Coumarinylmethyl esters for ultrafast release of high concentrations of cyclic nucleotides upon one- and two-photon photolysis

Article abstract of DOI:10.1002/anie.200502411

(Chemical Equation Presented) Shedding light: Efficient activation of cyclic nucleoside monophosphates (cNMPs) can be achieved upon one- and two-photon flash photolysis of novel photolabile coumarinylmethyl esters of cAMP and cGMP (A = adenosine, G = guanosine) as well as their 8-bromosubstituted derivatives. The phototriggers show high solubility in water and permit space- and time-resolved investigations of the molecular mechanisms of cyclic nucleotide dependent processes.

Full text of DOI:10.1002/anie.200502411

Angewandte
Chemie
Photoactivatable Compounds
DOI: 10.1002/anie.200502411
Coumarinylmethyl Esters for Ultrafast Release of
High Concentrations of Cyclic Nucleotides upon
One- and Two-Photon Photolysis**
Volker Hagen,* Brigitte Dekowski, Vasilica Nache,
Reinhard Schmidt, Daniel Geißler, Dorothea Lorenz,
Jenny Eichhorst, Sandro Keller, Hiroshi Kaneko,
Klaus Benndorf, and Burkhard Wiesner
Biologically inert, photoactivatable precursors (“caged” com-
pounds) of cyclic nucleoside monophosphates (cNMPs) are
powerful tools for studying the spatiotemporal dynamics of
cyclic nucleotide dependent processes.Among these com-
pounds, (coumarin-4-yl)methyl esters of cNMPs are most
useful because they show no background bioactivity, are
stable to solvolysis, and can be photolyzed efficiently and
extremely quickly.[1,2] Recently, we introduced [7-(diethyl-
amino)coumarin-4-yl]methyl (DEACM) esters of cNMPs as
caged compounds.[3,4] Compared with other coumarinyl-
methyl-caged cNMPs, the DEACM esters photorelease
cNMPs with higher photosensitivity at long-wavelength
irradiation (up to 436 nm), thus minimizing or even prevent-
ing damage to cellular components and chromophores by
photobleaching.
Herein, we describe the development of the 7-[bis(carb-
oxymethyl)amino]-substituted coumarinylmethyl building
blocks 1 and 2 (structures in Scheme 1) for the caging of
phosphates and other functionalities.With the axial and the
equatorial diastereomers of the {7-[bis(carboxymethyl)-
amino]coumarin-4-yl}methyl (BCMACM) esters of cAMP,
cGMP, 8-Br-cAMP, and 8-Br-cGMP 3–6 (Scheme 1), we
present new variants of the DEACM-caged cNMPs, which
maintain their favorable properties and additionally have
much higher aqueous solubility by virtue of their anionic
[*] Dr. V. Hagen, B. Dekowski, D. Geißler, D. Lorenz, J. Eichhorst,
S. Keller, Dr. B. Wiesner
Forschungsinstitut für Molekulare Pharmakologie
Robert-Rössle-Strasse 10, 13125 Berlin (Germany)
Fax: (+49)30-94793-159
E-mail: hagen@fmp-berlin.de
V. Nache, Prof. Dr. K. Benndorf
Institut für Physiologie II
Friedrich-Schiller-Universität Jena (Germany)
Prof. Dr. R. Schmidt
Institut für Physikalische und Theoretische Chemie
Johann-Wolfgang-Goethe-Universität Frankfurt (Germany)
H. Kaneko
Institut für Zoologie
Universität Heidelberg (Germany)
[**] This work was supported by the Fonds der Chemischen Industrie.
