 (ddd, 1 H, H-4′, J4′,3′ ) 2.6, J4′,5′a ) 3.9, J4′,5′b ) 4.1
Hz), 3.79 (dd, 1 H, H-5′a, J5′a,4′ ) 3.9, J5′a,5′b ) 11.2 Hz), 3.79 (dd, 1
H, H-5′′a, J5′′a,4′′ ) 3.9, J5′′a,5′′b ) 10.8 Hz), 3.74 (dd, 1 H, H-5′b, J5′b,4′
) 4.1, J5′b,5′a ) 11.2 Hz), 3.73 (dd, 1 H, H-5′′b, J5′′b,4′′ ) 4.1, J5′′b,5′′a
)
10.8 Hz), 2.57 (m, 1 H, H-6′′a), 2.52 (m, 1 H, H-4′′), 2.45 (m, 1 H,
H-6′′b), 1.58, 1.53, 1.36, 1.29 (each s, each 3 H, isopropyl CH3), 0.84
(s, 9 H, tert-butyl), 0.03, 0.02 (each s, each 3 H, dimethyl); NOE
(CDCl3, 400 MHz) irradiated H-2, observed H-1′′ (17.9%); 13C NMR
(CDCl3, 67.8 MHz) δ 153.8, 147.5, 140.7, 137.1, 124.0, 114.1, 111.8,
91.1, 87.0, 85.2, 83.5, 82.3, 81.2, 70.1, 64.6, 63.4, 45.0, 30.5, 28.0,
27.2, 25.8, 25.3, 18.3, -5.5, -5.6; HRMS (FAB, positive) calcd for
C28H46N5O7Si 592.3166 (MH+), found 592.3179; UV (MeOH) λmax 261
nm, sh 293 nm. Anal. Calcd for C28H45N5O7Si: C, 56.83; H, 7.66; N,
11.83. Found C, 56.65; H, 7.56; N, 11.83.
1
25.3, 25.3; 31P NMR (CDCl3, 202 MHz, decoupled with H) δ 51.0;
HRMS (FAB, positive) calcd for C54H57N5O9PS2 1014.3335 (MH+),
found 1014.3320; UV (MeOH) λmax 260 nm, sh 296 nm. Anal. Calcd
for C54H56N5O9PS2‚0.5H2O: C, 63.39; H, 5.62; N, 6.84. Found C, 63.32;
H, 5.69; N, 6.54.
N-1-[(1R,2S,3R,4R)-2,3-(Isopropylidenedioxy)-4-[(5-monometh-
oxytrityl)oxymethyl]cyclopentyl]-5′-O-(tert-butyldimethylsilyl)-2′,3′-
O-isopropylideneadenosine (28). A mixture of 16 (1.78 g, 3.0 mmol)
and MMTrCl (1.85 g, 6.0 mmol) in pyridine (20 mL) was stirred at
room temperature for 1.5 h. The mixture was evaporated, and the residue
was partitioned between EtOAc and H2O. The organic layer was washed
with brine, dried (Na2SO4), and evaporated. The residue was purified
by column chromatography (SiO2, 60% EtOAc in hexane) to give 28
(1.85 g, 71%) as a white form: 1H NMR (CDCl3, 500 MHz) δ 7.79 (s,
1 H, H-8), 7.70 (s, 1 H, H-2), 7.44-6.81 (m, 14 H, Ar-H), 6.02 (d, 1
H, H-1′, J1′,2′ ) 2.3 Hz), 5.11-5.08 (m, 2 H, H-2′, H-2′′), 5.02 (m, 1
N-1-[(1R,2S,3R,4R)-2,3-(Isopropylidenedioxy)-4-(hydroxy-
methyl)cyclopentyl]-5′-O-[bis(phenylthio)phosphoryl]-2′,3′-O-iso-
propylideneadenosine (31). A solution of 30 (1.19 g, 1.17 mmol) in
80% aqueous AcOH (15 mL) was stirred at room temperature for 6 h.
