Photolabile precursors of cyclic nucleotides with high aqueous solubility and stability

Article abstract of DOI:10.1021/jo020040g

Photolabile phosphotriester derivatives of cyclic AMP and cyclic GMP are described, where the additional group on the phosphate is a 2-nitrobenzyl that bears an electron-withdrawing and dianionic substituent. This confers high aqueous solubility and excel

Full text of DOI:10.1021/jo020040g

3474
J . Org. Chem. 2002, 67, 3474-3478
P h otola bile P r ecu r sor s of Cyclic Nu cleotid es w ith High Aqu eou s
Solu bility a n d Sta bility
Lijuan Wang,†,‡ J ohn E. T. Corrie,*,§ and J ohn F. Wootton†
Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University,
Ithaca, New York 14853, and National Institute for Medical Research, The Ridgeway,
Mill Hill, London NW7 1AA, U.K.
jcorrie@nimr.mrc.ac.uk
Received J anuary 18, 2002
Photolabile phosphotriester derivatives of cyclic AMP and cyclic GMP are described, where the
additional group on the phosphate is a 2-nitrobenzyl that bears an electron-withdrawing and
dianionic substituent. This confers high aqueous solubility and excellent resistance to hydrolysis
of the phosphotriester (t1/2 for hydrolysis at pH 7, 22 °C, is >3 months for the axial isomers 3a and
4a and >1 month for the equatorial isomers 3b and 4b). The photolysis quantum yields are in the
range 0.15-0.24, and the product release rate upon flash photolysis is 1.7 s-1 at pH 7.0, 20 °C.
Ch a r t 1
In tr od u ction
Cyclic adenine and guanine nucleotides have important
biological functions as second messengers in intracellular
signal transduction processes.1 One approach to studying
their role is to use photolabile derivatives that are inert
until irradiated by a brief pulse of near-UV light.2
A
number of such “caged” derivatives have been described,
in all of which the cyclic phosphate is esterified by a
photolabile group, including 2-nitrobenzyl,3a 3,4-dimethoxy-
2-nitrobenzyl,3b,c,e,f 1-(2-nitrophenyl)ethyl,3c,d 1-(3,4-di-
methoxy-2-nitrophenyl)ethyl,3c desyl,3g 2-anthraquinonyl-
methyl,3h 2-naphthylmethyl,3h 4-(7-methoxycoumarinyl)-
methyl,3h,i and 4-(7-acyloxycoumarinyl)methyl.3j Useful
generalizations can be drawn from these many deriva-
tives. First, axial esters 1 (Chart 1) are significantly more
resistant to hydrolysis than the comparable equatorial
esters 2. Second, stability is decreased by substituents
on the caging group at its benzylic position or by electron-
donating groups on its aromatic ring. Furthermore, most
† Cornell University.
‡ Current address: Merial Limited, PKDM, 631 US Route 1 South,
New Brunswick, NJ 08902.
§ National Institute for Medical Research.
(1) (a) Lincoln, T. M.; Cornwell, T. L. FASEB J . 1993, 7, 328. (b)
Kawasaki, H.; Springett, G. M.; Mochizuki, N.; Toki, S.; Nayaka, M.;
Matsuda, M.; Housman, D. E.; Graybiel, A. M. Science 1998, 282, 2275.
(c) Corbin, J . D., J ohnson, R. A., Eds. Methods in Enzymology;
Academic Press: San Diego, CA, 1988; Vol. 159.
of the above derivatives suffer from poor aqueous solubil-
ity that can limit their utility in biological systems (see
discussion in ref 3h). Their hydrophobicity is such that
they tend to partition into cell membranes and cannot
be assumed to retain a stable intracellular location, even
if introduced to a particular cell by microinjection. In an
endeavor to address these issues, we now describe new
caged cyclic nucleotides 3a ,b and 4a ,b that have good
aqueous solubility by virtue of the presence of two anionic
centers. As an additional benefit, these compounds have
been found to be unexpectedly stable with respect to
hydrolysis. While our work was in progress, Hagen et
al. described new variants of the 4-coumarinylmethyl
caged cyclic nucleotides referenced above,3h-j also bearing
anionic substituents, that have similarly good resistance
to hydrolysis but appear somewhat less soluble than the
compounds now described.3k
(2) Corrie, J . E. T.; Trentham, D. R. In Bioorganic Photochemistry;
Morrison, H., Ed.; Wiley: New York, 1993; Vol. 2, pp 243-305.
(3) (a) Engels, J .; Schlager, E. J . J . Med. Chem. 1977, 20, 907. (b)
Nerbonne, J . M.; Richard, S.; Nargeot, J .; Lester, H. A. Nature 1984,
310, 74. (c) Wootton, J . F.; Trentham, D. R. In Photochemical Probes
in Biochemistry; Nielsen, P. E., Ed.; NATO ASI Series C; Kluwer:
Dordrecht, The Netherlands, 1989; Vol. 272, pp 277-296. (d) Hagen,
V.; Dzeja, C.; Bendig, J .; Baeger, I.; Kaupp, U. B. J . Photochem.
Photobiol., B 1998, 42, 71. (e) Karpen, J . W.; Zimmerman, A. L.; Stryer,
L.; Baylor, D. A. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 1287. (f) Hagen,
V.; Dzeja, C.; Frings, S.; Bendig, J .; Krause, E.; Kaupp, U. B.
