Deactivation behavior and excited-state properties of (coumarin-4-yl)methyl derivatives. 2. Photocleavage of selected (coumarin-4-yl)methyl-caged adenosine cyclic 3′,5′-monophosphates with fluorescence enhancement

Article abstract of DOI:10.1021/jo010692p

A series of axial and equatorial diastereomers of (coumarin-4-yl)methyl-caged adenosine cyclic 3′,5′-monophosphates (cAMPs), 1-6, having methoxy, dialkylamino, or no substituent in the 6- and/or 7-positions, and their corresponding 4-(hydroxymethyl)coumarin photoproducts 7-12 have been synthesized. The photochemical and UV/vis spectroscopical properties (absorption and fluorescence) of 1-6 and 7-12 have been examined in methanol/aqueous HEPES buffer solution. Donor substitution in the 6-position causes a strong bathochromic shift of the long-wavelength absorption band, whereas substitution in the 7-position leads only to a weak red shift. The photochemical cleavage of the caged cAMPs was investigated, and the photoproducts were analyzed. Photochemical quantum yields, fluorescence quantum yields, and lifetimes of the excited singlet states were determined. The highest values of photochemical quantum yields (photo-S_N1 mechanism) were obtained with caged cAMPs having a donor substituent in the 7-position of the coumarin moiety, caused by electronic stabilization of the intermediately formed coumarinylmethyl cation. With donor substitution in the 6-position, the resulting moderate electronic stabilization of the coumarinylmethyl cation is overcompensated by the strong bathochromic shift, reducing the energy gap between the excited-state S₁ and the corresponding coumarinylmethyl cation. The rate constant for the ester cleavage and liberation of cAMP is about 10⁹ s^-1, estimated for the axial isomer of 6 by analysis of the fluorescence increase of the alcohol 12 formed upon laser pulse photolysis.

Full text of DOI:10.1021/jo010692p

J . Org. Chem. 2002, 67, 703-710
703
Dea ctiva tion Beh a vior a n d Excited -Sta te P r op er ties of
(Cou m a r in -4-yl)m eth yl Der iva tives. 2. P h otoclea va ge of Selected
(Cou m a r in -4-yl)m eth yl-Ca ged Ad en osin e Cyclic
3′,5′-Mon op h osp h a tes w ith F lu or escen ce En h a n cem en t
Torsten Eckardt,† Volker Hagen,† Bjo¨rn Schade,† Reinhardt Schmidt,‡
Claude Schweitzer,‡ and J u¨rgen Bendig*,§
Institute of Molecular Pharmacology, Robert-Ro¨ssle-Strasse 10, D-13125 Berlin, Germany, Institute of
Physical and Theoretical Chemistry, J ohann-Wolfgang-Goethe University Frankfurt/ Main, D-60439
Frankfurt/ Main, Germany, and Institute of Chemistry, Humboldt University Berlin, Hessische Strasse
1-2, D-10115 Berlin, Germany
juergen)bendig@chemie.hu-berlin.de
Received J uly 10, 2001
A series of axial and equatorial diastereomers of (coumarin-4-yl)methyl-caged adenosine cyclic 3′,5′-
monophosphates (cAMPs), 1-6, having methoxy, dialkylamino, or no substituent in the 6- and/or
7-positions, and their corresponding 4-(hydroxymethyl)coumarin photoproducts 7-12 have been
synthesized. The photochemical and UV/vis spectroscopical properties (absorption and fluorescence)
of 1-6 and 7-12 have been examined in methanol/aqueous HEPES buffer solution. Donor
substitution in the 6-position causes a strong bathochromic shift of the long-wavelength absorption
band, whereas substitution in the 7-position leads only to a weak red shift. The photochemical
cleavage of the caged cAMPs was investigated, and the photoproducts were analyzed. Photochemical
quantum yields, fluorescence quantum yields, and lifetimes of the excited singlet states were
determined. The highest values of photochemical quantum yields (photo-SN1 mechanism) were
obtained with caged cAMPs having a donor substituent in the 7-position of the coumarin moiety,
caused by electronic stabilization of the intermediately formed coumarinylmethyl cation. With donor
substitution in the 6-position, the resulting moderate electronic stabilization of the coumarinylmethyl
cation is overcompensated by the strong bathochromic shift, reducing the energy gap between the
excited-state S1 and the corresponding coumarinylmethyl cation. The rate constant for the ester
cleavage and liberation of cAMP is about 109 s-1, estimated for the axial isomer of 6 by analysis of
the fluorescence increase of the alcohol 12 formed upon laser pulse photolysis.
In tr od u ction
Several investigators have successfully employed caged
adenosine cyclic 3′,5′-monophoshates (cAMPs) to study
cAMP-dependent cellular processes.2-4 Recently Furuta
et al. introduced (7-methoxycoumarin-4-yl)methyl-caged
cAMP (7-MCM-caged cAMP) and described its favorable
properties such as a long half-life in the dark in physi-
ological buffer solutions and its relatively high efficiency
of photorelease.5 Using time-resolved fluorescence mea-
surements, we recently found that the analogues 7-MCM-
caged 8-Br-cAMP and 7-MCM-caged 8-Br-cGMP released
the corresponding cyclic nucleotides (8-Br-cAMP, 8-Br-
cGMP) rapidly within a few nanoseconds.6 The photo-
cleavage (Scheme 1) was shown to proceed similarly to
that of naphthylalkyl7,8 and benzyl9 phosphates and
involves heterolysis of the C-O ester bond (solvent-
The controlled photochemical release of bioactive ef-
fector molecules from masked inactive derivatives (caged
compounds) represents an exceptional method for inves-
tigating the mechanisms and kinetics of biomolecular
processes inside living cells.1,2 This technique allows the
generation of instantaneous concentration increases of
the biomolecule in the vicinity of activity, and the
biological response is triggered almost without delay.
* To whom correspondence should be addressed.
† Institute of Molecular Pharmacology.
‡ J ohann-Wolfgang-Goethe University Frankfurt/Main.
§ Humboldt University Berlin.
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10.1021/jo010692p CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/15/2002

704 J . Org. Chem., Vol. 67, No. 3, 2002
Eckardt et al.
Sch em e 1. Mech a n ism of th e P h otolysis of Ca ged
Sch em e 2. P h otolysis of th e Axia l a n d Equ a tor ia l
Isom er s of (Cou m a r in -4-yl)m eth yl Ester s of cAMP
(1-6), Liber a tin g th e (Cou m a r in -4-yl)Meth yl
Alcoh ols 7-12 a n d cAMP
cAMP s 1-6
assisted photoheterolysis) and subsequent ion-pair sepa-
ration by the polar solvent (formation of cAMP), followed
by trapping of the coumarinylmethyl carbocation by the
solvent (hydroxylation).10 This ionic mechanism is incon-
sistent with the radical pathway postulated by Furuta11
for (7-methoxycoumarin-4-yl)methyl diethyl phosphate.