The resulting mixture was evaporated, and the residue was partitioned
between EtOAc and aqueous saturated NaHCO3. The organic layer was
washed with H2O and then brine, dried (Na2SO4), and evaporated. The
residue was purified by column chromatography (SiO2, 3% MeOH in

8758 J. Am. Chem. Soc., Vol. 123, No. 36, 2001
Shuto et al.
CHCl3) to give 31 (665 mg, 77%) as a white form: 1H NMR (CDCl3,
500 MHz) δ 7.68 (br s, 2 H, H-2, H-8), 7.64-7.22 (m, 10 H, Ar-H),
5.98 (d, 1 H, H-1′, J1′,2′ ) 1.9 Hz), 5.31 (m, 1 H, H-2′′), 5.13 (dd, 1 H,
H-2′, J2′,1′ ) 1.9, J2′,3′ ) 6.1 Hz), 4.93 (m, 1 H, H-3′′), 4.73 (dd, 1 H,
H-3′, J3′,2′ ) 6.1, J3′,4′ ) 2.2 Hz), 4.53 (m, 1 H, H-1′′), 4.44-4.37 (m,
3 H, H-4′, H-5′), 3.78 (dd, 1 H, H-5′′a, J5′′a,4′′ ) 5.0, J5′′a,5′′b ) 10.6
Hz), 3.71 (m, 1 H, H-5′′b), 2.56 (m, 2 H, H-4′′, H-6′′a), 2.45 (m, 1 H,
H-6′′b), 1.60, 1.56 1.36, 1.31 (each s, each 3 H, isopropyl CH3); 13C
NMR (CDCl3, 67.8 MHz) δ 153.6, 147.6, 140.6, 137.9, 135.3, 135.2,
135.1, 135.0, 129.7, 129.6, 129.6, 129.4, 125.9, 125.8, 124.3, 114.6,
111.8, 90.8, 85.0, 84.9, 84.4, 83.4, 82.3, 81.2, 70.3, 66.4, 66.3, 64.6,
44.9, 30.3, 28.0, 27.1, 25.2; 31P NMR (CDCl3, 202 MHz, decoupled
with 1H) δ 51.1; HRMS (FAB, positive) calcd for C34H41N5O8PS2
742.2134 (MH+), found 742.2128; UV (MeOH) λmax 254 nm, sh 299
nm. Anal. Calcd for C34H40N5O8PS2‚0.5H2O: C, 54.39; H, 5.50; N,
9.33. Found C, 54.37; H, 5.48; N, 9.44.
raphy (1.1 cm × 11 cm, eluted with 20% aqueous CH3CN) to give 32
(7.7 mg, 10.5 µmol, 131 OD260 units, 93%) as a triethylammonium
salt: 1H NMR (D2O, 500 MHz) δ 8.77 (s, 1 H, H-2), 8.42 (s, 1 H,
H-8), 6.40 (br s, 1 H, H-1′), 5.81 (d, 1 H, H-2′, J2′,3′ ) 6.0 Hz), 5.02
(dd, 1 H, H-3′, J3′,2′ ) 6.0, J3′,4′ ) 2.4 Hz), 4.89-4.86 (m, 3 H, H-1′′,
H-2′′, H-3′′), 4.61 (m, 1 H, H-4′), 4.20-4.14 (m, 2 H, H-5′), 4.06 (m,
1 H, H-5′′a), 3.97 (m, 1 H, H-5′′b), 3.20 (q, 6 H, -CH2N, J ) 7.3
Hz), 3.14 (m, 1 H, H-6′′a), 2.91 (m, 1 H, H-4′′), 2.80 (m, 1 H, H-6′′b),
1.65, 1.64, 1.46, 1.43 (each s, each 3 H, isopropyl CH3), 1.28 (t, 9 H,
CH3CH2N, J ) 7.3 Hz); 13C NMR (D2O, 125 MHz) δ 153.7, 149.2,
148.0, 122.6, 117.4, 115.2, 94.3, 89.7, 89.2, 89.1, 87.3, 86.3, 84.2, 72.1,
69.2, 67.3, 49.5, 46.9, 46.8, 31.0, 28.9, 28.8, 27.1, 26.9, 11.0; 31P NMR
(D2O, 202 MHz, decoupled with 1H) δ -10.39 (d, J ) 15.3 Hz), -10.80
(d, J ) 15.3 Hz); HRMS (FAB, negative) calcd for C22H30N5O12P2
618.1366 [(M - H)-], found 618.1413; UV (H2O) λmax ) 258 nm.