Biochemistry 1996, 35, 7762. (g) Givens, R. S.; Athey, P. S.; Matuszew-
ski, B.; Kueper, L. W.; Xue, J . Y.; Fister, T. J . Am. Chem. Soc. 1993,
115, 6001. (h) Furuta, T.; Torigai, H.; Sugimoto, M.; Iwamura, M. J .
Org. Chem. 1995, 60, 3953. (i) Schade, B.; Hagen, V.; Schmidt, R.;
Herbrich, R.; Krause, E.; Eckhardt, T.; Bendig, J . J . Org. Chem. 1999,
64, 9109. (j) Furuta, T.; Momotake, A.; Sugimoto, M.; Hatayama, M.;
Torigai, H.; Iwamura, M. Biochem. Biophys. Res. Commun. 1996, 228,
193. (k) Hagen, V.; Bendig, J .; Frings, S.; Eckhardt, T.; Helm, S.;
Reuter, D.; Kaupp, U. B. Angew. Chem., Int. Ed. 2001, 40, 1046.
10.1021/jo020040g CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/25/2002

Photolabile Precursors of Cyclic Nucleotides
J . Org. Chem., Vol. 67, No. 10, 2002 3475
Sch em e 1
examples of such oxidations by DMSO have been re-
ported to be promoted by silver nitrate,6a barium
hydroxide,6b or sodium bicarbonate.6c Thus, the presence
of Ag2O under our conditions probably accounts for the
efficient oxidation.
An alternate esterification procedure, described by
Kataoka et al.,7 used the tri-n-butylammonium salt of
cAMP, benzyl bromide, and sodium carbonate in di-
methylacetamide. When applied to esterification of cAMP
with the substituted benzyl bromide 8, these conditions
successfully gave a mixture of 10a and its equatorial
isomer 11a (∼3:1 ratio) in overall 25-30% yield. The
acetate 12, prepared for purposes of quantification (see
below), was readily obtained from bromide 8 by similar
displacement with sodium acetate.
Stereochemical assignments for 10a and 11a were
made from the 31P NMR spectra by analogy with previous
data on cNMP esters, in which the axial isomer has been
assigned as having the more upfield 31P resonance.3a,b
Attempted cleavage of the TMSE esters from 10a by
treatment with (TBA)F caused extensive degradation, but
they were cleanly removed from 10a , 11a , and 12 by brief
exposure to TFA. This effective but little-used protocol
allows residues from the protecting group to be removed
cleanly during evaporation of TFA at the end of the
reaction.8,9 Final purification of the caged cyclic nucle-
otides was achieved by reverse-phase HPLC.
Ch a r t 2
The esterification of cyclic GMP gave 10b and its
equatorial isomer 11b in similar ratio but in much
reduced yield, apparently because of poor solubility of the
nucleotide. Separation of these isomers was not achieved,
and the mixture, after chromatography, was directly
subjected to TFA deprotection. The individual isomers
3b and 4b were then readily separated by preparative
reverse phase HPLC.
Accurate quantification of aqueous solutions of the
caged cyclic nucleotides requires the extinction coef-
ficients for the different compounds. These data were
estimated using the model compound 13, obtained as a
clean solid after deprotection of 12, to provide a spectrum
for the caging group (calibrated in terms of absorbance
coefficients at all wavelengths) that was added to com-
parable spectra of adenine or guanine nucleotides, thereby
giving computed spectra for 3a /4a and 3b/4b (see Sup-
porting Information). The calculated extinction coef-
ficients for 3a /4a and 3b/4b were 21 300 M-1 cm-1 (λmax
258 nm) and 20 300 M-1 cm-1 (λmax 251 nm), respectively.
Other important physicochemical properties of caged
compounds are their aqueous solubility and stability and
the rate and efficiency of release of the bioeffector species
upon flash photolysis. The limit of aqueous solubility was
not established, in part because of limited amounts of
the compounds, but dipotassium salts of all the com-
pounds were easily soluble to at least 55 mM concentra-
tion.
Resu lts a n d Discu ssion
Synthesis of the reagent 8 required for coupling with
cyclic nucleotides is shown in Scheme 1. N-BOC-imino-
diacetic acid 5 was esterified (DCC-DMAP) with 2-(tri-
methylsilyl)ethanol, and the protected diester 6 was
converted to amine 7 with anhydrous HCl-dioxane.
DCC-mediated coupling of 7 with 4-(bromomethyl)-3-
nitrobenzoic acid gave the amide 8. Furuta et al.4 have
reported formation of the benzyl ester of cAMP by
treating the free acid form of the nucleotide with benzyl
bromide and Ag2O in MeCN-DMSO, but under these
conditions, the substituted benzyl bromide 8 gave only
the nitroaldehyde 9 (Chart 2) in high yield, presumably
derived by DMSO-mediated oxidation of 8.5 Related
The stabilities of 3a ,b and 4a ,b in aqueous buffers
were determined by incubating solutions at pH values
(6) Yeh, M. Y. Ch’eng-kung Ta Hsueh Hsueh Pao 1979, 14, 31 (Chem.
Abstr. 1980, 92, 75352). (b) Climent, M. S.; Marinas, J . M.; Sinisterra,
J . V. React. Kinet. Catal. Lett. 1987, 34, 201. (c) Kornblum, N.; J ones,
W. J .; Anderson, G. J . J . Am. Chem. Soc. 1959, 81, 4113.
(7) Kataoka, S.; Uchida, R.; Yamaji, N. Heterocycles 1991, 32, 1351.
