Neurotransmitter amino acid-oxobenzo[f]benzopyran conjugates: synthesis and photorelease studies

Article abstract of DOI:10.1016/j.tet.2008.09.050

A series of fluorescent conjugates of neurotransmitter amino acids, such as β-alanine, tyrosine, 3,4-dihydroxyphenylalanine (DOPA) and glutamic acid, were prepared by reaction with a suitable fluorophore, namely 1-chloromethyl-9-methoxy-3-oxo-3H-benzo[f]benzopyran. The photophysical properties of the resulting ester bioconjugates were evaluated as well as the stability to photolysis at different wavelengths of irradiation (250, 300, 350 and 419 nm).

Full text of DOI:10.1016/j.tet.2008.09.050

Tetrahedron 64 (2008) 11175–11179
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Neurotransmitter amino aciddoxobenzo[f]benzopyran conjugates: synthesis
and photorelease studies
*
Maria J.G. Fernandes, M. Sameiro T. Gonçalves, Susana P.G. Costa
Centro de Qu´ımica, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of fluorescent conjugates of neurotransmitter amino acids, such as b-alanine, tyrosine, 3,4-di-
Received 8 August 2008
Received in revised form 17 September
2008
Accepted 18 September 2008
Available online 26 September 2008
hydroxyphenylalanine (DOPA) and glutamic acid, were prepared by reaction with a suitable fluorophore,
namely 1-chloromethyl-9-methoxy-3-oxo-3H-benzo[f]benzopyran. The photophysical properties of the
resulting ester bioconjugates were evaluated as well as the stability to photolysis at different wave-
lengths of irradiation (250, 300, 350 and 419 nm).
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Oxobenzobenzopyran
Benzocoumarin
Neurotransmitters
Amino acids
Photolysis
1. Introduction
because they can also act as temporary fluorescent labels. Fluo-
rescent labelling is suitable for analytical purposes, for being far
Photolabile protecting groupsdlight-sensitive molecules that
covalently bond to functional groups of molecules, which can then
be released photochemically, without the need for any chemical
reagent, have received remarkable interest in recent years, in or-
ganic synthesis as well as in life sciences.1
The use of photochemical methods as a deprotection procedure,
alternatively to conventional approaches, which employ acids, ba-
ses, reducing or oxidising reagents is an interesting tool in organic
synthesis.2 The protection of signalling biomolecules with a light-
sensitive group is also valuable in biological and medical research
fields, as photoactivable or caged biomolecules allow temporal and
spatially controlled delivery of bioactive molecules.3 Several
photoremovable protecting groups are now known for different
families of compounds including alcohols,4 amines,5 phosphates,6
aldehydes and ketones7 as well as carboxylic acids.8
more sensitive than common UV techniques, overcoming low de-
tection limits. In recent years, the direction of improvement on
photoreleasable groups has been towards the application of poly-
cyclic aromatic structures, which are fluorophores in most cases,
nonetheless the inconvenience of fluorescence deactivation in
some photochemical processes.
Compounds possessing the carboxylic acid group are of great
interest in organic chemistry and commonly need to be protected
in sequential synthesis against various reagents including reactive
nucleophiles, reducing agents and oxidants. Numerous carboxylic
acids are also bioactive molecules and so the photochemical release
of caged carboxylic compounds is of interest to basic biological
research and biomedical applications.
Among the most appealing carboxylic compounds are amino
acids, which play central roles both as building blocks of proteins
and as intermediates in metabolism. These molecules are of the
Recently, a number of moieties have been reported as new
photolabile protecting groups for carboxylic acids, including 2-
most abundant neurotransmitters in the brain.
b-Alanine is the
(dimethylamino)-5-nitrophenyl,9
a
-carboxy nitrobenzyl,10 3-nitro-
only naturally occurring beta amino acid and acts as a physiological
transmitter, being the rate-limiting precursor of carnosine, which is
2-naphthalenemethanol,11 p-hydroxyphenacyl,12
a
-keto amides,13
2-hydroxy-1,2,2-triphenylethanone,14
1-acyl-7-nitroindolines,15
a
b
-alanine–histidine dipeptide present in muscle and brain
tissues.17
The amino acid
and coumarin derivatives.16 Some of these groups are fluorescent,
which present advantages over those that are non-fluorescent,
L-tyrosine is a precursor of dopamineda neu-
rotransmitter involved in controlling movement, regulating
memory, sexual and reward-seeking behaviours and the regulation
of pituitary hormones. 3,4-Dihydroxyphenylalanine (DOPA) has
been historically considered an inert amino acid that alleviates the
* Corresponding author. Tel.: þ351 253 604054; fax: þ351 253 604382.
E-mail address: spc@quimica.uminho.pt (S.P.G. Costa).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.09.050

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M.J.G. Fernandes et al. / Tetrahedron 64 (2008) 11175–11179
sulfuric acid at room temperature, as reported earlier.20b This
fluorophore will be designated in this report by a three-letter code
(Bba) for simplicity of naming the various fluorescent conjugates, as
indicated in Table 1.
