Photorelease of amino acids from novel thioxobenzo[f]benzopyran ester conjugates

Article abstract of DOI:10.1007/s00726-011-0969-0

Aiming at the enhancement of the performance of (9-methoxy-3-oxo-3H- benzo[f]benzopyran-1-yl) methyl ester as photocleavable protecting group for the carboxylic acid function at long-wavelengths, 9-methoxy-3-thioxo-3H-benzo[f] benzopyran-l-valine and l-phenylalanine model conjugates were prepared through a thionation reaction of the corresponding oxo-benzobenzopyrans. These thioxobenzobenzopyran derivatives were subjected to photocleavage reactions in the same conditions as the parent oxo-benzobenzopyrans at different wavelengths of irradiation, and photocleavage data were obtained. It was found that the exchange of the carbonyl by a thiocarbonyl group enhanced the performance of the heterocyclic protecting group at 419 nm by improving the photolysis rates, making it an appropriate group for practical applications at long wavelengths.

Full text of DOI:10.1007/s00726-011-0969-0

Amino Acids (2012) 42:2275–2282
DOI 10.1007/s00726-011-0969-0
ORIGINAL ARTICLE
Photorelease of amino acids from novel thioxobenzo[f]benzopyran
ester conjugates
•
Ana M. Piloto Ana M. S. Soares
•
•
Susana P. G. Costa M. Sameiro T. Gonc¸alves
Received: 20 January 2011 / Accepted: 22 June 2011 / Published online: 8 July 2011
Ó Springer-Verlag 2011
Abstract Aiming at the enhancement of the performance
of (9-methoxy-3-oxo-3H-benzo[f]benzopyran-1-yl) methyl
ester as photocleavable protecting group for the carboxylic
acid function at long-wavelengths, 9-methoxy-3-thioxo-
3H-benzo[f]benzopyran-L-valine and L-phenylalanine
model conjugates were prepared through a thionation
reaction of the corresponding oxo-benzobenzopyrans.
These thioxobenzobenzopyran derivatives were subjected
to photocleavage reactions in the same conditions as the
parent oxo-benzobenzopyrans at different wavelengths of
irradiation, and photocleavage data were obtained. It was
found that the exchange of the carbonyl by a thiocarbonyl
group enhanced the performance of the heterocyclic pro-
tecting group at 419 nm by improving the photolysis rates,
making it an appropriate group for practical applications at
long wavelengths.
Photoremovable protecting groups (PRPGs) exhibit
numerous advantages, such as the relatively soft conditions
required for their deprotection and orthogonality with
respect to acid- or base-sensitive groups (Papageorgiou
et al. 1999; Pelliccioli and Wirz 2002; Bochet 2002; Singh
and Khade 2005a; Ma et al. 2005). PRPGs have become
broadly reported for various functional groups, including
carboxylic acids (Bochet 2000; Schaper et al. 2002; Jana
et al. 2010), amines (Peng et al. 1997; Fonseca et al. 2007),
alcohols (Loudwig and Goeldner 2001), phosphates (Din-
kel et al. 2003; Dinkel and Schultz 2003), aldehydes and
ketones (Wang et al. 2007; Yu et al. 2007) for convenient
and controlled release of molecules in a variety of envi-
ronments, including in materials science (Abelson et al.
1998; Bochet 2002), the caging and release of biologically
significant compounds (Givens et al. 2000; Grewer et al.
2000; Pelliccioli and Wirz 2002; Bochet 2002; Singh and
Khade 2005b; Mayer and Heckel 2006; Li et al. 2010a, b),
and in synthetic organic chemistry (Pillai 1980; Greene and
Wuts 1999).
Keywords Thioxobenzobenzopyran Á Benzocoumarin Á
Amino acids Á Photolabile protecting groups
The design and application of polyaromatic compounds,
as well as oxygen and nitrogen polycyclic heterocycles as
novel photocleavable protecting groups for the carboxylic
acid and amine functions of amino acids is part of our
current research interests. Carboxylic acid containing
molecules (amino acids, including neurotransmitters) have
been effectively protected by a variety of PRPGs, such as
1-pyrenylmethyl (Fernandes et al. 2007, 2008a), quino-
lones (Fonseca et al. 2010), benzoxazoles (Soares et al.
2010a), as well as 2-oxo-benzopyrans (trivially known as
coumarins) (Fonseca et al. 2007, 2010), and 2-oxo-benzo-
benzopyrans (benzocoumarins, Piloto et al. 2006; Fernan-
des et al. 2008a, b; Soares et al. 2010b). Their behaviour
towards photocleavage under UV irradiation was studied
at different wavelengths, in simulated physiological
Introduction
The choice of specific protecting groups remains of crucial
importance in the success of many steps of organic syn-
thesis and manipulation of polyfunctional molecules, since
they prevent the formation of undesired side products and
reactions (Isidro-Llobet et al. 2009).
A. M. Piloto Á A. M. S. Soares Á S. P. G. Costa Á
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

2276
A. M. Piloto et al.
environment in mixtures of aqueous buffer and different
organic solvents. In studies involving biomolecules,
photocleavage at 350 nm or longer wavelengths is pre-
ferable, as it avoids cell damage due to short-wave-
length light. According to recent results reported by us,
1-chloromethyl-9-methoxy-3-oxo-3H-benzo[f]benzopyran is
a suitable photocleavable protecting group for carboxylic
acids, with release at 350 nm occurring efficiently in short
times (Fernandes et al. 2008a, b). However, photocleavage
at longer wavelengths, namely at 419 nm usually resulted
in a large increase of the irradiation time, with a reduction
of the scope of its applications at this wavelength.