(8) (a) Asaoka, M.; Yanagida, N.; Takei, H. Tetrahedron Lett. 1980,
21, 4611. (b) Didier, E.; Horwell; D. C.; Pritchard, M. C. Tetrahedron
1992, 48, 8471.
(4) Furuta, T.; Torigai, H.; Osawa, T.; Iwamura, M. J . Chem. Soc.,
Perkin Trans. 1 1993, 3139.
(5) (a) Kornblum, N.; Powers, J . W.; Anderson, G. J .; J ones, W. J .;
Larson, H. O.; Levand, O.; Weaver, W. M. J . Am. Chem. Soc. 1957,
79, 6562. (b) Nace, H. R.; Monagle, J . J . J . Org. Chem. 1959, 24, 1792.
(9) J ansson, K.; Ahlfors, S.; Frejd, T.; Kihlberg, J .; Magnusson, G.;
Dahmen, J .; Noori, E.; Stenvall, K. J . Org. Chem. 1988, 53, 5629.

3476 J . Org. Chem., Vol. 67, No. 10, 2002
Wang et al.
Ta ble 1. Hyd r olysis Ra tes for Ca ged Cyclic Nu cleotid es
incorporate findings of recent studies by Wirz and col-
leagues11 regarding the mode of decay of aci-nitro anions.
Laser-initiated photolysis (320 nm) of 3a or 3b at 20 °C,
pH 7.0, in the presence or absence of 2 mM dithiothreitol
(DTT; added to sequester the liberated nitroso byproduct)
generated an “instantaneous” rise in 406 nm absorbance,
followed by a single-exponential decay of 400 ms half-
time, corresponding to a rate of 1.7 s-1 that is believed
to represent the rate of product release.
pseudo-first-order rate constant, h-1
compd
pH 7.00
pH 8.01
pH 9.04
3a
3b
4a
4b
2.3 × 10-4
2.0 × 10-4
6.8 × 10-4
7.0 × 10-4
3.2 × 10-4
3.0 × 10-4
9.6 × 10-4
8.7 × 10-4
8.0 × 10-4
3.4 × 10-4
1.3 × 10-3
9.4 × 10-4
Sch em e 2. Mech a n ism for P h otor elea se of Cyclic
Nu cleotid es fr om Th eir Ca ged P r ecu r sor s
Con clu sion
The properties of 3a ,b include high solubility, excellent
stability, and reasonably efficient photorelease under
appropriate conditions for intracellular applications. At
physiologically relevant pH values they should remain
localized in the cellular compartment to which they are
applied, since they will be present as biomembrane-
impermeant dianions. They share the relatively slow
rates of product release upon flash photolysis that are
common to other nitrobenzyl phosphotriesters of cyclic
nucleotides,3c but for many applications the favorable
properties of water solubility and membrane imper-
meance will likely be more significant than the slow
photorelease rate.
Exp er im en ta l Section
Gen er a l Meth od s. Caged ATP was prepared as described
previously,10 and all other reagents were obtained com-
mercially and used without further purification. Silica gel was
used for flash chromatography. Analytical reverse phase HPLC
used a 125 × 4.6 mm C18 column [mobile phase CH3CN-10
mM K phosphate, pH 5.5 (5:95 v/v)] and anion exchange HPLC
was on a 125 × 4.6 mm SAX column [mobile phase CH3CN-
10 mM K phosphate, pH 5.5 (15:85 v/v)]. Flow rates were 1.5
mL min-1 and detection was by absorbance at 254 nm.
Preparative reverse phase HPLC was on a 120 × 0.78 cm
column, with flow rate at 2 mL min-1. The composition of
mobile phases for preparative HPLC is described at relevant
points below. 1H and 13C NMR spectra were recorded in
deuteriochloroform with TMS as internal reference, unless
otherwise specified. 31P NMR spectra were referenced indi-
rectly to trimethyl phosphate, following IUPAC recommenda-
tions.12 On this scale, the chemical shift of 85% H3PO4 used
as the reference in previous work to assign stereochemistry
of cyclic nucleotide triesters3a,b is δ -3.52 ppm.13 Organic
extracts were dried over MgSO4, and solvents were removed
under reduced pressure. Buffer solutions were prepared from
solutions of appropriate acids at the specified molarities and
adjusted to the required pH by addition of concentrated KOH.
N-BOC-Im in od ia cetic Acid Bis[2-(tr im eth ylsilyl)eth yl]
Ester (6). A solution of DCC (1.07 g, 5.19 mmol) in CH2Cl2
(10 mL) was added to an ice-cold solution of N-(carboxy-
methyl)-N-[(1,1-dimethylethoxy)carbonyl]glycine14 (5) (0.55 g,
2.36 mmol), 2-(trimethylsilyl)ethanol (0.56 g, 4.75 mmol), and
DMAP (0.58 g, 4.75 mmol) in CH2Cl2 (40 mL). The mixture
was stirred at 0 °C for 1 h and then kept at room temperature
overnight and filtered. The filtrate was washed with 1 M
NaHSO4 (100 mL), saturated NaHCO3 (60 mL), and H2O (60
mL), dried, and evaporated. Flash chromatography (98:2 CH2-
Cl2-EtOAc) gave 6 as an oil (0.917 g, 90%): 1H NMR δ 4.13-
4.32 (m, 4H), 4.08 (s, 2H), 3.98 (s, 2H), 1.44 (s, 9H), 0.90-1.09
in the range 7-9 in the dark at 22 °C. Samples were
removed at time points up to 4 days and analyzed by
anion exchange HPLC. Only peaks corresponding to the
appropriate cyclic nucleotide and the expected alcohol 14
were detected. The estimated pseudo-first-order rate
constants for hydrolysis are shown in Table 1 and confirm
the expected higher stability of the axial isomers, which
have half-lives for hydrolysis in the region of 3000 h at
pH 7 and ambient temperature. This aqueous stability
is at least 5-fold better than for any previously reported
caged cyclic nucleotides. The data in Table 1 also show
that the hydrolysis rates increase only slowly with
increasing pH, presumably because of a combination of
spontaneous and base-catalyzed hydrolysis.
