2-Oxo-2H-benzo[h]benzopyran as a new light sensitive protecting group for neurotransmitter amino acids

Article abstract of DOI:10.1007/s00726-009-0383-z

Aiming at the development of new benzopyran-based photocleavable protecting groups, novel chloromethylated and hydroxymethylated 2-oxo-2H-benzo[h] benzopyran derivatives bearing a methoxy substituent were designed and used in the synthesis of a series of fluorescent bioconjugates, by linking through an ester or urethane bond to several model neurotransmitter amino acids (glycine, alanine, β-alanine and γ-aminobutyric acid, GABA). The resulting fluorescent bioconjugates with emission in the visible range and high fluorescent quantum yields, were subjected to photocleavage reaction in methanol/HEPES buffer (80:20) solution at different wavelengths of irradiation (250, 300, 350 and 419 nm) and photocleavage kinetic data were obtained.

Full text of DOI:10.1007/s00726-009-0383-z

Amino Acids (2010) 39:121–133
DOI 10.1007/s00726-009-0383-z
ORIGINAL ARTICLE
2-Oxo-2H-benzo[h]benzopyran as a new light sensitive protecting
group for neurotransmitter amino acids
•
Ana M. S. Soares Susana P. G. Costa
•
M. Sameiro T. Gonc¸alves
Received: 3 August 2009 / Accepted: 22 October 2009 / Published online: 12 November 2009
Ó Springer-Verlag 2009
Abstract Aiming at the development of new benzo-
pyran-based photocleavable protecting groups, novel chloro-
methylated and hydroxymethylated 2-oxo-2H-benzo[h]
benzopyran derivatives bearing a methoxy substituent were
designed and used in the synthesis of a series of fluorescent
bioconjugates, by linking through an ester or urethane bond
to several model neurotransmitter amino acids (glycine,
alanine, b-alanine and c-aminobutyric acid, GABA). The
resulting fluorescent bioconjugates with emission in the
visible range and high fluorescent quantum yields, were
subjected to photocleavage reaction in methanol/HEPES
buffer (80:20) solution at different wavelengths of irradi-
ation (250, 300, 350 and 419 nm) and photocleavage
kinetic data were obtained.
and light (Mayer and Heckel 2006); this later being an
example of a green chemical process as the release of the
relevant species occurs without the need for additional
chemical reagents, and as an alternative to classical
deprotection methods. Photoactivable molecules represent
a recent and fast developing methodology that can find
applications in drug delivery, enabling on demand release
of a well-defined amount of drug in a predetermined time
profile; in the elucidation of signalling processes involving
neurotransmitters (amino acids, physiological amines or
nucleotides) within cellular systems and in metabolic pro-
cesses; in multistep organic synthesis, with polyfunctional
reagents that require multiple protection/deprotection pro-
cedures; in combinatorial and analytical chemistry, among
other examples.
Keywords Oxobenzobenzopyran ꢀ Coumarin ꢀ
Neurotransmitters ꢀ Amino acids ꢀ
2-Oxo-benzopyrans (trivially known as coumarins) have
received considerable attention in applied photochemistry
due to their photocleavage capabilities. Coumarin deriva-
tives have been reported as suitable photocleavable pro-
tecting groups for alcohols (Gilbert et al. 2007), amines
(Takaoka et al. 2004), phosphates (Hagen et al. 2005),
carboxylic acids (Shembekar et al. 2005), aldehydes and
ketones (Lu et al. 2003) in cell biology and biophysical
studies. Benzopyrans possess additional features, from
which fluorescence stands out, with a broad spectral range
and high fluorescence quantum yields. This fact leads to
the assembly of fluorescent conjugates whenever benzo-
pyran derivatives are used as photocleavable protecting
groups (Piloto et al. 2006a, b; Fonseca et al. 2007;
Fernandes et al. 2008a, b).
Photocleavable protecting groups
Introduction
The control over the spatial and temporal sustained release
of chemically and biologically relevant species, in con-
nection with chemical, biological and medical research
fields, has known a great development in recent years. The
triggering agents usually employed can be enzymes
(Anderson et al. 2004), pH changes (Lee and Chmielewski
2005), reducing or oxidising agents (Khurana and Arora
2009; Vaxelaire et al. 2009), temperature (Han et al. 2005)
Neurotransmitter amino acids are a class of compounds
where the caging strategy has been largely applied, usually
through an ester linkage of a suitable photoreleasable
group (including coumarins) to their carboxylic terminal
(Fernandes et al. 2007, 2008a, b). Certain amino acids play
A. M. S. Soares ꢀ S. P. G. Costa ꢀ M. S. T. Gonc¸alves (&)
´
Centro de Quımica, Universidade do Minho, Campus de Gualtar,
4710-057 Braga, Portugal
e-mail: msameiro@quimica.uminho.pt
123

122
A. M. S. Soares et al.
an important role in neuronal communication at the central
nervous system (CNS), such as glycine (Gly) and c-ami-
nobutyric acid (GABA), which act as the main inhibitory
transmitters within the mammalian CNS (Banerjee et al.
2003; Curten et al. 2005). b-Alanine (b-Ala) is the only
naturally occurring beta amino acid and acts as a physio-
logical transmitter, being the rate-limiting precursor of
carnosine, which is a b-alanine-histidine dipeptide present
in muscle and brain tissues (Hill et al. 2007). Alanine (Ala)
is also an inhibitory neurotransmitter and an agonist of
ionotropic glycinergic and GABA receptors (Treffort et al.
2006; Legendre 2001).
determined on a BOMEM MB 104 spectrophotometer using
KBr discs. UV–visible absorption spectra (200–800 nm)
were obtained using a Shimadzu UV/2501PC spectropho-
tometer. 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°C. All chemical shifts are given in ppm
using dH Me4Si = 0 ppm as reference, and J values are
given in Hz. Assignments were made by comparison of
chemical shifts, peak multiplicities, and J values and were
supported by spin decoupling-double resonance and bidi-
mensional heteronuclear HMBC and HMQC correlation
techniques. Mass spectrometry analyses were performed at
the ‘‘C.A.C.T.I. - Unidad de Espectrometria de Masas’’, at
University of Vigo, Spain. Fluorescence spectra were col-
lected using a FluoroMax-4 spectrofluorometer. Photolyses
were carried out using a Rayonet RPR-100 chamber reactor
equipped with ten lamps of 254 (35 W), 300 (21 W), 350
(24 W) and 419 (14 W) nm. HPLC analyses were per-
formed using a Licrospher 100 RP18 (5 lm) 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.
In pursuit of our current research interests, which
involve the synthesis and application of novel oxygen and
nitrogen polycyclic heterocycles, as novel fluorescent tags
and photoreleasable protecting groups for the carboxylic
acid and amine functions of amino acids (Piloto et al. 2005,
2006a, b; Fonseca et al. 2007; Fernandes et al. 2007, 2008a,
b), we are interested in the evaluation and improvement of
their photophysical properties (as possible fluorogenic
derivatisation reagents) and also in the study of their
behaviour towards photocleavage under UV irradiation at
different wavelengths (254, 300, 350 and 419 nm), in
simulated physiological environment in a methanol/HEPES
buffer (80:20) solution. Recent results reported by us
indicated that a chloromethylated benzo[f]benzopyran was
an appropriate fluorescent tagging and photocleavable
protecting group for carboxylic acids, with photocleavage
at 350 nm occurring smoothly in short times (Fernandes
et al. 2008a, b), which is preferable in studies involving
biomolecules as it avoids cell damage due to short-wave-
length light.
Synthesis of 4-(chloromethyl)-6-methoxy-2-oxo-2H-
benzo[h]benzopyran (1)
To
a
solution of 4-methoxy-1-naphthol (0.501 g,
2.88 9 10-3 mol) in 70% aqueous sulphuric acid (3 mL),
ethyl chloroacetoacetate (0.6 mL, 4.36 9 10-3 mol) was
added and stirred at room temperature for 4 h. The reaction
mixture was poured into ice water and stirred for 2 h to
give a fine greenish precipitate. The solid was collected by
filtration, washed with cold water and dried in a vacuum
oven. After purification by dry flash chromatography, using
ethyl acetate/n-hexane, mixtures of increasing polarity as
eluent, compound 1 was obtained as a green solid (0.358 g,
45%). mp = 180.5–181.9°C. TLC (ethyl acetate/n-hexane,
In view of these facts, we now report the synthesis
of novel structurally related chloromethylated and hy-
droxymethylated 2-oxo-2H-benzo[h]benzopyrans and their
application in the synthesis of a set of fluorescent ester and
urethane conjugates of neurotransmitter amino acids, such
as glycine, alanine, b-alanine and c-aminobutyric acid. The
resulting bioconjugates were irradiated in a photochemical
reactor at the above-mentioned wavelengths of irradiation
and photocleavage kinetic data were obtained.
1
1:1): Rf = 0.69. H NMR (CDCl3, 400 MHz): dH = 4.07
(s, 3 H, OCH3), 4.73 (s, 2 H, CH2Cl), 6.63 (t, J = 0.8 Hz,
1 H, H-3), 6.84 (s, 1 H, H-5), 7.64–7.72 (m, 2 H, H-8 and
H-9), 8.25–8.33 (m, 1 H, H-7), 8.49–8.55 (m, 1 H, H-10).
13C NMR (CDCl3, 100.6 MHz): dC = 41.92 (CH2Cl),
55.85 (OCH3), 96.02 (C-5), 112.58 (C-4a), 115.27 (C-3),
122.27 (C-7), 122.45 (C-10), 124.03 (C-10a), 127.50
(C-6a), 127.94 (C-9), 128.55 (C-8), 146.11 (C-10b), 150.10
(C-4), 152.23 (C-6), 160.56 (C-2). IR (KBr 1%, cm-1):
m = 3,427, 2,934, 2,850, 1,709, 1,601, 1,565, 1,508, 1,476,
1,466, 1,422, 1,388, 1,274, 1,251, 1,175, 1,163, 1,143,
1,110, 1,077, 1,028, 986, 949, 922, 884, 845 and 730. UV/
Experimental
General
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 Kieselgel 60F254) and spots were visualised
under UV light. Chromatography on silica gel was carried
out on Merck Kieselgel (230–240 mesh). IR spectra were
123