Photolyses were carried out using a Rayonet RPR-100
chamber reactor equipped with 10 lamps of 350 and
419 10 nm. HPLC analyses were performed using a
Licrospher 100 RP18 (5 lm) column in a JASCO HPLC
system composed by a PU-2080 pump and a UV-2070
detector with ChromNav software. All reagents were used
as received.
Synthesis of N-(benzyloxycarbonyl)-L-valine (9-methoxy-
3-thioxo-3H-benzo[f]benzopyran-1-yl) methyl ester,
Z-Val-OTba 2a Lawesson’s reagent (0.231 g, 5.7 9 10-4
mol) was added to a solution of N-(benzyloxycarbonyl)-
L-valine (9-methoxy-3-oxo-3H-benzo[f]benzopyran-1-yl)
methyl ester, Z-Val-OBba 1a in toluene (5 mL), with stir-
ring, at room temperature. The reaction mixture was
refluxed for 48 h and the process was followed by TLC
(ethyl acetate/n-hexane, 2:8). The solvent was removed by
rotary evaporation under reduced pressure and the crude
residue was purified by column chromatography using
dichloromethane/n-hexane, with mixtures of increasing
polarity as eluent. Compound 2a was obtained as an orange
solid (0.081 g, 55%). mp = 131.7–132.0°C. TLC (ethyl
acetate/n-hexane, 2:8): Rf = 0.85. 1H NMR (CDCl3,
400 MHz): dH = 0.91 (d, J = 6.8 Hz, 3 H, c-CH3 Val),
1.04 (d, J = 6.8 Hz, 3 H, c-CH3 Val), 2.20-2.30 (m, 1 H,
b-CH Val), 3.96 (s, 3 H, OCH3), 4.40–4.50 (m, 1 H, a-CH
Val), 5.13 (s, 2 H, CH2 Z), 5.27 (d, J = 8.8 Hz, 1 H, a-NH
Val), 5.60–5.74 (m, 2 H, CH2), 7.26 (dd, J = 8.8 and
2.4 Hz, 1 H, H-8), 7.30–7.40 (m, 5 H, 59 Ar–H Z), 7.45
(s, 1 H, H-2), 7.48 (d, J = 7.2 Hz, 1 H, H-5), 7.49 (s, 1 H,
H-10), 7.85 (d, J = 8.8 Hz, 1 H, H-7), 7.96 (d, J = 8.8 Hz,
1 H, H-6). 13C NMR (CDCl3, 100.6 MHz): dC = 17.44
(c-CH3 Val), 19.21 (c-CH3 Val), 30.96 (b-CH Val), 55.51
(OCH3), 59.26 (a-CH Val), 64.62 (CH2), 67.28 (CH2 Z),
106.25 (C-10), 114.21 (C-4b), 114.93 (C-5), 117.21 (C-8),
126.60 (C-6a), 127.50 (C-2), 128.19 (19 Ar–C Z), 128.24
(29 Ar–C Z), 128.52 (29 Ar–C Z), 130.24 (C-6b), 131.46
(C-7), 134.36 (C-6), 136.02 (C-1 Z), 141.32 (C-1), 156.24
(CONH), 158.79 (C-4a), 159.96 (C-9), 171.55 (CO2CH2),
195.13 (C-3). IR (KBr 1%, cm-1): m = 3,339, 2,936, 2,964,
1,720, 1,623, 1,606, 1,591, 1,538, 1,455, 1,444, 1,432,
1,363, 1,346, 1,296, 1,230, 1,217, 1,181, 1,139, 1,096,
1,052, 1,026, 1,009, 985, 838. HRMS (ESI): calcd for
C28H28NO6S [M??H]: 506.16318; found: 506.16317.
Considering these facts, we now report the synthesis of
novel ester conjugates of 9-methoxy-3-thioxo-3H-
benzo[f]benzopyran-L-valine and L-phenylalanine obtained
by a thionation reaction of the 3-oxo-3H-benzo[f]benzo-
pyran bioconjugates, and compare their behaviour towards
irradiation with the parent compounds. To the best of our
knowledge, the use of thioxo derivatives as photocleavable
protecting groups has not been previously reported.
Photocleavage studies were carried out in a photochemical
reactor under irradiation at 350 and 419 nm, and kinetic
data was obtained.
Experimental section
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 determined on a BOMEM MB 104 spectro-
photometer using KBr discs. UV/visible absorption spectra
(200–700 nm) were obtained using a Shimadzu UV/
2501PC spectrophotometer. NMR spectra were obtained
on a Varian Unity Plus Spectrometer at an operating fre-
1
quency of 300 MHz for H NMR and 75.4 MHz for 13C
NMR or a Bruker Avance III 400 at an operating frequency
1
of 400 MHz for H 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 multi-
plicities and J values and were supported by spin decou-
pling-double resonance and bidimensional heteronuclear
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 collected using a FluoroMax-4 spectrofluorometer.