We thank Dr. E. Krause for mass-spectrometric and R. Lechler for
NMR measurements.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2005, 44, 7887 –7891
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7887

Communications
The synthesis of the key compounds 1 and 2 is outlined in
Scheme 2.7-Amino-4-methylcoumarin ( 7) was treated with
an excess of bromoacetic acid tert-butyl ester in the presence
of diisopropylethylamine to yield a mixture of 8 and 9, which
was separated by flash chromatography to provide the two
products in yields of 27% and 31%, respectively.SeO
2
oxidation of 9 gave the corresponding aldehyde 10 in a yield
of 80%.Condensation of 10 with tosylhydrazide furnished the
corresponding hydrazone (84% yield), which was trans-
formed into 1 by triethylamine-mediated Bamford–Stevens
reaction (87% yield).Compound 2 was best prepared (81%
yield) from the alcohol 11, which was synthesized in 74%
yield from 10 by reduction with NaBH4, by mesylation and
subsequent displacement of the mesyl group using LiBr in
THF.Our attempts to convert 9 directly into 2 by utilizing N-
bromosuccinimide were not successful as they resulted mainly
in bromo substitution in position 3 of the coumarin ring.
Alkylation of the cNMPs using Method A gave relatively
low product yields for the tert-butoxy-protected derivatives of
3 (12%) and 6 (7%) but an acceptable yield for the
corresponding analogue of 4 (25%).The reaction of 1 with
8-Br-cAMP yielded only traces of 5.However, the alternative
esterification by Method B gave relatively high product yields
for the tert-butoxy-protected derivatives of 3 (38%) and 5
(49%), an acceptable yield for the corresponding derivative
of 6 (18%), and only negligible amounts of the analogue of 4.
Compared with the syntheses of other caged cNMPs,[2,3,7] 3–6
can be prepared with good yields by Method A for the
synthesis of 4 and Method B for the preparation of 3, 5, and 6,
particularly because the product yields represent typical
rather than optimal conditions.
Both synthetic procedures resulted in diastereomeric
mixtures of the tert-butoxy-protected caged compounds.The
ratios of the axial to the equatorial diastereomers were
approximately 2:3 (Method A) and from 2:1 to 4:1 (Meth-
od B).The diastereomers were separated by reversed-phase
HPLC on a preparative scale.In all cases, the axial isomers
showed lower retention times than the equatorial isomers.
TFA deprotection of the tert-butoxy-protected diastereomers
of the esters resulted in the pure axial and equatorial isomers
of 3–6 with approximately quantitative yields (see the
Supporting Information for preparative details and analytical
characterization).The isomers were assigned by 31P NMR
spectroscopy.[8,9]
Scheme 1. Synthesis (I) and photolysis (II) of 3–6: a) DMSO/CH3CN (1:4),
608C, 24 h; b) CH3CN, 858C, 5 h; c) TFA/CH2Cl2/H2O (75:24:1), room
temperature, 20 min.
Like other coumarinylmethyl-caged cNMPs, compounds
3–6 are highly resistant to spontaneous hydrolysis in the dark.
HPLC monitoring of the pure diastereomers of the caged
compounds in aqueous HEPES buffer at pH 7.2 during a 24-h
period revealed no measurable formation (< 0.5%) of the
free cNMPs at room temperature (HEPES = 2-[4-(2-hydroxy-
ethyl)-1-piperazinyl]ethanesulfonic acid).All the compounds
are readily soluble in aqueous HEPES buffer at pH 7.2
(Table 1), which is a prerequisite for the administration of
large concentrations of phototriggers in cells or around
membrane patches at the tip of glass pipettes when the
patch–clamp technique is used.As expected, the hydrophilic
caged cNMPs do not measurably partition into model lipid
membranes at pH 7.2. This was demonstrated by the lack of
reaction heats when compounds 3 and 5 were titrated with
centers.Furthermore, we show that the new caged com-
pounds photorelease cNMPs upon one- as well as two-photon
excitation and that it is possible to determine quantitatively
the amount of cyclic nucleotide photoreleased inside a cell by
fluorescence measurements.
The compounds 3–6 were prepared as shown in
Scheme 1I by alkylation of the free acid form of the
respective cNMP with the 4-(diazomethyl)coumarin 1 (Meth-
od A) or by reaction of the tetra-n-butylammonium salts of
the cNMPs with the 4-(bromomethyl)coumarin 2 (Method B)
according to procedures described earlier for other coumar-
inylmethyl esters of cNMPs[5,6] and by subsequent removal of
the tert-butoxy protecting groups with trifluoroacetic acid
(TFA).