Cyclic ADP-carbocyclic-ribose (4). A solution of 32 (128 OD260
units) in 60% aqueous HCO2H (1 mL) was stirred at room temperature
for 3.5 h. After the solvent was evaporated, H2O (2 mL × 3) was added
to the residue and evaporated. To the residue was added TEAB buffer
(0.1 M, 30 µL) and H2O (2 mL), and the resulting solution was
lyophilized to give 4 [115 OD260 units, 90%] as a triethylammonium
salt: 1H NMR (D2O, 400 MHz, J value was measured by spin
decoupling) δ 9.14 (s, 1 H, H-2), 8.38 (s, 1 H, H-8), 6.06 (d, 1 H,
H-1′, J1′,2′ ) 6.3 Hz), 5.14 (dd, 1 H, H-2′, J2′,1′ ) 6.3, J2′,3′ ) 4.4 Hz),
4.95 (ddd, 1 H, H-1′′, J1′′,2′′ ) 4.4, J1′′,6′′a ) 10.2, J1′′,6′′b ) 2.9 Hz), 4.61
(dd, 1 H, H-3′, J3′,2′ ) 4.4, J3′,4′ ) 2.4 Hz), 4.54 (ddd, 1 H, H-5′a, J5′a,4′
) 7.7, J5′a,5′b ) 10.2, J5′a,P ) 2.4 Hz), 4.41 (dd, 1 H, H-2′′, J2′′,1′′ ) 4.4,
N-1-[(1R,2S,3R,4R)-2,3-(Isopropylidenedioxy)-4-(phosphonoxy-
methyl)cyclopentyl]-5′-O-[(phenylthio)phosphoryl]-2′,3′-O-isopro-
pylideneadenosine (14). POCl3 (186 µL, 2.0 mmol) was added to a
solution of 31 (148 mg, 0.2 mmol) in PO(OMe)3 (2 mL) at 0 °C, and
the mixture was stirred at the same temperature for 20 min. The reaction
was quenched by aqueous saturated NaHCO3 (4 mL), and the resulting
mixture was stirred at 0 °C for 10 min. To the mixture were added
TEAA buffer (2.0 M, 1 mL) and H2O (4 mL), and then the resulting
solution was applied to a C18 reversed phase column (1.1 × 18 cm).
The column was developed using a linear gradient of 0-60% CH3CN
in TEAA buffer (0.1 M, pH 7.0, 400 mL). Appropriate fractions were
evaporated, and excess TEAA was removed by C18 reversed phase
column chromatography (1.1 × 18 cm, eluted with 60% aqueous
CH3CN). Appropriate fractions were evaporated, and the residue was
coevaporated with pyridine (1 mL × 3). A mixture of the residue,
H3PO2 (80 µL, 1.6 mmol) and Et3N (104 µL, 0.75 mmol) in pyridine
(2.0 mL) was stirred at 0 °C for 1.0 h and then at room temperature
for 16 h in the dark. After TEAA buffer (2.0 M, 1.0 mL) was added,
the mixture was evaporated, and the residue was partitioned between
EtOAc and H2O. The aqueous layer was evaporated with TEAA buffer
(2.0 M, 1.0 mL), and the residue was dissolved in H2O (5 mL). The
solution was applied to a C18 reversed phase column (1.1 × 18 cm),
and the column was developed using a linear gradient of 0-40%
CH3CN in TEAA buffer (0.1 M, pH 7.0, 400 mL). Appropriate fractions
were evaporated, and excess TEAA was removed by C18 reversed phase