Synthesis
of
N-(benzyloxycarbonyl)-L-phenylalanine
(9-methoxy-3-thioxo-3H-benzo[f]benzopyran-1-yl) methyl
ester, Z-Phe-OTba 2b Starting from Lawesson’s reagent
(0.120 g, 2.97 9 10-4 mol), N-(benzyloxycarbonyl)-
L-phenylalanine (9-methoxy-3-oxo-3H-benzo[f]benzopyran-
1-yl) methyl ester, Z-Phe-OBba 1b and toluene (5 mL),
following the same procedure as described for 2a, com-
pound 2b was obtained as a yellow solid (0.035 g, 42%).
mp = 155.8–156.9°C. TLC (ethyl acetate/n-hexane, 2:8):
123

Photorelease of amino acids
2277
1
Rf = 0.87. H NMR (CDCl3, 400 MHz): dH = 3.16 (2 H,
m = 3,427, 2,968, 1,750, 1,739, 1,623, 1,591, 1,537, 1,501,
1,465, 1,444, 1,430, 1,372, 1,296, 1,231, 1,218, 1,198,
1,139, 1,096, 1,047, 1,028, 839. HRMS (ESI): calcd for
C20H22NO4S [M??H]: 372.12641; found: 372.12595.
d, J = 6.4 Hz, b-CH2 Phe), 3.94 (3 H, s, OCH3), 4.70–4.85
(1 H, m, a-CH Phe), 5.05–5.20 (m, 2 H, CH2 Z), 5.30 (d,
J = 5.6 Hz, 1 H, a-NH Phe), 5.56 (s, 2 H, CH2), 7.08–7.14
(m, 2 H, 1 9 Ar–H Phe and H-2), 7.15–7.23 (m, 4 H, 49
Ar–H Phe), 7.27 (dd, J = 9.2 and 2.4 Hz, 1 H, H-8),
7.30–7.38 (m, 5 H, 59 Ar–H Z), 7.42–7.47 (m, 2 H, H-5
and H-10), 7.84 (d, J = 8.8 Hz, 1 H, H-7), 7.95 (d,
J = 8.8 Hz, 1 H, H-6). 13C NMR (CDCl3, 100.6 MHz):
dC = 38.18 (b-CH2 Phe), 55.11 (a-CH Phe), 55.47
(OCH3), 64.71 (CH2), 67.22 (CH2 Z), 106.07 (C-10),
114.20 (C-4b), 114.85 (C-5), 117.26 (C-8), 126.53 (C-6a),
127.39 (19 Ar–C Phe), 127.98 (C-2), 128.13 (19 Ar–C Z),
128.20 (29 Ar–C Phe), 128.47 (29 Ar–C Z), 128.76 (29
Ar–C Phe), 128.98 (29 Ar–C Phe), 130.17 (C-6b), 131.38
(C-7), 134.27 (C-6), 135.02 (C-1 Phe), 135.95 (C-1 Z),
140.66 (C-1), 155.64 (CONH), 158.73 (C-4a), 159.90
(C-9), 171.16 (CO2CH2), 195.03 (C-3). IR (KBr 1%, cm-1):
m = 3,330, 3,063, 3,030, 2,934, 1,722, 1,623, 1,606, 1,592,
1,538, 1,520, 1,498, 1,455, 1,444, 1,432, 1,362, 1,343,
1,295, 1,230, 1,216, 1,178, 1,139, 1,096, 1,056, 1,027,
1,009, 838. HRMS (ESI): calcd for C32H28NO6S [M??H]:
554.16318; found: 554.16358.
Synthesis of L-phenylalanine (9-methoxy-3-thioxo-3H-
benzo[f]benzopyran-1-yl) methyl ester hydrobromide,
HBr.H-Phe-OTba 3b Starting from Z-Phe-OTba 2b
(0.015 g, 2.71 9 10-5 mol), 45% solution of hydrobromic
acid in acetic acid (65 lL), and acetic acid (1 mL),
following the same procedure as described for 3a (total
volume of hydrobromic acid in acetic acid 2.2 mL; reaction
time: 2 days), compound 3b was obtained as a yellow solid
(0.013 g, 93%). mp = 188.3–189.9°C. TLC (ethyl acetate/
1
methanol 1:1): Rf = 0.61. H NMR (CDCl3, 300 MHz):
dH = 3.0–3.20 (m, 2 H, b-CH2 Phe), 3.95 (s, 3 H, OCH3),
4.60 (broad s, 1 H, a-CH Phe), 5.90–6.0 (m, 2 H, CH2),
7.10–7.30 (m, 5 H, 59 Ar-Phe), 7.33 (s, 1 H, H-2), 7.38
(dd, J = 8.9 and 2.1 Hz, 1 H, H-8), 7.53 (d, J = 1.2 Hz, 1
H, H-10), 7.60 (d, J = 8.7 Hz, 1 H, H-5), 8.08 (d,
J = 9.0 Hz, 1 H, H-7), 8.26 (d, J = 9.0 Hz, 1 H, H-6),
8.53 (broad s, 3 H, NH3?). 13C NMR (CDCl3, 75.4 MHz):
dC = 36.13 (b-CH2 Phe), 53.21 (a-CH Phe), 55.42
(OCH3), 65.16 (CH2), 106.84 (C-10), 114.41 (C-5), 117.46
(C-8), 126.36 (C-4b), 126.62 (C-2), 127.33 (19 Ar–C Phe),
128.58 (29 Ar–C Phe), 129.26 (29 Ar–C Phe), 129.34
(C-6a), 129.75 (C-6b), 131.52 (C-7), 134.27 (C-1 Phe),
135.01 (C-6), 142.61 (C-1), 158.24 (C-4a), 159.43 (C-9),
168.73 (CO2CH2), 194.42 (C-3). IR (KBr 1%, cm-1):
m = 3,440, 2,924, 1,753, 1,623, 1,538, 1,455, 1,364, 1,296,
1,231, 1,139, 1,098, 840, 751, 701. HRMS (ESI): calcd for
C24H22NO4S [M??1]: 420.12641; found: 420.12568.