7888
www.angewandte.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2005, 44, 7887 –7891

Angewandte
Chemie
conversion proceeds by means of a pho-
tochemical SN1 mechanism (solvent-
assisted photoheterolysis) from S1 via a
singlet ion pair.This ion pair decays
mainly by recombination to the singlet
ground state.[10] Competitively, separa-
tion of the ion pair by the polar solvent
(formation of the cNMP anion) and
subsequent hydrolysis of the coumarinyl-
methyl carbocation or, alternatively,
direct reaction of a water molecule from
the solvent cage of the ion pair with its
carbocation followed by a very fast
deprotonation step may take place.[10,11]
Such a photolysis pathway implies that
product formation is very fast.Indeed,
time-resolved fluorescence measure-
ments upon single-pulse excitation
(0.5 ns half-width, 337 nm) of the axial
diastereomers of 3 and 4 revealed fluo-
rescence contributions from 12.Because
pure solutions of the caged com-
Scheme 2. Synthesis of 1 and 2: a) BrCH2COOtBu (3 equiv), iPr2EtN, NaI, CH3CN, reflux, 24 h;
b) SeO2, p-xylene, reflux, 6 h; c) NaBH4, MeOH, room temperature, 2 h; d) CH3SO2Cl, Et3N,
CH2Cl2, 58C, 20 min; e) LiBr, THF, room temperature, 1.5 h; f) p-tosylhydrazide, EtOH, room
temperature, 1.5 h; g) Et3N, MeOH, room temperature, 3 h.
pounds were irradiated, 12 must
Table 1: Long-wavelength absorption maxima (lmabasx), extinction coefficients, (emax), photochemical
quantum yields (fchem), fluorescence maxima (lmax), fluorescence quantum yields (f ), lifetimes of the
lowest excited singlet state (t), and solubilities (s) of the BCMACM esters 3–6 and of 12 in CH3CN/
HEPES-KCl buffer (5:95), pH 7.2.
have been formed and excited
during the photolysis pulse.
Actually, deconvolution of the fluo-
rescence signals with a biexponen-
tial decay function yielded rise
times of the fluorescence of 12 of
about 0.7 ns and 0.4 ns for the axial
diastereomers of 3 and 4, respec-
tively.The rate constants of cNMP
formation are the quotients of the
efficiencies of product formation
from the ion pairs and the fluores-
cence rise times of 12.[10] Consider-
ing the corresponding efficiencies of
product formation of 0.27 for 3 and
0.26 for 4, we obtain for the rate
constants values of 4 ” 108 sÀ1 and
6 ” 108 sÀ1, respectively.As the
quantum yields, fchem and ff, do
not vary much (Table 1), it is
expected that the equatorial dia-
f
f
[a]
Compound
lmabasx [nm]
e
max [mÀ1 cmÀ1
]
f
chem
lmf ax [nm]
f[b]
t [ns]
s [mm]
f
3 (axial)
3 (equatorial)
384
383
17000
17900
0.26
0.30
483
484
0.007
0.007
0.07[c]
0.06[c]
>2
>1
4 (axial)
4 (equatorial)
386
383
18000
18700
0.25
0.30
482
480
0.008
0.011
0.07[c]
0.10[c]
>2
>1
5 (axial)
5 (equatorial)
384
383
18500
18100
0.23
0.26
486
483
0.012
0.025
0.11[c]
0.23[c]
ꢀ1
ꢀ1
6 (axial)
6 (equatorial)
387
282
18100
18400
0.25
0.29
479
487
0.015
0.023
0.13[c]
0.21[c]
ꢀ1
ꢀ1
12
376
18400
–
479
0.17
1.59[d]
–
Estimated average uncertainties: [a] 15%, [b] 8%. [c] Calculated as t=t12 ”f/ꢀ12,[13] t12 =fluorescence
f
f
lifetime of 12, ꢀ1f 2 =fluorescence quantum yield of 12. [d] Measured fluorescence lifetime of 12.
lipid vesicles at different temperatures (see the Supporting
Information for details).Nevertheless, the caged compounds
rapidly enter live cells, probably using transport proteins or by
endocytosis, and HEK293 cells could be loaded with 6 by
incubation with a 50-mm buffered (pH 7.2) solution.
