{7-[Bis(carboxymethyl)amino]coumarin-4-yl}methoxycarbonyl derivatives for photorelease of carboxylic acids, alcohols/phenols, thioalcohols/thiophenols, and amines

Article abstract of DOI:10.1002/chem.200701142

Light-induced release of biomolecules from inactive precursor molecules represents a powerful method to study cellular processes with high temporal and spatial resolution. Here we report the synthesis and photochemistry of a series of {7-[bis(carboxymethyl)amino]coumarin-4-yl}methyl carboxylates, carbonates, carbamates, and thiocarbonates as potential phototriggers for compounds with COOH, OH, NH₂, and SH functions. The compounds are soluble in aqueous buffer, show low fluorescence, and are efficiently photolysed by irradiation with UV/Vis or IR light to release carboxylates, alcohols, phenols, amines, thioalcohols, or thiophenols.

Full text of DOI:10.1002/chem.200701142

FULL PAPER
DOI: 10.1002/chem.200701142
{7-[Bis(carboxymethyl)amino]coumarin-4-yl}methoxycarbonyl Derivatives
A
for Photorelease of Carboxylic Acids, Alcohols/Phenols, Thioalcohols/
Thiophenols, and Amines
Volker Hagen,* Brigitte Dekowski, Nico Kotzur, Ralf Lechler, Burkhard Wiesner,
Benoît Briand, and Michael Beyermann[a]
Abstract: Light-induced release of bio-
molecules from inactive precursor mol-
ecules represents a powerful method to
study cellular processes with high tem-
poral and spatial resolution. Here we
report the synthesis and photochemis-
carboxylates, carbonates, carbamates,
and thiocarbonates as potential photo-
triggers for compounds with COOH,
OH, NH2, and SH functions. The com-
pounds are soluble in aqueous buffer,
show low fluorescence, and are effi-
ciently photolysed by irradiation with
UV/Vis or IR light to release carboxy-
lates, alcohols, phenols, amines, thioal-
cohols, or thiophenols.
Keywords: coumarins · photoacti-
vatable compounds · photochemis-
try · photolysis · protecting groups
try of
a
series of {7-[bis(carboxy-
methyl)amino]coumarin-4-yl}methyl
À
Introduction
caged derivatives produces upon cleavage of a C O bond
an anion of the leaving group and a solvent-trapped
coumarin as a photo by-product.
Photolabile protecting groups have numerous applications
in chemistry. Their removal only requires light and allows a
very mild deprotection of sensitive molecules. The protec-
tion of biological signaling molecules by conversion into in-
active light-sensitive derivatives is also very attractive and
photoactivatable or caged biomolecules represent a power-
ful tool for cell biology, because they permit the controlled
delivery of bioactive molecules without physically disturbing
within organised biological systems with high temporal and
spatial precision.[1–3] When caged, the biomolecule is ren-
dered biologically inactive by covalent attachment of a pho-
tochemically removable protecting group (caging group) to
the key pharmacophoric functionality. Flash photolysis by
using light of a specific wavelength cleaves the modifying
group, that is, it triggers the uncaging and rapidly activates
the molecule. Among the available different caging groups
the coumarinylmethyl moieties have received increased at-
tention in recent years. They are members of arylalkyl-type
photo-removable protecting groups and photolysis of their
Herein, we describe the caging and uncaging of different
molecules with COOH, OH, NH2, and SH functionalities
(Scheme 1) by using our recently developed {7-[bis(carboxy-
methyl)amino]coumarin-4-yl}methyl (BCMACM) chromo-
phore,[4] and its {7-[bis(carboxymethyl)amino]coumarin-4-
yl}methoxycarbonyl (BCMACMOC) variant, respectively.
The BCMACM protecting group is a water-soluble version
of the earlier introduced 7-(dialkylamino)coumarinylmethyl
caging groups.[5–7] Caging of carboxylic acids, alcohols, and
amines by using coumarinylmethyl or coumarinylmethoxy-
carbonyl protecting groups has already been described.[4,8–19]
Coumarinylmethoxycarbonyl protected thioalcohols and
thiophenols have not yet been reported.
A
The special features of the BCMACM or BCMACMOC
protecting groups are the efficient photochemical release
upon irradiation in the visible wavelength region due to
large long-wavelength absorptivities, the sensitivity to two-
photon excitation, and the good solubility in aqueous buffer
by virtue of their anionic centres. The simultaneous appear-
ance of these properties is unique. Long-wavelength absorp-
tions allow uncaging to occur under non-damaging light con-
ditions to cells. Two-photon excitation is also favourable for
a less-damaging release of biomolecules, gives significantly
deeper tissue penetration, and allows more localised photo-
release. Caging of biomolecules frequently leads to very hy-
[a]Dr. V. Hagen, B. Dekowski, N. Kotzur, R. Lechler, Dr. B. Wiesner,
B. Briand, Dr. M. Beyermann
Leibniz-Institut für Molekulare Pharmakologie
Robert-Rçssle-Strasse 10, 13125 Berlin (Germany)
Fax : (+49)30-94793-159
E-mail: hagen@fmp-berlin.de
Chem. Eur. J. 2008, 14, 1621 – 1627
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1621

presence of DMAP to yield the
corresponding tert-butyl-pro-
tected derivatives, which were
purified by flash chromatogra-
phy, or by reverse-phase HPLC
on a preparative scale and then
deprotected with TFA to gener-
ate the carbonates 1c–e, the
carbamate 1 f, and the thiocar-
bonates 1g and 1h.
The BCMACM ester 1a, the
BCMACMOC-caged
alcohol
1e, the BCMACMOC-caged
amine 1 f, and the BCMAC-
MOC-caged thioalcohol 1g are
resistant to spontaneous hydro-
lysis in the dark. HPLC moni-
toring of the caged compounds
in aqueous HEPES buffer, at
pH 7.2, during a 24-hour period
revealed <0.5% of the free
uncaged compounds at room
temperature (HEPES=2-[4-(2-
hydroxyethyl)-1-piperazinyl]-
ethanesulfonic acid). More sus-
ceptibility to hydrolysis in the
dark was observed in the case
of the phenyl carbonates 1c,d
and the phenyl thiocarbonate
Scheme 1. A) Structures and B) photolysis of the phototriggers 1a–h.
drophobic derivatives and therefore a water-soluble protect-
ing group is favourable in most cases.
Results and Discussion
Scheme 2. Synthesis of 1a,b: a) 3a,b, DMAP, DCC, EtOAc, room tem-
perature, 4 h; b) TFA/CH2Cl2/H2O (75:24:1).