Synthesis of L-valine (9-methoxy-3-thioxo-3H-benzo[f]
benzopyran-1-yl) methyl ester hydrobromide, HBr.H-Val-
OTba 3a A 45% solution of hydrobromic acid in acetic
acid (60 lL), and acetic acid (1 mL) were added to Z-Val-
OTba 2a (0.017 g, 3.36 9 10-5 mol) with stirring, at room
temperature. The reaction mixture was maintained in these
conditions for 4 days, and the process was followed by TLC
(ethyl acetate/methanol, 1:1). During this time, additional
amounts of 45% solution of hydrobromic acid in acetic acid
were added until the total volume of 1.76 mL. When the
reaction was completed, cold diethyl ether was added
(2 mL), and the precipitate filtered off and washed with the
same solvent to give compound 3a as a brown solid
(0.010 g, 64%). mp = 181.7–183.4°C. TLC (ethyl acetate/
Synthesis of L-valine (9-methoxy-3-oxo-3H-benzo[f]benzo-
pyran-1-yl) methyl ester hydrobromide, HBr.H-Val-OBba
4a Starting from Z-Val-OBba 1a (0.030 g, 6.13 9
10-5 mol), 45% solution of hydrobromic acid in acetic
acid (32 lL), and acetic acid (1.5 mL), following the same
procedure as described for 3a (total volume of hydrobro-
mic acid in acetic acid 1.23 mL; reaction time: 3 days),
compound 4a was obtained as a yellow solid (0.019 g,
73%). mp = 218.9–220.1°C. TLC (ethyl acetate/methanol,
1
methanol, 1:1): Rf = 0.61. H NMR (CDCl3, 300 MHz):
dH = 0.94 (d, J = 2.1 Hz, 3 H, c-CH3 Val), 0.97 (d,
J = 2.1 Hz, 3 H, c-CH3 Val), 2.10–2.30 (m, 1 H, b-CH
Val), 3.98 (s, 3 H, OCH3), 4.23 (broad s, 1 H, a-CH Val),
6.01 (s, 2 H, CH2), 7.40 (dd, J = 9.3 and 1.8 Hz, 1 H, H-8),
7.49–7.60 (m, 3 H, H-10, H-5 and H-2), 8.09 (d,
1
1:1): Rf = 0.56. H NMR (CDCl3, 300 MHz): dH = 0.95
(d, J = 0.9 Hz, 3 H, c-CH3 Val), 0.98 (d, J = 0.9 Hz, 3 H,
c-CH3 Val), 2.19–2.26 (m, 1 H, b-CH Val), 3.95 (s, 3 H,
OCH3), 4.19 (broad s, 1 H, a-CH Val), 5.90–6.10 (m, 2 H,
CH2), 6.73 (s, 1 H, H-2), 7.31 (dd, J = 8.7 and 2.1 Hz, 1 H,
H-8), 7.39 (d, J = 9.0 Hz, 1 H, H-5), 7.49 (s, 1 H, H-10),
8.02 (d, J = 9.0 Hz, 1 H, H-7), 8.15 (d, J = 9.0 Hz, 1 H,
H-6), 8.48 (broad s, 3 H, NH3?). 13C NMR (CDCl3,
75.4 MHz): dC = 17.84 (c-CH3 Val), 18.15 (c-CH3 Val),
29.60 (b-CH Val), 55.54 (OCH3), 57.49 (a-CH Val), 65.46
(CH2), 106.51 (C-10), 111.53 (C-4b), 112.20 (C-2), 114.95
(C-5), 116.74 (C-8), 126.13 (C-6a), 130.18 (C-6b), 131.52
J = 9.0 Hz, 1 H, H-7), 8.27 (d, J = 9.0 Hz, 1 H, H-6). 13
C
NMR (CDCl3, 75.4 MHz): dC = 17.49 (c-CH3 Val), 19.31
(c-CH3 Val), 31.01 (b-CH Val), 55.60 (OCH3), 59.28 (a-CH
Val), 64.24 (CH2), 106.86 (C-10), 114.45 (C-5), 117.50
(C-8), 126.40 (C-4b), 126.58 (C-2), 127.31 (19 Ar–C Phe),
128.56 (29 Ar–C Phe), 129.24 (29 Ar–C Phe), 129.32
(C-6a), 129.73 (C-6b), 131.50 (C-7), 134.25 (C-1 Phe),
135.01 (C-6), 142.62 (C-1), 158.25 (C-4a), 159.44 (C-9),
168.75 (CO2CH2), 194.23 (C-3). IR (KBr 1%, cm-1):
123

2278
A. M. Piloto et al.
(C-7), 134.29 (C-6), 151.20 (C-1), 155.02 (C-4a), 159.31
(C-9), 159.48 (C-3), 168.53 (CO2CH2). IR (KBr 1%,
cm-1): m = 3,407, 2,969, 1,757, 1,694, 1,624, 1,550, 1,519,
1,495, 1,470, 1,447, 1,428, 1,378, 1,366, 1,340, 1,282,
1,251, 1,235, 1,214, 1,173, 1,140, 1,100, 1,062, 1,026, 973,
962, 844. HRMS (ESI): calcd for C20H22NO5 [M??1]:
356.14925; found: 356.14894.
Results and discussion
Synthesis of amino acid conjugates 2, 3 and 4
N-(Benzyloxycarbonyl)-L-valine
(9-methoxy-3-oxo-3H-
benzo[f]benzopyran-1-yl) methyl ester, Z-Val-OBba 1a and
N-(benzyloxycarbonyl)-L-phenylanaline (9-methoxy-3-oxo-
3H-benzo[f]benzopyran-1-yl) methyl ester, Z-Phe-OBba 1b
were prepared by reaction of 1-chloromethyl-9-methoxy-
3-oxo-3H-benzo[f]benzopyran with N-(benzyloxycarbonyl)-
L-valine and N-(benzyloxycarbonyl)-L-phenylanaline in the
presence of potassium fluoride in DMF, at room temperature,
as previously described (Piloto et al. 2006).