Photoactivation of the axial or equatorial diastereomers
of the (coumarin-4-yl)methyl esters 3–6 by irradiation with
wavelengths of 330–430 nm in aqueous buffer leads to the 7-
[bis(carboxymethyl)amino]-4-(hydroxymethyl)coumarin 12,
stereomers of 3 and 4 as well the diastereomers of 5 and 6
photorelease the cNMPs with similar rates.
The photochemical characteristics of the BCMACM
esters 3–6 in HEPES buffer (pH 7.2) are summarized in
Table 1.The absorption and the fluorescence spectra show
maxima at 382–387 nm and 479–487 nm, respectively, and
correspond to those of the DEACM esters.The single-photon
photochemical quantum yields, fchem, as well as the extinction
coefficients, emax, are high for caged compounds, which results
in excellent photosensitivity at long-wavelength irradiation.
As a consequence, even moderate UV/Vis irradiation is
sufficient to obtain large amounts of free cNMP.
a
proton, and the respective “free” cNMP anion
(Scheme 1II).The photoreaction is efficient and clean and
gives about 90–95% of the cNMP.Indications of a significant
triplet-state population were not found.In fact, the lifetimes
of the lowest excited singlet states S1 are extremely short,
about 0.1 ns (see Table 1). We assume that the photochemical
The combination of the high photosensitivity and the good
solubility of 3–6 also permits the generation of large
instantaneous cNMP concentration jumps.This could be
Angew. Chem. Int. Ed. 2005, 44, 7887 –7891
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 7889

Communications
demonstrated by means of cyclic nucleotide gated (CNG) ion
channels of the rod photoreceptor, which are activated by
cGMP.The CNGA1 subunit of these channels was expressed
heterologously in Xenopus oocytes, and the current con-
ducted by these channels was measured in inside-out patches
with the patch–clamp technique.For example, with 400 mm 4
in the bath solution, a 150-ms light flash generated concen-
tration jumps of free cGMP from zero to 72 mm.Figure 1
Figure 2. Time course of the Ca2+ influx through CNG channels into
HEK293 cells expressing the CNGA3 channel after photorelease of 8-
Br-cGMP from 6 (axial isomer) by exposure to UV light (364 nm, one-
&
photon excitation, ) and to IR light (745 nm, two-photon excitation,
*
), respectively. The cells were preincubated with 4 mm FLUO-4/AM
(30 min) and 50 mm 6 (20 min). The fluorescence intensity F of the
Ca2+ indicator FLUO-4 was recorded before and after irradiation (~).
There was no significant difference between the two curves.
F/F0 =relative fluorescence intensity (mean values Æ standard
deviations obtained from three cells).
Figure 1. Activation of homotetrameric CNGA1 channels by jumps in
cGMP concentration upon photolysis of 4 (axial isomer). The channels
were expressed heterologously in Xenopus oocytes, and the recordings
were performed in inside-out patches containing a high number of
channels. The concentration of free cGMP was determined from the
steady-state current of the traces referred to the steady-state current at
saturating cGMP and the concentration–response relationship for
these channels.[15] Before the flash, no current was present. The four
traces at the indicated voltages were recorded from the same patch
and were corrected for small leak currents by subtracting the respec-
tive traces recorded in the absence of cyclic nucleotide.
Figure 3. Fluorescence intensity of the Ca2+ indicator FLUO-4 in two
CNGA3-transfected HEK293 cells before and after photorelease of 8-
Br-cGMP from 6 (axial isomer) by exposure to UV (364 nm, upper
row) or to IR light (745 nm, lower row). The cells were preincubated
with 4 mm FLUO-4/AM (30 min)and 50 mm 6 (20 min). The indicated
times correspond to those given in Figure 2. The fluorescence intensity
is represented in an 8-bit gray scale.
illustrates the resulting activation time courses at four trans-
membrane voltages.The time courses are highly resolved and
are therefore most appropriate to give information on the
molecular mechanisms underlying the activation gating of
these channels.At maximum concentrations, 4 allowed us to
elicit larger cGMP concentration jumps than the DEACM
ester of cGMP.