Caged model compounds 1a,c,e,h, and the caged bioactive
molecules 1b,d,f,g (see Scheme 1)—compounds 1a and 1d
we have already recently introduced[4,18]—were synthesised
starting from the key building block (caging agent) 7-[bis-
(tert-butoxycarbonylmethyl)amino]-4-(hydroxymethyl)cou-
marin (6).[4] For synthesis of 1a,b phenylacetic acid or the
Boc-protected amino acid l-phenylalanine were esterified
with 6 in the presence of dimethylaminopyridine (DMAP)
and N,N’-dicyclohexylcarbodiimide (DCC) and upon purifi-
cation by using flash chromatography or preparative re-
verse-phase HPLC converted to 1a,b by removal of the tert-
butyl groups and, in the case of 1b, also of the tert-butoxy-
carbonyl (Boc) group by using trifluoroacetic acid (TFA)
(Scheme 2).
Scheme 3. Synthesis of 1c–h: a) 4-Nitrophenyl chloroformate, DMAP or
iPr2EtN, room temperature, 1 h; b) 5c–h, DMAP or iPr2EtN, room tem-
perature, 1–20 h; c) TFA/CH2Cl2/H2O (75:24:1), room temperature,
30 min.
Compounds 1c–h were prepared as shown in Scheme 3 by
reaction of 6 with 4-nitrophenyl chloroformate to the car-
bonate 7, reaction of 7 with phenol (5c), the vanilloid recep-
tor channel agonist capsaicin (5d), 2-phenylethanol (5e),
the a-adrenergic receptor ligand l-norepinephrine (5 f), the
N-(9-fluorenylmethoxycarbonyl) (Fmoc)-protected amino
acid l-cysteine (5g), or 4-acetamidothiophenol (5h) in the
1h. We found that approximately 7.5% of 1c, 3% of 1d and
10% of 1h were hydrolysed in HEPES buffer, at pH 7.2,
within 24 h. For 1h the estimated half-life time was about
140 h. Nevertheless, BCMACMOC-caged phenols and thio-
phenols should be sufficiently stable with respect to the time
needed for electrophysiological experiments. Only 1b was
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Chem. Eur. J. 2008, 14, 1621 – 1627

Coumarin Derivatives for Photorelease
FULL PAPER
max
Table 1. Long-wavelength absorption maxima,
l
, extinction coeffi-
abs
found to a large extent to be unstable in the dark in aqueous
HEPES buffer at pH 7.2. The percentages of 1b remaining
after one and six hours in the dark were 95 and 75%, re-
spectively. Hydrolysis of 1b is dependent upon pH. As ex-
pected[20] the compound is more stable at lower pH. We
found that 17% of 1b was hydrolysed in citrate/phosphate
buffer at pH 4.0 within 24 h. Because hydrolysis in the dark
is often a serious problem it seems that 1b is less suitable as
caged compound and requires some care to minimize con-
tamination by free amino acid. Spontaneous hydrolysis was
also described for the l-glutamic acid g-(6-bromo-7-hydrox-
yÀcoumarin-4-yl)methyl ester in aqueous buffer.[9]
cients at the absorption maxima and at 430 nm, emax and e430, photochemi-
cal quantum yields, fchem, fluorescence maxima, lmf ax, fluorescence quan-
tum yields, ff, of the BCMACM/BCMACMOC-caged compounds 1a–1h
and of 2 in CH3CN/HEPES-KCl buffer (5:95), pH 7.2.
max
abs
[a]
Compound
l
emax
[mÀ1 cmÀ1
e430
[mÀ1 cmÀ1
fhem
lmf ax
ff[b]
[nm]
G
]
N
]
[nm]
1a
1b
1c
1d
1e
1 f
1g
1h
2
381
383
381
383
382
380
383
383
376
20000
20000
19200
18750
19800
18300
18500
18700
18400
2100
3100
2300
2300
2200
1600
2000
2700
–
0.02
n.d.
0.15
0.12
0.08
0.01
0.06
0.09
–
488
n.d.
498
478
500
503
480
497
479
0.040
n.d.
0.038
0.021
0.041
0.087
0.052
0.011
0.170
As expected, compounds 1a–h were readily soluble in
aqueous buffer at pH 7.2. The limit of aqueous solubility
was not determined, but all the compounds were soluble to
at least 1 mm concentrations without any problem. This is
important for the administration of high concentrations of
the phototriggers under physiological conditions.
Estimated average uncertainties: [a]15%. [b]8%. n.d. =not determined.
extinction coefficients, e, are high. As shown exemplarily in
Figure 1 for compound 1 f the phototriggers 1a–h absorb
light up to 450 nm allowing uncaging at wavelengths
Photoactivation of the (coumarin-4-yl)methyl esters 1a,b
by irradiation with light of the wavelengths of 334–436 nm
in aqueous buffer leads to 7-[bis(carboxymethyl)amino]-4-
(hydroxymethyl)coumarin (2), a proton, and the phenylace-
tic acid anion 3a and phenylalanine anion 3b, respectively
(Scheme 1). Upon irradiation at 334–436 nm, the phototrig-
gers 1c–h release 2, CO2, and 5c–h (Scheme 1). As we have
shown earlier for other coumarinylmethyl-caged com-
pounds[8,21,22] we assume that the photochemical conversion
proceeds by means of a photochemical SN1 mechanism (sol-
vent-assisted photoheterolysis). Such a photolysis pathway
implies that the formation of 2 and, in the case of the photo-
cleavage of the compounds 1a,b, also the formation of 3a
and 3b is very fast. Using the linear free energy relation-
ships found for 7-(dimethylamino)coumarinylmethyl esters
between the rate of photocleavage and the pKa values of the
acids released during the photolysis,[22] we estimate that 1a–
h are photolysed within the nanosecond time-scale. Howev-
er, photolytic cleavage of the carbonates 1c–d, the carba-
mate 1 f, and the thiocarbonates 1g and 1h does not liberate
the compounds 5c–h directly, but unstable carbonate, carba-
mate, or thiocarbonate salt intermediates (4c–h) are
formed, which undergo decarboxylation in a thermal hydro-
Figure 1. UV/Vis spectrum of 1 f in CH3CN/HEPES-KCl buffer (5:95),
pH 7.2.