2-Oxo-2H-benzo[h]benzopyran as a new light sensitive protecting group
123
Vis (ethanol, nm): kmax (log e) = 379 (3.68). HRMS (EI):
calculated for C15H11O335Cl [M?]: 274.0397; found:
274.0395; calculated for C15H11O337Cl [M?]: 276.0367;
found: 276.0370.
1,020, 950, 881, 853, 781, 734 and 665. UV/Vis (ethanol,
nm): kmax (log e) = 380 (3.71). HRMS (EI): calculated for
C14H10O3 [M?]: 226.0630; found: 226.0633.
Compound 3 (0.098 g, 4.35 9 10-4 mol) was reacted
with potassium carbonate (0.064 g, 4.65 9 10-4 mol) and
methyl iodide (0.029 mL, 4.59 9 10-4 mol) in dry DMF
(10 mL), at reflux for 4 h under nitrogen atmosphere.
Distilled water (5 mL) was added to the reaction mixture
and the aqueous phase extracted with ethyl acetate
(4 9 10 mL). The organic phases were combined and
washed with NaCl saturated solution, dried with magne-
sium sulphate, filtered and evaporated under reduced
pressure. The methoxy derivative 2 was obtained as a dark
brown solid (0.072 g; 70%); the experimental data con-
firmed its structure and were in accordance with that
described above.
Synthesis of 6-methoxy-4-methyl-2-oxo-2H-
benzo[h]benzopyran (2)
To
a
solution of 4-methoxy-1-naphthol (0.612 g,
3.51 9 10-3 mol) in 70% aqueous sulphuric acid (6 mL),
ethyl acetoacetate (1.4 mL, 1.1 9 10-2 mol) was added
and stirred at room temperature for 3 h. The reaction
mixture was poured into ice water and stirred for 2 h to
give a fine pinkish precipitate. The solid was collected by
filtration, washed with cold water and dried in a vacuum
oven. After purification by dry flash chromatography, using
ethyl acetate/n-hexane, mixtures of increasing polarity as
eluent, compound 2 was obtained as a beige solid (0.130 g,
15%). mp = 131.1–133.4°C. TLC (ethyl acetate/n-hexane,
Synthesis of 6-methoxy-2-oxo-2H-benzo[h]
benzopyran-4-carbaldehyde (4)
1
1:1): Rf = 0.65. H NMR (CDCl3, 300 MHz): dH = 2.49
(s, 3 H, CH3), 4.04 (s, 3 H, OCH3), 6.35 (d, J = 1.2 Hz,
1 H, H-3), 6.73 (s, 1 H, H-5), 7.70–7.58 (m, 2 H, H-8 and
H-9), 8.20–8.28 (m, 1 H, H-7), 8.46–8.53 (m, 1 H, H-10).
13C NMR (CDCl3, 75.4 MHz): dC = 19.34 (CH3), 55.70
(OCH3), 96.55 (C-5), 114.46 (C-3), 114.94 (C-4a), 122.12
(C-7), 122.37 (C-10), 123.90 (C-10a), 127.22 (C-6a),
127.64 (C-9 or C-8), 128.04 (C-8 or C-9), 145.23 (C-10b),
151.93 (C-6), 153.17 (C-4), 161.13 (C-2). IR (KBr 1%,
cm-1): m = 3,417, 2,926, 1,725, 1,610, 1,596, 1,564, 1,506,
1,469, 1,453, 1,429, 1,386, 1,384, 1,272, 1,248, 1,234,
1,208, 1,176, 1,151, 1,110, 1,083, 1,032, 990, 944, 933,
873, 851 and 815. UV/Vis (ethanol, nm): kmax (log
e) = 371 (3.57). HRMS (EI): calculated for C15H12O3
[M?]: 240.0786; found: 240.0791.
Compound 2 (0.108 g, 4.50 9 10-4 mol) was reacted with
selenium dioxide (0.299 g, 2.70 9 10-3 mol) in chloro-
benzene (5 mL), at reflux for 36 h. The mixture was
filtered hot, and the solvent was removed by rotary evap-
oration. After purification by dry flash chromatography,
using ethyl acetate/n-hexane, mixtures of increasing
polarity as eluent, compound 4 was obtained as an orange
solid (0.090 g, 79%). mp = 222.5–224.8°C. TLC (chloro-
form/methanol, 9.5:0.5): Rf = 0.69. 1H NMR (CDCl3,
400 MHz): dH = 4.08 (s, 3 H, OCH3), 6.94 (s, 1 H, H-5),
7.65–7.72 (m, 2 H, H-8 and H-9), 7.89 (s, 1 H, H-3), 8.25–
8.32 (m, 1 H, H-7), 8.48–8.54 (m, 1 H, H-10) and 10.16
(s, 1 H, CHO). 13C NMR (CDCl3, 100.6 MHz): dC = 55.86
(OCH3), 97.28 (C-3), 110.48 (C-4a), 122.27 (C-10), 122.40
(C-7), 123.51 (C-10a), 125.19 (C-5), 127.62 (C-6a), 127.87
(C-9), 128.92 (C-8), 144.19 (C-10b), 147.09 (C-6), 152.66
(C-4), 160.51 (C-2) and 192.13 (CHO). IR (KBr 1%,
cm-1): m = 3,430, 3,066, 2,962, 2,926, 2,850, 1,731, 1,712,
1,630, 1,594, 1,561, 1,504, 1,472, 1,450, 1,418, 1,381,
1,272, 1,247, 1,183, 1,138, 1,110, 1,083, 1,028, 985, 928,
893, 867, 855 and 775. UV/Vis (ethanol, nm): kmax (log
e) = 377 (3.73). HRMS (EI): calculated for C15H10O6
[M?]: 254.0579; found: 254.0589.
6-Hydroxy-4-methyl-2-oxo-2H-benzo[h]
benzopyran (3)
In the same preparation, compound 3 was also obtained as
a greenish solid (0.095 g; 12%). mp = 289.0–291.5°C.
TLC (ethyl acetate/n-hexane, 1:1): Rf = 0.35. 1H NMR
(DMSO-d6, 400 MHz): dH = 2.49 (s, 3 H, CH3), 6.45
(d, J = 1.6 Hz, 1 H, H-3), 7.00 (s, 1 H, H-5), 7.63–7.73
(m, 2 H, H-8 and H-9), 8.19 (dt, J = 8.4 and 1.6 Hz, 1 H,
H-7), 8.30 (dt, J = 8.0 and 1.6 Hz, 1 H, H-10), 10.42 (s, 1
H, OH). 13C NMR (DMSO-d6, 100.6 MHz): dC = 12.63
(CH3), 100.90 (C-5), 114.07 (C-3), 115.40 (C-4a), 121.49
(C-10), 122.45 (C-7), 123.19 (C-10a), 126.32 (C-6a),
127.59 (C-9 or C-8), 127.61 (C-8 or C-9), 143.10 (C-10b),
149.51 (C-6), 153.67 (C-4) and 159.88 (C-2). IR (KBr 1%,
cm-1): m = 3,650-3,186, 2,956, 2,925, 2,854, 1,680, 1,598,
1,563, 1,482, 1,422, 1,403, 1,384, 1,372, 1,348, 1,296,
1,274, 1,259, 1,236, 1,206, 1,176, 1,148, 1,094, 1,078,
In the same preparation, 4-(hydroxymethyl)-6-methoxy-
2-oxo-2H-benzo[h]benzopyran (5) was also obtained in
10% yield. The experimental data confirmed its structure
and were in accordance with that described below.
4-(Hydroxymethyl)-6-methoxy-2-oxo-2H-
benzo[h]benzopyran (5)
Compound 4 (0.098 g, 3.85 9 10-4 mol) was reacted with
sodium borohydride (0.016 g, 4.28 9 10-4 mol) in
123