Furthermore, we reported that 7-MCM-caged cyclic
nucleotides are only weakly fluorescent, whereas the
photoproduct 4-(hydroxymethyl)-7-methoxycoumarin (7-
MCM-OH) shows strong fluorescence, which should
facilitate monitoring of the release process.6,10,12
In this paper we investigate the influence of substit-
uents in the 6- and 7-positions of the coumarin caging
group on (i) the spectroscopic properties (absorption and
fluorescence), (ii) the photophysical deactivation behavior
(fluorescence ability), and (iii) the photochemical reac-
tions of caged cAMPs.
Substituents influence the absorption and fluorescence
band positions of coumarin derivatives.13,14 Electron-
donating substituents in the 6- and/or 7-positions and
electron-withdrawing substituents in the 3-position of the
4-methylcoumarin moiety cause a significant bathochro-
mic shift of the S0-S1 transition. Most of the substituted
coumarins are characterized by large fluorescence quan-
tum yields. In the absence of heavy atoms15 and for the
6- and 8-bromo-substituted coumarins,16 the triplet popu-
lation is negligible and fluorescence competes with the
nonradiative deactivation (internal conversion).10 The
weak fluorescence is explained by an accelerated internal
conversion from the excited state caused by mixing of the
ππ* and the nπ* states.10,14 Additionally, the low fluo-
rescence quantum yields of some donor-acceptor-sub-
stituted coumarins are explained by a nonradiative
deactivation pathway via a twisted intramolecular charge-
transfer (TICT) state.16 Surprisingly, the influence of
coumarin structure on the efficiency of the photochemical
bond cleavage has not been investigated so far.
Photoactivation of the (coumarin-4-yl)methyl esters of
cAMP (1-6) leads to the corresponding 4-(hydroxymeth-
yl)coumarins 7-12, which are liberated during photolysis
of the phototrigger compounds (Scheme 2). To study the
influence of the electronic properties of donor substitu-
ents on the spectroscopic characteristics and photochemi-
cal efficiency, we synthesized the axial and equatorial
isomers of (coumarin-4-yl)methyl esters of cAMP (1-6)
(Scheme 3). 6 was already described in connection with
another series of caged cAMPs.13i In addition, the corre-
sponding 4-(hydroxymethyl)coumarins 7-12 were pre-
pared to investigate the photophysical deactivation be-
havior (Table 1). Furthermore, the phototriggers 2-4 and
their corresponding alcohols 8-10 were selected to
determine the influence of the number and position of
the substituents. Our final goal was to design suitable
(coumarin-4-yl)methyl-caged cAMPs with strong long-
wavelength absorption bands, high photochemical quan-
tum yields, and strong fluorescence enhancement during
photolysis.
(7) (a) Itoh, Y.; Gouki, M.; Goshima, T.; Hachimori, A.; Kojima, M.;
Karatsu, T. J . Photochem. Photobiol., A 1998, 117, 91-98. (b) Pincock,
J . A. Acc. Chem. Res. 1997, 30, 43-49. (c) Arnold, B.; Donald, L.;
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Am. Chem. Soc. 1994, 116, 3337-3346.
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Eckardt, T.; Bendig, J . J . Org. Chem. 1999, 64, 9109-9117.
(11) Furuta, T.; Wang, S. S.-H.; Dantzker, J . L.; Dore, T. M.; Bybee,
W. J .; Callaway, E. M.; Denk, W.; Tsien, R. Y. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 1193-1200.
(12) Bendig, J .; Helm, S.; Hagen, V. J . Fluoresc. 1997, 7, 357-361.
(13) (a) Ito, K.; Maruyama, J . Chem. Pharm. Bull. 1983, 31, 3014-
3023. (b) Giri, R.; Rathi, S. S.; Machwe, M. K.; Murti, V. V. S.
Spectrochim. Acta 1988, 44A, 805-807. (c) Arbeola, T. L.; Arbeola, F.
L.; Tapia, M. J .; Arbeola, I. L. J . Phys. Chem. 1993, 97, 4704-4707.
(d) Rechthaler, K.; Ko¨hler, G. Chem. Phys. 1994, 189, 99-116. (e)
Kaholek, M.; Hrdlovic, P. J . Photochem. Photobiol., A 1997, 108, 283-
288. (f) Raju, B. B.; Eliasson, B. J . Photochem. Photobiol., A 1998, 116,
135-142. (g) Fabian, W. M. F.; Niederreiter, K. S.; Uray, G.; Stadl-
bauer, W. J . Mol. Struct. 1999, 477, 209-220. (h) J oshi, H. S.;
J amshidi, R.; Tor, Y. Angew. Chem. 1999, 111, 2888-2891. (i) Hagen,
V.; Bendig, J .; Frings, S.; Eckardt, T.; Helm, S.; Reuter, D.; Kaupp, U.
B. Angew. Chem., Int. Ed. 2001, 40, 1046-1048.
(15) Li, L.-D.; Yang, S.-Z. Anal. Chim. Acta 1994, 296, 99-105.
(16) Corrie, J . E. T.; Munasinghe, V. R. N.; Rettig, W. J . Heterocycl.
Chem. 2000, 37, 1447-1455.
(14) Seixas de Melo, J . S.; Becker, R. S.; Macanita, A. L. J . Phys.
Chem. 1994, 98, 6054-6058.

Properties of (Coumarin-4-yl)methyl Derivatives
J . Org. Chem., Vol. 67, No. 3, 2002 705
Sch em e 3. Syn th esis of th e Axia l a n d Equ a tor ia l
Isom er s of (Cou m a r in -4-yl)m eth yl Ester s of cAMP
(1-6)
(Scheme 3). All synthetic procedures resulted in diaster-
eomeric mixtures of the caged compounds. The obtained
mixtures of 1-6 were separated into the pure diastere-
omers by RP-HPLC on a preparative scale. The axial
isomers eluted faster than the equatorial isomers. The
isomeric species were assigned by 31P NMR as reported
for the axial and equatorial isomers of known cAMP and
cGMP esters.18 The 31P NMR signals at high field
(between δ -5.1 and δ -4.0) correspond to the axial
isomers of 1-6 and those at low field (between δ -3.6
and δ -4.0) to the respective equatorial isomers. 4-(Hy-
droxymethyl)-7-methoxycoumarin (9) was prepared ac-
cording to Sehgal and Seshadri via hydrolysis of the
corresponding (coumarin-4-yl)methyl acetate.19 The al-
cohol 7, previously synthesized by Dutta et al., was
obtained by reduction of the corresponding aldehyde with
sodium borohydride.20 The unknown (hydroxymethyl)-
coumarins 8 and 10 were synthesized in analogous
fashion. The procedure of Sehgal and Seshadri failed for
the synthesis of both 7-(dialkylamino)-4-(hydroxymethyl)-
coumarins 11 and 12. These compounds were synthesized
via sodium borohydride reduction of the corresponding
7-(dialkylamino)-4-formylcoumarins,20 in contrast to the
method previously described by Kalmykowa et al.21 for
7-(diethylamino)-4-(hydroxymethyl)coumarin (12).
UV/Vis Absor p tion a n d F lu or escen ce Sp ectr a .
The absorption spectra of the caged compounds 1-6 are
characterized by two intensive absorption bands in the
region of λ ) 310-400 nm and around λ ) 260 nm
(Figure 1). The caged compounds are bichromophoric
systems, and the absorption spectra reflect the superim-
position of individual chromophores, with the long-
wavelength absorption band originating from the cou-
marin chromophore and the short-wavelength absorption
mainly caused by the purine base.12 The long-wavelength
absorption band properties of the caged compounds 1-6
are very similar to those of the corresponding alcohols
7-12 (Table 2). Therefore, the general influence of the
coumarin structure on the long-wavelength absorption
is discussed concerning the spectral properties of the
alcohols 7-12.