lysis step to yield 5c–h. The decarboxylation is rate-limiting
and results in a marked slowing of product release after
photolysis.[12] On the basis of the measured decarboxylation
rates upon photolysis of other carbonate-caged phenols,[23,24]
>430 nm under non-damaging light conditions. The single-
photon photochemical quantum yields for disappearance of
1a and 1e–g, fchem, are significantly lower than those of the
phosphates[4] probably because the corresponding carboxyl-
ic, carbamic, or thiocarbonic acid anions 3a and 4e–h have
lower leaving-group abilities. It appears that the strength of
the photoreleased acid influences the efficiency of product
formation. The photochemical quantum yields of disappear-
ance of 1c,d,h are comparatively high clearly due to the res-
onance stabilization of the leaving group by the phenyl
moiety. However, the compounds 1a and 1e–g allow effi-
cient uncaging too, because the product ef is proportional
to the amount of the product release for a given photon ex-
posure and the compounds have practically useful ef values
due to their high extinction coefficients. BCMACM- and
carbonate-caged
alcohols,[26]
and
carbamate-caged
amines,[27,28] the formation of 5c,d,f should occur with rate
constants of >100 sÀ1 and that of 5e with a rate constant of
about 0.4 sÀ1. Product-release rates from thiocarbonate inter-
mediates upon photolysis have not yet been determined.
The relatively slow kinetics in comparison to those of the
esters 1a,b and of phosphates will not be a problem in some
applications, but of course can be a potential drawback that
limits the applicability of these caged compounds.
The photochemical characteristics of the compounds 1a–h
in HEPES buffer (pH 7.2) are summarised in Table 1. The
absorption spectra show maxima at about 380 nm and the
Chem. Eur. J. 2008, 14, 1621 – 1627
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1623

V. Hagen et al.
BCMACMOC-caged compounds are sensitive not only to
one-photon, but also to two-photon excitation, that means
UV absorption can be replaced by the simultaneous absorp-
tion of two infrared (IR) photons. Two-photon uncaging
allows more localised photorelease with excellent three-di-
mensional resolution, and IR light gives significantly deeper
tissue penetration than UV/Vis light. Direct comparison of
the time course for two-photon uncaging of 1a and 1d,e
with that of N-[6-bromo-7-hydroxycoumarin-4-yl)methoxy-
carbonyl]-l-glutamic acid (Bhc-Glu)[9] by using a femtosec-
ond-pulsed, mode-locked Ti-sapphire laser at 740 nm reveals
that 1a photodecomposes initially less efficiently (factor
0.4), whereas 1d and 1e photodecompose initially 2.6 and
1.5 times more efficiently than Bhc-Glu under identical con-
ditions. Because Bhc-Glu displays a two-photon uncaging
action cross section, du, at 740 nm of about one Goeppert–
Mayer unit (GM; 1 GM=10À50 cm4 sphotonÀ1), the two-
photon action cross section of 1a should be lower and those
of 1d,e larger than 1 GM.
The fluorescence quantum yields, ff, of 1a–h are signifi-
cantly smaller than that of the photoreleased alcohol 2
(Table 1). Alcohol 2 also fluoresces inside cells.[4] Thus, fluo-
rescence spectroscopic visualization of the progress of the
photorelease from BCMACM- or BCMACMOC-caged
compounds within cells becomes possible, as earlier demon-
strated by using {6,7-[bis(carboxymethyl)amino]coumarin-4-
yl}-caged cyclic nucleotides.[29]
We have very recently described useful applications of the
caged capsaicin 1d as a research tool in pain physiology.[18]
The compound did not display measurable biological activi-
ty up to concentrations of 10 mm and also had high photo-
sensitivity under physiological conditions. Compared with
other caged vanilloid ligands,[24,25,30] 1d shows a lot of advan-
tages, only the relatively slow photorelease kinetics is not
optimal. Compound 1h and its tert-butyl-protected precursor
are useful as active esters for the effective introduction of
the BCMACMOC protecting group to SH or NH2 functions
of peptides. Applications by using 1 f,g are in progress.
hol/phenol, amine, and thioalcohol/thiophenol leaving
groups by using visible light in aqueous media are unique.
Experimental Section
Materials: N-Boc-l-phenylalanine (acid form of 3b), phenol (5c), 2-phe-
nylethanol (5e), and l-norepinephrine (5 f) were purchased from Acros
Organics (Belgium). N-Fmoc-(S)-trityl-l-cysteine, 4-nitrophenyl chloro-
formate,
4-acetamidothiophenol
(5h),
4-dimethylaminopyridine
(DMAP), N,N’-dicyclohexylcarbodiimide (DCC), and iPr2EtN were ob-
tained from Fluka (Germany). Trifluoroacetic acid (TFA) was from Lan-
caster (UK). Capsaicin (5d) was obtained from Sigma (Germany). The
remaining chemicals were of the highest grade commercially available
and were used without further purification. 7-[Bis(carboxymethyl)-
AHCTREUNG
AHCTREUNG
amino]coumarin-4-yl}methyl phenylacetate (1a) were prepared as de-
scribed previously.[4] TLC plates (silica gel 60 F254) were purchased from
E. Merck (Germany). Silica gel for flash chromatography was from J. T.
Baker (The Netherlands). CH3CN from Riedel-de Han (Germany) was
of HPLC grade. Water was purified with a Milli-Q-Plus system (Milli-
pore, Germany). All reactions were carried out under N2. The synthetic
and analytical procedures with caged compounds were performed under
yellow light provided by sodium vapor lamps.
1
Instrumentation: H and 13C NMR spectra were recorded by using a Bru-
ker AV 300 or Bruker DRX 600 spectrometer. Chemical shifts are
a
given in parts per million (ppm) by using the residue solvent peaks as ref-
erence relative to TMS. The J values are given in Hz. Mass spectra were
measured by using electrospray ionization (ESI) mass spectrometry in
the positive ionization mode by using an Agilent 6210 ESI-TOF spec-
trometer (Agilent Technologies, USA). UV/Vis spectra were recorded by
using a UV/Vis spectrophotometer Lambda 9 (Perkin–Elmer). Fluores-
cence spectra were recorded by using a Jasco FP-6500 spectrometer. Ana-
lytical reverse-phase HPLC (RP-HPLC) was carried out by using a Shi-
madzu LC-20 system (flow rate: 1 mLminÀ1) equipped with a DAD-UV
detector and a fluorescence detector (lexc =380 nm, lem =495 nm) by
using a PLRP-S column, 300 Š, 8 mm, 250”4.6 mm from Polymer Labo-
ratories (UK) or a Nucleodur 100-5 C18 ec column, 100 Š, 5 mm, 250”
4 mm, Macherey–Nagel (Germany). Preparative RP-HPLC was conduct-
ed by using a Shimadzu LC-8 A system (flow rate: 10 mLminÀ1) with a
UV/Vis detector (SPD-6 AV, lexc =254 nm) over a Nucleogel RP 100-10
(100 Š, 10 mm, 300”25 mm) or a Nucleodur 100-5 C18 ec column (100 Š,
5 mm, 250”21 mm) from Macherey–Nagel (Germany). One-photon pho-
tolysis of all synthesised photoprotected compounds in solution was per-
formed by using a high-pressure mercury lamp (HBO 500, Oriel, USA)
with controlled light intensity and metal interference transmission filters
(365 or 436 nm, Oriel, USA). For all experiments, UV and fluorescence
quartz cuvettes with a path length of 1 cm and a cross-sectional area of
1 cm2 were used. During irradiation, the solutions in the cuvettes were
mixed by a magnetic stirrer. All synthetic and analytical procedures were
performed in darkness or under yellow light provided by sodium vapor
lamps. The melting points were uncorrected.