BCMACM-caged cNMPs are highly sensitive not only to
one-photon but also to two-photon excitation.Images
obtained by confocal laser scanning microscopy (Figures 2
and 3) indicate that upon photolysis of 6, 8-Br-cGMP
mediates the entry of Ca2+ ions into HEK293 cells expressing
the cGMP-gated ion channel of the cone photoreceptor
(CNGA3).UV (argon laser, 364 nm) as well as IR light
(Cameleon diode-pumped laser, 745 nm) evoked a saturable
increase in the fluorescence intensity of the Ca2+ indicator
fate.[13] Direct comparison of the time course for two-
photon uncaging of 6 (axial isomer) with that of N-[(6-
bromo-7-hydroxycoumarin-4-yl)methoxycarbonyl]-l-glutamic
acid (Bhc-glu)[12] reveals that 6 photodecomposes initially 1.3
times faster than Bhc-glu under identical conditions
(Figure 4).Because Bhc-glu displays a two-photon uncag-
ing action cross-section, du, at 740 nm of about
10À50 cm4 sphotonÀ2, the two-photon action cross-section of
6 should be slightly larger than 10À50 cm4 sphotonÀ2.Similar du
values are expected also for the equatorial isomer of 6 as well
as for the diastereomers of 3–5.
The fluorescence quantum yields ff of 3–6 are one order
of magnitude smaller than that of the photoreleased alcohol
12 (Table 1).Compound 12 fluoresces also inside cells (see
the Supporting Information), in contrast to 7-(diethylamino)-
4-(hydroxymethyl)coumarin whose fluorescence is totally
quenched inside HEK293 cells.[4] Thus, fluorescence spectro-
FLUO-4, which indicates Ca2+-ion influx through CNG
2+
channels.Addition of excess Mg
to the used Ca2+ buffer
blocked Ca2+ influx, and neither UV nor IR irradiation in the
absence of 6 led to changes in the fluorescence signal of the
cells (not shown).High sensitivity to two-photon photolysis
has been described earlier for (6-bromo-7-hydroxycoumarin-
4-yl)methyl-caged compounds[2,12] and recently for (6,7-
dimethoxycoumarin-4-yl)methyl diethyl phosphate as well
as sodium [7-(dimethylamino)coumarin-4-yl]methyl sul-
7890 www.angewandte.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2005, 44, 7887 –7891

Angewandte
Chemie
within cells under physiological conditions.This approach can
extend decisively the tools available for the study of space-
and time-dependent aspects of cNMP-triggered processes.
Finally, the novel protecting group should be useful in the
caging and uncaging of other biomolecules with phosphate,
carboxylate, and other functionalities.Exemplarily, the
BCMACM ester of phenylacetic acid has already been
prepared.The caged compound shows high solubility in
aqueous buffer and can be photolyzed rapidly and efficiently
(fchem = 0.02, emax = 20000) in the visible-wavelength region.
Received: July 11, 2005
Revised: August 22, 2005
Published online: November 4, 2005
Figure 4. Initial time course of the two-photon photolysis of 6 (axial
isomer) and Bhc-glu upon 740-nm irradiation in HEPES buffer, pH 7.2.
The concentrations of 6 and Bhc-glu (starting concentrations 100 mm)
were determined by RP-HPLC. Solid lines are linear regression lines. 6:
c/mm=À0.0049t/s + 99.65 (R2 =0.987); Bhc-glu: c/mm=À0.0037t/s
+ 99.74 (R2 =0.991). c=concentrations of the caged compounds.
Keywords: ion channels · nucleotides · photoactivatable
.
compounds · photolysis · signal transduction
[1] V.Hagen, K.Benndorf, UB. .Kaupp in
Dynamic Studies in
Biology: Phototriggers, Photoswitches and Caged Biomolecules
(Eds.: M.Goeldner, R. S.Givens), Wiley-VCH, Weinheim, 2005,
pp.155 – 178.
scopic visualization of the progress of cNMP photorelease
from BCMACM-caged cNMPs within cells becomes possible,
as demonstrated in Figure 5.The generation of 8-Br-cGMP
[2] T.Furuta, H.Takeuchi, M.Isozaki, Y.Takahashi, M.Kanehara,
M.Sugimoto, T.Watanabe, K.Noguchi, TM. .Dore, T.