Synthesis of L-phenylalanine (9-methoxy-3-oxo-3H-
benzo[f]benzopyran-1-yl) methyl ester hydrobromide,
HBr.H-Phe-OBba 4b Starting from Z-Phe-OBba 1b
(0.058 g, 1.08 9 10-3 mol), 45% solution of hydrobromic
acid in acetic acid (72 lL), and acetic acid (1.5 mL), fol-
lowing the same procedure as described for 3a (total volume
of hydrobromic acid in acetic acid 2.9 mL; reaction time:
3 days), compound 4b was obtained as a yellow solid
(0.032 g, 61%). mp = 231.1–232.9°C. TLC (ethyl acetate/
Conjugates 1a and 1b were reacted with Lawesson’s
reagent in toluene (Jesberger et al. 2003), under reflux
conditions, followed by column chromatography purifica-
tion affording corresponding thioxobenzopyran conjugates
2a (55%) and 2b (42%). In addition to the use of this
heterocycle for molecules that require multiple protecting
groups, it was intended to evaluate its usefulness in the
release of unprotected moieties in a biological perspective.
Therefore, the N-benzyloxycarbonyl-protecting group was
removed by acidolysis with hydrobromic acid in acetic acid
yielding conjugates 3a and 3b bearing the amino acid and
the photosensitive tag.
1
methanol, 1:1): Rf = 0.58. H NMR (CDCl3, 300 MHz):
dH = 3.0–3.26 (m, 2 H, b-CH2 Phe), 3.94 (s, 3 H, OCH3),
4.57 (t, J = 6.9 Hz, 1 H, a-CH Phe), 5.80–6.0 (2 H, m, CH2),
6.49 (s, 1 H, H-2), 7.15–7.28 (m, 5 H, 59 Ar–H Phe), 7.31
(dd, J = 8.7 and 2.1 Hz, 1 H, H-8), 7.41 (d, J = 8.7 Hz, 1 H,
H-5), 7.44 (s, 1 H, H-10), 8.02 (d, J = 9.0 Hz, 1 H, H-7),
8.16 (d, J = 9.3 Hz, 1 H, H-6), 8.60 (broad s, 3 H, NH3?).
13C NMR (CDCl3, 75.4 MHz): dC = 36.14 (b-CH2 Phe),
53.28 (a-CH Phe), 55.31 (OCH3), 65.21 (CH2), 106.13
(C-10), 111.41 (C-4b), 112.17 (C-2), 114.82 (C-5), 116.75
(C-8), 126.00 (C-6a), 127.31 (19 Ar–C Phe), 128.56 (29
Ar–C Phe), 129.31 (29 Ar–C Phe), 130.04 (C-6b), 131.32
(C-7), 134.06 (C-1 Phe), 134.48 (C-6), 150.56 (C-1), 154.86
(C-4a), 159.14 (C-3 and C-9), 168.67 (CO2CH2). IR (KBr
1%, cm-1): m = 3,419, 2,914, 1,749, 1,715, 1,626, 1,594,
1,552, 1,519, 1,498, 1,456, 1,445, 1,428, 1,377, 1,363, 1,337,
1,280, 1,234, 1,216, 1,204, 1,141, 1,121, 1,082, 1,063, 1,021,
855. HRMS (ESI): calcd for C24H22NO5 [M??H]:
404.14925; found: 404.14858.
In order to compare the behaviour towards irradiation of
the thioxobenzopyran conjugates 3a and 3b with the rela-
ted oxobenzopyran derivatives, cleavage of the N-benzyl-
oxycarbonyl-protecting group was also carried out in
compounds 1a and 1b, following the same conditions as
mentioned above, to provide compounds 4a and 4b
(Scheme 1; Table 1). The oxo-benzobenzopyran (Bba) and
the thioxo-benzobenzopyran (Tba) moieties will be desig-
nated in this report by a three-letter code for simplicity of
naming the various fluorescent conjugates, as indicated in
Tables 1 and 2.
All new compounds were fully characterised by high
1
resolution mass spectrometry, IR, H and 13C NMR spec-
General photolysis procedure
troscopy. 1H NMR spectra showed signals of the amino acid
residues, such as the a-CH (d 4.19–4.85 ppm), b-CH
(d 2.10–2.30 ppm), b-CH2 (d 3.0–3.30 ppm), as well as
c-CH3 (d 0.91–1.04 ppm). The heterocycle methylene
group was also visible for all conjugates (d 5.56–6.01 ppm).
The effect of the thiocarbonyl group in compounds 2a, 2b,
3a and 3b was notorious in the chemical shift of the pyran
proton H-2, which appeared downfield in the range d
7.08–7.60 ppm, whereas in compounds 4a and 4b, having a
carbonyl group, it occurred at d 6.49 or 6.73 ppm.
The confirmation of the presence of the new C=S bond
(C-3) at the heterocyclic ring (2a, 2b, 3a and 3b) was also
supported by 13C NMR spectra signals at d 194.23–
195.13 ppm, instead of the carbonyl group, which occurred at
d 159.14 or 159.48 ppm, in conjugates 4a and 4b. The
A 1 9 10-4 M methanol/HEPES (80:20) solution of com-
pounds 1–4 (5 mL) were placed in a quartz tube and irra-
diated in a Rayonet RPR-100 reactor at the desired
wavelength. The lamps used for irradiation were of 350 and
419 10 nm.
Aliquots of 100 lL were taken at regular intervals and
analysed by RP-HPLC. The eluent was acetonitrile/water,
75:25 or methanol/water, 64:36 (4a and 4b), at a flow rate
of 0.8 mL/min, previously filtered through a Millipore,
type HN 0.45-lm filter and degassed by ultra-sound for
30 min. The chromatograms were traced by detecting UV
absorption at the wavelength of maximum absorption for
each compound (retention time: 1a, 7.1; 1b, 7.4; 2a, 9.7;
2b, 10.6; 3a and 3b, 4.2; 4a and 4b, 8.0 min).