Conclusion
We evaluated the usefulness of the BCMACM/BCMAC-
MOC-caging moiety for the design of phototriggers for bio-
molecules with different classes of functional groups. Our
results demonstrate that BCMACM and BCMACMOC are
very useful caging groups. They allow caging and uncaging
of the most important functional groups of biological rele-
vance as shown exemplarily for the derivatives 1a–h. The
phototriggers possess favourable properties. They are, with
the exception of 1b, sufficiently resistant to hydrolysis and
are soluble in aqueous buffer. Photocleavage is efficient
upon long-wavelength UV/Vis excitation, as well as, expo-
sure to IR light.
N-Boc-l-phenylalanine {7-[bis(tert-butoxycarbonylmethyl)amino]coumar-
in-4-yl}methyl ester: Compound 6 (64.3 mg, 0.15 mmol) was dissolved in
EtOAc (3 mL), cooled to 108C, and then N-Boc-l-phenylalanine
(39.8 mg, 0.15 mmol), some DMAP, and DCC (38.8 mg, 0.19 mmol) were
added with stirring. The mixture was allowed to warm to room tempera-
ture, stirred for 4 h, filtered, evaporated, and the residue purified by
preparative RP-HPLC. The desired product was eluted by use of a linear
gradient of 40–95% B in 60 min (eluent A, H2O; eluent B, CH3CN). The
fraction with a retention time of 59 min was collected, evaporated in
vacuo, redissolved in CH3CN/H2O, and lyophilised to give the desired
product (45 mg, 45%) as a pale yellow solid. M.p. 76–788C (decomp);
TLC: Rf =0.53 (THF/hexane 1:2 v/v); tR =19.45 min (analytical HPLC,
20–95% B in A in 20 min, eluent A, H2O; eluent B, CH3CN); 1H NMR
(300 MHz, [D6]DMSO, 278C, TMS): d=1.33 (s, 9H), 1.42 (s, 18H), 2.89–
Other applications, for instance, in organic chemistry are
conceivable, because hydrophilic photochemical protecting
groups that allow efficient release of carboxylic acid, alco-
1624
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Chem. Eur. J. 2008, 14, 1621 – 1627

Coumarin Derivatives for Photorelease
FULL PAPER
3.09 (m, 2H), 4.19 (s, 4H), 4.27–4.35 (m, 1H), 5.26–5.38 (m, 2H), 6.12 (s,
1H), 6.47 (d, J=1.9 Hz, 1H), 6.55 (dd, J=9.0 and 1.9 Hz, 1H), 7.20–7.30
(m, 5H), 7.47 ppm (d, J=9.0 Hz, 1H); 13C NMR (75.5 MHz, [D6]DMSO,
278C): d=27.7, 28.1, 36.2, 53.5, 55.4, 61.6, 78.5, 81.1, 98.1, 106.7, 106.9,
109.1, 125.3, 126.5, 128.2, 129.1, 137.3, 150.0, 151.3, 155.0, 155.5, 160.2,
168.8, 171.6 ppm; HRMS (ESI): m/z: calcd for C36H46N2O10 [M+Na]+:
689.3050; found: 689.3041.
calcd for C21H17N1O9 [M+H]+: 428.0982; found: 428.0991; elemental
analysis calcd (%) for C21H17N1O9·0.5H2O (436.38): C 57.80, H 4.16, N
3.21; found: C 57.44, H 4.42, N 3.12.
(E)-{7-[Bis(tert-butoxycarbonylmethyl)amino]coumarin-4-yl}methyl
2-
methoxy-4-[(8-methylnon-6-enamido)methyl]phenyl carbonate: This
compound was prepared by modification of our previously reported pro-
cedure[18] by stirring of
6 (103 mg, 0.24 mmol), DMAP (36.7 mg,
0.3 mmol), and 4-nitrophenyl chloroformate (60.5 mg, 0.3 mmol) in THF
(5 mL) for 1 h, filtration, evaporation of the filtrate, and reaction of the
formed 7 with capsaicin (91.6 mg, 0.3 mmol) in CH2Cl2 (5 mL) in the
presence of DMAP (36.7 mg, 0.3 mmol) for 3.5 h. Working up as de-
scribed gave the pure product (65 mg, 36%).