column chromatography (1.1 × 18 cm, eluted with 40% aqueous
CH3CN) to give 14 (125 mg, 75%) as a triethylammonium salt: 1H
NMR (D2O, 500 MHz) δ 8.66 (s, 1 H, H-2), 8.41 (s, 1 H, H-8), 7.28-
7.11 (m, 5 H, Ar-H), 6.33 (d, 1 H, H-1′, J1′,2′ ) 2.2 Hz), 5.41 (dd, 1
H, H-2′, J2′,1′ ) 2.2, J2′,3′ ) 6.0 Hz), 5.02 (dd, 1 H, H-3′, J3′,2′ ) 6.0,
J3′,4′ ) 1.7 Hz), 4.96-4.85 (m, 3 H, H-1′′, H-2′′, H-3′′), 4.75 (m, 1 H,
H-4′), 4.23 (m, 1 H, H-5′a), 4.15 (m, 1 H, H-5′b), 4.06 (m, 2 H, H-5′′),
3.19 (q, 6 H, -CH2N, J ) 7.3 Hz), 2.64 (m, 2 H, H-4′′, H-6′′a), 2.45
(m, 1 H, H-6′′b), 1.66, 1.65, 1.42, 1.42 (each s, each 3 H, isopropyl
CH3), 1.28 (t, 9 H, CH3CH2N, J ) 7.3 Hz); 13C NMR (D2O, 125 MHz)
δ 153.8, 148.7, 147.0, 146.5, 135.0, 135.0, 132.3, 131.7, 130.4, 122.1,
118.4, 117.5, 94.3, 89.0, 88.9, 86.8, 86.3, 84.2, 83.2, 68.6, 68.5, 67.9,
67.8, 49.4, 46.2, 46.1, 35.5, 28.7, 27.0, 26.7, 11.0; 31P NMR (D2O,
202 MHz, decoupled with 1H) δ 1.05 (s), 17.10 (s); HRMS (FAB,
negative) calcd for C28H36N5O12P2S 728.1556 [(M - H)-], found
728.1576; UV (H2O) λmax ) 260 nm.
J2′′,3′′ ) 3.9 Hz), 4.41 (ddd, 1 H, H-4′, J4′,3′ ) 2.4, J4′,5′a ) 7.7, J4′,5′b
)
3.4 Hz), 4.24 (dd, 1 H, H-3′′, J3′′,2′′ ) 3.9, J3′′,4′′ ) 3.9 Hz), 4.19 (m, 2
H, H-5′′), 4.11 (ddd, 1 H, H-5′b, J5′b,4′ ) 3.4, J5′b,5′a ) 10.2, J5′b,P ) 3.4
Hz), 3.18 (q, 6 H, -CH2N, J ) 7.3 Hz), 3.07 (ddd, 1 H, H-6′′a, J6′′a,1′′
) 10.2, J6′′a,4′′ ) 10.7, J6′′a,6′′b ) 15.1 Hz), 2.55 (m, 1 H, H-4′′), 2.41
(ddd, 1 H, H-6′′b, J6′′b,1′′ ) 2.9, J6′′b,4′′ ) 3.4, J6′′b,6′′a ) 15.1 Hz), 1.28
(t, 9 H, CH3CH2N, J ) 7.3 Hz); NOE (D2O, 400 MHz) irradiated H-2,
observed H-2′′ (4.8%), H-3′′ (3.9%), H-6′′b (7.2% %), irradiated H-8,
observed H-1′ (14.4%), irradiated H-6′′b, observed H-2 (14.5%); 13C
NMR (D2O, 125 MHz) δ 154.0 (C-6), 149.2 (C-4), 147.9 (C-8), 147.0
(C-2), 122.8 (C-5), 93.3 (C-1′), 87.7 ‘C-4′), 81.1 (C-2′′), 76.6 (C-3′′),
76.2 (C-2′), 73.5 (C-3′), 68.1 (C-5′′), 67.5 (C-5′), 66.8 (C-1′′), 49.5
(-CH2N), 45.6 (C-4′′), 31.1 (C-6′′), 11.0 (CH3CH2N); 31P NMR (D2O,
1
202 MHz, decoupled with H) δ -9.20 (d, J ) 11.4 Hz), -10.26 (d,
J ) 11.4 Hz); HRMS (FAB, negative) calcd for C16H22N5O12P2
538.0740[(M - H)-], found 538.0723; UV (H2O) λmax ) 259 nm (ꢀ )
11100, based on the total phosphate analysis), UV (pH 11.5, 25 mM
phosphate buffer) λmax ) 261 nm, sh 291 nm. The potassium salt of 4
was prepared by the successive treatments of the above triethyl-
amomonium salt with Diaion PK-212 resign (H+ form) and with the
same resign (K+ form).