Our purpose being the investigation of the behaviour to pho-
tolysis conditions of the ester linkage between fluorophore 1 and
the different neurotransmitters, we prepared the corresponding
conjugates for their evaluation under irradiation at 254, 300, 350
and 419 nm. Derivatisation at the carboxylic acid group of N-ben-
zyloxycarbonyl-protected amino acid neurotransmitters, such as
Table 1
Yields, UV–vis and fluorescence data for fluorescent neurotransmitter conjugates
3a–e in absolute ethanol
Compound
Yield (%) UV–vis
lmax (nm) log
Fluorescence
3
lmax (nm) FF
Stokes’ shift
(nm)
3a Z-
3b Z-Tyr(OtBu)–OBba 76
3c Z-DOPA–OBba 36
b
-Ala–OBba
80
347
348
349
349
346
3.88 466
3.95 465
3.83 463
3.88 469
4.00 464
0.75 119
0.69 117
0.14 114
0.74 120
0.75 118
3d Z-Glu(OMe)–OBba 76
3e Z-Glu(OBba)–OMe 29
b-alanine 2a, tyrosine 2b, 3,4-dihydroxyphenylalanine (DOPA) 2c
and glutamic acid, at the main and side chain, 2d and 2e, re-
spectively, with fluorophore 1 was carried out in DMF, at room
temperature, by using potassium fluoride.21
symptoms of Parkinson’s disease by its conversion to dopamine via
the enzyme aromatic -amino acid decarboxylase. Misu et al. have
L
proposed that DOPA itself is a neurotransmitter and/or neuro-
modulator in addition to being a precursor of dopamine. It fulfils
the several criteria that must be satisfied before a compound is
accepted as a neurotransmitter in the homeostatic mechanism for
maintaining blood pressure.18 Glutamate is the major excitatory
neurotransmitter in the mammalian central nervous system,
largely distributed in all regions of the brain and being involved in
cognitive functions like learning and memory. Furthermore, it is
also a precursor for the synthesis of the major inhibitory neuro-
The expected bioconjugates 3a–e (Scheme 1, Table 1) were
obtained in low to good yields (29–80%) and characterised by the
usual spectroscopic techniques.
TheIR spectra ofconjugates 3a–eshowed bands duetostretching
vibrations of the ester carbonyl group of the fluorophore-amino acid
linkage from 1712 to 1729 cmꢀ1. 1H NMR spectra showed signals of
the amino acid residues, such as the
e) or -CH2 ( 2.76 ppm, for 3a), as well as the characteristic protons
of the fluorophore methylene group ( 5.58–5.67 ppm). The confir-
a-CH (d 4.42–4.55 ppm, for 3b–
a
d
d
transmitter, the g
-aminobutyric acid (GABA).19
mation of the presence of the newly formed ester linkage was also
supported by 13C NMR spectra signals of the carbonyl group, which
Our recently reported results involve the design and application
of fluorescent oxygen heterocycles, as novel labels and photo-
releasable protecting groups for the carboxylic acid function of
amino acids.16c–d,20 In previous work, we prepared conjugates of
GABA-neurotransmitter amino acid, with a set of polyaromatic and
polyheteroaromatic fluorescent reagents, based on naphthalene,
pyrene and oxobenzopyran derivatives (trivially known as cou-
marins).20c The evaluation of their photophysical properties and
their behaviour towards photocleavage revealed that an angular
fused oxobenzopyran derivative proved to be the most fluorogenic
derivatisation reagent and the most effective as photocleavable
protecting group under irradiation at 350 nm. Photolysis at around
350 nm is preferable in studies involving biomolecules as it avoids
cell damage due to short-wavelength light, and so we decided to
study the caging and uncaging of several other relevant neuro-
transmitter amino acids, using the 1-chloromethyl-9-methoxy-
were found between d 171.18 and 171.80 ppm.
2.2. Evaluation of the photophysical properties
of conjugates 3a–e
The UV–vis absorption and emission spectra of degassed 10ꢀ5 to
10ꢀ6 M solutions in absolute ethanol of conjugates 3a–e were
measured, absorption and emission maxima, molar absorptivities
and fluorescence quantum yields are also reported (Table 1).
Fluorescence quantum yields were calculated using 9,10-dipheny-
lanthracene as standard (FF¼0.95 in ethanol).22 For the FF de-
termination, the fluorescence standard was excited at the
wavelengths of maximum absorption found for each one of
the compounds to be tested and in all fluorimetric measurements
the absorbance of the solution did not exceed 0.1. Conjugates 3a–e
exhibited moderate to excellent fluorescence quantum yields
3-oxo-3H-benzo[f]benzopyran fluorophore, in
a MeOH/HEPES
solution (80:20).
(0.14<FF<0.75), and large Stokes’ shifts between 114 and 120 nm.
