Novel 3',5'-cyclic nucleotide analogue: adenosine 3',5'-cyclic boranomonophosphate.

Article abstract of DOI:10.1021/ol0003230

A general procedure for the first synthesis of a 3',5'-cyclic boranomonophosphate was established. Specifically, adenosine 3',5'-cyclic boranomonophosphosphate (cyclic AMPB, 4c), a P-borane (BH(3)) analogue of adenosine 3',5'-cyclic monophosphate (cAMP),

Full text of DOI:10.1021/ol0003230

ORGANIC
LETTERS
2001
Vol. 3, No. 6
795-797
Novel 3′,5′-Cyclic Nucleotide Analogue:
Adenosine 3′,5′-Cyclic
Boranomonophosphate
Jinlai Lin, Kaizhang He, and Barbara Ramsay Shaw*
Department of Chemistry, Box 90346, Duke UniVersity,
Durham, North Carolina 27708-0346
brshaw@chem.duke.edu
Received October 20, 2000
ABSTRACT
A general procedure for the first synthesis of a 3′,5′-cyclic boranomonophosphate was established. Specifically, adenosine 3′,5′-cyclic
boranomonophosphosphate (cyclic AMPB, 4c), a P-borane (BH ) analogue of adenosine 3′,5′-cyclic monophosphate (cAMP), was synthesized
3
via a phosphite approach in good yield. The method is also applicable for syntheses of natural cAMP and its phosphorothioate analogue. The
two diasteromers of cyclic AMPB 4c were separated, and their chemical structures were established via spectroscopic methods.
3′,5′-Cyclic adenosine monophosphate (cAMP) is ubiquitous
in all forms of life and vital in the regulation of various
biological processes.1 For example, cAMP is an ancient
hunger signal and a second messenger in the action of many
hormones; it is central to the coordinated control of glycogen
synthesis and breakdown, and stimulates protein kinases and
the transcription of several inducible catabolic operons.1 To
better understand biological processes involving cAMP,
various modified analogues have been prepared as enzyme
substrates or inhibitors.1-4 Among cAMP analogues, the
N6,2′-O-dibutyryl cyclic AMP2 and cyclic AMP phosphoro-
thioate (cyclic AMPS)4 are most commonly used. However,
N6,2′-O-dibutyryl cyclic AMP releases butyric acid on
hydrolysis, which can cause a number of reactions unrelated
to the action of cyclic AMP that might not be evident by
applying butyric acid to the system as a control.2,4d Cyclic
AMPS has many special properties such as high nuclease
resistance, but its relativly poor lipophilicity4g and loss of
sulfur during incubation4c greatly limit its applications.
Boronated nucleotides,5 in which one of two nonbridging
oxygen atoms of phosphate is replaced by a borane group
(BH3), might be useful because they are more lipophilic and
nuclease resistant than the normal congener and phosphoro-
thioates.5f Here we report the first synthesis of a boronated
cyclic nucleotide analogue, specifically adenosine 3′,5′-cyclic
boranomonophosphate (cyclic AMPB).
Commonly, nucleoside 3′,5′-cyclic phosphates are prepared
from the nucleoside monophosphate through intramolecular
(4) (a) Pereira, M. E.; Segaloff, D. L.; Ascoli, M.; Eckstein, F. J. Biol.
Chem. 1987, 262, 6093-6100. (b) Eckstein, F. Annu. ReV. Biochem. 1985,
54, 367-402. (c) Eckstein, F.; Bar, H. P. Biochim. Biophys. Acta 1969,
191, 316-321. (d) Eckstein, F.; Eimerl, S.; Schiramm, M. FEBS Lett. 1976,
64, 92-94. (e) Baraniak, J.; Kinas, R. W.; Lesiak, K.; Stec, W. J. J. Chem.
Soc., Chem. Commun. 1979, 940-941. (f) Eckstein, F.; Kutzke, U.