The quantum yield for photorelease of cyclic nucleo-
tides from the more stable axial isomers was evaluated
by comparison in mixed solution with the well-character-
ized P3-1-(2-nitrophenyl)ethyl ester of adenosine 5′-tri-
phosphate (caged ATP).10 The values determined for 3a ,b
were 0.16 and 0.24, respectively. Stoichiometric formation
of free cyclic nucleotides upon photolysis was confirmed
by RP-HPLC analysis. The rate of product release fol-
lowing flash irradiation was also determined. It is
important to recall that the 2-nitrobenzyl photochemistry
involves dark reactions after the photochemical event
that are rate limiting for release of the bioeffector.2 Thus,
the rate of release of bioactive products from 1-(2-
nitrophenyl)ethyl esters of cyclic nucleotides is controlled
by the decay rate of the light-induced aci-nitro anion
intermediate,3c which can be monitored by time-resolved
absorption spectrophotometry at 406 nm. The expected
photolysis pathway, including the aci-nitro intermediate,
is shown in Scheme 2. This mechanism is based on that
previously established for substituted 2-nitrobenzyl es-
ters of other nucleoside phosphates10 but modified to
(11) (a) Il’chev, Y. V.; Wirz, J . J . Phys. Chem A 2000, 104, 7856. (b)
Schwo¨rer, M.; Wirz, J . Helv. Chim. Acta 2001, 84, 1441.
(12) Markley, J . L.; Bax, A.; Arata, Y.; Hilbers, C. W.; Kaptein, R.;
Sykes, B. D.; Wright, P. E.; Wu¨thrich, K. Pure Appl. Chem. 1998, 70,
117.
(13) Maurer, T.; Kalbitzer, H. R. J . Magn. Reson. B 1996, 113, 177.
(14) Kurth, M. J .; Milco, L. A.; Miller, R. B. Tetrahedron 1992, 48,
1407.
(10) Walker, J . W.; Reid, G. P.; McCray, J . A.; Trentham, D. R. J .
Am. Chem. Soc. 1988, 110, 7170.

Photolabile Precursors of Cyclic Nucleotides
J . Org. Chem., Vol. 67, No. 10, 2002 3477
(m, 4H), 0.04 (s, 18H); 13C NMR δ 170.2, 170.0, 155.3, 81.1,
63.62, 63.58, 49.9, 49.3, 28.4, 17.6, 17.5, -1.30; HRMS (CI)
m/z 432.2238 (M - H), calcd for C19H38NO6Si2 432.2230.
mg) and pure 11a (17 mg). Data for 10a : 1H NMR (9:1 CDCl3-
MeOH-d4, acetone reference) δ 8.19 (d, J ) 1.5 Hz, 1H), 8.13
(s, 1H), 7.86 (s, 1H), 7.82 (d, J ) 7.9 Hz, 1H), 7.76 (dd, J ) 7.9
Hz, 1.5 Hz, 1H), 5.91 (s, 1H), 5.56 (d, J H,P ) 7.3 Hz, 2H), 5.27
(dd, J ) 8.7 Hz, 4.7 Hz, 1H), 4.75 (d, J ) 4.9 Hz, 1H), 4.59-
4.64 (m, 1H), 4.36-4.40 (m, 2H), 4.16-4.22 (m, 4H), 4.00 (s,
2H), 3.37 (s, 2H), 0.97 (t, J ) 8.7 Hz, 2H), 0.91 (t, J ) 8.7 Hz,
2H), -0.03 (s, 9H), -0.06 (s, 9H); 31P NMR (9:1 CDCl3-MeOH-
d4, TMP reference) δ -8.65; HRMS (FAB) m/z 824.2511 (M +
1), calcd for C32H47N7O13PSi2 824.2508. Data for 11a : 1H NMR
(9:1 CDCl3-MeOH-d4, acetone reference) δ 8.19 (d, J ) 1.4
Hz, 1H), 8.18 (s, 1H), 7.87 (s, 1H), 7.81 (d, J ) 8.1 Hz, 1H),
Im in od ia cetic Acid Bis[2-(Tr im eth ylsilyl)eth yl] Ester
(7). A solution of 4 M HCl in anhydrous dioxane (7 mL) was
added to 6 (0.34 g, 0.78 mmol), and the mixture was stirred
at room temperature for 1 h. The solvent was evaporated, and
the residue was dissolved in water (60 mL). The solution was
adjusted to pH 8 with 5% aqueous ammonia, saturated with
NaCl, and extracted with CH2Cl2 (3 × 60 mL). The organic
layer was washed with brine (60 mL), dried, and evaporated.