The long-wavelength absorption band maximum is
related to the ππ* transition. The nπ* state is very close
to the ππ* state,10,14 but the corresponding transition is
not observable because of the low transition probability.
In accordance, for the coumarin derivative 7 our molec-
ular orbital calculations (ZINDO/S-CI) predict the nπ*
transition to be the lowest in energy (Figure 2). S0 f S1-
(nπ*) is mainly characterized by the transitions HOMO
- 2 f LUMO (coefficient 0.51) and HOMO - 2 f LUMO
+ 2 (coefficient 0.36). The specific symmetry of the n
orbital HOMO - 2 (Figure 3a) illustrates the nπ*
character of S1 most clearly. The weak oscillator strength
(f ) 0.0004) is also in accordance with an nπ* transition.
S0 f S2(ππ*) is dominantly characterized by the transi-
tion HOMO f LUMO (coefficient 0.59). The HOMO and
LUMO have typical π symmetry (Figure 3b). The calcu-
lated oscillator strength is f ) 0.22, and thus, the ππ*
absorption band completely masks the nπ* transition.
Ta ble 1. P h otop h ysica l Da ta of th e
(Cou m a r in -4-yl)m eth yl Alcoh ols 7-12 in MeOH/HEP ES,
1:4
compound
CM-OH (7)
6-MCM-OH (8)
7-MCM-OH (9)
DMCM-OH (10)
DMACM-OH (11)
DEACM-OH (12)
τf/ns
τfn/ns
kf/(108 s-1
)
<0.4
2.67
3.5
4.9
3.42
3.35
<80
16.7
5.4
>0.125
0.6
1.86
1.20
1.77
2.03
8.3
5.66
4.93
Resu lts a n d Discu ssion
Syn th esis. The caged cAMPs 1-4 were synthesized
by two different methods, involving substitution of the
respective 4-(bromomethyl)coumarins using either the
tetra-n-butylammonium salt of cAMP following our re-
cently developed approach6,10 (Scheme 3) or silver(I) oxide
activated cAMP as described by Furuta et al.5,17 Among
the advantages of the tetra-n-butylammonium salt pro-
cedure are higher product yields, shorter reaction times,
larger diastereomeric excess of the axial isomers of caged
cAMPs 1-4, and facilitated workup in comparison with
the silver(I) oxide route.10 Contrary to the preparation
of 1-4, attempts to obtain (7-(dialkylamino)coumarin-4-
yl)methyl-caged cAMPs 5 and 6 under these reaction
conditions failed. These caged cAMP derivatives were
therefore synthesized by alkylation of the free acid of
cAMP with 4-(diazomethyl)-7-(dimethylamino)coumarin
(17) and 4-(diazomethyl)-7-(diethylamino)coumarin (18)
(18) Engels, J .; Schlaeger, E. J . J . Med. Chem. 1977, 20, 907-911.
Engels, J . J . Bioorg. Chem. 1979, 8, 9-16.
(19) Sehgal, J . M.; Seshadri, T. R. J . Sci. Ind. Res. 1953, 12B, 346-
349.
(20) Dutta, L. N.; Bhattacharyya, M.; Sarkar, A. K. Can. J . Chem.
1995, 73, 1556-1562.
(21) Kalmykova, E. A.; Kuznetsova, N. A.; Kaliya, O. L.; Tavrizova,
M. A. J . Gen. Chem. USSR 1991, 61, 911-916.
(17) Furuta, T.; Torigai, H.; Osawa, T.; Iwamura, M. J . Chem. Soc.,
Perkin Trans. 1 1993, 3139-3142.

706 J . Org. Chem., Vol. 67, No. 3, 2002
Eckardt et al.
F igu r e 1. UV/vis spectra of the axial isomers of the (coumarin-4-yl)methyl-caged cAMPs in MeOH/HEPES, 1:4 (an asterisk
indicates MeOH/HEPES, 1:1).
Ta ble 2. P r op er ties of th e In vestiga ted
Cou m a r in ylm eth yl-Ca ged cAMP s 1-6 a n d th e
4-(Hyd r oxym eth yl)cou m a r in s 7-12 in MeOH/HEP ES, 1:4a
λabsmax/nm
compound
(ꢀ/M-1 cm-1
)
λfmax/nm æchem
æf
CM-cAMP (1)
ax 314 (5900)
eq 310 (5500)
ax 346 (4500)
eq 345 (4200)
ax 328 (13200)
eq 325 (13300)
ax 349 (11000)
eq 346 (11500)
396
0.085 0.0005
0.055 0.0005
0.03 0.008
0.02 0.0085
0.13 0.030
0.07 0.040
0.04 0.021
0.04 0.023
0.28 0.0085
0.26 0.0070
0.21 0.0055
0.23 0.0060
0.005
392
6-MCM-cAMP (2)
7-MCM-cAMP (3)
DMCM-cAMP (4)
450
452
400.5
400.5
444
443
DMACM-cAMP (5)a ax 394 (17200)
eq 387 (16100)
482
480
DEACM-cAMP (6)a ax 402 (18600)
eq 396 (20200)
483
485
F igu r e 2. Energies of the lowest excited singlet states of 7-10
based on ZINDO/S-CI calculations.
CM-OH (7)
310 (5100)
337 (4800)
317 (13300)
341 (11700)
378 (17800)
387 (20900)
397
6-MCM-OH (8)
7-MCM-OH (9)
DMCM-OH (10)
DMACM-OH (11)
DEACM-OH (12)
465.5
394
0.16
0.65
437.5
491
484
0.59
is similar to that of 8. The effect of the substituent in
the 6-position dominates, but the extinction coefficient
is about 2 times higher.
0.21
0.082
a
In MeOH/HEPES, 1:1.
The spectroscopic properties of the 7-dialkylamino-
substituted compounds 11 and 12 are qualitatively
similar to those of 9, but the stronger donor ability of
the dimethylamino and the diethylamino groups causes
a red shift up to the visible spectral region (Table 2)
combined with increasing extinction coefficients.
The fluorescence spectra of the caged compounds 1-6
and the alcohols 7-12 (Figure 4) correspond to the
respective absorption spectra. The Stokes shifts vary from
75.5 nm (equatorial isomer of 3) to 107 nm (equatorial
isomer of 2) (Table 2). The integral fluorescence intensi-
ties of the spectra shown in Figure 4 are equivalent to
the fluorescence quantum yields. Generally speaking, the
absorption and fluorescence spectra of the axial and
equatorial isomers of 1-6 are very similar, and no
indications of a ground-state interaction between the
coumarin moiety and the purine chromophore were
observed.
Because of the dominance of the HOMO f LUMO
transition, the influence of substituents on the long-
wavelength absorption band (S0 f S2) is described
approximately by structural influences on the HOMO and
LUMO. Figure 3b shows the 2D contour orbital plotting
(orbital squared) of the HOMO and LUMO.