l-Phenylalanine {7-[bis(carboxymethyl)amino]coumarin-4-yl}methyl ester
trifluoroacetate, (O-BCMACM-caged phenylalanine, 1b):
A sample
(40 mg, 0.06 mmol) of the product described above was stirred in a mix-
ture (5 mL) of TFA/CH2Cl2/H2O (75:24:1) at room temperature for
30 min. The solvents were evaporated and the residue purified by prepa-
rative RP-HPLC (PLRP-S). Compound 1b was eluted by use of a linear
gradient of 5–95% B in 75 min (eluent A, H2O/0.1% TFA; eluent B,
CH3CN). The fraction with a retention time of 35.4 min was collected,
evaporated in vacuo, redissolved in CH3CN/H2O, and lyophilised to give
1b (16 mg, 58.3%) as a pale-yellow solid. M.p. >1108C (decomp);
[a]2D5 =+3.6 (c=0.67 in DMSO); tR =11.8 min (analytical HPLC, PLRP-
S, 5–95% B in A in 20 min, eluent A, H2O/0.1% TFA; eluent B,
(E)-{7-[Bis(carboxymethyl)amino]coumarin-4-yl}methyl 2-methoxy-4-[(8-
methylnon-6-enamido)methyl]phenyl carbonate (BCMACMOC-caged
capsaicin, 1d): Compound 1d was prepared by deprotection of the bis-
tert-butyl ester of 1d (60 mg, 0.08 mmol) with a mixture (10 mL) of TFA/
CH2Cl2/H2O (50:50:1) by stirring at room temperature for 20 min. Work-
ing up as described[18] gave the pure product (54 mg, 100%). The spectral
and analytical data for 1d were in accordance with those earlier de-
scribed.[18]
1
CH3CN); H NMR (300 MHz, [D6]DMSO, 278C, TMS): d=3.08–3.22 (m,
2H), 4.21 (s, 4H), 4.52 (t, J=6.8 Hz, 1H) 5.29 (d, J=15.2 Hz, 1H), 5.43
(d, J=15.2 Hz, 1H), 6.07 (s, 1H), 6.46 (s, 1H), 6.54 (d, J=9.0 Hz, 1H),
7.23–7.34 (m, 5H), 7.40 (d, J=8.9 Hz, 1H), 8.53 ppm (brs, 2H);
13C NMR (75.5 MHz, [D6]DMSO, 278C): d=36.1, 53.2, 53.5, 62.6, 97.9,
106.6, 107.1, 109.0, 125.4, 127.4, 128.6, 129.3, 134.4, 148.9, 151.3, 155.1,
160.2, 168.6, 171.5 ppm; 19F NMR (282.5 MHz, [D6]DMSO, 278C, with
neat CF3COOH as external reference relative to neat CFCl3): d=
À73.9 ppm; HRMS (ESI): m/z: calcd for C23H22N2O8 [M+H]+: 455.1454;
{7-
ACHTREUNG
ethyl carbonate: The active ester
7
6
0.24 mmol) and 4-nitrophenyl chloroformate (60.5 mg, 0.3 mmol) in THF
(5 mL) in the presence of DMAP (36.7 mg, 0.3 mmol) as described for
the bis-tert-butyl ester of 1c. Dissolution of 7 in CH2Cl2 (5 mL), addition
of DMAP (36.7 mg, 0.3 mmol) and 2-phenylethanol (5e, 36.7 mg,
0.3 mmol), stirring at room temperature for 3.5 h, evaporation, purifica-
tion by preparative RP-HPLC (PLRP-S) by using a linear gradient 50–
95% B in 80 min (eluent A, H2O; eluent B, CH3CN, retention time=
56.9 min), and lyophilization gave the desired product (50 mg, 37%) as a
pale yellow solid. M.p. 45–478C; TLC: Rf =0.75 (THF/hexane, 1:1 v/v);
tR =16.5 min (analytical HPLC, PLRP-S, 50–95% B in A in 22 min,
eluent A, H2O; eluent B, CH3CN); 1H NMR (300 MHz, [D6]DMSO,
278C, TMS): d=1.42 (s, 18H), 2.95 (t, J=6.7 Hz, 2H), 4.20 (s, 4H), 4.37
(t, J=6.7 Hz, 2H), 5.33 (s, 2H), 6.01 (s, 1H), 6.48 (d, J=1.9 Hz, 1H),
6.58 (dd, J=9.0 and 1.9 Hz, 1H), 7.21–7.32 (m, 5H), 7.50 ppm (d, J=
9.0 Hz, 1H); 13C NMR (75.5 MHz [D6]DMSO, 278C): d=27.7, 34.2, 53.5,
64.4, 68.5, 81.1, 98.2, 106.9, 107.0, 109.2, 125.4, 126.4, 128.4, 128.8, 137.4,
149.9, 151.4, 153.9, 155.1, 160.2, 168.7 ppm; HRMS (ESI): m/z: calcd for
C31H37N1O9 [M+Na]+: 590.2366; found: 590.2360.
found
455.1468;
elemental
analysis
calcd
(%)
for
C23H22N2O8·1H2O·CF3COOH (586.48): C 51.20, H 4.30, N 4.73; found: C
50.92, H 4.10, N 4.98.
{7-
A
phenyl
carbonate: DMAP (36.7 mg, 0.3 mmol), and 4-nitrophenyl chloroformate
(60.5 mg, 0.3 mmol) were added with stirring to a solution of 6 (103 mg,
0.24 mmol) in THF (5 mL). The reaction mixture was stirred at room
temperature for 1.0 h, filtered, and evaporated. The residue containing
the intermediately formed active ester 7 was dissolved in CH2Cl2 (5 mL),
after which DMAP (36.7 mg, 0.3 mmol) and phenol (5c, 28.2 mg,
0.3 mmol) were added. The reaction mixture was stirred for 3.5 h, evapo-
rated, and purified by preparative RP-HPLC (Nucleogel 100-10). The de-
sired product was eluted by using a linear gradient 50–95% B in 80 min
(eluent A, H2O; eluent B, CH3CN). The main fraction with a retention
time of 54.3 min was collected, evaporated in vacuo, redissolved in
CH3CN/H2O, and lyophilised to give the product (65 mg, 50%) as pale
yellow crystals. M.p. 79–818C; TLC: Rf =0.16 (hexane/EtOAc, 16:6 v/v);
tR =14.97 min (analytical HPLC, PLRP-S, 5–95% B in A in 20 min,
eluent A, H2O; eluent B, CH3CN); 1H NMR (600 MHz, [D6]DMSO,
278C, TMS): d=1.42 (s, 18H), 4.20 (s, 4H), 5.49 (s, 2H), 6.17 (s, 1H),
6.49 (d, J=2.1 Hz, 1H), 6.61 (dd, J=8.9 and 2.2 Hz, 1H), 7.28–7.33 (m,
3H), 7.43–7.48 (m, 2H), 7.59 ppm (d, J=8.9 Hz, 1H); 13C NMR
(75.5 MHz [D6]DMSO, 278C): d=27.7, 53.5, 65.4, 81.1, 98.2, 106.9, 107.4,
109.2, 121.2, 125.5, 126.3, 129.6, 149.3, 150.7, 151.5, 152.5, 155.1, 160.2,
168.7 ppm; HRMS (ESI): m/z: calcd for C29H33N1O9 [M+H]+: 540.2234;
found: 540.2226.