Chemical Stability of cADPR (1) and cADPcR (4). A buffer (pH
2.0, 50 mM KCl-HCl buffer; pH 7.0, 100 mM TEAA buffer; pH 12.0,
50 mM KCl-NaOH buffer) containing cADPR (free acid, Yamasa,
Tokyo, Japan) or cADPcR (potassium salt) (1.0 OD260 unit) was
incubated at 37 °C. The reaction mixtures (10 µL) were sampled after
2.0, 4.0, 6.0, 8.0, 24, 48, 72 h, and were then added to a TEAA buffer
(pH 7.0, 100 mM, 90 µL) at 0 °C. The resulting solutions (50 µL)
were analyzed by reverse phase and ion exchange HPLC (TSK-GEL
DEAE-2SW, 4.6 mm × 250 mm; 0-300 mM HCO2NH4/20% MeCN,
20 min; 260 nm).
Cyclic ADP-carbocyclic-ribose Diacetonide (32). To a mixture of
AgNO3 (41 mg, 241 µmol), Et3N (33 µL, 241 µmol), and molecular
sieves (3 Å) (1.0 g) in pyridine (8 mL), was added a solution of 14
(9.5 mg, 11.4 µmol) in pyridine (8 mL) slowly over 15 h, using a
syringe-pump, at room temperature in the dark. The molecular sieves
were filtered off with Celite and washed with H2O. To the combined
filtrate and washings was added TEAA buffer (2.0 M, pH 7.0, 1 mL),
and the resulting solution was evaporated. The residue was partitioned
between EtOAc and H2O, and the aqueous layer was evaporated. The
residue was dissolved in 0.1 M TEAA buffer (5 mL), which was applied
to a C18 reverse phase column (1.1 × 11 cm). The column was
developed using a linear gradient of 0-40% CH3CN in TEAA buffer
(0.1 M, pH 7.0, 200 mL). Appropriate fractions were evaporated, and
excess TEAA was removed by C18 reverse phase column chromatog-
The Dimroth Rearrangement of cADPcR (4) Forming N6-Cyclic
ADP-carbocyclic-ribose (34). A solution of 4 (potassium salt, 115
OD260 units, 9.3 µmol) in a KCl-NaOH buffer (50 mM, pH 12.0, 4
mL) was stirred at 37 °C for 4 days. After addition of H2O (90 mL),
the pH of the mixture was adjusted about 4.0 with AcOH, and the
resulting solution was applied to a DEAE-Sephadex A-25 column
-
(HCO3 form, 1.5 cm × 20 cm). The column was developed using a
linear gradient of 0.1-0.5 M TEAB buffer (pH 7.8, 400 mL).