To compare the spectroscopic consequences of 6- and
7-methoxy substitution on the coumarin moiety, it is
necessary to compare the respective MO coefficients. The
MO coefficients of the HOMO and LUMO differ much
more strongly at the 6- than at the 7-position, and
consequently the HOMO-LUMO energy difference, which
approximately corresponds to the long-wavelength exci-
tation energy, decreases more strongly in the case of
6-methoxy substitution as compared to 7-methoxy sub-
stitution (8 and 9 in Figure 2). However, the influence
of the methoxy substituent on the energy of the nπ* state
is almost negligible; therefore, a state inversion takes
place in the case of methoxy substitution for both 8 and
9.
Dea ctiva tion P r op er ties a n d P h otoch em istr y.
The ability of the caged compounds to fluoresce is
significantly lowered (Table 2). Both photochemical reac-
tion (ester cleavage) and nonradiative deactivation (in-
ternal conversion) represent the main deactivation path-
After 6,7-dimethoxy substitution (10) the bathochromic
shift of the long-wavelength absorption band (Figure 1)

Properties of (Coumarin-4-yl)methyl Derivatives
J . Org. Chem., Vol. 67, No. 3, 2002 707
F igu r e 3. 2D contour orbital plotting (orbital squared) for the mainly participating molecular orbitals of 7: (a) S0 f S1 transition
(HOMO - 2 f LUMO and HOMO - 2 f LUMO + 2), (b) S0 f S2 transition (HOMO f LUMO).
F igu r e 4. Fluorescence spectra of the photochemically liber-
F igu r e 5. Fluorescence decay curves of 6 and of the liberated
ated coumarinylmethyl alcohols 7-12 in MeOH/HEPES, 1:4.
alcohol 12.
ways of the singlet excited states. In accordance with
tokinetic property is in agreement with its minimal
previous results,6,10,16 no indications of a significant
transition probability (extinction coefficient) according to
triplet-state population were found. Therefore, an inter-
system crossing process as shown in Figure 1 does not
the Strickler-Berg equation.22
On photolysis of the caged compounds, only the forma-
play a significant role in describing the deactivation
tion of cAMP and the corresponding coumarinylmeth-
behavior.
yl alcohol may be observed quantitatively. In analogy
The fluorescence quantum yields æf of the alcohols
7-12 are up to 2 magnitudes higher than those of the
caged compounds. The significantly lower value for the
unsubstituted coumarinylmethyl alcohol 7 is the result
of the lower energy, fluorescing nπ* state (Figure 2),
causing a decreased radiative rate constant kf. In this
to previously discussed coumarinylmethyl-caged com-
pounds,6,10 the mechanism of the photolytic ester cleavage
(photo-SN1) is described in Scheme 1. Information con-
cerning the magnitude of the rate constant of the product
formation can be gleaned from the fluorescence decay
curves of the caged compounds after single-pulse pho-
case the fluorescence cannot compete successfully with
tolysis. In Figure 5 the fluorescence decay curves of 6
the internal conversion. An estimation of kf using eq 1
and of the corresponding alcohol 12 are shown together
was not possible because the fluorescence decay time τf
with the time profile of the response function of the laser/
was too short to be measured with the equipment used.
detection system used. At low excitation intensity the
kf ) (τfn)-1 ) æf(τf)-1
(1)
fluorescence decay curve of the caged compound 6 (axial
isomer) follows the single-exponential decay law. The
time delay is relatively very close to the response func-
tion, and an exact deconvolution is not possible because
Table 1 shows the decay times τf, the radiative lifetimes
τfn, and the radiative rate constants kf for the alcohols
investigated. Apart from that of 7 discussed above, the
radiative rate constant for 8 is the smallest. This pho-
(22) Strickler, S. J .; Berg, R. A. J . Chem. Phys. 1962, 37, 814-819.

708 J . Org. Chem., Vol. 67, No. 3, 2002
Eckardt et al.
SeO2 were purchased from Sigma (Germany). 7-(Diethyl-
amino)-4-methylcoumarin, p-toluenesulfonyl hydrazide, and
triethylamine were obtained from Lancaster (Germany). 4-(Bro-
momethyl)coumarin and 4-(bromomethyl)-6-methoxycoumarin
were synthesized according to Woodruff23 via bromination of
the 4-methyl group with NBS. 4-(Diazomethyl)-7-(diethylami-
no)coumarin was prepared according to Ito et al.24 by SeO2
oxidation of 7-(diethylamino)-4-methylcoumarin to the corre-
sponding coumarin-4-carbaldehyde followed by triethylamine-
mediated Bamford-Stevens reaction of its tosylhydrazone.
4-(Diazomethyl)-7-(dimethylamino)coumarin was synthesized
following the same procedure starting from 7-(dimethylamino)-
4-methylcoumarin.
of the limited time resolution of the equipment (less than
200 ps). High-intensity pulse photolysis of the axial
isomer 6 produces a more complex decay curve. In the
first time interval (0-2 ns), the low- and high-intensity
decay curves are very similar and are determined by the
kinetics of the weak fluorescence of the caged compound.
In the second interval (2 ns and later) an additional
fluorescence signal appears, prolonging the global fluo-
rescence decay. The delay of the fluorescence decay (high-
intensity excitation) corresponds with the fluorescence
decay curve of the alcohol 12 (Figure 5). The complex
fluorescence decay behavior is the result of the formation
of the strong fluorescent alcohol during the laser pulse
and the excitation of the liberated alcohol by the same
(actinic) laser pulse. The observed delay increases with
the single-pulse intensity. Considering the pulse dura-
tion, the rate constant of the formation of the coumari-
nylmethyl alcohols 7-12 and, therefore, of the liberation
7-MCM-caged cAMP (3) and 7-(diethylamino)-4-(hydroxy-
methyl)coumarin (12) were prepared as previously described.10,25
TLC plates, silica 60-F254, were purchased from E. Merck
(Germany). Silica gel (30-60 µm) for flash chromatography
was from J . T. Baker (The Netherlands). Acetonitrile from
Riedel-deHae¨n (Germany) was HPLC grade. All other chemi-
cals and solvents were reagent grade and were used without
further purification. Water was purified with a Milli-Q system
(Millipore, Germany). For analytical HPLC a Hewlett-Packard
HP 1100 system with DAD and fluorescence detection was
used. A C18 column (Spherisorb ODS 2, 5 µm, 250 × 4 mm,
Polymer Laboratories Ltd., U.K.) was used for HPLC analysis
at a flow rate of 1 mL/min at 20 °C with an injection volume
of 20 µL. Preparative RP-HPLC using a Shimadzu LC-8A
system with UV detection (SPD-6AV) was carried out over a
PLRP-S 100-10 column (300 × 25 mm, Polymer Laboratories)
at a flow rate of 10 mL/min using a linear gradient of 5-45%
(1-5) or 15-45% (6) over 105 min; eluent A was water, and
eluent B was acetonitrile.
of the cyclic nucleotide cAMP is about 109 s-1
.