The crude product was purified on a short silica gel column
(92:8 CH2Cl2-EtOAc) to give 7 as an oil (0.25 g, 95%). In
subsequent runs the crude material was used without chro-
matography: 1H NMR δ 4.25-4.13 (m, 4H), 3.41 (s, 4H), 2.24
(s, 1H), 0.86-1.09 (m, 4H), 0.00 (s, 18H); 13C NMR δ 169.4,
60.7, 47.8, 14.9, -3.9; HRMS (CI) m/z 334.1870 (M + H), calcd
for C14H32NO4Si2 334.1856.
7.75 (dd, J ) 8.1 Hz, 1.4 Hz, 1H), 5.93 (s, 1H), 5.67 (d, J H,P
)
7.7 Hz, 2H), 5.31 (dd, J ) 8.8 Hz, 4.9 Hz, 1H), 4.70 (d, J ) 4.9
Hz, 1H), 4.63-4.68 (m, 1H), 4.48-4.53 (m, 2H), 4.15-4.22 (m,
4H), 3.99 (s, 2H), 3.37 (s, 2H), 0.97 (t, J ) 8.6 Hz, 2H), 0.91 (t,
J ) 8.6 Hz, 2H), -0.02 (s, 9H), -0.05 (s, 9H); 31P NMR (9:1
CDCl3-MeOH-d4, TMP reference) δ -6.89; HRMS (FAB) m/z
824.2511 (M + 1), calcd for C32H47N7O13PSi2 824.2508.
N-(4-(Br om om eth yl)-3-n itr oben zoyl)im in odiacetic Acid
Bis[2-(tr im eth ylsilyl)eth yl] Ester (8). A solution of 7 (130
mg, 0.39 mmol) and 4-(bromomethyl)-3-nitrobenzoic acid15 (101
mg, 0.39 mmol) in anhydrous CH2Cl2 (10 mL) at 0 °C was
treated with a solution of DCC (88.6 mg, 0.43 mmol) in CH2-
Cl2 (4 mL). The reaction was stirred at 0 °C for 30 min and
then left at room temperature overnight and filtered. The
evaporated filtrate was flash chromatographed (98:2 CH2Cl2-
EtOAc) to give 8 as an oil (168 mg, 75%): 1H NMR δ 8.12 (d,
J ) 1.8 Hz, 1H), 7.70 (dd, J ) 7.8, 1.8 Hz, 1H), 7.62 (d, J )
7.8 Hz, 1H), 4.80 (s, 2H), 4.21-4.28 (m, 6H), 4.05 (s, 2H), 0.95-
1.05 (m, 4H), 0.03 (s, 9H), 0.01 (s, 9H); 13C NMR δ 166.7, 166.4,
166.2, 145.4, 133.9, 132.2, 130.7, 129.7, 121.8, 62.2, 61.6, 49.4,
45.5, 25.6, 15.0, -3.97; HRMS (CI) m/z 574.1166, calcd for
Ad en osin e 3′,5′-Cyclic([RP ]-{4-[N,N-bis(ca r boxym eth -
yl)ca r ba m oyl]-2-n itr op h en yl}m eth yl P h osp h a te (3a ) a n d
[SP ] Isom er (4a ). TFA (1 mL) was added to 10a (107 mg, 130
µmol), and the solution was stirred at room temperature for
30 min. The TFA was removed in vacuo, the residue was
reevaporated with MeOH (×3), and solid K2HPO4 (97.4 mg)
was added to provide a 2-fold molar excess relative to car-
boxylate groups. The material was suspended in water,
adjusted to pH 6.7 with dilute KOH, whereupon the solid
dissolved. The solution was washed with CH2Cl2 and lyophi-
lized. The residue was dissolved in water (2 mL) and applied
to the preparative reverse phase HPLC column (preequili-
brated with 10 mM K phosphate, pH 5.5) and eluted with the
same buffer. After passage of 50 mL, the mobile phase was
changed to water. The product began to elute (single peak)
after 150 mL of H2O and was collected in a further 250 mL
and lyophilized to give 3a (113 µmol, 87%) that was redissolved
in water and stored at -20 °C. The elution protocol, originally
developed for purification of other caged compounds,16 enables
recovery as its potassium salt but free of extraneous ions.
Analytical RP-HPLC showed a single peak, tR 7.7 min, and
10.2 min on anion exchange HPLC: 1H NMR (D2O) δ 8.37 (d,
J ) 1.6 Hz, 1H), 8.18 (s, 1H), 8.14 (s, 1H), 7.94 (d, J ) 8.1 Hz,
1H), 7.90 (dd, J ) 8.1 Hz, 1.6 Hz, 1H), 6.18 (s, 1H), 5.68 (d,
J H,P ) 7.7 Hz, 2H), 5.08 (ddd, J ) 9.9 Hz, 5.1 Hz, J H,P ) 1.3
Hz, 1H), 4.82 (ddd, J gem ) 9.3 Hz, J vic ) 5.1 Hz, J H,P ) 22.2
Hz, 1H), 4.79 (d, J ) 5.0 Hz, 1H), 4.62 (t, J ) 9.8 Hz, 1H),
4.49 (dt, J ) 10.3 Hz, 4.8 Hz, 1H), 4.12 (s, 2H), 3.87 (s, 2H);
31P NMR (D2O, TMP reference) δ -7.65; HRMS (FAB) m/z
624.1092 (M + 1), calcd for C22H23N7O13P 624.1091.