The DOPA conjugate 3c showed the lower fluorescence quantum
yield, through fluorescence deactivation probably due to the possi-
bility of establishing extra H-bonds through the free hydroxyl
groups to the solvent. All the studied neurotransmitters are
not easily detectable by UV–vis absorption and have no or low in-
trinsic fluorescence and thus, the introduction of a fluorophore like
the considered oxobenzobenzopyran 1 proved to be a very appro-
priate strategy to enhance the photophysical properties of the
2. Results and discussion
2.1. Synthesis
1-Chloromethyl-9-methoxy-3-oxo-3H-benzo[f]benzopyran
was synthesised through Pechmann reaction, between 7-
methoxy-2-naphthol and ethyl 4-chloroacetoacetate, catalysed by
1
a
O
a n = 2, R = H
R
OtBu
OH
O
b n = 1, R = H2C
O
O
n
O
Ph
O
N
H
c n = 1, R =
O
H2C
d n = 1, R = H2C
R
O
3a-d
OH
+
OH
O
n
O
Ph
Ph
O
N
H
O
Cl
O
KF
2a-d
O
or
O
DMF
r.t.
or
OH
O
O
O
O
O
O
O
O
O
N
H
O
O
Ph
O
N
H
2e
O
3e
O
Scheme 1. Synthesis of fluorescent neurotransmitter ester conjugates 3a–e.

M.J.G. Fernandes et al. / Tetrahedron 64 (2008) 11175–11179
11177
resulting conjugates. Moreover, the fluorescence quantum yield of
14
13
12
11
10
9
the fluorophore in its free form was low (FF¼0.03)16c and had
a dramatic increase upon connection via a ester linkage to the
amino acid.
2.3. Photolysis studies of neurotransmitter conjugates 3a–e
Considering that the main goal of this work was to study the
possibility of using fluorophore 1 as a fluorescent photocleavable
protecting group in the caging of relevant biomolecules like neu-
rotransmitters, photolysis studies of conjugates 3a–e were carried
out. Solutions of the mentioned compounds in methanol/HEPES
buffer 80:20 were irradiated in a Rayonet RPR-100 reactor, at 254,
300, 350 and 419 nm, in order to determine the best cleavage
conditions. The course of the photocleavage reaction was followed
by reverse phase HPLC with UV detection.
The plots of peak area (A) of the starting material versus irra-
diation time were obtained for each compound, at the considered
wavelengths. Peak areas were determined by HPLC, which revealed
a gradual decrease with time, and were the average of three runs.
The determined irradiation time represents the time necessary for
the consumption of the starting materials until less than 5% of the
initial area was detected (Table 2).
0
20
40
60
80 100 120 140 160 180 200
Irradiation time (min)
Figure 1. Plot of ln A versus irradiation time for the photolysis of conjugates 3a (-), 3b
(:), 3c (C), 3d (ꢁ) and 3e (A) at 350 nm in MeOH/HEPES (80:20) solution.
the side chain, respectively, revealed that cleavage of the ester
linkage at the side chain was always faster, in the conditions tested.
As reported before,16c the N-benzyloxycarbonyl urethane-type
blocking group was stable in the tested conditions, no cleavage
being detected. The photochemical quantum yields were calculated
as previously described23 and were lower than 0.01. The efficiency
of the photocleavage process was not high, due to fluorescence
deactivation and other photophysical processes, which limit the
overall quantum yield of deprotection, as well as the low power of
the lamps used and the fact that using an open chamber reactor can
result in a larger dissipation of radiation. Nevertheless, considering
the short irradiation times, 1-chloromethyl-9-methoxy-3-oxo-3H-
benzo[f]benzopyran 1 can be considered an additional alternative
to other established photocleavable protecting groups with appli-
cation in neurotransmitter chemistry.
For each compound and based on HPLC data, the plot of ln A
versus irradiation time showed a linear correlation for the disap-
pearance of the starting material, which suggested a first order
reaction, obtained by the linear least squares methodology for
a straight line (Fig. 1). The corresponding rate constants were cal-
culated and are presented in Table 2.
Concerning the influence of the wavelength of irradiation on the
rate of photocleavage reactions of conjugates 3a–e in methanol/
HEPES buffer 80:20 solution, it was found that the irradiation times
were quite comparable at 254 and 350 nm, irradiation at 350 nm
being preferable for reasons explained earlier. For all the wave-
lengths of irradiation considered, the irradiation times for conju-
gate 3b were always shorter and so the photocleavage at 419 nm
was also tested. Although a large increase (ca. 7 h) in the irradiation
time was observed (473 min, k¼6.3ꢁ10ꢀ3 minꢀ1), the wavelength
of 419 nm can still be considered useful for certain practical
applications.
Regarding our synthesised conjugates, which are benzyl-type
carboxylic acid esters and if we consider the conventional depro-
tection methods usually employed in the deprotection of carboxylic
benzyl esters, such as catalytic hydrogenolysis and acid or base
hydrolysis, we can suggest that photocleavage is an attractive
method to achieve deprotection of this type of compounds. In
terms of reaction time, chemical methods can take longer than the
irradiation times presented in this report, with additional reagents
being necessary.