124
A. M. S. Soares et al.
chloroform (7 mL) and ethanol (17 mL) for 3 days at room
temperature. After purification by dry flash chromatogra-
phy, using ethyl acetate/n-hexane, mixtures of increasing
polarity as eluent, compound 5 was obtained as a light
yellow solid (0.046 g, 47%). mp = 219.1–220.7°C. TLC
(ethyl acetate/n-hexane, 7:3): Rf = 0.60. 1H NMR
(DMSO-d6, 400 MHz): dH = 4.02 (s, 3 H, OCH3), 4.86
(dd, J = 4.2 and 1.2 Hz, 2 H, CH2OH), 5.73 (t, J = 4.2 Hz,
1 H, OH), 6.55 (t, J = 1.2 Hz, 1 H, H-3), 6.93 (s, 1 H, H-5),
7.67–7.75 (m, 2 H, H-8 and H-9), 8.19 (dd, J = 8.0 and
1.5 Hz, 1 H, H-7), 8.32 (dd, J = 7.6 and 1.6 Hz, 1 H,
H-10). 13C NMR (DMSO-d6, 100.6 MHz): dC = 56.01
(OCH3), 59.58 (CH2OH), 97.12 (C-5), 110.22 (C-3),
112.78 (C-4a), 121.60 (C-10), 121.90 (C-7), 123.03
(C-10a), 126.28 (C-6a), 127.92 (C-9), 128.23 (C-8), 144.17
(C-10b), 151.08 (C-6), 157.37 (C-4) and 160.12 (C-2). IR
(KBr 1%, cm-1): m = 3,410, 2,924, 2,853, 1,707, 1,602,
1,567, 1,506, 1,474, 1,451, 1,422, 1,384, 1,355, 1,315,
1,275, 1,250, 1,143, 1,117, 1,097, 1,061, 987, 950, 856,
762, 736 and 666. UV/Vis (ethanol, nm): kmax (log
e) = 365 (3.69). HRMS (EI): calculated for C15H12O4
[M?]: 256.0736; found: 256.0745.
112.05 (C-4a), 112.95 (C-3), 122.27 (C-7), 122.41 (C-10),
123.97 (C-10a), 127.41 (C-6a), 127.99 (C-9 and Ar–C),
128.15 (Ar–C), 128.28 (Ar–C), 128.52 (Ar–C), 128.55 (C-
8 and Ar–C), 135.99 (Ar–C), 145.79 (C-10b), 148.86 (C-4),
152.38 (C-6), 156.32 (C=O urethane), 160.55 (C-2) and
169.47 (C=O ester). IR (KBr 1%, cm-1): m = 3,315, 2,925,
1,735, 1,687, 1,600, 1,538, 1,475, 1,455, 1,422, 1,386,
1,291, 1,197, 1,110, 1,085, 1,060, 989 and 750. UV/Vis
(ethanol, nm): kmax (log e) = 376 (3.72). HRMS (EI):
calculated for C25H22NO7 [M?]: 448.14095; found:
448.13908.
N-(Benzyloxycarbonyl)-L-alanine (6-methoxy-2-oxo-
2H-benzo[h]benzopyran-4-yl) methyl ester (7b)
Compound 1 (0.070 g, 2.55 9 10-4 mol), DMF (3 mL),
KF (0.045 g, 7.75 9 10-4 mol) and Z-Ala-OH (0.058 g,
2.59 9 10-4 mol) were used and the reaction time was
1 day. Mixtures of increasing polarity of ethyl acetate/
n-hexane were used as the dry flash chromatography eluent
to give compound 7b as a yellow solid (0.101 g, 86%).
mp = 153.3–154.7°C. TLC (ethyl acetate/n-hexane, 1:1):
Rf = 0.87. 1H NMR (CDCl3, 400 MHz): dH = 1.53
(d, J = 7.2 Hz, 3 H, b-CH3 Ala), 4.00 (s, 3 H, OCH3),
4.49–4.59 (m, 1 H, a-CH Ala), 5.07–5.17 (m, 2 H, CH2 Z),
5.28–5.47 (m, 3 H, CH2 and a-NH Ala), 6.52 (s, 1 H, H-3),
6.53 (s, 1 H, H-5), 7.25–7.40 (m, 5 H, 5 9 Ar–H Z), 7.60–
7.70 (m, 2 H, H-8 and H-9), 8.23 (d, J = 9.2 Hz, 1 H, H-7)
and 8.45 (d, J = 9.2 Hz, 1 H, H-10). 13C NMR (CDCl3,
100.6 MHz): dC = 18.23 (b-CH3 Ala), 49.77 (a-CH Ala),
55.79 (OCH3), 62.36 (CH2), 67.09 (CH2 Z), 95.11 (C-5),
111.98 (C-4a), 112.78 (C-3), 122.21 (C-10), 122.27 (C-7),
123.83 (C-10a), 127.27 (C-6a), 127.88 (C-9 and Ar–C),
128.09 (Ar–C), 128.18 (C-8 and Ar–C), 128.40 (Ar–C),
128.48 (Ar–C), 136.02 (Ar–C), 145.59 (C-10b), 148.94 (C-
4), 152.20 (C-6), 155.67 (C=O urethane), 160.47 (C-2) and
172.37 (C=O ester). IR (KBr 1%, cm-1): m = 3,312, 2,928,
1,758, 1,714, 1,598, 1,566, 1,551, 1,506, 1,473, 1,455,
1,422, 1,387, 1,345, 1,311, 1,276, 1,252, 1,211, 1,178,
1,161, 1,116, 1,085, 1,073, 1,030, 986, 959, 856 and 815.
UV/Vis (ethanol, nm): kmax (log e) = 375 (3.72). HRMS
(EI): calculated for C26H24NO7 [M?]: 462.15467; found:
462.15473.
General procedure for the synthesis of conjugates 7a–d
4-(Chloromethyl)-6-methoxy-2-oxo-2H-benzo[h]benzopy-
ran 1 (1 equiv) was dissolved in dry DMF (2 or 3 mL),
potassium fluoride (*3 equiv) and the corresponding
amino acid neurotransmitter 6a–d (1 equiv) was added.
The reaction mixture was stirred at room temperature for 1
or 3 days. The solvent was removed by rotary evaporation
under reduced pressure, and the crude residue was purified
by column chromatography using mixtures of ethyl acetate
and light petroleum or n-hexane as eluent.
N-(Benzyloxycarbonyl)-L-glycine (6-methoxy-2-oxo-
2H-benzo[h]benzopyran-4-yl) methyl ester (7a)
Compound 1 (0.063 g, 2.30 9 10-4 mol), DMF (2 mL),
KF (0.040 g, 6.85 9 10-3 mol) and Z-Gly-OH (0.054 g,
1.21 9 10-4 mol) were used, and the reaction time was
1 day. Mixtures of increasing polarity of ethyl acetate/light
petroleum were used as the dry flash chromatography
eluent to give compound 7a as a yellow solid (0.054 g,
53%). mp = 173.5–175.1°C. TLC (ethyl acetate/light
N-(Benzyloxycarbonyl)-L-b-alanine (6-methoxy-2-oxo-
2H-benzo[h]benzopyran-4-yl) methyl ester (7c)
1
petroleum, 4:6): Rf = 0.48. H NMR (CDCl3, 400 MHz):
dH = 4.04 (s, 3 H, OCH3), 4.16 (d, J = 6.0 Hz, 2 H, CH2
Gly), 5.16 (s, 2 H, CH2 Z), 5.34 (br s, 1 H, a-NH Gly), 5.43
(s, 2 H, CH2), 6.56 (s, 1 H, H-3), 6.61 (s, 1 H, H-5), 7.28–
7.40 (m, 5 H, 5 9 Ar–H Z), 7.64–7.72 (m, 2 H, H-8 and
H-9), 8.25–8.30 (m, 1 H, H-7), 8.48–8.54 (m, 1 H, H-10).
13C NMR (CDCl3, 100.6 MHz): dC = 42.80 (CH2 Gly),
55.90 (OCH3), 62.37 (CH2), 67.35 (CH2 Z), 95.17 (C-5),
Compound 1 (0.087 g, 3.16 9 10-4 mol), DMF (2 mL),
KF (0.055 g, 9.49 9 10-4 mol) and Z-b-Ala-OH (0.074 g,
3.29 9 10-4 mol) were used and the reaction time was
3 days. Mixtures of increasing polarity of ethyl acetate/
n-hexane were used as the dry flash chromatography eluent
to give compound 7c as a yellow solid (0.099 g, 70%).
123