The equatorial isomer 11a (36 mg, 44 µmol) was processed
similarly to give 4a (32 µmol, 73%). Its retention time on
analytical RP-HPLC was 8.5 min, with 9.8 min on anion
exchange HPLC: 1H NMR (D2O) δ 8.39 (d, J ) 0.8 Hz, 1H),
8.22 (s, 1H), 8.21 (s, 1H), 7.90-7.94 (m, 2H), 6.20 (s, 1H), 5.70
(d, J H,P ) 8.3 Hz, 2H), 5.26 (dd, J ) 9.0 Hz, 5.1 Hz, 1H), 4.84-
4.90 (m, 2H), 4.69 (t, J ) 10.3 Hz, 1H), 4.64 (dt, J ) 10.4 Hz,
5.4 Hz, 1H), 4.13 (s, 2H), 3.90 (s, 2H); 31P NMR (D2O, TMP
reference) δ -6.48; HRMS (FAB) m/z 624.1095 (M + 1), calcd
for C22H23N7O13P 624.1091.
Gu a n osin e 3′,5′-Cyclic([RP ]-{4-[N,N-bis(ca r boxym eth -
yl)ca r ba m oyl]-2-n itr op h en yl}m eth yl P h osp h a te (3b) a n d
[SP ] Isom er (4b). Cyclic GMP (Na+ salt) was converted to its
triethylammonium salt by ion-exchange chromatography over
DEAE cellulose eluted with a gradient of triethylammonium
bicarbonate (10-250 mM). Fractions containing the nucleotide
were rotary evaporated under reduced pressure (1 mmHg), and
residual triethylammonium bicarbonate was removed by
repeated evaporation of methanol. The nucleotide was redis-
solved in water-methanol (1:9 v/v) and stored at -20 °C until
used. An aliquot of this solution containing cGMP (0.49 mmol)
was mixed with a solution of tri-n-butylamine (0.139 mL, 0.58
C
22H35BrN2O7Si2 574.1160.
N-(4-F or m yl-3-n itr oben zoyl)im in od ia cetic Acid Bis[2-
(tr im eth ylsilyl)eth yl] Ester (9). A solution of cyclic AMP
monohydrate (20 mg, 0.056 mmol) and 8 (32.4 mg, 0.056 mmol)
in CH3CN (2 mL) and DMSO (2 mL) was stirred under N2,
and Ag2O (26.2 mg, 0.113 mmol) was added. The mixture was
stirred at 65 °C in the dark for 16 h and then filtered, and the
solids were washed with CH2Cl2. The filtrate was evaporated
and then mixed with H2O (20 mL), saturated with NaCl, and
extracted with CH2Cl2 (3 × 10 mL). The organic extract was
washed with brine (3 × 10 mL), dried, and evaporated. Flash
chromatography (95:5 CH2Cl2-EtOAc) gave 9 as a gum (25.7
mg, 90%): 1H NMR δ 10.41 (s, 1H), 8.21 (d, J ) 1.6, 1H), 7.98
(d, J ) 7.5, 1H), 7.84-7.87 (m, 1H), 4.21-4.30 (m, 6H) 4.01
(s, 2H), 0.94-1.06 (m, 4H), 0.05 (s, 9H), 0.02 (s, 9H); 13C NMR
δ 187.3, 168.6, 168.6, 168.5, 149.4, 140.2, 132.3, 132.1, 130.3,
123.3, 64.7, 64.1, 51.65, 47.85, 17.4, -1.55; HRMS (CI) m/z
510.1856, calcd for C22H34N2O8Si2 510.1854.
Ad en osin e 3′,5′-Cyclic([RP ]-{4-[N,N-b is(2-(t r im et h yl-
silyl)eth oxyca r bon ylm eth yl)ca r ba m oyl]-2-n itr op h en yl}-
m eth yl P h osp h a te (10a ) a n d [SP ] Isom er (11a ). Cyclic
AMP (free acid, 108 mg, 0.33 mmol) was suspended in EtOH
(2 mL) and treated with tri-n-butylamine (95 µL, 0.4 mmol)
in EtOH (2 mL). The mixture was evaporated to dryness,
suspended in EtOH (4 mL), and evaporated to dryness again
and then dried in vacuo overnight. The solid residue was
dissolved in N,N-dimethylacetamide (10 mL), treated with Na2-
CO3 (69.6 mg, 0.66 mmol), and stirred under N2 at 80 °C. A
solution of 8 (196 mg, 0.33 mmol) in N,N-dimethylacetamide
(1 mL) was added, and after 1 h at 80 °C the solvent was
evaporated in vacuo. The residue was suspended in acetonitrile
and filtered, and the insoluble material was washed with
EtOH. The combined filtrates were evaporated, and the
residue was redissolved in MeOH (1 mL), diluted with CH2-
Cl2 (15 mL), and filtered to remove a flocculent precipitate.
Flash chromatography of the residue (step gradient from CH2-
Cl2 to 9:1 CH2Cl2-MeOH) gave axial isomer 10a (44 mg) as
the faster-migrating product and two mixed fractions that were
enriched in the axial or equatorial isomers. These fractions
were each chromatographed again to give additional 10a (9
(16) Thirlwell, H.; Corrie, J . E. T.; Reid, G. P.; Trentham, D. R.;
Ferenczi, M. A. Biophys. J . 1994, 67, 2436.
(15) Rich, D. H.; Gurwara, S. K. J . Am. Chem. Soc. 1975, 97, 1575.