{7-[Bis(carboxymethyl)amino]coumarin-4-yl}methyl 2-phenylethyl car-
A
bonate (1e): The bis-tert-butyl ester of 1e (43 mg, 0.075 mmol) was
stirred in a mixture (7.5 mL) of TFA/CH2Cl2/H2O (74:25:1) at room tem-
perature for 20 min. The solvents were evaporated, and the residue was
co-evaporated two times with diethyl ether, dissolved in CH3CN/H2O,
and lyophilised to give 1e (35 mg, 100%). Because the product was not
absolutely pure it was purified by preparative RP-HPLC (Nucleodur 100-
5). The desired product was eluted by use of a linear gradient of 30–95%
B in 70 min (eluent A, H2O/0.1% TFA; eluent B, CH3CN). The fraction
with a retention time of 32.4 min was collected, evaporated in vacuo, re-
dissolved in CH3CN/H2O, and lyophilised to give pure 1e (25 mg, 72%)
as a pale yellow solid. M.p. >758C (decomp); tR =14.2 min (analytical
HPLC, Nucleodur 100-5, 30–95% B in A in 20 min, eluent A, 0.1%
TFA/H2O; eluent B, CH3CN); 1H NMR (300 MHz, [D6]DMSO, 278C,
TMS): d=2.95 (t, J=6.7 Hz, 2H), 4.23 (s, 4H), 4.37 (t, J=6.7 Hz, 2H),
5.33 (s, 2H), 6.01 (s, 1H), 6.48 (d, J=2.4 Hz, 1H), 6.60 (dd, J=9.2 and
2.4 Hz, 1H), 7.18–7.32 (m, 5H), 7.48 (d, J=8.9 Hz, 1H), 13.00 ppm (brs,
2H); 13C NMR (75.5 MHz [D6]DMSO, 278C): d=34.2, 52.8, 64.5, 68.5,
98.1, 106.7, 106.9, 109.2, 125.4, 126.5, 128.4, 128.8, 137.5, 149.9, 151.5,
154.0, 155.1, 160.2, 171.3 ppm; HRMS (ESI): m/z: calcd for C23H21N1O9
[M+H]+: 456.1295; found: 456.1291; elemental analysis calcd (%) for
C23H21N1O9·0.5H2O (464.43): C 59.48, H 4.77, N 3.02; found: C 59.09, H
4.84, N 3.31.
{7-[Bis(carboxymethyl)amino]coumarin-4-yl}methyl phenyl carbonate
R
(1c): The bis-tert-butyl ester of 1c (54.0 mg, 0.1 mmol) was stirred in a
mixture (5 mL) of TFA/CH2Cl2/H2O (74:25:1) at room temperature for
20 min. The solvents were evaporated, and the residue was coevaporated
two times with diethyl ether, dissolved in CH3CN/H2O, and lyophilised to
give 1c (40.6 mg, 93.5%) as a pale yellow solid. M.p. >1108C (decomp);
TLC: Rf =0.34 (CHCl3/MeOH/1% TFA, 95:5:1 v/v); tR =8.10 min (ana-
lytical HPLC, PLRP-S, 30–95% B in A in 20 min, eluent A, 0.1% TFA/
H2O; eluent B, CH3CN); 1H NMR (600 MHz, [D6]DMSO, 278C, TMS):
d=4.24 (s, 4H), 5.48 (s, 2H), 6.17 (s, 1H), 6.50 (d, J=2.2 Hz, 1H), 6.63
(dd, J=9.0 and 2.2 Hz, 1H), 7.30–7.33 (m, 3H), 7.43–7.48 (m, 2H), 7.57
(d, J=9.0 Hz, 1H), 12.93 ppm (s, 2H); 13C NMR (75.5 MHz [D6]DMSO,
278C): d=52.9, 65.4, 98.1, 106.7, 107.3, 109.2, 121.2, 125.6, 126.4, 129.7,
149.3, 150.7, 151.5, 152.5, 155.2, 160.2, 171.3 ppm; HRMS (ESI): m/z:
N-{{7-[Bis(tert-butoxycarbonylmethyl)amino]coumarin-4-yl}methoxycar-
bonyl}-l-norepinephrine: The active ester
(103 mg, 0.24 mmol) and 4-nitrophenyl chloroformate (60.5 mg,
0.3 mmol) in THF (5 mL) in the presence of DMAP (36.7 mg, 0.3 mmol)
as described for the bis-tert-butyl ester of 1c. Dissolution of 7 in DMF
7 was prepared from 6
Chem. Eur. J. 2008, 14, 1621 – 1627
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1625

V. Hagen et al.
(4 mL), addition of DMAP (36.7 mg, 0.3 mmol) and l-norepinephrine hy-
drochloride (5 f, 51.4 mg, 0.25 mmol), again addition of DMAP (36.7 mg,
0.3 mmol) stirring at room temperature for 3.5 h, evaporation, purifica-
tion by preparative RP-HPLC (Nucleogel 100-10) by using a linear gradi-
ent 30–95% B in 60 min (eluent A, H2O; eluent B, CH3CN, retention
time=37.2 min) and lyophilization gave the desired product (70 mg,
47.4%) as a pale-yellow solid. M.p. 115–1168C; TLC: Rf =0.38 (THF/
hexane, 1:1 v/v); tR =11.2 min (analytical HPLC, PLRP-S, 30–95% B in
A in 20 min, eluent A, H2O; eluent B, CH3CN); 1H NMR (300 MHz,
[D6]DMSO, 278C, TMS): d=1.42 (s, 18H) 3.04–3.15 (m, 2H), 4.19 (s,
4H), 4.41–4.45 (m, 1H), 5.19–5.22 (m, 3H), 6.10 (s, 1H), 6.47 (d, J=
1.9 Hz, 1H), 6.56 (m, 2H), 6.66 (d, J=8.0 Hz, 1H), 6.74 (s, 1H), 7.46–
7.52 (m, 2H), 8.67 (s, 1H), 8.78 ppm (s, 1H); 13C NMR (75.5 MHz
[D6]DMSO, 278C): d=27.7, 48.7, 53.5, 60.8, 71.1, 81.1, 98.2, 105.9, 107.0,
109.1, 113.4, 115.1, 116.9, 125.1, 134.5, 144.3, 144.9, 151.3, 151.9, 155.0,
155.4, 160.5, 168.8 ppm; HRMS (ESI): m/z: calcd for C31H38N2O11
[M+Na]+: 637.2373; found: 615.2361.
789.2693; found: 789.2697; elemental analysis calcd (%) for
C41H44N2O12S1 (788.26): C 62.42, H 5.62, N 3.55, S 4.06; found: C 62.27,
H 5.91, N 3.76, S 4.04.
(S)-{{7-[Bis(carboxymethyl)amino]coumarin-4-yl}methoxycarbonyl}-N-
Fmoc-l-cysteine ((S)-BCMACMOC-caged N-Fmoc-l-cysteine, 1g): The
bis-tert-butyl ester of 1g (19.9 mg, 0.025 mmol) was stirred in a mixture
(4 mL) of TFA/CH2Cl2/H2O (75:24:1) at room temperature for 30 min.