Taking into consideration the influence of the structure of the
neurotransmitter on the photocleavage rates, we could see that the
3. Conclusions
b
-alanine conjugate 3a had the largest irradiation times for all
Fluorescent neurotransmitter amino acid ester conjugates 3a–e
were prepared in moderate to good yields by using a simple syn-
thetic method involving a chloromethylated oxobenzobenzopyran
precursor and the carboxylic acid group of a series of N-benzylox-
ycarbonyl-protected neurotransmitter amino acids. The photophysical
studies confirmed that the 1-chloromethyl-9-methoxy-3-oxo-3H-
benzo[f]benzopyran 1 is a suitable fluorogenic reagent for the
derivatisation of non-fluorescent molecules: the relative fluores-
cence quantum yields of the bioconjugates were determined and
found to be moderate to excellent.
wavelengths of irradiation. The presence of polar side chains in the
other studied neurotransmitters appeared to have an influence on
the decrease of the irradiation times, as shorter irradiation times
were necessary for all the other conjugates 3b–e compared to that
of conjugate 3a. The O-blocked tyrosine conjugate 3b cleaved faster
in all wavelengths of irradiation, while that the closely related
DOPA conjugate 3c cleaved slower (ca. 3–6 times), suggesting
a detrimental effect of the free hydroxyl groups (3b) on the irra-
diation time, when compared to the hydroxyl-protected compound
(3c). Comparison of the data obtained for the glutamic acid con-
jugates 3d and 3e, bearing the fluorophore at the main chain and at
Regarding the photocleavage studies of the fluorescent conju-
gates, in methanol/HEPES buffer solution (80:20), at 254, 300 and
Table 2
Irradiation times (in min) and rate constants (ꢁ10ꢀ2 minꢀ1) for the photolysis of compounds 3a–e at different wavelengths in MeOH/HEPES (80:20) solution
Compound
254 nm
Irr time
300 nm
Irr time
350 nm
Irr time
k
k
k
3a
3b
3c
3d
3e
Z-
b
-Ala–OBba
418
53
302
217
178
0.72
5.69
0.94
1.26
1.72
957
64
250
547
271
0.31
4.80
1.15
0.53
1.13
513
44
183
233
135
0.59
6.70
1.57
1.24
2.24
Z-Tyr(OtBu)–OBba
Z-DOPA–OBba
Z-Glu(OMe)–OBba
Z-Glu(OBba)–OMe

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M.J.G. Fernandes et al. / Tetrahedron 64 (2008) 11175–11179
350 nm, it was possible to conclude that the structure of the neu-
rotransmitter had an influence on the irradiation times and, espe-
cially in the case of the tyrosine conjugate 3b, can be considered
useful for practical applications. Owing to the irradiation times,
photolysis at 350 nm would be the most convenient for the
uncaging of the compounds studied.
In summary, the synthesised conjugates required short irradi-
ation times for photocleavage to occur, making them appropriate
for using as photolabile protecting groups for organic molecules,
including amino acids and other relevant biomolecules, in addition
to their usefulness in fluorescent labelling due to the high Stokes’
shifts and good fluorescent quantum yields.
7.90 (d, J 8.8 Hz, 1H, H-6). 13C NMR (CDCl3):
d
¼34.45 (CH2), 36.46
(CH2), 55.43 (OCH3), 64.44 (CH2 Bba), 66.82 (CH2 Z), 105.79 (C-10),
111.81 (C-4b), 113.11 (C-2), 115.30 (C-5), 116.45 (C-8), 126.35 (C-6a),
128.49 (2ꢁPh-C), 130.17 (3ꢁPh-C), 130.53 (C-6b), 131.34 (C-7),
133.80 (C-6), 136.29 (C1 Ph), 150.50 (C-1), 155.56 (C-4a), 156.25
(C]O urethane), 159.63 (C-9), 160.13 (C-3), 171.53 (C]O ester). IR
(KBr 1%, cmꢀ1):
1519, 1455, 1445, 1364, 1337, 1231, 1168, 1139, 1075, 1059, 1020, 935,
n
¼3378, 3064, 3033, 2926, 2854, 1722, 1625, 1552,
838, 737, 697. UV–vis (MeOH/HEPES 80:20, nm): lmax (log
3
)¼349
(4.08), 277 (3.94), 235 (4.54). HRMS (EI): calcd for C26H23NO7 [Mþ]
461.1475; found 461.1476.
4.2.2. N-(Benzyloxycarbonyl)-O-(tert-butyloxycarbonyl)-L-tyrosine
4. Experimental section
4.1. General
(9-methoxy-3-oxo-3H-benzo[f]benzopyran-1-yl) methyl ester,
Z-Tyr(OtBu)–OBba (3b)
Starting from compound
1 (0.100 g, 0.364 mmol) and Z-
Tyr(OtBu)–OH$DCHA (2b) (0.201 g, 0.364 mmol), compound 3b
All melting points were measured on a Stuart SMP3 melting
point apparatus and are uncorrected. TLC analyses were carried out
on 0.25 mm thick precoated silica plates (Merck Fertigplatten Kie-
selgel 60F254) and spots were visualised under UV light. Chroma-
tography on silica gel was carried out on Merck Kieselgel (230–240
mesh). IR spectra were determined on a BOMEM MB 104 spectro-
photometer using KBr discs. UV–vis absorption spectra (200–800 nm)
were obtained using a Shimadzu UV/2501PC spectrophotometer.