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Bar, H.-P. Biochemistry 1974, 13, 3806-3810. (h) Burgers, P. M.;
Eckstein, F.; Hunneman, D. H.; Baraniak, J.; Kinas, R. W.; Lesiak, K.;
Stec, W. J. J. Biol. Chem. 1979, 254, 9959-9961. (i) Stec. W. J. Acc. Chem.
Res. 1983, 16, 411-417. (j) Senter, P. D.; Eckstein, F.; Mulsch, A.; Bohme,
E. J. Biol. Chem. 1983, 258, 6741-6745. (k) Genleser, H-G.; Dostmann,
W.; Bottin, U.; Butt, E. Tetrahedron Lett. 1988, 29, 2803-2804.
(1) (a) Jost, J. P. et al. Annu. ReV. Biochem. 1971, 40, 741. (b) Robison,
G. A. et al. In Cyclic AMP; Academic Press: New York, 1971. (c) AdVances
in Cyclic Nucleotide Research; Greengard, P., Robison, G. A., Eds.; Raven
Press Books, Ltd.: Hagerstown, MD, 1972; Vol. 1.
(2) Ito, F.; Chou, J. Y. J. Biol. Chem. 1984, 259, 2526-2530.
(3) (a) Bottka, S.; Tomasz, J.; Cruse, W. B. T.; Zhang, S.; Kennard, O.
Nucleosides Nucleotides 1997, 16, 2123-2131. (b) Regan, A. C.; Sciam-
metta, N.; Tattersall, P. I. Tetrahedron Lett. 2000, 41, 8211-8215.
10.1021/ol0003230 CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/23/2001

dehydration or from a nucleoside monophosphate derivative
via intramolecular cyclization.6,7 For example, treatment of
5′-AMP with 1,3-dicyclohexylcarbodiimide (DCC)6 or reac-
tion of 5′-AMP p-nitrophenyl ester with t-BuOK7a provides
convenient methods to synthesize normal 3′,5′-cyclic AMP.
Yet, neither the treatment of adenosine 5′-boranomonophos-
phate (5′-AMPB) 1 with DCC in dilute pyridine solution at
room temperature, nor treatment of adenosine 5′-borano-
monophosphate p-nitrophenyl ester (5′-AMPB-NPE) 2 under
strongly basic anhydrous conditions (t-BuOK), yielded
compound 4c. Further, adenosine 5′-boranomonophosphate
p-diisopropylamidate8 (5′-AMPB-DIPA) 3 treated with tet-
razole (4 equiv) in DMF at 55 °C also failed to give the
desired product 3′,5′-cyclic AMPB 4c. After several unsuc-
cessful attempts to prepare the cyclic boronated phosphodi-
ester compounds by direct methods in Scheme 1, we were
Scheme 2. Synthesis of Adenosine 3′,5′-Cyclic
P-Boranomonophosphatea
Scheme 1
a (a) [(i-Pr)2N]2POCH2CH2CN/diisopropylamine hydrotetrazolide/
DMF; (b) tetrazole; (c) (Et)(i-Pr)2N:BH3 or Me2S:BH3; (d) from 7
to 9a, I2/H2O/pyridine, from 7 to 9b, sulfur (S8); (e) NH4OH/
CH3OH; (f) Bu4NF.
7 was treated either with excess (4 equiv) borane-N,N-
diisopropylethylamine complex (DIPEA-BH3) with stirring
for 2 h at room temperature or with 1 M borane-dimethyl
sulfide in CH2Cl2 at 0 °C with stirring for 10 min to give
the borane-phosphite intermediate 8. After evaporation and
extraction with ethyl acetate and water, the organic layer
was concentrated and treated with a mixture of ammonium
hydroxide and methanol (v:v ) 1:1) to give compound 10c.
Without purification, 10c was treated with 1 M tetrabutyl-
ammonium fluoride to give cyclic AMPB 4c. Compound 4c
was purified by ion-exchange chromatography on a column
forced to explore a synthetic method that involved the
phosphite approach in Scheme 2.