Appropriate fractions were evaporated and coevaporated with H2O
(three times) to remove excess TEAB. The residue was lyophilized to
give 34 (106 OD265 units, 6.5 µmol, 70%; the yield was calculated
using the molar absorption coefficient of N6-methyladenosine (ꢀ
)16300, λmax ) 265 nm) 4b as a triethylammonium salt: 1H NMR (D2O,

Cyclic ADP-Carbocyclic-Ribose
J. Am. Chem. Soc., Vol. 123, No. 36, 2001 8759
500 MHz) δ 8.51, 8.38 (each s, each 1 H, H-2, H-8), 6.17 (s, 1 H,
H-1′), 4.75 (m, 1 H, H-2′), 4.64 (m, 1 H, H-3′), 4.60 (m, 1 H, H-2′′),
4.42 (m, 1 H, H-3′′), 4.36 (m, 1 H, H-4′), 4.24 (m, 1 H, H-1′′), 4.10-
3.94 (m, 4 H, H-5′, H-5′′), 3.20 (q, 6 H, -CH2N, J ) 7.3 Hz), 2.38
(m, 1 H, H-6′′a), 2.30 (m, 1 H, H-4′′), 2.11 (m, 1 H, H-6′′b), 1.28 (t,
9 H, CH3CH2N, J ) 7.3 Hz); 31P NMR (D2O, 202 MHz, decoupled
12.5 mM Tris, pH 8.2) and were finally resuspended in fresh stdASW.
Sperm were likewise shed by injecting 0.5 M KCl, collected directly
from the genital pores, stored at 4 °C until use, and diluted 5000-fold
into stdASW just before use. [Ca2+]i was measured with a Ca2+-sensitive
fluorescent dye, indo-1 (Dojindo, Kumamoto, Japan). Each egg cell
was fastened to a poly-L-lysine (Mr ) 1000-4000; Sigma) coated glass
coverslip at the bottom of 150 µL stdASW in a Lucite-frame chamber,
mounted on the stage of a TMD epifluorescence microscope (Nikon,
Tokyo, Japan). The dye was pressure-injected into eggs at a final
concentration of 100 µM, from a 10 mM stock solution containing
100 mM potassium aspartate and 10 mM Hepes, titrated to pH 7.0
with Tris, according to the method of Hiramoto.22 For measuring the
fluorescence of indo-1, the selected wavelengths were 355 nm (band-
pass) at the excitation filter, 380 nm (cutoff) at the dichroic mirror,
and simultaneously 405 nm (band-pass) and 485 nm (band-pass) at
the emission detectors, which were Hamamatsu R647-01 photomulti-
pliers (Hamamatsu Photonics, Hamamatsu, Japan). Fluorescence from
the whole egg was measured, and [Ca2+]i was calculated from the ratio
of 405-nm fluorescence intensity to the corresponding 485-nm intensity,
according to the previously reported method.23 Under the monitoring
of [Ca2+]i, cADPR or cADPcR was injected into eggs in the dark. These
chemicals were dissolved in injection buffer, were filled in the tip of
micropipet, and were pressure-injected ∼1 min after impalement of
the micropipet into eggs. Experiments were performed at 20-25 °C.
1
with H) δ -9.25 (m), -9.69 (m); HRMS (FAB, negative) calcd for
C16H22N5O12P2 538.0740 [(M - H)-], found 538.0752; UV (H2O) λmax
) 271 nm (pH 7.0), UV (pH 11.5, 25 mM phosphate buffer) λmax
)
271 nm; 13C NMR (potassium salt, D2O, 125 MHz) δ 156.6 (C-6),
154.3 (C-2), 151.2 (C-4), 143.4 (C-8), 122.1 (C-5), 93.0 (C-1′), 85.8
(C-4′), 78.8 (C-2′′), 76.1 (C-2′), 75.4 (C-3′′), 72.1 (C-3′), 68.4 (C-5′′),
65.9 (C-5′), 60.1 (C-1′′), 44.9 (C-4′′), 32.3 (C-6′′).
Stabilitiy of cADPR (1) and cADPcR (4) in Membrane Fraction
of Rat Brain Extract. Rat brain extract was prepared by a procedure
according to the previous method.16 cADPR (free acid) or cADPcR
(potassium salt) (1.0 OD260 unit) was preincubated in 8 mM Hepes-
Pipes-Tris buffer (pH 7.0, 8 mM, 150 µL) at 37 °C for 5 min. This
was added to the solution of the membrane fraction of rat brain extract
(100 µL), and the mixture was incubated at 37 °C. The reaction mixture
was sampled (25 µL) at every 30 min afterward and diluted with water
(175 µL), which was frozen in liquid nitrogen to stop the reaction.