2-Oxo-2H-benzo[h]benzopyran as a new light sensitive protecting group
125
mp = 119.9–122.6°C. TLC (ethyl acetate/n-hexane, 1:1):
Rf = 0.64. 1H NMR (CDCl3, 300 MHz): dH = 2.74
(t, J = 6.0 Hz, 2 H, a-CH2 b-Ala), 3.56 (q, J = 6.0 Hz, 2 H,
b-CH2 b-Ala), 3.94 (s, 3 H, OCH3), 5.10 (s, 2 H, CH2 Z),
5.26 (s, 2 H, CH2), 5.41 (br s, 1 H, NH), 6.44 (s, 1 H, H-3),
6.46 (s, 1 H, H-5), 7.24–7.38 (m, 5 H, 5 9 Ar–H Z), 7.56–
7.66 (m, 2 H, H-8 and H-9), 8.12–8.20, (m, 1 H, H-7) and
8.34–8.42 (m, 1 H, H-10). 13C NMR (CDCl3, 75.4 MHz):
dC = 34.34 (a-CH2 b-Ala), 36.46 (b-CH2 b-Ala), 55.68
(OCH3), 61.55 (CH2), 66.71 (CH2 Z), 94.90 (C-5), 111.85
(C-4a), 112.25 (C-3), 122.11 (C-7 and C-10), 123.66
(C-10a), 127.09 (C-6a), 127.77 (Ar–C), 128.00 (C-9),
128.05 (C-8), 128.27 (2 9 Ar–C), 128.41 (2 9 Ar–C),
136.26 (Ar–C), 145.36 (C-10b), 149.20 (C-4), 152.02 (C-
6), 156.23 (C=O urethane), 160.45 (C-2), 171.42 (C=O
ester). IR (KBr 1%, cm-1): m = 3,346, 2,936, 1,721, 1,643,
1,614, 1,599, 1,565, 1,529, 1,507, 1,474, 1,455, 1,422,
1,385, 1,312, 1,274, 1,249, 1,170, 1,143, 1,112, 1,087,
1,029, 989, 951, 860 and 770. UV/Vis (ethanol, nm): kmax
(log e) = 375 (3.75). HRMS (EI): calculated for
C26H24NO7 [M?]: 462.15600; found: 462.15473.
HRMS (EI): calculated for C27H26NO7 [M?]: 476.1717;
found: 476.1704.
General procedure for the removal
of N-benzyloxycarbonyl group
in conjugates 7a–c
A 45% solution of hydrobromic acid in acetic acid (20 lL)
and acetic acid (1 mL) were added to the amino acid
conjugates 7a–c, with stirring, at room temperature. The
reaction mixture was maintained in these conditions for
3 days, and the process was followed by TLC (ethyl ace-
tate/n-hexane; 6:4). During this time, additional amounts of
45% solution of hydrobromic acid in acetic acid were
added, and the total volume for each compound was
mentioned below. When the reaction was completed, cold
diethyl ether was added (0.5 mL), and the precipitate fil-
tered off and washed with the same solvent to give a light
yellow solid.
L-Glycine (6-methoxy-2-oxo-2H-benzo[h]benzopyran-
4-yl) methyl ester hydrobromide (8a)
N-(Benzyloxycarbonyl)-L-c-aminobutyric acid
(6-methoxy-2-oxo-2H-benzo[h]benzopyran-4-yl)
methyl ester (7d)
Starting from conjugate 7a (0.025 g, 5.59 9 10-5 mol)
and using 45% solution of hydrobromic acid in acetic acid
(110 lL), compound 8a was obtained (0.004 g, 18%).
mp = 194.8–195.9°C. TLC (ethyl acetate/methanol, 4:3):
Rf = 0.54. 1H NMR (CD3OD, 400 MHz): dH = 4.13
(s, 5 H, a-CH2 Gly and OCH3), 5.74 (s, 2 H, CH2), 6.66
(s, 1H, H-3), 6.95 (s, 1 H, H-5), 7.70–7.80 (m, 2 H, H-8 and
H-9), 8.31–8.37 (m, 1 H, H-7) and 8.46–8.51 (m, 1 H,
H-10). 13C NMR (CD3OD, 100.6 MHz): dC = 41.07
(a-CH2 Gly), 56.60 (OCH3), 64.28 (CH2), 97.09 (C-5),
113.03 (C-3), 113.72 (C-4a), 122.94 (C-10), 123.45 (C-7),
125.08 (C-10a), 128.82 (C-6a), 129.23 (C-9), 129.73 (C-8),
146.65 (C-10b), 151.92 (C-4), 153.84 (C-6), 162.66 (C-2)
and 168.32 (C=O ester). IR (KBr 1%, cm-1): m = 3,515,
3,414, 2,992, 2,926, 2,719, 2,651, 1,729, 1,596, 1,567,
1,505, 1,475, 1,453, 1,423, 1,387, 1,317, 1,276, 1,253,
1,172, 1,143, 1,111, 1,086, 1,061, 985, 963, 923, 871, 840,
733 and 666. UV/Vis (ethanol, nm): kmax (log e) = 374
(3.49). HRMS (EI): calculated for C17H15NO5 [M?]:
313.09505; found: 313.09504.
Compound 1 (0.097 g, 3.55 9 10-4 mol), DMF (3 mL),
KF (0.065 g, 1.12 9 10-3 mol) and Z-GABA-OH (0.087 g,
3.67 9 10-4 mol) were used, and the reaction time was
3 days. Mixtures of increasing polarity of ethyl acetate/light
petroleum were used as the dry flash chromatography eluent
to give compound 7d as a yellow solid (0.095 g, 56%).
mp = 144.5–146.4°C. TLC (ethyl acetate/light petroleum,
1:1): Rf = 0.74. 1H NMR (CDCl3, 400 MHz): dH = 1.84–
2.0 (m, 2 H, b-CH2 GABA), 2.56 (t, J = 7.2 Hz, 2 H,
a-CH2 GABA), 3.30 and 3.33 (2 9 d, J = 6.4 and 6.8 Hz,
2 H, c-CH2 GABA), 4.01 (s, 3 H, OCH3), 4.96 (br s, 1 H,
a-NH GABA), 5.10 (s, 2 H, CH2 Z), 5.34 (s, 2 H, CH2), 6.55
(s, 1 H, H-3), 6.58 (s, 1 H, H-5), 7.28–7.38 (m, 5 H, 5 9 Ar–
H Z), 7.62–7.70 (m, 2 H, H-8 and H-9), 8.26 (d, J = 9.2 Hz,
1 H, H-7) and 8.49 (d, J = 9.6 Hz, 1 H, H-10). 13C NMR
(CDCl3, 100.6 MHz): dC = 25.17 (b-CH2 GABA), 31.16
(a-CH2 GABA), 40.22 (c-CH2 GABA), 55.85 (OCH3),
61.51 (CH2), 66.72 (CH2 Z), 95.15 (C-5), 112.08 (C-4a),
112.36 (C-3), 122.22 (C-7), 122.34 (C-10), 123.92 (C-10a),
127.31 (C-6a), 127.91 (Ar–C), 128.05 (Ar–C), 128.09 (C-9
and Ar–C), 128.40 (Ar–C), 128.47 (C-8 and Ar–C), 136.41
(Ar–C), 145.63 (C-10b), 149.58 (C-4), 152.24 (C-6), 156.45
(C=O urethane), 160.68 (C-2) and 172.36 (C=O ester). IR
(KBr 1%, cm-1): m = 3,310, 2,927, 1,737, 1,715, 1,692,
1,652, 1,565, 1,556, 1,505, 1,455, 1,385, 1,321, 1,266,
1,249, 1,174, 1,137, 1,110, 1,083, 1,030, 945, 828, 772, 737
and 665. UV/Vis (ethanol, nm): kmax (log e) = 373 (3.66).
L-Alanine (6-methoxy-2-oxo-2H-benzo[h]benzopyran-
4-yl)methyl ester hydrobromide (8b)
Starting from conjugate 7b (0.035 g, 7.59 9 10-5 mol)
and using 45% solution of hydrobromic acid in acetic acid
(130 lL), compound 8b was obtained (0.022 g, 71%).
mp = 198.2–198.8°C. TLC (ethyl acetate/methanol, 4:3):
Rf = 0.74. 1H NMR (CD3OD, 400 MHz): dH = 1.70
(d, J = 7.2 Hz, 3 H, b-CH3 Ala), 4.11 (s, 3 H, OCH3),
123