123

Photorelease of amino acids
2279
Scheme 1 Synthesis of
thioxobenzo[f]benzopyran and
oxobenzo[f]benzopyran
conjugates 2–4
O
R
O
O
R
O
R
O
O
O
O
N
O
Ph
NH2.HBr
N
O
Ph
H
b
a
H
O
S
O
S
O
O
O
O
O
2
3
1
b
1- 4 a R = CH(CH3)2
b R = CH2Ph
O
R
O
NH2.HBr
O
O
a) Lawesson´s reagent/toluene; b) HBr/CH3CO2H
O
4
Table 1 Yields, UV/visible and fluorescence data for conjugates 1–4 in absolute ethanol and methanol/HEPES buffer (80:20) solution
Compound
Yield (%) EtOH
kabs (nm) log e kem (nm) UF
MeOH/HEPES (80:20)
Dk (nm) kabs (nm) log e kem (nm) UF
Dk (nm)
1a
Z-Val-OBbaa
75
348
347
407
408
407
407
344
345
4.07
4.08
3.70
3.83
3.74
3.75
3.76
3.79
478
478
458
464
461
461
468
468
0.58 130
0.59 131
351
349
417
417
417
416
348
345
4.19
4.09
4.14
4.19
4.44
4.13
4.35
3.94
490
490
476
477
477
476
478
473
0.23
139
141
59
1b Z-Phe-OBbaa 94
0.25
2a
2b Z-Phe-OTba
3a H-Val-OTba
3b H-Phe-OTba
4a H-Val-OBba
Z-Val-OTba
55
42
64
93
72
61
0.01
0.02
0.02
0.04
51
56
54
54
0.002
0.004
0.004
0.004
0.36
60
60
60
0.71 124
0.55 123
130
128
4b H-Phe-OBba
0.29
a
Data in ethanol was previously reported (Piloto et al. 2006)
Table 2 Irradiation times (tirr in min), rate constants (k, 910-2 min-1) and photochemical quantum yields (UP, 910-3) for the photolysis of
conjugates 1–4 at 350 and 419 nm in methanol/HEPES buffer (80:20) solution
Compound
350 nm
419 nm
tirr
k
UP
tirr
k
UP
1a
1b
2a
2b
3a
3b
4a
4b
Z-Val-OBba
Z-Phe-OBba
Z-Val-OTba
Z-Phe-OTba
H-Val-OTba
H-Phe-OTba
H-Val-OBba
H-Phe-OBba
33
38
9.08
8.28
4.76
0.84
1.97
2.12
0.49
0.58
0.098
0.093
0.130
0.020
0.015
0.043
0.004
0.011
462
301
30
0.66
1.0
0.079
0.144
0.119
0.062
0.041
0.078
0.006
0.004
66
9.68
5.80
6.07
6.49
0.09
0.04
354
162
146
607
520
52
48
49
3304
7478
chemical shift of the pyran carbon C-2 was also influenced by
the proximity of the carbon–sulphur double bond, being in the
range d 126.58–127.98 ppm for compounds 2a, 2b, 3a and
3b, and d 112.17 or 112.20 ppm for compounds 4a and 4b.
HEPES buffer (80:20) solution of thioxobenzopyran
conjugates 2a, 2b, 3a, 3b, in comparison with the
corresponding oxobenzopyran conjugates 1a, 1b, 4a, 4b
were measured; absorption and emission maxima, molar
absorptivities and relative fluorescence quantum yields are
also reported (Table 1). Relative fluorescence quantum
yields were calculated using 9,10-diphenylanthracene as
standard (UF = 0.95 in ethanol) (Morris et al. 1976). For
the UF determination, the fluorescence standard was exci-
ted at the wavelengths of maximum absorption found for
Evaluation of the photophysical properties of amino
acid conjugates 1, 2, 3 and 4
The UV/visible absorption and emission spectra of degas-
sed 10-5 M solutions in absolute ethanol and in a methanol/
123

2280
A. M. Piloto et al.
O
R
R
1a,b
2a,b
HO
HO
N
H
O
Ph
hν
photo
by-products
O
O
+
3a,b
4a,b
NH2
Scheme 2 Photocleavage reactions of conjugates 1–4
Fig. 1 Normalised UV/visible absorption and fluorescence spectra of
conjugates 3a (full line) and 4a (spaced line) in methanol/HEPES
(80:20) solution (3a, kexc = 417 nm; 4a, kexc = 348 nm)
each one of the compounds to be tested and in all fluori-
metric measurements the absorbance of the solution did not
exceed 0.1.
By comparison of absorption maxima for all compounds
in both solvents, a slight bathochromic shift (2–10 nm) was
observed in methanol/HEPES buffer (80:20) solution
(except for compound 4b, no variation) (kmax 344–408 nm,
in ethanol, and 345–417 nm, in methanol/HEPES buffer).
Furthermore, upon exchange of the carbonyl group at the
heterocycle in compounds 1a and 1b for the thiocarbonyl
group in 2a and 2b, a considerable bathochromic shift
(59–68 nm) was observed. The same trend occurred for
Z-deprotected conjugates 4a, 4b in comparison with 3a, 3b
(62–71 nm), being the highest kmax values in methanol/
HEPES buffer, as in the latter conjugates. Absorption
maxima for thioxo- and oxo-conjugates were independent
of the amino acid residue.