After the samples were a centrifuged at 12000 rpm at 4 °C for 15 min,
supernatants were filtered using a centrifugal filter at 12000 rpm at 4
°C for 15 min, and the resulting filtrates (50 µL) were analyzed by
ion-exchange HPLC (TSK-GEL DEAE-2SW, 4.6 mm × 250 mm;
0-300 mM HCO2NH4/20% MeCN, 20 min; 260 nm).
Acknowledgment. This investigation was supported by a
Grant-in-Aid for Creative Scientific Research (13NP0401) from
the Japan Society for Promotion of Science. We also thank the
Japan Society for Promotion of Sciences for support of M.F.
We are grateful to Professor T. Katada and Dr. Kontani for their
kind gift of CD38 plasmid, to Dr. S. Sakata for his kind gift of
cADPR, to Professor H. Harashima for his suggestion on the
rat brain experiment, and to Professor B. V. L. Potter for helpful
discussion. We are also grateful to Ms. H. Matsumoto, A.
Maeda, S. Oka, and N. Hazama (Center for Instrumental
Analysis, Hokkaido University) for technical assistance with
NMR, MS, and elemental analysis.
Treatments of cADPR (1) and cADPcR (4) by CD38 cADPR
Hydrolase. ThioHis-CD38 cADPR hydrolase was expressed in Es-
cherichia coli BL21(DE3) cells and purified from crude cell extract
using Ni2+-chelating column (Invitrogen) according to the reported
method.18 cADPR (free acid) or cADPcR (potassium salt) (0.5 OD260
unit) was preincbated in Hepes-Pipes-Tris buffer (pH 7.0, 5 mM, 230
µL) at 37 °C for 5 min. To the solution was added CD38 solution (20
µL),18 and the resulting mixture was incubated at 37 °C. The reaction
mixture was sampled (40 µL) at every 15 min, added to water (160
µL), and frozen in liquid nitrogen to stop the reaction. After the samples
were centrifuged at 12000 rpm at 4 °C for 15 min, the supernatants
were filtered using a centrifugal filter at 12000 rpm at 4 °C for 30
min, and the resulting filtrates (125 µL) were analyzed by ion-exchange
HPLC (TSK-GEL DEAE-2SW, 4.6 mm × 250 mm; 0-300 mM
HCO2NH4/20% MeCN, 20 min; 260 nm).
Supporting Information Available: Figures showing stabil-
ity of cADPcR (4) and cADPR (1) in aqueous media, stability
of cADPcR (4) and cADPR (1) in rat brain extract, and
susceptibility of cADPcR (4) and cADPR (1) to CD38 cADPR
hydrolase; 1H NMR data table of cADPR (1) and cADPcR (4)
in D2O; 1H NMR spectral charts of 22, 30, 14, 32, and 4 (PDF).
This material is available free of charge via the Internet at
http://pubs.acs.org.
Measurement of Ca2+-Mobilizing in Sea Urchin Eggs. Eggs of a
species of Japanese sea urchin, Anthocidaris crassispina, were shed
into filtrated seawater (FSW) by injection of 0.5 M KCl into the
coelomic cavity, and the shed eggs were washed three times with FSW.
The eggs were stripped of their jelly coats by washing twice with Ca2+
-
and Mg2+-free artificial seawater [CaMgFASW; 520 mM NaCl, 10 mM
KCl, 10 mM 3-[4-(2-hydroxyethyl)-1-piperazinyl]-propanesulfonic acid
(Epps), titrated to pH 8.2 with tris(hydroxymethyl)aminomethane (Tris),
and 2 mM ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA)], then
were rinsed twice more with standard artificial seawater (stdASW; 430
mM NaCl, 10 mM KCl, 50 mM MgCl2, 10 mM CaCl2, 10 mM Epps,
JA010756D
(22) Hiramoto, Y. Exp. Cell Res. 1974, 87, 403-406.
(23) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1985,
260, 3440-3450.