126
A. M. S. Soares et al.
4.40–4.50 (m, 1 H, a-CH Ala), 5.70 (s, 2 H, CH2), 6.61 (s,
1 H, H-3), 6.90 (s, 1 H, H-5), 7.70–7.78 (m, 2 H, H-8 and
H-9), 8.28–8.34 (m, 1 H, H-7) and 8.42–8.47 (m, 1 H, H-
10). 13C NMR (CD3OD, 100.6 MHz): dC = 16.29 (b-CH3
Ala), 49.90 (a-CH Ala), 56.90 (OCH3), 64.53 (CH2), 97.06
(C-5), 113.14 (C-3), 113.68 (C-4a), 122.91 (C-10), 123.44
(C-7), 125.01 (C-10a), 128.77 (C-6a), 129.22 (C-9), 129.72
(C-8), 146.62 (C-10b), 151.80 (C-4), 153.78 (C-6), 162.59
(C-2) and 170.67 (C=O ester). IR (KBr 1%, cm-1):
m = 3,426, 2,924, 1,727, 1,698, 1,638, 1,613, 1,597, 1,566,
1,505, 1,474, 1,453, 1,423, 1,386, 1,315, 1,274, 1,249,
1,204, 1,176, 1,145, 1,111, 1,085, 1,031, 987, 960, 857,
815, 787, 733 and 666. UV/Vis (ethanol, nm): kmax (log
e) = 373 (3.49). HRMS (EI): calculated for C18H17NO7
[M?]: 327.11071; found: 327.11072.
purification by dry flash chromatography, using chloroform/
methanol, mixtures of increasing polarity as eluent,
gave compound 10a as a light yellow solid (0.038 g,
42%). mp = 175.7–177.9°C. TLC (chloroform/methanol,
9.5:0.5): Rf = 0.75. 1H NMR (CDCl3, 300 MHz): dH = 2.62
(t, J = 5.7 Hz, 2 H, a-CH2 b-Ala), 3.50–3.62 (m, 2 H, b-CH2
b-Ala), 3.74 (s, 3 H, OCH3 b-Ala), 4.04 (s, 3 H, OCH3), 5.38
(s, 2 H, CH2), 5.59 (t, J = 5.7 Hz, 1 H, NH b-Ala), 6.56 (s, 1
H, H-3), 6.65 (s, 1 H, H-5), 7.64–7.72 (m, 2 H, H-8 and H-9),
8.24–8.32 (m, 1 H, H-7) and 8.48–8.56 (m, 1 H, H-10). 13
C
NMR (CDCl3, 75.4 MHz): dC = 34.02 (a-CH2 b-Ala),
36.74 (b-CH2 b-Ala), 51.9 (OCH3 b-Ala), 55.87 (OCH3),
62.01 (CH2), 95.29 (C-5), 112.10 (C-4a), 112.21 (C-3),
122.22 (C-10), 122.40 (C-7), 123.97 (C-10a), 127.31 (C-6a),
127.88 (C-9), 128.37 (C-8), 145.63 (C-10b), 150.52 (C-4),
152.24 (C-6), 155.27 (C=O urethane), 160.90 (C-2) and
172.68 (C=O ester). IR (KBr 1%, cm-1): m = 3,324, 3,081,
2,925, 2,854, 1,739, 1,711, 1,612, 1,598, 1,562, 1,554, 1,505,
1,474, 1,451, 1,420, 1,382, 1,324, 1,312, 1,252, 1,197, 1,174,
1,145, 1,110, 1,083, 1,052, 1,016, 986, 952, 890, 876, 811
and 734. UV/Vis (ethanol, nm): kmax (log e) = 374 (3.70).
HRMS (EI): calculated for C20H20NO7 [M?]: 386.12455;
found: 386.12343.
L-b-Alanine (6-methoxy-2-oxo-2H-
benzo[h]benzopyran-4-yl) methyl ester hydrobromide
(8c)
Starting from conjugate 7c (0.040 g, 8.67 9 10-5 mol) and
using 45% solution of hydrobromic acid in acetic acid
(60 lL), compound 8c was obtained (0.0158 g, 45%).
mp = 204.3–205.4°C. TLC (ethyl acetate/methanol, 4:3):
Rf = 0.26. 1H NMR (CD3OD, 400 MHz): dH = 3.02
(t, J = 6.4 Hz, 2 H, a-CH2 b-Ala), 3.30–3.40 (m, 2 H, b-CH2
b-Ala), 4.08 (s, 3 H, OCH3), 5.58 (s, 2 H, CH2), 6.57 (s, 1 H,
H-3), 6.85 (s, 1 H, H-5), 7.66–7.77 (m, 2 H, H-8 and H-9),
N-[(6-Methoxy-2-oxo-2H-benzo[h]benzopyran-4-
yl)methyloxycarbonyl]-L-b-alanine (10b)
The same procedure described before for the synthesis
of compound 10a was followed using CDI (0.046 g,
2.84 9 10-4 mol) in DMF (1 mL), 4-(hydroxymethyl)-6-
methoxy-2-oxo-2H-benzo[h]benzopyran (5) (0.020 g,
7.81 9 10-5 mol) in DMF (1 mL) (stirring time 24 h), and
b-alanine (9b) (0.014 g, 1.56 9 10-4 mol) (stirring time
24 h). After evaporation of the solvent, the crude mixture
was dissolved in methanol and the white precipitated fil-
tered off. The methanol solution evaporated and purified by
preparative chromatography (ethyl acetate/methanol, 9:1)
to give compound 10b as a light yellow solid (0.0242 g,
82%). mp = 224.9–225.9°C. TLC (ethyl acetate/methanol,
8:2): Rf = 0.61. 1H NMR (DMSO-d6, 400 MHz):
dH = 2.43 (t, J = 6.8 Hz, 2 H, a-CH2 b-Ala), 3.20-3.30
(m, 2 H, b-CH2 b-Ala), 4.05 (s, 3 H, OCH3), 5.44 (s, 2 H,
CH2), 6.50 (s, 1 H, H-3), 7.00 (s, 1 H, H-5), 7.66 (t,
J = 5.2 Hz, 1 H, a-NH), 7.71–7.79 (m, 2 H, H-8 and H-9),
8.23–8.30 (m, 1 H, H-7) and 8.37–8.45 (m, 1 H, H-10). 13
C
NMR (CD3OD, 100.6 MHz): dC = 32.20 (a-CH2 b-Ala),
36.33 (b-CH2 b-Ala), 56.55 (OCH3), 63.42 (CH2), 97.02
(C-5), 112.81 (C-3), 113.77 (C-4a), 122.88 (C-10), 123.41
(C-7), 124.97 (C-10a), 128.69 (C-6a), 129.15 (C-9), 129.05
(C-8), 146.49 (C-10b), 152.49 (C-4), 153.69 (C-6), 162.71
(C-2) and 171.59 (C=O ester). IR (KBr 1%, cm-1):
m = 3,437, 3,010, 2,923, 1,741, 1,699, 1,631, 1,607, 1,592,
1,565, 1,504, 1,473, 1,444, 1,422,1,383, 1,336, 1,312, 1,275,
1,251, 1,197, 1,172, 1,150, 1,114, 1,088, 1,032, 1,017, 992,
959, 927, 877, 729 and 666. UV/Vis (ethanol, nm): kmax
(log e) = 371 (3.33). HRMS (EI): calculated for C18H17NO5
[M?]: 327.11071; found: 327.11074.
N-[(6-Methoxy-2-oxo-2H-benzo[h]benzopyran-4-
yl)methyloxycarbonyl]-L-b-alanine methyl ester (10a)
8.20–8.26 (m, 1 H, H-7) and 8.33–8.38 (m, 1 H, H-10). 13
C
To a solution of CDI (0.059 g, 3.64 9 10-4 mol) in dry
DMF (1 mL), 4-(hydroxymethyl)-6-methoxy-2-oxo-2H-
benzo[h]benzopyran (5) (0.060 g, 2.43 9 10-4 mol) dis-
solved in dry DMF (3 mL) was added, and the reaction
mixture was stirred at room temperature for 3 h. After the
addition of b-alanine methyl ester (9a) (0.025 g,
2.44 9 10-4 mol) the mixture was kept reacting in the
same conditions for 12 h. The solvent was evaporated and
NMR (DMSO-d6, 100.6 MHz): dC = 34.07 (a-CH2
b-Ala), 36.66 (b-CH2 b-Ala), 56.11 (OCH3), 61.44 (CH2),
97.36 (C-5), 111.25 (C-3), 112.50 (C-4a), 121.70 (C-10),
122.00 (C-7), 123.04 (C-10a), 126.46 (C-6a), 128.15 (C-9),
128.57 (C-8), 144.51 (C-10b), 151.27 (C-6), 152.44 (C-4),
155.31 (C=O urethane), 159.76 (C-2) and 172.72 (CO2H).
IR (KBr 1%, cm-1): m = 3,269, 3,081, 2,992, 2,972, 2,933,
1,741, 1,714, 1,690, 1,613, 1,572, 1,505, 1,476, 1,461,
123

2-Oxo-2H-benzo[h]benzopyran as a new light sensitive protecting group
127
1,426, 1,386, 1,305, 1,274, 1,254, 1,197, 1,180, 1,147,
1,114, 1,085, 1,046, 992, 956, 888, 844, 771, 738 and 666.
UV/Vis (ethanol, nm): kmax (log e) = 373 (3.52). HRMS
(EI): calculated for C19H17NO7 [M?]: 339.11071; found:
339.11072.
reaction, catalysed by sulphuric acid at room temperature,
yielding 4-(chloromethyl)-6-methoxy-2-oxo-2H-benzo[h]
benzopyran 1 or 6-methoxy-4-methyl-2-oxo-2H-benzo[h]
benzopyran 2 (Gangadasu et al. 2004, synthesised by a
different method) together with the unexpected demethy-
lated derivative, 6-hydroxy-4-methyl-2-oxo-2H-benzo[h]
benzopyran 3, the latter being methylated with methyl
iodide to obtain the desired methoxy derivative 2.
By reaction of compound 2 with selenium dioxide, the
methyl group was oxidised to the aldehyde 4, which was
then reacted with sodium borohydride, affording the
4-(hydroxymethyl)-6-methoxy-2-oxo-2H-benzo[h]benzopy-
ran 5 (Scheme 1; Table 1). It should be noted that com-
pound 5 was also obtained as a by-product (10%) in the
reaction with selenium dioxide by an allylic hydroxylation
(Paquete 1995). Fluorophores 1 and 5 will be designated in
this report by a three letter code (Flu) for simplicity of
naming the various fluorescent conjugates, as indicated in
Table 3.
General photolysis procedure
A 1 9 10-4 M methanol/HEPES (80:20) solution of
compounds 7a–d, 8a–c and 10a, b (5 mL) were placed in a
quartz tube and irradiated in a Rayonet RPR-100 reactor at
the desired wavelength. The lamps used for irradiation
were of 254, 300, 350 and 419 10 nm.
Aliquots of 100 lL were taken at regular intervals and
analysed by RP-HPLC. The eluent was acetonitrile/water,
3:1, at a flow rate of 0.8 mL/min for all compounds, pre-
viously filtered through a Millipore, type HN 0.45-lm filter
and degassed by ultrasound for 30 min. The chromato-
grams were traced by detecting UV absorption at the
wavelength of maximum absorption for each compound
(retention time: 7a, 6.6; 7b, 6.5; 7c, 6.7; 7d, 6.0; 8a, 4.2;
8b, 4.2; 8c, 4.2; 10a, 4.7; 10b, 2.3 min).
The functionalised fluorophores 1 and 5 were used in the
preparation of several neurotransmitter amino acids con-
jugates through different linkages, namely ester and ure-
thane, by reaction at their carboxylic acid or amino
functions, respectively.
Results and discussion
N-Benzyloxycarbonyl-protected amino acids, such as
glycine (6a), alanine (6b), b-alanine (6c) and c-aminobu-
tyric acid (6d), were derivatised at the C-terminus with
chloromethylated benzo[h]benzopyran 1 in the presence of
potassium fluoride (Tjoeng and Heavner 1981) in DMF, at
room temperature resulting in the expected bioconjugates
Synthesis
4-Methoxy-1-naphthol was reacted with ethyl 4-chloro-
acetoacetate or ethyl acetoacetate through a Pechmann
Scheme 1 Synthesis of
functionalised 6-methoxy-2-
oxo-2H-benzo[h]benzopyrans
1–5
O
O
O
O
Cl
Cl
O
OH
O
O
O
1
H2SO4 70%
rt
O
O
O
O
O
O
O
+
OH
O
O
O
SeO2
2
3
chlorobenzene,
reflux
O
MeI/ K2CO3
DMF, N2, reflux
OH
NaBH4
CHO
EtOH, rt
O
O
5
4
123