NMR spectra were obtained on a Varian Unity Plus Spectrometer at
an operating frequency of 300 MHz for 1H NMR and 75.4 MHz for
13C NMR or a Bruker Avance III 400 at an operating frequency of
400 MHz for 1H NMR and 100.6 MHz for 13C NMR using the solvent
peak as internal reference at 25 ꢂC. All chemical shifts are given in
parts per million using dH Me4Si¼0 ppm as reference and J values
are given in hertz. Assignments were made by comparison of
chemical shifts, peak multiplicities and J values and were supported
by spin decoupling-double resonance and bidimensional hetero-
nuclear HMBC and HMQC correlation techniques. Mass spectrom-
etry analyses were performed at the ‘C.A.C.T.I.dUnidad de
Espectrometria de Masas’, at University of Vigo, Spain. Fluorescence
spectra were collected using a FluoroMax-4 spectrofluorometer.
Photolyses were carried out using a Rayonet RPR-100 chamber
reactor equipped with 10 lamps of 254 (35 W), 300 (21 W), 350
(24 W) and 419 (14 W) nm. HPLC analyses were performed using
was obtained as a yellow solid (0.169 g, 76%). Mp¼73.0–74.1 ꢂC. 1H
NMR (CDCl3):
d
¼1.30 (s, 9H, C(CH3)3), 3.10–3.19 (m, 2H,
b-CH2), 3.95
(s, 3H, OCH3), 4.74–4.79 (m, 1H, a-H), 5.09 (m, 2H, CH2 Z), 5.13 (d, J
7.6 Hz, 1H, NH), 5.66 (m, 2H, CH2 Bba), 6.63 (s, 1H, H-2), 6.88 (d, J
8.4 Hz, 2H, H-30and H-50), 7.02 (d, J 8.4 Hz, 2H, H-20and H-60), 7.23
(dd, J 8.8 and 2.4 Hz, 1H, H-8), 7.32–7.34 (m, 6H, 5ꢁPh-H and H-5),
7.41 (br s,1H, H-10), 7.83 (d, J 9.2 Hz,1H, H-7), 7.91 (d, J 8.8 Hz,1H, H-
6). 13C NMR (CDCl3):
d
¼28.76 (C(CH3)3), 37.35 (
b-CH2), 55.13 (a-C),
55.44 (OCH3), 64.96 (CH2 Bba), 67.21 (CH2 Z), 78.47 (C(CH3)3),105.50
(C-10), 111.76 (C-4b), 113.32 (C-2), 115.29 (C-5), 116.65 (C-8), 124.26
(C-30 and C-50),126.33 (C-6a),128.17 (2ꢁPh-C),128.23 (Ph-C),128.49
(2ꢁPh-C), 129.53 (C-20and C-60), 129.75 (C10), 130.50 (C-6b), 131.35
(C-7), 133.81 (C-6), 135.96 (C-1 Ph), 149.86 (C-1), 154.74 (C-40),
155.58 (C-4a), 155.69 (C]O urethane), 159.71 (C-9), 159.97 (C-3),
171.18 (C]O ester). IR (KBr 1%, cmꢀ1):
2929, 2853, 1725, 1625, 1553, 1519, 1507, 1455, 1445, 1427, 1365,
1339, 1232, 1162, 1105, 1061, 1025, 898, 838, 696. UV–vis (MeOH/
n
¼3336, 3065, 3032, 2975,
HEPES 80:20, nm): lmax (log
3
)¼351 (4.03), 273 (3.82), 235 (4.55).
HRMS (EI): calcd for C36H35NO8 [Mþ] 609.2364; found 609.2363.
4.2.3. N-(Benzyloxycarbonyl)-3,4-dihydroxy-L-phenylalanine
(9-methoxy-3-oxo-3H-benzo[f]benzopyran-1-yl) methyl ester,
Z-DOPA–OBba (3c)
Starting from compound 1 (0.100 g, 0.364 mmol) and Z-DOPA–
OH (2c) (0.121 g, 0.364 mmol), compound 3c was obtained as
a Licrospher 100 RP18 (5 mm) column in a HPLC system composed
by a Jasco PU-980 pump, a UV/Vis Shimadzu SPD-GAV detector and
a Shimadzu C-RGA Chromatopac register. All reagents were used as
received.
a yellow oily solid (0.087 g, 36%). 1H NMR (CDCl3):
d
¼2.90–2.97 (m,
-CH2), 3.92 (s, 3H, OCH3), 4.70–4.77
-H), 5.10 (s, 2H, CH2 Z), 5.49 (d, J 8.7 Hz, 1H, NH), 5.58 (d, J
1H,
b-CH2), 3.08–3.15 (m, 1H, b
(m, 1H,
a
5.1 Hz, 2H, CH2 Bba), 6.29 (s, 1H, H-2), 6.55 (dd, J 8.1 and 1.5 Hz, 1H,
H-60), 6.70 (d, J 1.8 Hz, 1H, H-20), 6.76 (d, J 7.8 Hz, 1H, H-50), 7.19 (d, J
7.2 and 2.1 Hz,1H, H-8), 7.27–7.40 (m, 6H, 5ꢁPh-H and H-5), 7.37 (d,
J 2.1 Hz, H-10), 7.78 (d, J 9.0 Hz, H-7), 7.85 (d, J 8.7 Hz, H-6). 13C NMR
4.2. General procedure for the synthesis of compounds 3a–e
1-Choromethyl-9-methoxy-3-oxo-3H-benzo[f]benzopyran, Bba-
Cl (1 equiv) was dissolved in dry DMF (2 mL), potassium fluoride
(3 equiv) and the corresponding amino acid neurotransmitters
1a–e (1 equiv) were added. The reaction mixture was stirred at
room temperature for 3–4 days. The solvent was removed by rotary
evaporation under reduced pressure and the crude residue was
purified by column chromatography in silca gel using dichloro-
methane as eluent.