Fig. 2 Plot of ln A versus irradiation time for the photolysis at
419 nm of conjugates 1a (diamond), 2a (triangle), 3a (square) and 4a
(circle) in methanol/HEPES buffer (80:20) solution (for better
visualisation, time scale is shown only up to 150 min, although
photolysis for 4 proceeded until ca. 7,500 min)
(80:20) solution in a Rayonet RPR-100 reactor at 350 and
419 nm (Scheme 2). 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 average of three runs. The determined irra-
diation time represents the time necessary for the con-
sumption of the starting materials until \5% of the initial
area was detected (Table 2). 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 (Fig. 2). The corresponding rate constants
were calculated and are presented in Table 2. The photo-
chemical quantum yields (UP) were calculated based on
half-lives (t1/2), molar absorptivities (e) and the incident
photon flux (I0), which was determined by potassium
ferrioxalate actinometry (Muller et al. 2001). The calcu-
lated photochemical quantum yields indicated that the
photocleavage process was not as efficient as desirable,
probably due to the dissipation of part of the absorbed
Bioconjugates 1a, 1b, 4a and 4b exhibited high fluo-
rescence quantum yields in ethanol (0.55 \ UF \ 0.71)
and in methanol/HEPES buffer (0.23 \ UF \ 0.36), con-
trarily to the minor fluorescent thioxo analogues 2a, 2b, 3a
and 3b.
Compounds 2a, 2b, 3a and 3b displayed emission
maxima between 458 and 464 nm (in ethanol) and at about
477 nm (in methanol/HEPES buffer), with moderate
Stokes’ shifts (Dk 51–60 nm), but inferior to those of the
corresponding oxobenzopyran conjugates 1a, 1b, 4a and
4b (123–141 nm) (Table 1; Fig. 1).
Photolysis studies of amino acid conjugates 1–4
The sensitivity of thioxo-conjugates 2a, 2b, 3a and 3b in
comparison with oxo-conjugates 1a, 1b, 4a and 4b towards
UV/visible irradiation was evaluated by exposing solutions
of the mentioned compounds in methanol/HEPES buffer
123

Photorelease of amino acids
2281
energy via fluorescence pathways that compete with the
photochemical reaction, as well as the type of reactor used
(open chamber reactor).
UV/visible characterization was carried out in absolute
ethanol and methanol/HEPES buffer (80:20) solution.
Photocleavage studies of the oxo- and thioxo-conju-
gates, in methanol/HEPES buffer (80:20) solution at
350 nm revealed that the amino acid-heterocycle ester
bond was efficiently photolised, releasing the N-protected
and free amino acids from both types of precursors.
However, the most interesting results were obtained at
419 nm for the thioxoconjugates, revealing that the pres-
ence of the thiocarbonyl clearly improved the photolysis
rates, giving practicable irradiations times (30–52 min).
Although the parent oxo-protecting group still repre-
sents an interesting alternative as photocleavable group for
the carboxylic acid, the newly reported thioxo-protecting
group, (9-methoxy-3-thioxo-3H-benzo[f]benzopyran-1-yl)
methyl ester, emerges as an innovation for the release at
longer wavelengths, since it photolyses with short irradia-
tion times at wavelengths that are not detrimental to a
variety of applications.
The release of N-benzyloxycarbonyl valine and phenyl-
alanine required shorter irradiation times than the cor-
responding free amino acids at 350 nm (except for 3b). The
presence of the thiocarbonyl group at the heterocycle
clearly affected the photolysis rate of the amino acid-het-
erocycle ester bond. Comparison of thioxo-conjugates 3
with the corresponding oxo-conjugate 4, showed that irra-
diation times were shorter for 3 revealing the significance
of the thioxo-group in the release of the free amino acids.
However, this trend was reversed when the N-protecting
group was present (compare data for conjugates 2 and 1).
It was expected that the bathochromic shift in the
maximum absorption wavelengths related to the presence
of the C=S bond would result in an increase in the effi-
ciency of the photolysis at longer wavelengths, leading to
shorter irradiation times. The obtained results confirmed
this expectation, as the photocleavage at 419 nm of thioxo-
conjugates 2a, 2b, 3a and 3b occurred rapidly
(30–52 min), in comparison with the corresponding oxo-
conjugates 1a, 1b, 4a and 4b (301–7,478 min). For these
compounds and at this wavelength, the release of N-pro-
tected or free valine and phenylalanine occurred in similar
irradiation times. The results indicated that the release of
the amino acids at 419 nm from thioxobenzobenzopyrans
is practicable for organic synthesis and also for caging
applications, considering the short irradiation times and the
fact that it minimises the side reactions for the remaining
functionalities of the molecule.
Acknowledgments Thanks are due to the Foundation for Science
and Technology (Portugal) for financial support through project
PTDC/QUI/69607/2006 (FCOMP-01-0124-FEDER-007449), and a
PhD grant to A.M.P. (SFRH/BD/61459/2009). The NMR spectro-
meter Bruker Avance III 400 is part of the National NMR Network
and was purchased in the framework of the National Program for
Scientific Re-equipment, contract REDE/1517/RMN/2005 with funds
from POCI 2010 (FEDER) and FCT.
References
Abelson J, Simon M, Marriott G (1998) Caged compounds. Academic
Press, New York
Bochet CG (2000) Wavelength-selective cleavage of photolabile
protecting groups. Tetrahedron Lett 41:6341–6346
Bochet CG (2002) Photolabile protecting groups and linkers. J Chem
Soc Perkin Trans 1:125–142
Dinkel C, Schultz C (2003) Synthesis of caged myo-inositol 1, 3, 4,
5-tetrakisphosphate. Tetrahedron Lett 44:1157–1159
Dinkel C, Wichmann O, Schultz C (2003) Versatile reagents to
introduce caged phosphates. Tetrahedron Lett 44:1153–1155
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: synthesis
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
Given the interest in the development of novel protecting
groups cleavable with UV A or even visible radiation, it
was found that a simple modification involving the
exchange of the carbonyl group of the light sensitive
9-methoxy-3-oxo-3H-benzo[f]benzopyran-1-yl) methyl ester
reported before by us, for a thiocarbonyl group by a
thionation reaction, lead to a enhancement of the photo-
lysis performance at 419 nm. This newly reported
thioxobenzo[f]benzopyran can be considered an addition to
the collection of photolabile protecting groups for applica-
tion at long wavelengths.