128
A. M. S. Soares et al.
approach constitutes the most suitable model to evaluate
the usefulness of the new fluorescent heterocycle as
photoreleasable protecting group for neurotransmitter
amino acids.
Table 1 Yields and UV/visible data for precursors 1–5 and conju-
gates 7a–d, 8a–c and 10a, b in absolute ethanol and methanol/HEPES
buffer (80:20) solution
Compound Yield EtOH
(%)
MeOH/HEPES buffer (80:20)
4-(Hydroxymethyl)-6-methoxy-2-oxo-2H-benzo[h]benzo-
pyran 5 was reacted with the N-terminus of b-alanine
methyl ester (9a) and free b-alanine (9b), by a 1,10-car-
bonyldiimidazole (CDI) carbonyl transfer reaction
(D’Addona and Bochet 2001), in DMF, at room tempera-
ture, yielding the corresponding urethane-linked conjugates
10a (42%) and 10b (82%) (Scheme 2; Table 1). All
compounds synthesised were fully characterised by high
resolution mass spectrometry, IR, 1H and 13C NMR
spectroscopy.
kmax (nm) log e kmax (nm)
log e
1
45
15
12
79
47
53
86
70
56
18
71
45
42
82
379
371
380
377
365
376
375
375
373
374
373
371
374
373
3.68 383
3.57 371
3.71 377
3.73 380
3.69 371
3.72 373
3.72 374
3.75 376
3.66 370
3.39 372
3.49 373
3.33 370
3.70 374
3.52 371
3.70
3.68
3.73
3.61
3.73
3.73
3.72
3.78
3.60
3.62
3.64
3.63
3.71
3.62
2
3
4
5
7a
7b
7c
7d
8a
8b
8c
10a
10b
The IR spectra of conjugates 7a–d, 8a–c, 10a, b
showed bands due to stretching vibrations of the different
carbonyl groups present at the fluorophore-amino acid
1
conjugates from 1,643 to 1,758 cm-1. H NMR spectra
showed signals of the amino acid residues, such as the
a-CH2 (d 4.16 ppm, 7a or 4.13 ppm, 8a), a-CH (d
4.49–4.59 ppm, 7b or d 4.40–4.50 ppm, 8b); a-, b-CH2 (d
2.72, 3.57 ppm, 7c; d 3.02, 3.30–3.40 ppm, 8c), as well as
a-, b-, c-CH2 (d 2.56, 1.84–2.0, 3.30–3.33 ppm, 7d). The
fluorophore methylene group was also visible for all
conjugates (d 5.26–5.74 ppm). Also the characteristic
aromatic protons, H-3 and H-5, of the oxobenzopyran
ring were present at 6.44–6.66 and 6.46–6.95 ppm,
respectively.
7a–d (Scheme 2; Table 1) in good yields (53–86%). Fur-
thermore, the N-benzyloxycarbonyl-protecting group was
removed by acidolysis with hydrobromic acid in acetic acid
yielding the corresponding conjugates 8a–c bearing the
neurotransmitter and the photosensitive fluorophore. This
Scheme 2 Synthesis of
fluorescent neurotransmitter
conjugates 7a–d, 8a–c, 10a, b
O
O
O
O
R
O
R
KF
DMF, rt
OH
O
1 +
Ph
O
N
H
Ph
O
N
H
n
n
O
O
a n = 1, R = H
6a-d
b n = 1, R = Me
c n = 2, R = H
d n = 3, R = H
7a-d
HBr / CH3CO2H
O
O
R
O
HBr.H2N
n
O
O
8a-c
O
O
O
O
H
N
CDI
DMF, rt
O
OR
5 +
H2N
OR
O
O
9a,b
a R = Me
b R = H
10a,b
123

2-Oxo-2H-benzo[h]benzopyran as a new light sensitive protecting group
129
The confirmation of the presence of the newly formed
ester (7a–d) and urethane (10a, b) linkages were also
supported by 13C NMR spectra signals of the carbonyl
group, which were found at d 169.47–172.37 ppm and at
about 155.30 ppm, respectively.
For benzobenzopyran 5 and conjugates 10a, b, no shift
(for 10b in methanol/HEPES) or a bathochromic shift was
observed both in ethanol and in methanol/HEPES buffer
solution.
Bioconjugates 7a–d and 8a–c exhibited good fluores-
cence quantum yields in ethanol (0.43 \ UF \ 0.63) and in
methanol/HEPES buffer (0.28 \ UF \ 0.53), which are
higher than the value obtained for the corresponding
chloromethylated precursor 1, being at least ten times (9–
13) in ethanol and up to fifty times (28–53) in methanol/
HEPES buffer. In the case of conjugates 10a, b, when
compared to the fluorophore 5, it was observed that the
higher value was displayed by compound 10b (UF = 0.71)
in ethanol.
Evaluation of the photophysical properties
of conjugates 7a–d, 8a–c and 10a, b
The UV/visible absorption and emission spectra of
degassed 10-5 M solutions in absolute ethanol and in a
methanol/HEPES buffer (80:20) solution of conjugates
7a–d, 8a–c and 10a, b, in comparison with precursors 1
and 5 were measured, absorption and emission maxima,
molar absorptivities and relative fluorescence quantum
yields are also reported (Tables 1, 2). Relative fluorescence
quantum yields were calculated using 9,10-diphenylan-
thracene as standard (UF = 0.95 in ethanol) (Morris et al.
1976). For the UF determination, 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.
Compounds 7a–d, 8a–c and 10a, b displayed emission
maxima at about 472 nm (in ethanol) and in the range 469–
484 nm (in methanol/HEPES buffer), with large Stokes’
shifts (ca. 100 nm), which is an important feature in fluo-
rescent labelling for bioapplications. Moreover, compari-
son between precursors 1, 5 and the corresponding
conjugates 7c, 10a and 10b, in methanol/HEPES buffer
solution revealed longer wavelengths of maximum emis-
sion than in ethanol.
By comparison of absorption maxima for all compounds
in both solvents, no significant changes were observed
(kmax in the range 365–380 nm, in ethanol, and 370–
383 nm, in methanol/HEPES buffer). Furthermore, upon
linkage of benzobenzopyran 1 to amino acids 6a–d, a slight
hypsochromic shift (up to 8 nm in ethanol and up to 13 nm
in methanol/HEPES buffer) was observed for conjugates
7a–d and their corresponding Z-deprotected form 8a–c.
By comparison of fluorescence maxima for benzobenz-
opyrans 1, 5 and the corresponding conjugates 7a–d, 8a–c
and 10a, b in ethanol, a shift to longer wavelengths (9–
12 nm) occurred. In the case of methanol/HEPES buffer
there was no considerable variation except for a batho-
chromic shift for conjugates 7c (13 nm), 10a and 10b
(8 nm). Figure 1 illustrates the absorption and fluorescence
normalised spectra of tags 1, 5 and the b-alanine
Table 2 Fluorescence data for precursors 1–5 and conjugates 7a–d, 8a–c and 10a, b in absolute ethanol and methanol/HEPES buffer (80:20)
solution
Compound
EtOH
MeOH/HEPES buffer (80:20)
kem (nm)
UF
Stokes’ shift (nm)
kem (nm)
UF
Stokes’ shift (nm)
1
461
463
459
461
460
473
472
471
473
471
471
470
472
471
0.05
0.33
0.17
0.49
0.45
0.48
0.56
0.63
0.60
0.43
0.50
0.63
0.31
0.71
82
72
79
84
95
97
98
96
100
97
98
99
98
98
471
447
466
472
469
474
472
484
470
469
471
469
477
477
0.01
0.32
0.02
0.30
0.45
0.34
0.31
0.28
0.38
0.51
0.53
0.43
0.44
0.47
88
76
2
3
89
4
92
5
98
7a
7b
7c
7d
8a
8b
8c
10a
10b
101
98
108
100
97
98
99
103
106
123