The general procedure for the synthesis of cyclic AMPB
is outlined in Scheme 2. Phosphitylation of N4-benzoyl-2′-
O-(tert-butyldimethylsilyl)adenosine 5 in DMF by 2-cyano-
ethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (i-Pr2N)2-
P(OCH2CH2CN) with diisopropylamine hydrotetrazolide as
catalyst gave 6. After 1 h, tetrazole was added. Without
stirring, the intramolecular cyclization occurred to yield
intermediate 7 after 3 h. Without purification, intermediate
-
packed with QA-52 cellulose (HCO3 ) eluted with a linear
(5) (a) Sood, A.; Shaw, B. R.; Spielvogel, B. F. J. Am. Chem. Soc. 1990,
112, 9000-9001. (b) Tomasz, J.; Shaw, B. R.; Porter, K.; Spielvogel, B.
F.; Sood, A. Angew. Chem., Int. Ed. Engl. 1992, 31, 1373-1375. (c) Shaw,
B. R.; Madison, J.; Sood, A.; Spielvogel, B. F. Methods Mol. Biol. 1993,
20, 225-243. (d) Li, H.; Hardin, C.; Shaw, B. R. J. Am. Chem. Soc. 1996,
118, 6606-6614. (e) Porter, K. W.; Briley, J. D.; Shaw, B. R. Nucleic
Acids Res. 1997, 25, 1611-1617. (f) Sergueev, D. S.; Shaw, B. R. J. Am.
Chem. Soc. 1998, 120, 9417-9427. (g) Rait, V.; Shaw, B. R. Antisense
Nucleic Acid Drug DeV. 1999, 9, 53-60. (h) Lin, J-L.; Shaw, B. R. Chem.
Commun. 1999, 1517-1518. (i) Lin, J-L.; Shaw, B. R. Tetrahedron Lett.
2000, 41, 6701-6704. (j) Lin, J-L.; Shaw, B. R. Chem. Commun. 2000,
2115-2116.
gradient of 0.005 and 0.2 M ammonium bicarbonate buffer,
pH 9.6. Compound 4c was obtained in 48% overall yield
(from 5 to 4c) and identified by 31P NMR (typical broad
peaks at 92 and 97 ppm), 1H NMR spectroscopy, and FAB--
MS [M- (m/e) C10H14O5N5PB, calcd 326.07, found, 326.08].
Successful separation of the two diastereomers (Rp and Sp)
of 4c was achieved by reverse-phase HPLC; chemical
structures were established via spectroscopic methods. The
two diastereomers of 4c have considerably different 31P
chemical shifts: 92 ppm (br) for cyclic AMPB isomer I (first
eluted diastereomer, 4c-I), and a broad tetramer from 95.7
to 98.2 ppm for cyclic AMPB isomer II (second eluted
diastereomer, 4c-II). The chemical shift difference arises
because the BH3 group can assume an equitorial or axial
position in the 3′,5′-cyclic six-membered ring of cyclic
(6) (a) Tener, G. M.; Khorana, H. G.; Markham, R.; Pol, E. H. J. Am.
Chem. Soc. 1958, 80, 6623-6230. (b) Smith, M.; Drummond, G. T.;
Khorana, H. G. J. Am. Chem. Soc. 1961, 83, 698-706. (c) Sagi G.; Szucs
K.; Vereb, G.; Otvos, L. J. Med. Chem. 1992, 35, 4549-4556.
(7) (a) Borden, R. K.; Smith. M. J. Org. Chem. 1966, 31, 3247-3253.
(b) Genieser, H.-G.; Butt, E.; Bottin, U.; Dostmann, W.; Jastorff, B. Synthesis
1989, 53-54.
(8) Lin, J.-L.; He, K.; Shaw, B. R. HelV. Chim. Acta 2000, 83, 1392-
1397.