The photochemical ester cleavage of the [(dialkylami-
no)coumarinyl]methyl-caged compounds 5 and 6 is more
efficient than the photolysis of the other compounds
investigated (compare the photochemical quantum yields
in Table 2). Following the reaction pathway shown in
Scheme 1, the photochemical quantum yield is deter-
mined by (a) the ratio of the rate constants of the
competing photophysical deactivation on the depopula-
tion of the singlet excited state (fluorescence and nonra-
diative, kfn and knr) and bond heterolysis (k1) and (b) the
competition of the ion pair recombination (k0) and the
escape process (kesc). On stabilization of the intermedi-
1H NMR spectra were recorded on a Gemini 200 spectrom-
eter (Varian) using TMS as internal standard. 31P NMR
spectra were recorded using a Bruker DRX 600 spectrometer
with 85% phosphoric acid as external standard.
+
ately formed carbocation Cou-CH2 by electron donor
ESI mass spectrometry was performed on a triple quadru-
pole instrument (TSQ 700, Finnigan MAT, Germany) equipped
with an electrospray ion source (API-ESI) operating in the
positive mode with a capillary temperature of 200 °C at a
voltage of 4.5 kV.
All melting points are uncorrected.
Syn th eses. (Cou m a r in -4-yl)m eth yl Ad en osin e Cyclic
3′,5′-Mon op h osp h a te (1).
groups, the recombination (k0) is less efficient and the
escape process (kesc) whereby cAMP and the correspond-
ing alcohol are formed predominates.
The decreased photoreactivity of 2 and 4 compared
with 1 and 3 probably results from the low-lying S1 state
(compare æchem and λf in Table 2). In this case the energy
gap between the excited-state S1 and the ion pair state
[Cou-CH2+ -OH] is less exergonic, decreasing k1 and
reducing the ratio k1/(kfn + knr).
Via t h e Tet r a -n -b u t yla m m on iu m Sa lt P r oced u r e
(Meth od A). A mixture of 497 mg (2 mmol) of 13 and 606.8
mg (1 mmol) of the dihydrate of the tetra-n-butylammonium
salt of cAMP (prepared from cAMP via ion exchange with
tetra-n-butylammonium hydroxide) was refluxed in 50 mL of
acetonitrile in the dark for 5 h. The solvent was evaporated
in vacuo, and the residue was washed with 20 mL of water,
dried, and dissolved in chloroform/methanol. The mixture was
purified by flash chromatography. Elution using chloroform/
methanol (19:1 to 4:1, v/v) yielded 140 mg (28.9%) of the
diastereomeric mixture of 2 in an 85:15 ratio (axial/equatorial)
as a colorless solid after evaporation and lyophilization.
Preparative RP-HPLC permitted separation of the axial and
equatorial isomers.
Via th e Silver (I) Oxid e P r oced u r e (Meth od B). A
mixture of 197.5 mg (0.6 mmol) of cAMP and 447 mg (1.8
mmol) of 13 was stirred in 5 mL of DMSO and 30 mL of
acetonitrile in the dark. After addition of 277.7 mg (1.2 mmol)
of silver(I) oxide the resulting black suspension was stirred
at 60 °C in the dark for 45 h. The reaction mixture was filtered
and washed with chloroform. The combined filtrates were
evaporated under reduced pressure. DMSO was removed by
repeated extraction with ether. The residue was purified by
flash chromatography. Elution with chloroform/methanol (19:1
Con clu sion s
Depending on the chemical structure of the coumari-
nylmethyl caging group (R, R′), the caged cAMPs can be
excited in a wavelength region from 300 up to 450 nm.
Using specific donor substituents in defined positions, it
is possible to tune the absorbance to given excitation
wavelengths (light source). The moderate fluorescence
properties of the caged compounds in comparison with
the corresponding strongly fluorescent 4-(hydroxymeth-
yl)coumarin photoproducts allow the indirect estimation
of the amount of photolytically released cyclic nucleotides
in aqueous buffer solutions using fluorescence measure-
ments. The rate constant of the liberation of cAMP is on
the nanosecond (3, 4) or subnanosecond (5, 6) time scale,
and therefore orders of magnitude less than 1 ms (rise
time of well-known o-nitrobenzyl-caged compounds).1e,h,4c
These ultrafast jumps in the biological activity can be
used for studying fast physiological responses.
(23) Woodruff, H. In Organic Synthesis; Drake, N. L., Ed.; Wiley &
Sons: New York, 1944; Vol. 24, pp 69-72.
(24) Ito, K.; Maruyama, J . Chem. Pharm. Bull. 1983, 31 (9), 3014-
3023.
(25) Scho¨nleber, R. O.; Bendig, J .; Hagen, V.; Giese, B. Bioorg. Med.
Chem., in press.
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. Phenol, 4-hydroxyanisole, 4-(bro-
momethyl)-7-methoxycoumarin, 4-(bromomethyl)-6,7-dimethoxy-
coumarin, 7-(dimethylamino)-4-methylcoumarin, cAMP, and

Properties of (Coumarin-4-yl)methyl Derivatives
J . Org. Chem., Vol. 67, No. 3, 2002 709
to 4:1, v/v) yielded 74.5 mg (25.5%) of the diastereomeric
mixture of 1 in a 57:43 ratio (axial/equatorial) as a colorless
solid after evaporation and lyophilization. The axial and
equatorial isomers were separated from each other by pre-
parative RP-HPLC.
31P NMR (DMSO-d6) heteronuclear decoupled δ -4.99; 1H
NMR (DMSO-d6) δ 3.86 (3H, s), 3.88 (3H, s), 4.30 (1H, dt, J )
10.0 and 5.0 Hz), 4.48 (1H, t, J ) 10.0 Hz), 4.69-4,76 (2H,
m), 5.41 (1H, dd, J ) 10.0 and 5.0 Hz), 5.51 (2H, d, J ) 6.0
Hz), 6.07 (s, 1H), 6.40 (1H, d, J ) 5.0 Hz), 6.49 (1H, s), 7.15
(1H, s), 7.19 (1H, s), 7.35 (2H, s), 8.11 (1H, s), 8.35 (1H, s);
ESI MS 548.2 [M + H]+. Anal. Calcd for C22H22N5O10P·3H2O
(601.46): C, 43.93; H, 4.69; N, 11.64. Found: C, 44.10; H, 4.43;
N, 11.22.
Da ta for a xia l (cou m a r in -4-yl)m eth yl cAMP (a xia l 1):
TLC Rf 0.55 (chloroform/methanol, 5:1, v/v); 31P NMR (DMSO-
1
d6) heteronuclear decoupled δ -5.06; H NMR (DMSO-d6) δ
4.30 (1H, dt, J ) 10.0 and 5.0 Hz), 4.48 (1H, t, J ) 10.0 Hz),
4.52-4.55 (1H, m), 4.65-4.76 (2H, m), 5.39 (1H, dd, J ) 10.0
and 5.0 Hz), 5.52 (1H, d, J ) 6.0 Hz), 6.07 (1H, s), 6.40 (1H, d,
J ) 7 Hz), 6.68 (1H, s), 7.35-7.42 (3H, m), 7.48 (1H, d, J )
8.2 Hz), 7.67 (1H, t, J ) 8.3 Hz), 7.81 (1H, d, J ) 7.7 Hz), 8.10
(1H, s), 8.34 (1H, s); ESI MS m/e 488.3 [M + H]+. Anal. Calcd
for C20H18N5O8P·H2O (505.38): C, 47.53; H, 3.99; N, 13.86.