130
A. M. S. Soares et al.
1,2
1
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 irradiation 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 aver-
age of 3 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 3).
A
F
0,8
0,6
0,4
0,2
0
330
380
430
480
530
580
630
Wavelength (nm)
For each compound and based on HPLC data, the plot of
ln A versus irradiation time showed a linear correlation for
the disappearance of the starting material, which suggested
a first order reaction, obtained by the linear least squares
methodology for a straight line. The corresponding rate
constants were calculated and are presented in Table 3.
Concerning the influence of the wavelength of irradia-
tion on the rate of photocleavage reactions of conjugates
7a–d and 10a in methanol/HEPES buffer (80:20) solution,
it was found that irradiation times at 300 and 350 nm were
comparable, the best results being obtained at 350 nm.
Taking into consideration the influence of the structure
of the neurotransmitter on the photocleavage rates, we
could see that the b-alanine conjugate 7c had the largest
irradiation times for 300 and 350 nm. In contrast, the
irradiation times for alanine and GABA conjugates 7b and
7d were always shorter and very similar, and as a result,
photocleavage at 419 nm was also tested for these two
compounds. Although for compound 7b the irradiation
time was at about two times larger (57 min) than that
obtained at 350 nm and that for 7d was six times larger
(245 min), these values indicate that for these neurotrans-
mitter conjugates the wavelength of 419 nm still represents
a practical possibility if photodeprotection should be
considered.
Fig. 1 Normalised UV/visible absorption (A) and fluorescence (F)
spectra of precursors 1, 5 and conjugates 8c, 10b in ethanol (1,
kexc = 379 nm; 5, kexc = 365 nm; 8c, kexc = 375 nm; 10b,
kexc = 374 nm) (1, black full line; 5, grey full line; 8c, black spaced
line; 10b, grey spaced line)
bioconjugates 8c and 10b in ethanol. The shape of the
spectra in methanol/HEPES buffer solution was similar.
The neurotransmitters used as models in this report are
representative of aliphatic amino acids, which do not
absorb or emit in the visible region, not being easily
detectable by absorption and fluorescence techniques. As
such, the linkage of a fluorophore like the considered
oxobenzo[h]benzopyran proved to be a very suitable
approach to enhance the photophysical properties of bio-
molecules of this type.
Photolysis studies of neurotransmitter conjugates 7a–d,
8a–c and 10a, b
The sensitivity of benzo[h]benzopyran conjugates 7a–d,
8a–c and 10a, b towards UV–visible irradiation was
evaluated by exposing solutions of the mentioned com-
pounds in methanol/HEPES buffer (80:20) solution in a
Rayonet RPR-100 reactor at 254, 300, 350 and 419 nm.
Table 3 Irradiation times (in minutes) and rate constants (910-2 min-1) for the photolysis of conjugates 7a–d, 8a–c, 10a, b and 11a, b
(Fernandes et al. 2008a, b) at different wavelengths in methanol/HEPES buffer (80:20) solution
Compound
254 nm
Irr time
300 nm
Irr time
350 nm
Irr time
419 nm
Irr time
k
k
k
k
7a
Z-Gly-OFlu
93
48
52
52
–
3.24
5.45
6.16
5.35
–
144
49
592
45
–
2.09
6.17
0.48
6.62
–
60
30
5.02
9.94
0.62
6.77
0.67
0.66
0.80
1.87
3.91
0.59
3.78
–
57
–
–
7b
Z-Ala-OFlu
5.36
7c
Z-b-Ala-OFlu
Z-GABA-OFlu
H-Gly-OFlu
438
39
–
7d
245
–
1.21
–
8a
469
441
370
140
63
8b
H-Ala-OFlu
–
–
–
–
–
–
8c
H-b-Ala-OFlu
Flu-b-Ala-OMe
Flu-b-Ala-OH
Z-b-Ala-OBba
Z-GABA-OBba
–
–
–
–
–
–
10a
10b
11a
11b
75
–
4.10
–
140
–
1.50
–
–
–
–
–
418
169
0.72
1.75
957
124
0.31
2.51
513
84
–
–
–
–
123

2-Oxo-2H-benzo[h]benzopyran as a new light sensitive protecting group
131
At this point, a comparison could be made between the
data collected for benzo[h]benzopyran conjugates 7c and
7d here reported and previously described benzo[f]benzo-
pyran b-alanine and GABA conjugates 11a and 11b
(Fernandes et al. 2008a, b). These compounds differ in the
fused framework of the tricyclic heterocycle system
(Table 3; Fig. 2). Considering the results for compounds
7c, d and 11a, b presented in Table 3, photorelease of
conjugates 7c and d occurred with shorter irradiation times
at 300 and 350 nm (up to three times) as well as at 254 nm
(up to eight times). In both series, b-alanine conjugates
were associated with slower cleavages. These results
confirmed that the newly synthesised 4-(chloromethyl)-
6-methoxy-2-oxo-2H-benzo[h]benzopyran 1 has advanta-
ges as a photocleavable protecting group when compared
with the previously reported one.
13
12
11
0
50
100
150
200
250
300
350
Irradiation time (min)
Fig. 3 Plot of ln A versus irradiation time for the photolysis at
350 nm of conjugates 8a (triangles), 8b (squares), 8c (circles) and
10b (multiplication symbol) in methanol/HEPES buffer (80:20)
solution
Upon irradiation at the usual wavelengths, a new set of
signals appeared related to aliphatic protons of the amino
acid residue and the N- or C-termini protecting groups (in
case of cleavage of conjugates 7a–c and 10a). After a
variable irradiation period (depending on the wavelength of
irradiation and structure of the conjugate) the signals due to
the amino acid residue in the conjugate form disappeared
completely and were replaced by the corresponding set of
signals of Z-protected amino acids (Z-Aaa-OH, for 7a–d),
b-alanine methyl ester (H-b-Ala-OMe, for 10a) or the free
neurotransmitter amino acids (H-Aaa-OH, for 8a–c and
10b). These observations confirmed the quantitative release
of the amino acid, without removal of the Z-protecting
group, when it was present, which is an interesting result
considering its linkage similarity with the new reported
urethane-bonded benzopyran. Moreover, signals due to
by-products related to the fluorophore were also detected
As a continuation of this research, photolysis of conju-
gates 8a–c and 10b, which have only the light-sensitive
moiety linked to the neurotransmitter, was carried out in
the same conditions at 350 nm (Fig. 3). It was found that
for compounds 8a–c irradiation times were large (370–
469 min), being for b-alanine conjugate 8c inferior to that
of the release of Z-protected amino acid from ester con-
jugate 7c. Cleavage of compound 10b was the fastest; the
free b-alanine was released within at about half irradiation
time than the b-alanine methyl ester from conjugate 10a.
By comparison of b-alanine conjugates 7c and 10a, as
well as 8c and 10b which differ in the type of linkage
between the fluorophore and the neurotransmitter, it was
found that at 300 and 350 nm the urethane bond (10a, b)
cleaved significantly faster than the ester bond (7c and 8c).
Furthermore, the release of Z-protected amino acids
(6a–d), b-alanine methyl ester (9a), as well as the free
residues, as the expected photolysis products of conju-
gates 7a–d, 10a and 8a–c, 10b, respectively, was also
followed by 1H NMR in a methanol-d4/D2O (80:20)
solutions with concentrations ranging from 1.05 9 10-2
to 1.47 9 10-2 M.
1
in the H NMR spectra.
1
As example, Figs. 4 and 5 show H NMR spectra for
photolysis at 350 nm of b-alanine conjugates 8c and 10b,
in comparison with that of the expected released b-alanine.
As mentioned before, upon irradiation it was visible a
decrease in triplets related to a-CH2 and b-CH2 (d 3.04 and
3.33 ppm, 8c; 2.60 and 3.48, 10b), as well as the singlet for
the benzylic-type CH2 of the fluorophore (d 5.62 ppm, 8c;
5.49 ppm, 10b). In both cases, the released b-alanine gave
rise to a new set of signals, namely a-CH2 (d at about
2.70 ppm) and b-CH2 (d at about 3.20 ppm).
O
O
O
O
Ph
O
N
H
Conclusions
n
O
The synthesis of a novel fused benzopyran, namely a
6-methoxy-2-oxo-2H-benzo[h]benzopyran, was achieved
in good yield by a one-step procedure, in the case of the
4-chloromethyl derivative 1, and through a few steps of
simple synthetic modifications in the case of the
4-hydroxymethyl derivative 5. In order to evaluate the
behaviour of this polyaromatic heterocycle as new
O
11 a n = 2 Z-
β
-Ala-OBba
b n = 3 Z-GABA-OBba
Fig. 2 Structure of 2-oxo-2H-benzo[f]benzopyran neurotransmitter
conjugates 11a, b (Fernandes et al. 2008a, b)
123