3478 J . Org. Chem., Vol. 67, No. 10, 2002
Wang et al.
mmol) in EtOH (2.4 mL), evaporated to dryness and reevapo-
rated twice from EtOH (2 × 6 mL), and then kept under
vacuum overnight. A solution of the dried residue in dry N,N-
dimethylacetamide (14 mL) was mixed with Na2CO3 (80 mg,
0.75 mmol) and stirred under N2 at 80 °C. A solution of 8 (0.425
g, 0.74 mmol) in N,N-dimethylacetamide (1.5 mL) was added,
the mixture was heated for 30 min and then cooled, and the
solvent was evaporated in vacuo. The residue was dissolved
in MeOH (6 mL), diluted with CHCl3 (120 mL), washed with
water and brine, dried, and evaporated. Flash chromatography
(91:9 CH2Cl2-MeOH) gave a pale gum (68.6 mg). This
contained a mixture of the isomers 10b and 11b that was not
further characterized but directly deprotected by TFA treat-
ment. The crude product was neutralized as potassium salts,
as described for the individual cAMP derivatives. The aqueous
solution (pH 6.5-7.0) was applied to the preparative reverse
phase HPLC column [equilibrated with CH3CN-10 mM K
phosphate, pH 5.5 (5:95 v/v)] and developed isocratically with
the same solvent. The axial isomer 3b eluted after ∼160 mL
in a volume of 110 mL followed, with essentially no overlap,
by the equatorial isomer 4b in a further 380 mL. After pH
adjustment to 6.5-7.0 with 1 M KOH, the eluates were
lyophilized and the dried residues were each dissolved in water
(3.2 mL) and separately desalted on the same preparative
HPLC column, as described above for the cyclic AMP deriva-
tives. Recoveries of the purified isomers were 0.021 and 0.013
mmol of the axial 3b and equatorial 4b isomers, respectively.
The elution patterns of the two isomers under the desalting
conditions are essentially identical. Analytical HPLC (anion
exchange) showed each isomer as a single peak of almost
identical retention time (13.1 and 12.8 min for 3b and 4b,
respectively) and reverse phase HPLC gave tR 3.0 and 5.0 min
for 3b and 4b, respectively.
2H), 4.08 (s, 2H), 2.19 (s, 3H); HRMS (FAB) m/z 355.0755 (M
+ 1), calcd for C14H15N2O9 355.0778. A portion was converted
to the alcohol 14 by brief alkaline hydrolysis and isolated by
anion-exchange chromatography as the sodium salt. The
product had 1H NMR (D2O; acetone reference) δ 8.28 (d, J )
1.5 Hz, 1H), 7.87 (d, J ) 8.0 Hz, 1H), 7.84 (dd, J ) 8.0 Hz, 1.5
Hz, 1H), 5.03 (s, 2H), 4.11 (s, 2H), 3.88 (s, 2H). On anion
exchange HPLC it had tR 13.1 min, coincident with the axial
isomer 3b of caged cGMP.
Hyd r olytic Sta bility of Ca ged Cyclic Nu cleotid es.
Solutions of 3a ,b and 4a ,b (each 50 µM) were prepared in 71
mM aqueous buffers of the following compositions: K phos-
phate, pH 7.0 and pH 8.0, and K CHES, pH 9.0. The solutions
were incubated at 22 °C, and at recorded times, aliquots (5
µL) were withdrawn and analyzed by anion exchange HPLC.
Retention time data for the caged cyclic nucleotides and the
hydrolysis product 14 are given above: values for cAMP and
cGMP were 1.9 and 2.3 min, respectively. The proportions of
free and caged nucleotide were estimated from the peak areas,
normalized for differences in ꢀ254, and the pseudo-first-order
rate constants for the disappearance of caged nucleotide were
determined by linear regression analysis of ln(% caged) vs
time. No peaks for products other than the alcohol 14 and the
cyclic nucleotides were detected.
P h otolytic P r op er ties of Ca ged Cyclic Nu cleotid es. (a )
P r od u ct Qu a n tu m Yield (Qp). Solutions containing caged
ATP and caged cNMP (each ∼40 µM) and 1 mM DTT were
prepared in 50 mM K phosphate, pH 7.0. Aliquots (400 µL)
were irradiated in a 1-cm path-length quartz cuvette, using
light from a 100 W xenon arc lamp that passed through a band-
pass filter before illuminating the cell. Portions (50 µL) were
removed for analysis at time zero and after total irradiation
times of 2, 4, 8, 16, 32, and 64 s for replicate analysis of caged
and uncaged cNMP by anion exchange HPLC using the
conditions described above and for caged ATP by the same
procedure but by employing 100 mM K phosphate rather than
10 mM in the HPLC mobile phase. The Qp value for the
photorelease of cNMP was calculated from the known Qp (0.63)
for caged ATP10 and the ratio of the slopes of the linear
regression plots of ln(% caged nucleotide) vs total time of
irradiation.
(b) Stoich iom etr y of P r od u ct F or m a tion . Solutions of
3a or 3b (each 0.50 mM) in 10 mM K phosphate, pH 7.0, with
DTT (2 mM) were irradiated in 0.1 cm path-length quartz
cuvettes in a photochemical reactor (16 × 350 nm lamps) for
3-5 min and analyzed by anion exchange HPLC. Conversions
of starting material were ∼30% under these conditions, and
the only nucleotide products observed were the respective free
cyclic nucleotides. Measured yields of each free cyclic nucleo-
tide were in the range 102-105% of that expected from the
amount of starting material consumed.