Found: C, 47.44; H, 3.80; N, 13.30.
Da t a for eq u a t or ia l (6,7-d im et h oxycou m a r in -4-yl)-
m eth yl cAMP (equ a tor ia l 4): TLC Rf 0.58 (chloroform/
methanol, 5:1, v/v); 31P NMR (DMSO-d6) heteronuclear decou-
pled δ -3.32; 1H NMR (DMSO-d6) δ 3.86 (3H, s), 3.88 (3H, s),
4.50-4.55 (2H, m), 4.71 (1H, t, J ) 4.0 Hz), 4.76-4.79 (1H,
m), 5.36-5.38 (1H, m), 5.46 (2H, d, J ) 7.0 Hz), 6.09 (1H, s),
6.38-6.39 (2H, m), 7.13 (1H, s), 7.16 (1H, s), 7.38 (2H, s), 8.20
(1H, s), 8.39 (1H, s); ESI MS 548.2 [M + H]+. Anal. Calcd for
Da t a for eq u a t or ia l (cou m a r in -4-yl)m et h yl cAMP
(equ a tor ia l 1): TLC Rf 0.53 (chloroform/methanol, 5:1, v/v);
31P NMR (DMSO-d6) heteronuclear decoupled δ -3.66; 1H
NMR (DMSO-d6) δ 4.09 (1H, dt, J ) 11.0 and 5.5 Hz), 4.31
(1H, t, J ) 11.0 Hz), 4.55-4.77 (3H, m), 5.38 (1H, dd, J )
10.0 and 5.5 Hz), 5.48 (1H, t, J ) 6.4 Hz), 6.09 (1H, s), 6.37
(1H, s), 6.57 (1H, s), 7.36 (2H, br s), 7.42-7.48 (2H, m), 7.68
(1H, t, J ) 7.8 Hz), 7.78 (1H, d, J ) 7.8 Hz), 8.26 (1H, s), 8.39
(1H, s); ESI MS m/e 488.2 [M + H]+. Anal. Calcd for
C
22H22N5O10P·4H2O (619.48): C, 42.66; H, 4.88; N, 11.31.
Found: C, 42.28; H, 4.36; N, 10.81.
[7-(Dim eth yla m in o)cou m a r in -4-yl]m eth yl Ad en osin e
Cyclic 3′,5′-Mon op h osp h a te (5).
A mixture of 114.6 mg (0.5 mmol) of 4-(diazomethyl)-7-
(dimethylamino)coumarin (17) and 164.6 mg (0.5 mmol) of the
free acid of cAMP was stirred in 10 mL of acetonitrile and 10
mL of DMSO at 60 °C in the dark for 7 h. An additional
quantity (114.6 mg, 0.5 mmol) of the diazo compound 17 was
added, and the mixture was stirred at 60 °C for a further 17
h. Acetonitrile was evaporated under reduced pressure, and
DMSO was removed by repeated extraction with ether/pentane
(1:1). The residue which contained the axial and the equatorial
isomers of 5 in a 48:52 ratio was dissolved in a small volume
of chloroform/methanol (1:1, v/v) and separated by flash
chromatography on a silica gel column. Elution with methanol/
chloroform (1:24, v/v) and methanol/chloroform (2:23, v/v) gave
fractions containing mixtures of the axial and the equatorial
isomers of 5. The fractions were dried on a rotary evaporator.
Lyophilization yielded mixtures of the two isomers of 5 (44.5
mg, 16.0%) as solids. The axial and equatorial isomers were
purified by preparative RP-HPLC.
C
20H18N5O8P·H2O (505.38): C, 47.53; H, 3.99; N, 13.86.
Found: C, 47.31; H, 3.72; N, 13.52.
(6-Meth oxycou m ar in -4-yl)m eth yl Aden osin e Cyclic 3′,5′-
Mon op h osp h a te (2). This compound was synthesized follow-
ing the same procedures previously described for 1 from 14
(537 mg, 2 mmol) and the dihydrate of the tetra-n-butylam-
monium salt of cAMP (608 mg, 1 mmol) or from cAMP (197.5
mg, 0.6 mmol), 14 (483 mg, 1.2 mmol), and silver(I) oxide
(277.7 mg, 1.2 mmol). Method A yielded 161 mg (31.3%) of the
diastereomeric mixture of 2 in an 85:15 ratio (axial/equatorial)
as a colorless solid after evaporation and lyophilization. On
the other hand, method B yielded 75 mg (25.3%, axial:
equatorial ) 55:45).
Da ta for a xia l (6-m eth oxycou m a r in -4-yl)m eth yl cAMP
(a xia l 2): TLC Rf 0.64 (chloroform/methanol, 5:1, v/v); 31P
Da t a for a xia l [7-(d im et h yla m in o)cou m a r in -4-yl]-
m eth yl cAMP (a xia l 5): TLC Rf 0.61 (chloroform/methanol,
5:1, v/v); 31P NMR (DMSO-d6) heteronuclear decoupled δ
1
NMR (DMSO-d6) heteronuclear decoupled δ -5.04; H NMR
(DMSO-d6) δ 3.83 (3H, s), 4.30 (1H, dt, J ) 10.0 and 5.0 Hz),
4.49 (1H, t, J ) 10.0 Hz), 4.70-4.75 (2H, m), 5.41 (1H, dd, J
) 10.0 and 5.0 Hz), 5.53 (2H, d, J ) 6.0 Hz), 6.07 (1H, s), 6.41
(1H, d, J ) 4.2 Hz), 6.67 (1H, s), 7.25-7.28 (2H, m), 7.35 (2H,
br s), 7.43 (1H, d, J ) 8.9 Hz), 8.09 (1H, s), 8.34 (1H, s); ESI
MS m/e 518.4 [M + H]+. Anal. Calcd for C21H20N5O9P·H2O
(535.41): C, 47.11; H, 4.14; N, 13.08. Found: C, 47.44; H, 3.76;
N, 13.26.
1
-4.96; H NMR (DMSO-d6) δ 3.02 (6H, s), 4.28 (1H, dt, J )
10.0 and 4.0 Hz), 4.42 (1H, t, J ) 10.0 Hz), 4.67-4.72 (2H,
m), 5.38 (1H, dd, J ) 9.0 and 5.0 Hz), 5.41 (2H, d, J ) 7.0 Hz),
6.03 (1H, s), 6.26 (1H, s), 6.39 (1H, d, J ) 4.0 Hz), 6.61 (1H, d,
J ) 2.0 Hz), 6.71 (1H, dd, J ) 9.0 and 2.0 Hz), 7.35 (2H, s),
7.56 (1H, d, J ) 9.0 Hz), 8.11 (1H, s), 8.34 (1H, s); ESI MS
531.3 [M + H]+. Anal. Calcd for C22H23N6O8P·1H2O (548.45):
C, 48.18; H, 4.59; N, 15.32. Found: C, 48.51; H, 4.33; N, 15.20.