132
A. M. S. Soares et al.
Fig. 5 1H NMR spectra in methanol-d4/D2O (80:20) of the photolysis
of conjugate Flu-b-Ala-OH 10b (C = 1.47 9 10-2 M) at 350 nm:
a before irradiation; b after irradiation for 20 min; c after irradiation
for 40 min; d after irradiation for 140 min; e H-b-Ala-OH
A study of the photophysical properties of the conju-
gates was carried out in ethanol and methanol/HEPES
buffer (80:20) solution, and the results suggested that
precursors 2-oxo-2H-benzo[h]benzopyran 1 and 5 are
suitable fluorophores for the derivatisation of non-fluores-
cent molecules, yielding bioconjugates with high relative
fluorescence quantum yields.
Fig. 4 1H NMR spectra in methanol-d4/D2O (80:20) of the photolysis
of conjugate H-b-Ala-OFlu 8c (C = 1.05 9 10-2 M) at 350 nm:
a before irradiation; b after irradiation for 35 min; c after irradiation
for 60 min; d after irradiation for 90 min; e H-b-Ala-OH
photoreleasable protecting group, tags 1 and 5 were effi-
ciently used in the synthesis of fluorescent neurotransmitter
amino acid ester 7a–d and urethane 10a, b conjugates, by
reaction with the carboxylic acid or amino terminals of
N-benzyloxycarbonyl-protected and the methyl ester, or the
free form (in case of 10b), of excitatory and inhibitory
neurotransmitters such as glycine, alanine, b-alanine and
c-aminobutyric acid.
Regarding the photocleavage studies of the fluorescent
conjugates, in methanol/HEPES buffer (80:20) solution, at
254, 300 and 350 nm, it was possible to conclude that
the structure of the neurotransmitter had an influence on
the irradiation times. Although a suitable wavelength of
irradiation could be chosen for all neurotransmitters
123

2-Oxo-2H-benzo[h]benzopyran as a new light sensitive protecting group
133
Gangadasu B, Narender P, Raju BC, Rao VJ (2004) ZrCl4 catalysed
solvent free synthesis of coumarins. J Chem Res 7:480–481
conjugates, Z-protected alanine and GABA amino acids
cleaved faster in all wavelengths considered. Owing to the
irradiation times, photolysis at 419 nm can also be appli-
cable for some of the compounds studied. In order to
restore the biological activity as neurotransmitter, conju-
gates having only the amino acid linked to the light-sen-
sitive protecting group 8a–c (Z-protecting groups were
removed before irradiation in compounds 7a–c) and 10b
were irradiated at 350 nm, and it was found that the best
results were observed for the uncaging of b-alanine from
the urethane-conjugate.
¨
Gilbert D, Funk K, Dekowski B, Lechler R, Keller S, Mohrlen F,
Frings S, Hagen V (2007) Caged capsaicins: new tools for the
examination of TRPV1 channels in somatosensory neurons.
ChemBioChem 8:89–97
Hagen V, Dekowski B, Nache V, Schmidt R, Geissler D, Lorenz D,
Eichhorst J, Keller S, Kaneko H, Benndorf K, Wiesner B (2005)
Coumarinylmethyl esters for ultrafast release of high concentra-
tions of cyclic nucleotides upon one- and two-photon photolysis.
Angew Chem Int Ed 44:7887–7891
Han HD, Kim TW, Shin BC, Choi HS (2005) Release of calcein from
temperature-sensitive liposomes in a poly(N-isopropylacrylam-
ide) hydrogel. Macromol Res 13:54–61
Although there are differences in photolysis times,
quantitative release of all the neurotransmitters studied was
proved to occur in the tested conditions. Furthermore, the
newly reported benzo[h]benzopyran can be considered an
enhancement of our previously fluorescent fused hetero-
cycles studied as photolabile protecting groups.
Hill CA, Harris RC, Kim HJ, Harris BD, Sale C, Boobis LH, Kim CK,
Wise JA (2007) Influence of beta-alanine supplementation on
skeletal muscle carnosine concentrations and high intensity
cycling capacity. Amino Acids 32:225–233
Khurana JM, Arora R (2009) Rapid chemoselective deprotection of
benzyl esters by nickel boride. Synthesis 7:1127–1130
Lee HM, Chmielewski J (2005) Liposomal cargo unloading induced
by pH-sensitive peptides. J Pept Res 65:355–363
Legendre P (2001) The glycinergic inhibitory synapse. Cell Mol Life
Sci 58:760–793
Lu M, Fedoryak OD, Moister BR, Dore TM (2003) Bhc-diol as a
photolabile protecting group for aldehydes and ketones. Org Lett
5:2119–2122
Acknowledgments Thanks are due to the Foundation for Science
and Technology (Portugal) for financial support through project
PTDC/QUI/69607/2006. The NMR spectrometer Bruker Avance III
400 is part of the National NMR Network and was acquired with
funds from FCT and FEDER.
Mayer G, Heckel A (2006) Biologically active molecules with a
‘‘light switch’’. Angew Chem Int Ed 45:4900–4921
Morris JV, Mahaney MA, Huber JR (1976) Fluorescence quantum
yield determinations. 9, 10-Diphenylanthracene as a reference
standard in different solvents. J Phys Chem 80:969–974
Paquete LA (ed) (1995) Encyclopedia of reagents for organic
synthesis, vol 6. Wiley, New York, pp 4437–4438
Piloto AM, Costa SPG, Gonc¸alves MST (2005) A naphtho[2,
1-b]furan as a new fluorescent label: synthesis and spectral
characterization. Tetrahedron Lett 46:4757–4760
References
Anderson TL, Davidsen J, Begtrup M, Mouritsen OG, Jorgensen K
(2004) Enzymatic release of antitumor ether lipids by specific
phospholipase A2 activation of liposome-forming prodrugs.
J Med Chem 47:1694–1703
Piloto AM, Fonseca ASC, Costa SPG, Gonc¸alves MST (2006a)
Carboxylic fused furans for amino acid fluorescent labeling.
Tetrahedron 62:9258–9267
Piloto AM, Rovira D, Costa SPG, Gonc¸alves MST (2006b)
Oxobenzo[f]benzopyrans as new fluorescent photolabile protect-
ing groups for the carboxylic function. Tetrahedron 62:11955–
11962
Shembekar VR, Chen Y, Carpenter BK, Hess GP (2005) A protecting
group for carboxylic acids that can be photolyzed by visible
light. Biochemistry 44:7107–7114
Banerjee A, Grewer C, Ramakrishnan L, Jager J, Gameiro A,
Breitinger HGA, Gee KR, Carpenter BK, Hess GP (2003)
Toward the development of new photolabile protecting groups
that can rapidly release bioactive compounds upon photolysis
with visible light. J Org Chem 68:8361–8367
Curten B, Kullmann HMP, Bier ME, Kandler K, Schmidt BF (2005)
Synthesis, photophysical, photochemical and biological proper-
ties of caged GABA, 4-[[(2H–1-benzopyran-2-one-7-amino-4-
methoxy)carbonyl] amino] butanoic acid. Photochem Photobiol
81:641–648
Takaoka K, Tatsu Y, Yumoto N, Nakajima T, Shimamoto K (2004)
Synthesis of carbamate-type caged derivatives of a novel
glutamate transporter blocker. Bioorg Med Chem 12:3687–3694
Tjoeng FS, Heavner GA (1981) Improved preparation of 4-(Boc-
aminoacyloxymethyl)-phenylacetic acids for use in peptide-
D’Addona D, Bochet CG (2001) Preparation of carbamates from
amines and alcohols under mild conditions. Tetrahedron Lett
42:5227–5229
Fernandes MJG, Gonc¸alves MST, Costa SPG (2007) Photorelease of
amino acid neurotransmitters from pyrenylmethyl ester conju-
gates. Tetrahedron 63:10133–10139
Fernandes MJG, Gonc¸alves MST, Costa SPG (2008a) Comparative
study of polyaromatic and polyheteroaromatic fluorescent
photocleavable protecting groups. Tetrahedron 64:3032–3038
Fernandes MJG, Gonc¸alves MST, Costa SPG (2008b) Neurotrans-
mitter amino acid—oxobenzo[f]benzopyran conjugates: synthe-
sis and photorelease studies. Tetrahedron 64:11175–11179
Fonseca ASC, Gonc¸alves MST, Costa SPG (2007) Photocleavage
studies of fluorescent amino acid conjugates bearing different
types of linkages. Tetrahedron 63:1353–1359
synthesis on solid supports utilizing a protecting group
removable by photolysis or reduction. Synthesis 897–899
´
Treffort N, Dubreucq G, Canu MH, Guerardel Y, Falempin M,
Picquet F (2006) Variations in amino acid neurotransmitters in
the rat ventral spinal cord after hindlimb unloading. Neurosci
Lett 403:147–150
Vaxelaire C, Souquet F, Lannou MI, Ardisson J, Royer J (2009)
Anodic oxidation: an attractive alternative to CAN-mediated
cleavage of para-methoxyphenyl ethers. Eur J Org Chem
19:3138–3140
123