796
Org. Lett., Vol. 3, No. 6, 2001

AMPB. The spectra are broad because both 11B (spin 3/2)
and 10B (spin 5/2) contribute to the spectra.5d
is expected to neither form classical hydrogen bonds nor
coordinate metal ions10 as well as oxygen in the parental
cyclic NMP. Like cyclic AMPS, the two diastereomers of
cyclic AMPB are anticipated to have different substrate
properties toward transferases and hydrolases and should be
useful for investigating the roles of phosphate and metal ions
in biological processes and elucidating the stereochemical
and metal requirements of the enzymatic reactions involving
cAMP. (iii) Finally, boronated nucleotides may offer a unique
advantage over other modified congeners because they could
The method described in this paper is also applicable for
synthesis of normal adenosine 3′,5′-cyclic monophosphate
4a and adenosine 3′,5′-cyclic phosphorothioate 4b. Upon
oxidation of intermediate 7 with iodine and sulfur, com-
pounds 9a and 9b were obtained, respectively. After remov-
ing protecting groups, 3′,5′-cAMP 4a (55% overall yield
from 5) and 3′,5′-cyclic AMPS 4b (52% overall yield from
5) were obtained.
be used for boron neutron capture therapy (BNCT),11
a
Our current synthetic approach (Scheme 2) is based on a
cyclic phosphite triester 7 intermediate, prepared by the
intramolecular cyclization of a 5′-phosphoramidite 6. In this
step, a competitive side-reaction can be intermolecular
coupling to form diadenosine p-cyanoethyl phosphite; how-
ever, under appropriate conditions (such as low temperature,
low concentration of reactant, and without stirring), the
desired intramolecular cyclization was the predominant
reaction. By boronating, oxidizing, or sulfurizing intermediate
7, followed by deprotection, the 3′,5′-cyclic AMPB 4c,
normal 3′,5′-cyclic AMP 4a, or 3′,5′-cyclic AMPS 4b were
obtained, respectively (Scheme 2). This method could also
be useful for the synthesis of base-modified nucleoside 3′,5′-
cyclic monophosphates since the conditions avoid strong
basic or other harsh reaction conditions previously used.6,7
radiation therapy that can selectively destroy cells which have
preferentially taken up boron.
To summarize, we have synthesized a new type of
modified 3′,5′-cyclic AMP in which one of the two non-
bridging oxygen atoms of a phosphodiester group has been
replaced with a borane group. The boronated 3′,5′-cyclic
AMPB 4c is stable under a broad range of pH (3-11)
conditions and is expected to be highly resistant to enzymatic
cleavage. The borane may impart a greater membrane
permeability than cAMP. The increased lipophilicity and
nuclease resistance, in conjunction with the potential utility
as a carrier of 10B in boron neutron capture therapy (BNCT)
for the treatment of cancer, make cyclic AMPB analogues
promising candidates for therapeutic applications. The two
diastereomers of boronated cyclic NMPB should be very
useful for investigating biochemical processes involving the
normal cyclic NMP.
The highly lipophilic (less-polar) boron analogue of cAMP
should be useful in many ways. (i) Boron-containing
compounds facilitate transport of organic molecules through
membranes.9 The isoelectronic substitution of borane for one
of the nonbridging oxygens in phosphate diesters should
impart an increase in lipophilicity and change in polarity
relative to cAMP. The borane should enhance the hydro-
phobicity of cyclic AMPB and may enable the compound
to penetrate the plasma membrane and enter cells. (ii) The
BH3 group in a boronated cyclic nucleoside monophosphate
Acknowledgment. This work was supported by NIH
grant 1R01-GM57693 to B.R.S.
Supporting Information Available: Experimental pro-
1
cedures and 31P NMR, H NMR, MS (FAB-), and HRMS
(FAB-) spectra for compound 4c, cyclic AMPB isomer I
(4c-I), and cyclic AMPB isomer II (4c-II). This material is
available free of charge via the Internet at http://pubs.acs.org
OL0003230
(9) (a) Smith, B. D.; Gardiner, S. J.; Munro, T. A.; Paugam, M.-F.;
Riggs, J. A. J. Inclusion Phenom. Mol. Recognit. Chem. 1998, 32, 121-
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