Da ta for equ a tor ia l [7-(d im eth yla m in o)cou m a r in -4-yl]-
m eth yl cAMP (equ a tor ia l 5): TLC Rf 0.59 (chloroform/
methanol, 5:1, v/v); 31P NMR (DMSO-d6) heteronuclear decou-
Da ta for equ a tor ia l (6-m eth oxycou m a r in -4-yl)m eth yl
cAMP (equ a tor ia l 2): TLC Rf 0.62 (chloroform/methanol, 5:1,
v/v); 31P NMR (DMSO-d6) heteronuclear decoupled δ -3.50;
1H NMR (DMSO-d6) δ 3.86 (3H, s), 4.51-4.54 (2H, m), 4.72
(1H, t, J ) 5.0 Hz), 4.76-4.79 (1H, m), 5.37 (1H, q, J ) 5.0
Hz), 5.47-5.49 (2H, m), 6.09 (1H, s), 6.36 (1H, s), 6.56 (1H, s),
7.22 (1H, d, J ) 2.6 Hz), 7.28 (1H, dd, J ) 9.1 and 2.7 Hz),
7.36 (2H, br s), 7.42 (1H, d, J ) 9.0 Hz), 8.19 (1H, s), 8.39 (1H,
s); ESI MS m/e 518.3 [M + H]+. Anal. Calcd for C21H20N5O9P·
H2O (535.41): C, 47.11; H, 4.14; N, 13.08. Found: C, 46.94;
H, 3.85; N, 13.21.
1
pled δ -3.50; H NMR (DMSO-d6) δ 3.03 (6H, s), 4.49-4.52
(2H, m), 4.72 (1H, t, J ) 5.0 Hz), 4.73-4.77 (1H, m), 5.34-
5.38 (3H, m), 6.09 (1H, s), 6.15 (1H, s), 6.37 (1H, d, J ) 4.0
Hz), 6.61 (1H, d, J ) 2.0 Hz), 6.76 (1H, dd, J ) 9.0 and 4.0
Hz), 7.36 (2H, s), 7.52 (1H, d, J ) 9.0 Hz), 8.19 (1H, s), 8.39
(1H, s); ESI MS 531.4 [M + H]+. Anal. Calcd for C22H23N6O8P·
2H2O (566.46): C, 46.65; H, 4.80; N, 14.84. Found: C, 46.68;
H, 4.65; N, 14.37.
(6,7-Dim eth oxycou m a r in -4-yl)m eth yl Ad en osin e Cy-
clic 3′,5′-Mon op h osp h a te (4).
[7-(Dieth ylam in o)cou m ar in -4-yl]m eth yl Aden osin e Cy-
clic 3′,5′-Mon op h osp h a te (6). Following the same procedure
described above for 5, this compound was synthesized from
the free acid of cAMP (164.6 mg, 0.5 mmol) and 4-(diazo-
methyl)-7-(diethylamino)coumarin (18) (257.3 mg, 1 mmol) in
16 mL of acetonitrile and 4 mL of DMSO.
A 59 mg yield of a mixture of the two isomers of 6 was
obtained after flash chromatography and lyophilization. The
yield was 20.5%, and the isomers were formed in a 45:55 ratio
(axial/equatorial). Preparative RP-HPLC as described for 5
permitted separation of the axial form from the equatorial
form.
Following the same procedures described above for 1, this
compound was synthesized by reaction of 16 (448.6 mg, 1.5
mmol) with the dihydrate of the tetra-n-butylammonium salt
of cAMP (303.4 mg, 0.5 mmol) or from cAMP (164.6 mg, 0.5
mmol), 16 (448.6 mg, 1.5 mmol), and silver(I) oxide (231.8 mg,
1.0 mmol).
Method A yielded 175 mg (approximately 57.3%) and
method B 57 mg (approximately 18.7%) of a mixture of the
two diastereomers after flash chromatography and lyophiliza-
tion. The isomers were formed in an 85:15 ratio (axial/
equatorial) (method A) and in a 55:45 ratio (method B).
Da ta for a xia l (6,7-d im eth oxycou m a r in -4-yl)m eth yl
cAMP (a xia l 4): TLC Rf 0.59 (chloroform/methanol, 5:1, v/v);
Da ta for a xia l [7-(d ieth yla m in o)cou m a r in -4-yl]m eth yl
cAMP (a xia l 6): TLC Rf 0.86 (chloroform/methanol, 5:1, v/v);

710 J . Org. Chem., Vol. 67, No. 3, 2002
Eckardt et al.
31P NMR (DMSO-d6) heteronuclear decoupled δ -4.96; 1H
NMR (DMSO-d6) δ 1.10 (6H, t, J ) 7.0 Hz), 3.42 (4H, q, J )
7.0 Hz), 4.28 (1H, dt, J ) 10.0 and 5.0 Hz), 4.41 (1H, t, J )
10.0 Hz), 4.66-4.72 (2H, m), 5.37-5.41 (3H, m), 6.06 (1H, s),
6.22 (1H, s), 6.37 (1H, d, J ) 4.0 Hz), 6.57 (1H, d, J ) 2.0 Hz),
6.66 (1H, dd, J ) 9.0 and 2.0 Hz), 7.34 (2H, s), 7.53 (1H, d, J
) 9.0 Hz), 8.12 (1H, s), 8.33 (1H, s); ESI MS 559.3 [M + H]+.
Anal. Calcd for C24H27N6O8P·1H2O (576.50): C, 50.00; H, 5.07;
N, 14.58. Found: C, 49.70; H, 4.78; N, 14.09.
aqueous HEPES buffer solution (0.01 M HEPES/NaOH, 0.12
M NaCl, 3 mM KCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.2)/30%
acetonitrile or 20% methanol were placed in quartz cuvettes
with a path length of 1 cm and irradiated for periods ranging
from 3 to 90 s in steps of 3-10 s. Before and after each
irradiation, the absorption spectra were recorded and the
concentrations of the remaining caged compounds were mea-
sured by HPLC. The photochemical quantum yields Φchem were
determined as described6 using potassium ferrioxalate acti-
Da ta for equ a tor ia l [7-(d ieth yla m in o)cou m a r in -4-yl]-
m eth yl cAMP (equ a tor ia l 6): TLC Rf 0.84 (chloroform/
methanol, 5:1, v/v); 31P NMR (DMSO-d6) heteronuclear decou-
nometry26 according to the equation Φchem ) (dc/dt)Iabs 1 V.
-
The initial slope dc/dt of the experimentally obtained concen-
tration/irradiation time function was determined directly using
the HPLC peak areas of the caged compounds. The value of
Iabs (absorbed light intensity in moles of photons per second
at t ) 0 and at λ ) 313, 333, or 365 nm) was calculated by
multiplying the absorption factor R (taken from the UV spectra
at t ) 0 and λ ) 313, 333, or 365 nm) by the light intensity of
the HBO 500 exposure tool (determined using the actinometer
compound). The volume V of the solution irradiated was 3 mL.
F lu or escen ce Mea su r em en ts. Fluorescence spectra were
measured using an MPF-2A fluorescence spectrometer (Hita-
chi-Perkin-Elmer) combined with a correction and digitaliza-
tion unit. Solutions (10 µM) of the compounds under investi-
gation were placed in quartz cuvettes of path length 1 cm and
excited perpendicularly.