Kurahashi, M.Iwamura, R.Y.Tsien,
1119 – 1128.
ChemBioChem 2004, 5,
[3] V.Hagen, J.Bendig, S.Frings, T.Eckardt, S.Helm, D.Reuter,
U.B. Kaupp, Angew. Chem. 2001, 113, 1078 – 1080; Angew.
Chem. Int. Ed. 2001, 40, 1046 – 1048.
[4] V.Hagen, S.Frings, B.Wiesner, S.Helm, U.B.Kaupp, J.Bendig,
ChemBioChem 2003, 4, 434 – 442.
[5] T.Furuta, M.Iwamura, R.Y.Tsien,
Methods Enzymol. 1998,
291, 50 – 63.
[6] V.Hagen, J.Bendig, S.Frings, B.Wiesner, B.Schade, S.Helm, D.
Lorenz, U.B.Kaupp, J. Photochem. Photobiol. B 1999, 53, 91 –
102.
[7] L.Wang, J.E.T.Corrie, J.F.Wootton,
3474 – 3478.
[8] J.Engels, E.J.Schlaeger, J. Med. Chem. 1977, 20, 907 – 911.
[9] J.Engels, Bioorg. Chem. 1979, 8, 9 – 16.
J. Org. Chem. 2002, 67,
Figure 5. Monitoring the photorelease of 8-Br-cGMP from 6 within a
CNGA3-transfected HEK293 cell by measuring the total whole-cell
current and the fluorescence emitted from liberated 12 using excitation
at 360 nm. The cell was loaded in the whole-cell configuration of the
patch–clamp technique with 80 mm 6 and irradiated. The change in
current amplitude and the fluorescence intensity increase (&) show
photolytic liberation of 8-Br-cGMP.
[10] R.Schmidt, D.Geißler, V.Hagen, J.Bendig,
J. Phys. Chem. A
2005, 109, 5000 – 5004.
[11] B.Schade, V.Hagen, R.Schmidt, R.Herbrich, E.Krause, T.
Eckardt, J.Bendig, J. Org. Chem. 1999, 64, 9109 – 9117.
[12] T.Furuta, S.S.-H.Wang, J.L.Dantzker, T.M.Dore, W.J.Bybee,
E.M. Callaway, W. Denk, R.Y. Tsien,
USA 1999, 96, 1193 – 1200.
Proc. Natl. Acad. Sci.
from 6 in CNGA3-transfected HEK293 cells was monitored
simultaneously by current recordings resulting from CNG-
channel activation and by the fluorescence emitted from the
liberated photoproduct 12 upon excitation at 360 nm.Since
the fluorescence signal can be calibrated,[14] it is possible to
quantify cNMP release from BCMACM esters of cNMPs
using fluorescence spectroscopy.In addition to the high
solubility of BCMACM-caged cNMPs in aqueous buffer, the
possibility of visualizing and quantifying the release process
within cells is a considerable advantage of BCMACM-caged
cNMPs over the DEACM-caged analogues.
[13] D.Geißler, YN. .Antonenko, R.Schmidt, S.Keller, OO. .
Krylova, B.Wiesner, J.Bendig, P.Pohl, V.Hagen, Angew. Chem.
2005, 117, 1219 – 1223; Angew. Chem. Int. Ed. 2005, 44, 1195 –
1198.
[14] V.Hagen, S.Frings, J.Bendig, D.Lorenz, B.Wiesner, U.B.
Kaupp, Angew. Chem. 2002, 114, 3775 – 3777; Angew. Chem. Int.
Ed. 2002, 41, 3625 – 3628.
[15] V.Nache, J.Kusch, V.Hagen, K.Benndorf, unpublished results.
In conclusion, we have developed a photolabile coumar-
inylmethyl protecting group bearing a dianionic substituent
and used this protecting group for the caging of cNMPs.The
caged cNMPs exhibit high photosensitivities at long-wave-
length irradiation and allow large and instantaneous cNMP
concentration jumps upon one- and two-photon excitation
Angew. Chem. Int. Ed. 2005, 44, 7887 –7891
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7891