1
The axial isomer 3b had H NMR (D2O; acetone reference)
δ 8.37 (d, J ) 1.5 Hz, 1H), 7.94 (d, J ) 8.1 Hz, 1H), 7.90 (dd,
J ) 8.1 Hz, 1.5 Hz, 1H), 7.86 (s, 1H), 6.03 (s, 1H), 5.69 (d of
AB quartet, J gem ) 13.6 Hz, J H,P ) 6.7 Hz, 2H), 5.18 (dd, J )
10.0 Hz, 5.2 Hz, 1H), 4.83 (d, J ) 5.5 Hz, 1H), 4.65 (t, J ) 10.0
Hz, 1H), 4.45 (dt, J ) 10.4 Hz, 4.9 Hz, 1H), 4.13 (s, 2H), 3.89
(s, 2H). Signals for one further proton were obscured by the
HOD peak. 31P NMR (D2O; TMP reference): -7.66. HRMS
(FAB): m/z 662.0885 (M + 2H + Na); calcd for C22H20N7O14
P
1
+ 2H + Na, 662.0860. The equatorial isomer 4b had H NMR
(D2O, acetone reference) δ 8.39 (d, J ) 1.5 Hz, 1H), 7.93 (d, J
) 8.2 Hz, 1H), 7.91 (dd, J ) 8.2 Hz, 1.5 Hz, 1H), 7.88 (s, 1H),
6.04 (s, 1H), 5.71 (d, J H,P ) 8.5 Hz, 2H), 5.35 (dd, J ) 9.9 Hz,
5.2 Hz, 1H), 4.85 (d, J ) 5.5 Hz, 1H), 4.83 (ddd, J H,P ) 22 Hz,
J ) 12.1 Hz, 6.5 Hz, 1H), 4.64 (m, 1H), 4.55 (dt, J ) 10.1, 5.5
Hz, 1H), 4.12 (s, 2H), 3.89 (s, 2H). 31P NMR (D2O; TMP
reference): -6.24. HRMS (FAB): m/z 678.0585 (M + 2H + K);
calcd for C22H20N7O14P + 2H + K, 678.0599.
(c) Ra tes of P r od u ct Relea se. Solutions of 3a or 3b (∼0.3
mM) in 50 mM K phosphate, pH 7.0, ( DTT (2 or 11 mM)
were flash irradiated in an absorption spectrophotometer
linked to a dye laser (320 nm). Details of the apparatus have
been described previously.10 Absorption transients were moni-
tored at 406 nm.
N-(4-(Acet oxym et h yl)-3-n it r ob en zoyl)im in od ia cet ic
Acid Bis[2-(tr im eth ylsilyl)eth yl] Ester (12). Na2CO3 (8.5
mg, 0.08 mmol) was added to a solution of acetic acid (3.8 µL,
0.067 mmol) in DMF (1 mL), followed by a solution of 8 in
DMF (0.5 mL), and the mixture was stirred at room temper-
ature overnight. The solution was diluted with brine and
extracted with EtOAc (3 × 10 mL). The combined organic
solution was washed with brine (×3), dried, and evaporated.
Flash chromatography (95:5 CH2Cl2-EtOAc) gave 12 as an
oil, 35.2 mg (95%): 1H NMR δ 8.20 (d, J ) 1.6 Hz, 1H), 7.75
(dd, J ) 8.1 Hz, 1.6 Hz, 1H), 7.64 (d, J ) 8.1 Hz, 1H), 5.51 (s,
2H), 4.21-4.28 (m, 6H), 4.04 (s, 2H), 2.16 (s, 3H), 0.95-1.05
(m, 4H), 0.04 (s, 9H), 0.01 (s, 9H); 13C NMR δ 170.4, 169.6,
169.1, 168.9, 147.5, 135.8, 134.5, 132.45, 129.7, 124.05, 64.8,
64.3, 62.8, 52.1, 48.2, 21.0, 17.7, -1.3.
N-(4-(Acet oxym et h yl)-3-n it r ob en zoyl)im in od ia cet ic
Acid (13). The diester 12 (136 mg, 0.24 mmol) was dissolved
in TFA (2.25 mL) and kept for 0.5 h at room temperature. TFA
was evaporated, and the residue was triturated with Et2O and
stored at -20 °C. The solid material was filtered out, resus-
pended in boiling Et2O (10 mL), and filtered out again to give
13 as a colorless solid (70 mg, 82%), mp 56-57 °C: 1H NMR
(methanol-d4) δ 8.25 (d, J ) 1.2 Hz, 1H), 7.81 (dd, J ) 7.9 Hz,
1.2 Hz, 1H), 7.68 (d, J ) 7.9 Hz, 1H), 5.53 (s, 2H), 4.30 (s,
Ack n ow led gm en t. We are grateful to Drs. V. R. N.
Munasinghe and G. Kelly for recording NMR spectra,
to Ms. Carolyn Coghill for technical assistance, and to
Dr. D. R. Trentham for the flash photolysis data. We
thank the MRC Biomedical NMR Centre for access to
facilities and Drs. K. Welham and S. L. Mullen for
HRMS data. This work was supported by the College
of Veterinary Medicine of Cornell University, the Medi-
cal Research Council in the U.K., and in part by NIH
Grant 2P01 HL 19242-24.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra for
compounds 3a ,b, 4a ,b, 6-9, 10a , 11a , and 12-14 and com-
puted UV spectra for 3a ,b and 4a ,b. This material is available
free of charge via the Internet at http://pubs.acs.org.
J O020040G