(CDCl3):
d
¼37.76 (
b-CH2), 55.42 (a-C), 55.49 (OCH3), 64.78 (CH2
Bba), 67.29 (CH2 Z), 105.84 (C-10), 111.83 (C-4b), 112.16 (C-2), 115.05
(C-5), 116.10 (C-50), 116.26 (C-20), 116.66 (C-8), 121.57 (C-60), 127.47
(C-10), 128.16 (2ꢁPh-C), 128.26 (Ph-C), 128.50 (2ꢁPh-C), 130.31 (C-
6b), 131.33 (C-7), 134.00 (C-6), 135.89 (C1 Ph), 143.79 (C-40), 144.45
(C-30), 151.04 (C-1), 155.22 (C-4a), 155.89 (C]O urethane), 159.70
(C-9), 161.27 (C-3), 171.55 (C]O ester). IR (KBr 1%, cmꢀ1):
(br), 2927, 1712, 1702, 1625, 1552, 1519, 1445, 1363, 1340, 1284, 1233,
1216, 1177, 1114, 1062, 1025, 839, 751, 697, 666. UV–vis (MeOH/
n
¼3415
4.2.1. N-(Benzyloxycarbonyl)-
benzo[f]benzopyran-1-yl) methyl ester, Z-
Starting from compound 1 (0.100 g, 0.364 mmol) and Z-
L
-
b
-alanine (9-methoxy-3-oxo-3H-
b
-Ala–OBba (3a)
HEPES, 80:20, nm): lmax (log
3
)¼350 (3.91), 280 (3.84), 236 (4.55).
b-Ala–
HRMS (EI): calcd for C32H27NO9 [Mþ] 569.1686; found 569.1690.
OH (2a) (0.081 g, 0.364 mmol), compound 3a was obtained as
a yellow solid (0.135 g, 80%). Mp¼121.4–123.0 ꢂC. 1H NMR (CDCl3):
4.2.4. 2-(N-Benzyloxycarbonyl)amino-5-methyl-1-(9-methoxy-3-
oxo-3H-benzo[f]benzo pyran-1-yl) methyl pentanedioate,
Z-Glu(OMe)–OBba (3d)
d
¼2.76 (t, J 6.0 Hz, 2H, CH2), 3.53 (q, J 6.0 Hz, 2H, CH2), 3.95 (s, 3H,
OCH3), 5.10 (s, 2H, CH2 Z), 5.35 (br s, 1H, NH), 5.67 (s, 2H, CH2 Bba),
6.63 (s, 1H, H-2), 7.21 (dd, J 8.8 and 2 Hz, 1H, H-8), 7.27–7.35 (m, 6H,
5ꢁPh-H and H-5), 7.42 (br s, 1H, H-10), 7.82 (d, J 8.8 Hz, 1H, H-7),
Starting from compound
1 (0.100 g, 0.364 mmol) and Z-
Glu(OMe)–OH (2d) (0.107 g, 0.364 mmol), compound 3d was

M.J.G. Fernandes et al. / Tetrahedron 64 (2008) 11175–11179
11179
obtained as a light brown solid (0.147 g, 76%). Mp¼83.2–84.5 ꢂC. 1H
0.6 (compound 3c), 0.8 (compounds 3a,d–e) or 1.2 (compound
3b) mL/min, previously filtered through a Millipore, type HN
0.45 mm filter and degassed by ultra-sound for 30 min. The chro-
matograms were traced by detecting UV absorption at the wave-
length of maximum absorption for each compound (retention time:
3a, 5.3; 3b, 8.1; 3c, 5.6; 3d, 5.3; 3e, 5.4 min).