The solvents were evaporated, and the residue was coevaporated two
times with diethyl ether, dissolved in CH3CN/H2O, and purified by prepa-
rative RP-HPLC (Nucleogel RP 100-10). The desired product was eluted
by use of a linear gradient of 30–95% B in 60 min (eluent A, H2O/0.1%
TFA; eluent B, CH3CN). The fraction with a retention time of 35.1 min
was collected, evaporated in vacuo, redissolved in CH3CN/H2O, and
lyophilised to give pure 1g (16.9 mg, 98.6%) as a pale-yellow solid. M.p.
>1578C (decomp); [a]2D5 =À35.7 (c=0.67 in DMSO); tR =10.93 min (ana-
lytical HPLC, PLRP-S, 30–95% B in A in 20 min, eluent A, 0.1% TFA/
H2O; eluent B, CH3CN); 1H NMR (300 MHz, [D6]DMSO, 278C, TMS):
d=3.10–3.14 (m, 1H), 3.42–3.45 (m, 1H), 4.23 (s, 4H), 4.23–4.35 (m,
4H), 5.47 (s, 2H), 6.04 (s, 1H), 6.48 (d, J=1.9 Hz, 1H), 6.58 (dd, J=9.0
and J=2.0 Hz, 1H), 7.32 (t, J=7.3 Hz, 2H), 7.40–7.41 (m, 2H), 7.46 (d,
J=8.9 Hz, 1H), 7.71–7.72 (m, 2H), 7.87 (d, J=8.0 Hz, 1H) 7.89 (d, J=
7.2 Hz, 2H), 12.88 ppm (brs, 3H); 13C NMR (75.5 MHz [D6]DMSO,
278C): d=32.2, 46.6, 52.7, 53.3, 64.2, 65.7, 98.1, 106.6, 107.1, 109.2, 120.1,
125.2, 125.4, 127.0, 127.6, 140.7, 143.7, 149.5, 151.5, 155.1, 156.0, 160.2,
169.6, 171.2, 171.5 ppm; HRMS (ESI): m/z: calcd for C33H28N2O12S1
[M+H]+: 677.1451; found: 677.1450; elemental analysis calcd (%) for
C33H28N2O12S1·0.5H2O (685.66): C 57.81, H 4.26, N 4.09, S 4.68; found: C
57.78, H 4.27, N 4.14, S 4.49.
N-{{7-[Bis(carboxymethyl)amino]coumarin-4-yl}methoxycarbonyl}-l-nor-
epinephrine (N-BCMACMOC-caged l-norepinephrine, 1 f): The bis-tert-
butyl ester of 1 f (45 mg, 0.073 mmol) was stirred in a mixture (15 mL) of
TFA/CH2Cl2/H2O (50:50:1) at room temperature for 10 min. The solvents
were evaporated, and the residue was co-evaporated two times with di-
ethyl ether, dissolved in CH3CN/H2O, and lyophilised to give 38.7 mg
(0.073 mmol, 100%) of 1 f. Because the product was not absolutely pure
it was purified by preparative RP-HPLC (Nucleogel 100-10). The desired
product was eluted by use of a linear gradient of 5–60% B in 70 min
(eluent A, H2O/0.1% TFA; eluent B, CH3CN). The fraction with a reten-
tion time of 35.5 min was collected, evaporated in vacuo, redissolved in
CH3CN/H2O, and lyophilised to give pure 1 f (29.8 mg, 77%) as pale-
yellow solid. M.p. >1558C (decomp); [a]2D5 =+29.4 (c=0.67 in DMSO);
tR =10.5 min (analytical HPLC, (PLRP-S), 5–60% B in A in 20 min,
eluent A, 0.1% TFA/H2O; eluent B, CH3CN); 1H NMR (300 MHz,
[D6]DMSO, 278C, TMS): d=3.04–3.12 (m, 2H), 4.22 (s, 4H), 4.42 (t, J=
6.1 Hz, 1H), 5.22 (s, 3H), 6.09 (s, 1H), 6.47 (d, J=1.7 Hz, 1H), 6.53–6.61
(m, 2H), 6.69 (dd, J=7.1 and J=1.7 Hz, 1H), 6.73 (d, J=1.2 Hz, 1H),
7.48–7.53 (m, 2H), 8.71 (s, 1H), 8.82 (s, 1H), 12.89 ppm (brs, 2H);
13C NMR (75.5 MHz [D6]DMSO, 278C): d=48.7, 52.7, 60.8, 71.1, 98.0,
105.9, 106.8, 109.1, 113.4, 115.1, 116.9, 125.2, 134.5, 144.3, 144.9, 151.4,
151.8, 155.0, 155.5, 160.5, 171.3 ppm; HRMS (ESI): m/z: calcd for
C23H22N2O11 [M+Na]+: 525.1121; found: 525.1147; elemental analysis
calcd (%) for C23H22N2O11·1.5H2O (464.43): C 52.18, H 4.76, N 5.29;
found: C 52.19, H 4.98, N 5.42.