The fluorescence quantum yields were determined at 298
K by the relative method27 using quinine sulfate as a standard
(Φf ) 0.545 in 0.1 N H2SO4). The absorbance values of the
solutions of the standard and the investigated compound were
identical at the excitation wavelengths (313, 333, and 365 nm;
see above). The different refractive indices of the solutions were
taken into account.
1
pled δ -3.51; H NMR (DMSO-d6) δ 1.13 (6H, t, J ) 7.0 Hz),
3.45 (4H, q, J ) 7.0 Hz), 4.50-4.53 (2H, m), 4.71 (1H, t, J )
5.0 Hz), 4.75 (1H, d, J ) 12.0 Hz), 5.34-5.35 (3H, m), 6.08
(1H, s), 6.11 (1H, s), 6.36 (1H, d, J ) 4.0 Hz), 6.56 (1H, d, J )
2.0 Hz), 6.72 (1H, dd, J ) 9.0 and 3.0 Hz), 7.35 (2H, s), 7.49
(1H, d, J ) 9.0 Hz), 8.19 (1H, s), 8.39 (1H, s); ESI MS 559.4
[M + H]+. Anal. Calcd for C24H27N6O8P·H2O (576.50): C, 50.00;
H, 5.07; N, 14.58. Found: C, 49.89; H, 4.74; N, 14.04.
4-(Hyd r oxym eth yl)cou m a r in (7). A mixture of 1.6 g (6.7
mmol) of 13 and 5.5 g (67 mmol) of sodium acetate in 30 mL
of acetic anhydride was refluxed for 4 h. After filtration and
washing of the residue with 20 mL of boiling acetic anhydride,
the resulting filtrate was poured into ice-water after being
cooled. The resulting white precipitate of 4-(acetoxymethyl)-
coumarin was refluxed in 100 mL of a mixture of ethanol and
hydrochloric acid (1:1) for 2 h. After the reaction mixture was
cooled to room temperature, it yielded 0.8 g (71%) of 7 as a
colorless solid: mp 136 °C; NMR data available in ref 23; ESI
MS 177.2 [M + H]+. Anal. Calcd for C10H8O3 (176.17): C, 68.18;
H, 4.58. Found: C, 67.86; H, 4.56.
4-(Hyd r oxym eth yl)-6-m eth oxycou m a r in (8). Following
the same procedure described above for 7, this compound was
synthesized from 1.8 g (6.7 mmol) of 14. The yield was 590
The time-resolved fluorescence decay measurements (pulse
sampling method) were performed using a nitrogen laser (λ )
337 nm) as excitation source and a transient recorder.28 The
fluorescence detection wavelength was λf ) 405 nm (interfer-
ence filter). The fluorescence decay curves of the (photochemi-
cally stable) coumarinylmethyl alcohols were measured using
the pulse sampling method (20 pulses using the same solution).
The fluorescence decay curves of the caged compounds were
measured on single-pulse excitation. The solution was renewed
after each single-pulse photolysis.
1
mg (64%): mp 183 °C; H NMR (CDCl3) δ 3.85 (3H, s), 4.90
(2H, s), 6.64 (1H, s), 6.93 (1H, d, J ) 3 Hz), 7.11 (1H, dd, J )
9.0 and 3.0 Hz), 7.30 (1H, d, J ) 4.2 Hz); ESI MS 207.4 [M +
H]+. Anal. Calcd for C11H10O4 (206.20): C, 64.07; H, 4.89.
Found: C, 63.89; H, 4.74.
6,7-Dim eth oxy-4-(h yd r oxym eth yl)cou m a r in (10). Com-
pound 10 was synthesized as described for 7 from 2 g (6.7
mmol) of 16. The reaction yielded 1.138 g (72%) of 10: mp
188 °C;
Details of the equipment and the deconvolution procedure
of the experimental decay curve are described in ref 28. The
time resolution achieved was about 200 ps.
1H NMR (CDCl3) δ 3.90 (3H, s), 3.92 (3H, s), 4.85 (2H, s),
6.46 (1H, s), 6.82 (1H, s), 6.88 (1H, s); ESI MS 237.0 [M +
H]+. Anal. Calcd for C12H12O5 (236.22): C, 61.01; H, 5.12.
Found: C, 60.47; H, 5.01.
Molecu la r Or bita l Ca lcu la tion s. Molecular orbital cal-
culations were performed using the semiempirical method
ZINDO/S-CI, a component of the HyperChem 5.1 Pro software
package,29 considering 10 singly excited configurations (10
occupied, 10 unoccupied). Atomic coordinates for the ZINDO/S
calculations were obtained from minimum energies determined
using the molecular mechanics force field method MM+. The
HOMO/LUMO data in Figures 2 and 3 were taken from the
spectroscopy program of the software package.
7-(Dim eth yla m in o)-4-(h yd r oxym eth yl)cou m a r in (11).
A mixture of 1.77 g (8.1 mmol) of 7-(dimethylamino)coumarin-
4-carbaldehyde and 587 mg (15.5 mmol) of sodium borohydride
in 50 mL of THF and 50 mL of 2-propanol was refluxed
overnight. After decomposition of borohydride by addition of
hydrochloric acid (1 M) and following neutralization, the
organic solvents were removed in vacuo. After extraction with
chloroform, drying, and purification via flash chromatography
(elution with chloroform), 910 mg (51%) of an orange-yellow
Ack n ow led gm en t. We thank S. Helm, B. Dekowski,
and J . Lossmann for technical assistance and S. Hecht,
University of California, Berkeley, for fruitful discus-
sions. We are grateful to R. Winter for NMR, E. Krause
for MS investigations, and J . Dickson for critical reading
of the manuscript. We thank the Deutsche Forschungs-
gemeinschaft (DFG) and the Fonds der Chemischen
Industrie for financial support.
1
solid was obtained: mp 175-185 °C; H NMR (CDCl3) δ 3.03
(6H, s), 4.64 (2H, s), 5.95 (1H, s), 6.47 (1H, d, J ) 2.5 Hz),
6.60 (1H, dd, J ) 8.9 and 2.6 Hz), 7.35-7.40 (1H, m); ESI MS
220.1 [M + H]+. Anal. Calcd for C12H13NO3 (219.24): C, 65.74;
H, 5.98; N, 6.39. Found: C, 65.24; H, 5.98; N, 6.41.
UV/Vis Sp ectr oscop y, P h otolysis Exp er im en ts, a n d
Mea su r em en ts of th e P h otoch em ica l Qu a n tu m Yield s.
UV/vis spectra were recorded on a U-3410 spectrophotometer
(Hitachi, J apan). Photolysis was carried out using a high-
pressure mercury lamp (HBO 500, Oriel) with controlled light
intensity and metal interference filters (Schott, Germany) of
313 nm (1), 333 nm (2-4), and 365 nm (5, 6). The irradiated
solutions were analyzed by HPLC for quantum yield deter-
minations. Solutions (50 µM) of the caged compounds in
J O010692P
(26) Kuhn, H. J .; Braslawsky, S. E.; Schmidt, R. Pure Appl. Chem.
1989, 61, 187-210.
(27) Demas, J . N.; Crosby, G. A. J . Phys. Chem. 1971, 75, 991-999.
(28) Grewer, C.; Brauer, H.-C. J . Phys. Chem. 1993, 97, 5001-5006.
(29) Hypercube Inc., Gainsville, FL.