NMR (CDCl3):
d
¼2.02–2.15 (m, 1H,
g-CH2), 2.24–2.33 (m, 1H, g-CH2),
2.45–2.50 (m, 2H,
Bba), 4.52–4.59 (m, 1H,
b
-CH2), 3.64 (s, 3H, OCH3 Glu), 3.92 (s, 3H, OCH3
-H), 5.10 (s, 2H, CH2 Z), 5.63 (s, 2H, CH2
a
Bba), 5.75 (d, J 8.1 Hz, 1H, NH), 6.62 (s, 1H, H-2), 7.18 (dd, J 9.0 and
2.1 Hz, 1H, H-8), 7.22–7.31 (m, 7H, 5ꢁPh-H, H-5 and H-10), 7.77 (d, J
9.3 Hz, 1H, H-7), 7.84 (d, J 8.7 Hz, 1H, H-6). 13C NMR (CDCl3):
d
¼26.88 (
g-CH2), 29.84 (b-CH2), 51.83 (OCH3 Glu), 53.54 (a-C),
Acknowledgements
55.36 (OCH3 Bba), 64.94 (CH2 Bba), 67.14 (CH2 Z), 105.53 (C-10),
111.57 (C-4b), 112.83 (C-2), 115.12 (C-5), 116.49 (C-8), 126.19 (C-6a),
128.12 (3ꢁPh-C), 128.40 (2ꢁPh-C), 130.35 (C-6b), 131.26 (C-7),
133.71 (C-6), 135.91 (C1 Ph), 150.02 (C-1), 155.39 (C-4a), 156.00
(C]O urethane), 159.59 (C-9), 159.97 (C-3), 171.25 (C]O ester),
Thanks are due to the Foundation for Science and Technology
(Portugal) for financial support through project PTDC/QUI/69607/
2006 and a Ph.D. grant to M.J.G.F. (SFRH/BD/36695/2007).
172.96 (C]O Glu). IR (KBr 1%, cmꢀ1):
1729, 1692, 1625, 1595, 1553, 1519, 1445, 1381, 1365, 1267, 1231,
1124, 1055, 1026, 985, 941, 840, 821, 728, 697. UV–vis (MeOH/
n
¼3329, 3067, 3033, 2952,
References and notes
1. (a) Bochet, C. G. J. Chem. Soc., Perkin Trans. 1 2002, 125; (b) Pelliccioli, A. P.; Wirz,
J. Photochem. Photobiol. Sci. 2002, 1, 441.
2. (a) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 3rd ed.;
Wiley: New York, NY, 1999; (b) Kocienski, P. J. Protecting Groups; Georg Thieme:
New York, NY, 2000.
3. (a) Goeldner, M.; Givens, R. S. Dynamic Studies in Biology: Phototriggers, Pho-
toswitches and Caged Biomolecules; Wiley-VCH: Weinheim, 2005; (b) Mayer, G.;
Heckel, A. Angew. Chem., Int. Ed. 2006, 45, 4900.
HEPES, 80:20, nm): lmax (log
3
)¼351 (4.00), 274 (3.79), 239.0 (4.54).
HRMS (EI): calcd for C29H27NO9 [Mþ] 533.1686; found 533.1683.
4.2.5. 4-(N-Benzyloxycarbonyl)amino-5-methyl-1-(9-methoxy-3-
oxo-3H-benzo[f]benzo pyran-1-yl) methyl pentanedioate,
Z-Glu(OBba)–OMe (3e)
Starting from compound
1 (0.070 g, 0.255 mmol) and Z-
4. (a) Loudwig, S.; Goeldner, M. Tetrahedron Lett. 2001, 42, 7957.
5. Peng, L.; Wirz, J.; Goeldner, M. Tetrahedron Lett. 1997, 38, 2961.
6. (a) Dinkel, C.; Wichmann, O.; Schultz, C. Tetrahedron Lett. 2003, 44, 1153; (b)
Dinkel, C.; Schultz, C. Tetrahedron Lett. 2003, 44, 1157; (c) Li, W.-h.; Llopis, J.;
Whitney, M.; Zlokarnik, G.; Tsien, R. Y. Nature 1998, 392, 936.
7. (a) Wang, P.; Hu, H.; Wang, Y. Org. Lett. 2007, 9, 1533; (b) Yu, J.-Y.; Tang, W.-J.;
Wang, H.-B.; Song, Q.-H. J. Photochem. Photobiol., A 2007, 185, 101; (c) Kantevari,
S.; Narasimhaji, C. V.; Mereyala, H. B. Tetrahedron 2005, 61, 5849.
8. (a) Bochet, C. G. Tetrahedron Lett. 2000, 41, 6341; (b) Schaper, K.; Mobarekah, S.
A. M.; Grewer, C. Eur. J. Org. Chem. 2002, 1037.
9. Banerjee, A.; Grewer, C.; Ramakrishnan, L.; Ja¨ger, J.; Gameiro, A.; Breitinger,
H.-G. A.; Gee, K. R.; Carpenter, B. K.; Hess, G. P. J. Org. Chem. 2003, 68, 8361.
10. Grewer, C.; Ja¨ger, J.; Carpenter, B. K.; Hess, G. P. Biochemistry 2000, 39, 2063.
11. Singh, A. K.; Khade, P. K. Tetrahedron 2005, 61, 10007.
12. Givens, R. S.; Weber, J. F. W.; Conrad, P. G.; Orosz, G.; Donahue, S. L.; Thayer, S. A.
J. Am. Chem. Soc. 2000, 122, 2687.
Glu(OMe)–OH (2e) (0.076 g, 0.255 mmol), compound 3e was
obtained as a light brown solid (0.040 g, 29%). Mp¼101.1–103.6 ꢂC.