O-{{7-[Bis(carboxymethyl)amino]coumarin-4-yl}methyl}
(S)-(4-
acetamidophenyl) thiocarbonate (BCMACMOC-caged 4-acetamidothio-
AHCTREUNG
phenol, 1h): The active ester 7 was prepared following the procedure de-
scribed for the bis-tert-butyl ester of 1c from 6 (103 mg, 0.24 mmol) and
4-nitrophenyl chloroformate (75 mg, 0.38 mmol) in CH2Cl2 (5 mL) in the
presence of iPr2EtN (48 mg, 0.38 mmol). To the solution of 7 were added
a
solution of 4-acetamidothiophenol (62 mg, 0.38 mmol) in CH2Cl2
(5 mL) and iPr2EtN (48 mg, 0.38 mmol). The mixture was stirred at room
temperature for 1 h and the formed O-{{7-[bis(tert-butoxycarbonylmethyl)-
amino]coumarin-4-yl}methyl} (S)-(4-acetamidophenyl) thiocarbonate de-
protected without separation by treatment with TFA (3 mL) and stirring
for 30 min. Compound 1h was precipitated by addition of Et2O and puri-
fied by preparative RP-HPLC (Nucleodur 100-5) by using a linear gradi-
ent 10–50% B in 60 min (eluent A, 0.1% TFA/H2O; eluent B, CH3CN;
retention time=50.6 min). Lyophilization gave 1h (61 mg, 49%) as a
pale-yellow solid. M.p. >1358C (decomp); tR =6.54 min (analytical
HPLC, Nucleodur 100-5, 30–95% B in A in 20 min, eluent A, 0.1%
TFA/H2O; eluent B, CH3CN); 1H NMR (300 MHz, [D6]DMSO, 278C,
TMS): d=2.07 (s, 3H), 4.23 (s, 4H), 5.48 (s, 2H), 6.01 (s, 1H), 6.48 (d,
J=2.0 Hz, 1H), 6.61 (dd, J=9.0 and J=2.0 Hz, 1H), 7.46 (d, J=8.9 Hz,
1H), 7.51 (d, J=8.7 Hz, 1H), 7.68 (d, J=8.6 Hz, 2H), 10.17 (s, 1H),
12.78 ppm (brs, 2H); 13C NMR (75.5 MHz, [D6]DMSO, 278C): d=24.1,
52.7, 64.6, 98.1, 106.7, 107.2, 109.2, 119.3, 119.5, 125.5, 135.7, 141.1, 149.5,
151.5, 155.1, 160.2, 168.7, 171.2 ppm; HRMS (ESI): m/z: calcd for
C23H20N2O9S1 [M+H]+: 501.0968; found: 501.0978; elemental analysis
calcd (%) for C23H20N2O9S1·H2O (518.50): C 53.28, H 4.28, N 5.40, S 6.18;
found: C 52.90, H 4.25, N 5.51, S 6.00.
(S)-{{7-[Bis(tert-butoxycarbonylmethyl)amino]coumarin-4-yl}methoxycar-
bonyl}-N-Fmoc-l-cysteine: The active ester 7 was prepared by following
the procedure described for the bis-tert-butyl ester of 1c from
6
(154.3 mg, 0.36 mmol) and 4-nitrophenyl chloroformate (86 mg,
0.43 mmol) in CH2Cl2 (5 mL) in the presence of DMAP (52 mg,
0.3 mmol). To the solution of 7 were added a solution of N-Fmoc-l-cys-
teine (148 mg, 0.43 mmol; prepared by treatment of N-Fmoc-(S)-trityl-l-
cysteine (252 mg, 0.43 mmol) with a mixture (4 mL) of TFA/CH2Cl2/H2O
(74:25:1) in the presence of triisopropylsilane (0.088 mL, 0.43 mmol) at
room temperature, for 30 min, and evaporation) in CH2Cl2 (5 mL) and
DMAP (150 mg, 1.23 mmol). The mixture was stirred at room tempera-
ture for 20 h, was evaporated, and purified by preparative RP-HPLC
(Nucleodur 100-5) by using a linear gradient 30–95% B in 60 min (eluent
A, 0.1% TFA/H2O; eluent B, CH3CN; retention time=54.1 min). Lyo-
philization gave the desired product (49 mg, 17%) as a pale-yellow solid.
M.p. 108–1108C (decomp); TLC: Rf =0.22 (CH2Cl2/CH3OH, 9:1 v/v);
tR =18.37 min (analytical HPLC, Nucleodur 100-5, 30–95% B in A in
20 min, eluent A, H2O; eluent B, CH3CN); 1H NMR (300 MHz,
[D6]DMSO, 278C, TMS): d=1.41 (s, 18H), 3.08–3.15 (m, 1H), 3.41–3.47
(m, 1H), 4.19 (s, 4H), 4.21–4.34 (m, 4H), 5.48 (s, 2H), 6.05 (s, 1H), 6.47
(d, J=1.9 Hz, 1H), 6.58 (dd, J=9.0 and J=2.3 Hz, 1H), 7.31 (t, J=
7.3 Hz, 2H), 7.40 (t, J=7.4 Hz, 2H), 7.47 (d, J=8.9 Hz, 1H), 7.71 (d, J=
7.3 Hz, 2H), 7.87 (d, J=8.0 Hz, 1H), 7.89 (d, J=7.3 Hz, 2H), 12.88 ppm
(brs, 1H); 13C NMR (75 MHz [D6]DMSO, 278C): d=27.7, 32.3, 46.6,
53.3, 53.5, 64.3, 65.8, 81.1, 98.2, 106.8, 107.2, 109.2, 120.1, 125.2, 125.4,
127.0, 127.6, 140.7, 143.7, 149.5, 151.5, 155.1, 156.0, 160.2, 168.7, 169.6,
171.5 ppm; HRMS (ESI): m/z: calcd for C41H44N2O12S1 [M+H]+:
Solubility: The solubilities of 1a–h in HEPES buffer (10 mm HEPES and
120 mm KCl adjusted to pH 7.2 with 2n KOH) were estimated by using
analytical RP-HPLC at room temperature.
Hydrolytic stability: Freshly prepared solutions of 1a–h in HEPES
buffer, pH 7.2, and of 1b in citrate/phosphate buffer, pH 4.0, were left in
the dark at room temperature and monitored over a period of 24 h by
using analytical RP-HPLC.
Photochemical quantum yields: The differential photochemical quantum
yields (fchem) were determined for 1a and 1c–h at 365 nm in 5% CH3CN/
0.01m HEPES/KOH buffer (pH 7.2) by the relative method as previously
described[31] by using (6,7-dimethoxycoumarin-4-yl)methyl diethyl phos-
phate (fchem =0.08)[32] in 5% CH3CN/0.01m HEPES/KOH buffer
(pH 7.2) as standard. Identical absorbances for the references and 1a or
1626
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 1621 – 1627

Coumarin Derivatives for Photorelease
FULL PAPER
1c–h were used during photolysis. For kinetic investigations the irradiat-
ed solutions of 1a, 1c–h, and (6,7-dimethoxycoumarin-4-yl)methyl diethyl
phosphate were analysed by using analytical HPLC.
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Fluorescence quantum yields: The fluorescence quantum yields (ff) of
1a, 1c–h, and of 6 were determined at 258C in CH3CN/HEPES buffer
(5:95), pH 7.2, by the relative method[33] versus quinine sulfate in 0.1n
H2SO4 as a standard (ff =0.545). At the excitation wavelength used, the
absorbance values of the standard and the investigated compounds were
identical.
[17]B. Cürten, P. H. M. Kullmann, M. E. Bier, K. Kandler, B. F. Schmidt,
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This work was supported by the Deutsche Forschungsgemeinschaft (HA
2694/3), and the Fonds der Chemischen Industrie.
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Received: July 24, 2007
Published online: November 28, 2007
Chem. Eur. J. 2008, 14, 1621 – 1627
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