Conclusions
The synthesis of novel thioxobenzo[f]benzopyran ester
conjugates was achieved in good yields through a simple
thionation reaction with Lawesson’s reagent from the
corresponding oxobenzo[f]benzopyrans. In order to obtain
the parameters necessary for monitoring the photolysis
reaction, as well as the fluorescence properties, the
Fonseca ASC, Gonc¸alves MST, Costa SPG (2010) Light-induced
cleavage of phenylalanine model conjugates based on coumarins
and quinolones. Amino Acids 39:699–712
Givens RS, Weber JFW, Conrad PG, Orosz G, Donahue SL, Thayer
SA (2000) New phototriggers 9: p-hydroxyphenacyl as a
123

2282
A. M. Piloto et al.
C-terminus photoremovable protecting group for oligopeptides.
J Am Chem Soc 122:2687–2697
Greene TW, Wuts GM (1999) Protective groups in organic chemistry,
3rd edn. John Wiley, New York
Papageorgiou G, Ogden DC, Barth A, Corrie JET (1999) Photorelease
of carboxylic acids from 1-acyl-7-nitroindolines in aqueous
solution: a reagent for rapid and efficient photorelease of
L-glutamate. J Am Chem Soc 121:6503–6504
Grewer C, Jager J, Carpenter BK, Hess GP (2000) A new photolabile
precursor of glycine with improved properties: a tool for
chemical kinetic investigations of the glycine receptor. Bio-
chemistry 39:2063–2070
Isidro-Llobet A, Alvarez M, Albericio F (2009) Amino acid-
protecting groups. Chem Rev 109:2455–2504
Jana A, Atta S, Sarkar SK, Singh NDP (2010) 1-Acetylpyrene with
dual functions as an environment-sensitive fluorophore and
fluorescent photoremovable protecting group. Tetrahedron
66:9798–9807
Jesberger M, Davis TP, Barner L (2003) Applications of Lawesson’s
reagent in organic and organometallic syntheses. Synthesis
13:1929–1958
Pelliccioli AP, Wirz J (2002) Photoremovable protecting groups:
reaction mechanisms and applications. Photochem Photobiol Sci
1:441–458
Peng L, Wirz J, Goeldner M (1997) 2-Nitrobenzyl quaternary
ammonium derivatives photoreleasing nor-butyrylcholine in the
microsecond time range. Tetrahedron Lett 38:2961–2964
Pillai VNR (1980) Photoremovable protecting groups in organic
synthesis. Synthesis 9:1–26
Piloto AM, Rovira D, Costa SPG, Gonc¸alves MST (2006)
Oxobenzo[f]benzopyrans as new fluorescent photolabile protecting
groups for the carboxylic function. Tetrahedron 62:11955–11962
Schaper K, Mobarekah SAM, Grewer C (2002) Synthesis and
photophysical characterization of a new, highly hydrophilic
caging group. Eur J Org Chem 2002:1037–1046
Singh AK, Khade PK (2005a) 3-Nitro-2-naphthalenemethanol: a
photocleavable protecting group for carboxylic acids. Tetrahe-
dron 61:10007–10012
Li YM, Shi J, Caia R, Chen XY, Guo QX, Liu L (2010a)
Development of new quinoline-based photo-labile groups for
photo-regulation of bioactive molecules. Tetrahedron Lett
51:1609–1612
Li YM, Shi J, Caia R, Chen XY, Luo ZF, Guo QX (2010b) New
quinoline-based caging groups synthesized for photo-regulation
Singh AK, Khade PK (2005b) Anthracene-9-methanol—a novel
fluorescent phototrigger for biomolecular caging. Tetrahedron
Lett 46:5563–5566
of aptamer activity.
211:129–134
J Photochem Photobiol A Chem
Soares AMS, Costa SPG, Gonc¸alves MST (2010a) Oxazole light
triggered protecting groups: synthesis and photolysis of fused
heteroaromatic conjugates. Tetrahedron 66:8189–8195
Soares AMS, Costa SPG, Gonc¸alves MST (2010b) 2-Oxo-2H-
benzo[h]benzopyran as a new light sensitive protecting group
for neurotransmitter amino acids. Amino Acids 39:121–133
Wang P, Hu H, Wang Y (2007) Novel photolabile protecting group
for carbonyl compounds. Org Lett 9:1533–1535
Yu J-Y, Tang W-J, Wang H-B, Song Q-H (2007) Anthraquinon-2-
ylethyl-1’, 2’-diol (Aqe-diol) as a new photolabile protecting
group for aldehydes and ketones. J Photochem Photobiol A
Chem 185:101–105
Loudwig S, Goeldner M (2001) N-Methyl-N-(o-nitrophenyl)carba-
mates as photolabile alcohol protecting groups. Tetrahedron Lett
42:7957–7959
Ma C, Steinmetz MG, Kopatz EJ, Rathore R (2005) Photochemical
cleavage and release of carboxylic acids from a-keto amides.
J Org Chem 70:4431–4442
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
´
Muller C, Even P, Viriot M-L, Carre M-C (2001) Protection and
labeling of thymidine by a fluorescent photolabile group. Helv
Chim Acta 84:3735–3741
123