1H NMR (CDCl3):
CH2), 2.53–2.71 (m, 2H,
OCH3 Bba), 4.50 (q, J 5.1 Hz 1H,
d
¼1.98–2.10 (m, 1H,
g-CH2), 2.28–2.39 (m, 1H, g-
b
-CH2), 3.78 (s, 3H, OCH3 Glu), 3.95 (s, 3H,
-H), 5.11 (s, 2H, CH2 Z), 5.50 (d, J
a
8.1 Hz,1H, NH), 5.66 (s, 2H, CH2 Bba), 6.65 (s,1H, H-2), 7.22 (dd, J 8.7
and 2.4 Hz,1H, H-8), 7.27–7.35 (m, 6H, 5ꢁPh-H and H-5), 7.40 (s,1H,
H-10), 7.82 (d, J 9.0 Hz, 1H, H-7), 7.90 (d, J 8.7 Hz, 1H, H-6). 13C NMR
(CDCl3):
d
¼27.63 (
g-CH2), 29.90 (b-CH2), 52.65 (OCH3 Bba), 53.01
(a
-C), 55.39 (OCH3 Glu), 64.28 (CH2 Bba), 67.12 (CH2 Z), 105.71 (C-
10), 111.82 (C-4b), 112.61 (C-2), 115.26 (C-5), 116.53 (C-8), 126.31 (C-
6a), 128.05 (Ph-C), 128.17 (2ꢁPh-C), 128.46 (2ꢁPh-C), 130.54 (C-6b),
131.29 (C-6), 133.72 (C-7), 135.99 (C1 Ph), 150.75 (C-1), 155.48 (C-
4a), 155.94 (C]O urethane), 159.61 (C-9), 160.18 (C-3), 171.80 (C]O
13. Ma, C.; Steinmetz, M. G.; Kopatz, E. J.; Rathore, R. J. Org. Chem. 2005, 70, 4431.
14. Ashraf, M. A.; Russell, A. G.; Wharton, C. W.; Snaith, J. S. Tetrahedron 2007,
63, 586.
15. Papageorgiou, G.; Ogden, D. C.; Barth, A.; Corrie, J. E. T. J. Am. Chem. Soc. 1999,
121, 6503.
ester), 172.09 (C]O Glu). IR (KBr 1%, cmꢀ1):
n
¼3328, 3081, 2954,
1724, 1693, 1625, 1553, 1521, 1455, 1446, 1407, 1380, 1365, 1277,
1230, 1179, 1053, 1024, 985, 958, 942, 902, 838. UV–vis (MeOH/
16. (a) Shembekar, V. R.; Chen, Y.; Carpenter, B. K.; Hess, G. P. Biochemistry 2005, 44,
7107; (b) Hagen, V.; Dekowski, B.; Kotzur, N.; Lechler, R.; Wiesner, B.; Briand, B.;
Beyermann, M. Chem.dEur. J. 2008, 14, 1621; (c) Piloto, A. M.; Rovira, D.; Costa,
S. P. G.; Gonçalves, M. S. T. Tetrahedron 2006, 62, 11955; (d) Fonseca, A. S. C.;
Gonçalves, M. S. T.; Costa, S. P. G. Tetrahedron 2007, 63, 1353.
17. Hill, C. A.; Harris, R. C.; Kim, H. J.; Harris, B. D.; Sale, C.; Boobis, L. H.; Kim, C. K.;
Wise, J. A. Amino Acids 2007, 32, 225.
18. Misu, Y.; Goshima, Y.; Miyamae, T. Trends Pharmacol. Sci. 2002, 23, 262.
19. Kreibich, T. A.; Chalasani, S. H.; Raper, J. A. J. Neurosci. 2004, 24, 7085.
20. (a) Piloto, A. M.; Costa, S. P. G.; Gonçalves, M. S. T. Tetrahedron Lett. 2005, 46,
4757; (b) Piloto, A. M.; Fonseca, A. S. C.; Costa, S. P. G.; Gonçalves, M. S. T.
Tetrahedron 2006, 62, 9258; (c) Fernandes, M. J. G.; Gonçalves, M. S. T.; Costa, S.
P. G. Tetrahedron 2008, 64, 3032.
HEPES 80:20, nm): lmax (log
3
)¼347 (4.00), 273 (3.94), 235 (4.55).
HRMS (EI): calcd for C29H27NO9 [Mþ] 533.1686; found 533.1689.
4.3. General photolysis procedure
A 1ꢁ10ꢀ3 M (compound 3c) or 1ꢁ10ꢀ4 M MeOH/HEPES (80:20)
solution of compounds 3a–b,d–e (5 mL) was placed in a quartz tube
and irradiated in a Rayonet RPR-100 reactor at the desired wave-
length. The lamps used for irradiation were of 254, 300, 350 and
419ꢃ10 nm.
21. Tjoeng, F. S.; Heavner, G. A. Synthesis 1981, 897.
22. Morris, J. V.; Mahaney, M. A.; Huber, J. R. J. Phys. Chem. 1976, 80, 969.
´
23. (a) Muller, C.; Even, P.; Viriot, M.-L.; Carre, M.-C. Helv. Chim. Acta 2001, 84,
Aliquots of 100 mL were taken at regular intervals and analysed
3735; (b) Fernandes, M. J. G.; Gonçalves, M. S. T.; Costa, S. P. G. Tetrahedron
2007, 63, 10133.
by RP-HPLC. The eluent was acetonitrile/water, 3:1, at a flow rate of