A photoactivable amino acid based on a novel functional coumarin-6-yl-Alanine

Article abstract of DOI:10.1007/s00726-012-1310-2

A novel fluorescent amino acid, L-4-chloromethylcoumarin-6-yl-Alanine, was obtained from tyrosine by a Pechmann reaction. The assembly of the heterocyclic ring at the tyrosine side chain could be achieved before or after incorporation of tyrosine into a d

Full text of DOI:10.1007/s00726-012-1310-2

Amino Acids (2012) 43:2329–2338
DOI 10.1007/s00726-012-1310-2
ORIGINAL ARTICLE
A photoactivable amino acid based on a novel functional
coumarin-6-yl-alanine
•
Andrea S. C. Fonseca M. Sameiro T. Gonc¸alves
•
Susana P. G. Costa
Received: 18 January 2012 / Accepted: 20 April 2012 / Published online: 9 May 2012
Ó Springer-Verlag 2012
Abstract A novel fluorescent amino acid, L-4-chlor-
omethylcoumarin-6-yl-alanine, was obtained from tyrosine
by a Pechmann reaction. The assembly of the heterocyclic
ring at the tyrosine side chain could be achieved before or
after incorporation of tyrosine into a dipeptide, and amino
acid and dipeptide ester conjugates were obtained by
coupling to a model N-protected alanine. The behaviour of
one of the fluorescent conjugates towards irradiation was
studied in a photochemical reactor at different wavelengths
(254, 300, 350 and 419 nm). The photoreaction course in
methanol/HEPES buffer solution (80:20) was followed by
HPLC/UV monitoring. It was found that the novel unnat-
ural amino acid could act as a fluorescent label, due to its
fluorescence properties, and, more importantly, as a
photoactivable unit, due to the short irradiation times
necessary to cleave the ester bond between the model
amino acid and the coumarin-6-yl-alanine.
construction of chromophore-labelled peptides and pro-
teins, which can be easily studied by fluorescence spec-
troscopy-based techniques (Katritzky and Narindoshvili
2009). Moreover, if an extra functional group is introduced
into the amino acid residue, it can also be used for coupling
purposes. This feature is quite appealing when a strategy
for peptide conformation restriction (for example, in the
synthesis of cyclic peptides or peptidomimetics) and cross-
linking is to be considered. Using certain photoactive
groups that are cleaved by the action of light (Guillier et al.
2000; Corrie et al. 2005; Mayer and Heckel 2006), the
ability to remove the constriction by photolysis may be an
interesting feature. Photochemical cleavage is widely used
to obtain control over the availability of specific molecules
in areas of research such as solution and solid-phase
organic synthesis, biochemical and biophysical research
(for e.g. in caging strategies). Such a methodology has
obvious advantages for not requiring additional chemical
reagents, thus being compatible with acid or base sensitive
groups, and for providing spatial and temporal resolution of
the cleavage process. Reports have been published with
examples such as the photoregulation of thrombin aptamer
activity (Li et al. 2009), the photocontrol of helix content in
short peptides (Kumita et al. 2000), and in the light-trig-
gered assembly of protein complexes (Grunwald et al.
2010).
Keywords Coumarin-6-yl-alanine ꢀ Coumarin ꢀ
Fluorescence ꢀ Amino acid conjugates ꢀ Photolysis
Introduction
The development of functional amino acid analogues is an
area of expanding interest due to their potential use as
intrinsic and extrinsic probes in peptide and protein con-
formational studies and in bioactivity and pharmacological
research. The design of fluorescent amino acids allows the
Coumarin derivatives have been reported as photo-
cleavable protecting groups for molecules bearing different
functional groups (Hagen et al. 2005; Geissler et al. 2005;
Lin and Lawrence 2002; Shembekar et al. 2007; Gilbert
et al. 2007) and as coumarins exhibit fluorescence, they may
also act as fluorescent labels, enabling the direct monitoring
of processes during synthesis, or the visualisation of mol-
ecules inside living cells as new extrinsic and intrinsic
fluorescent probes for biologically active molecules.
A. S. C. Fonseca ꢀ M. S. T. Gonc¸alves ꢀ S. P. G. Costa (&)
´
Centro de Quımica, Universidade do Minho, Campus de Gualtar,
4710-057 Braga, Portugal
e-mail: spc@quimica.uminho.pt
123

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A. S. C. Fonseca et al.
The synthesis of coumarinyl-alanines has been reported
were obtained on a Varian Unity Plus Spectrometer at an
1
earlier by diastereoselective alkylation of chiral glycine
equivalents, using different chiral auxiliaries and in mul-
tiple step synthesis with low global yields (Bennett et al.
1997; Kele et al. 2000) or using protected aspartic and
glutamic acids as chiral starting materials, through a
Pechmann condensation reaction between a b-ketoester
intermediate with phenol derivatives, in fair yields (Brun
et al. 2004).
operating frequency of 300 MHz for H and 75.4 MHz for
13C or a Bruker Avance III 400 at an operating frequency
of 400 MHz for 1H and 100.6 MHz for 13C using the
solvent peak as internal reference at 25 °C. All chemical
shifts are given in ppm using dH Me4Si = 0 ppm as ref-
erence 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 bidimensional heteronuclear correlation
techniques. Low and high resolution 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. Photolyses were carried out using a
Rayonet RPR-100 chamber reactor equipped with 10 lamps
of 254 (35 W), 300 (21 W), 350 (24 W) and 419 (14 W)
nm. HPLC analyses were performed using a Licrospher
100 RP18 (5 lm) column in a HPLC system composed by
a Jasco PU-980 pump, a Shimadzu SPD-GAV UV/Vis
detector and a Shimadzu C-RGA Chromatopac register. All
commercial reagents were used as received. Protected
tyrosine 1a,b and alanine 4a–c derivatives were obtained
by standard amino acid protecting group protocols. The
L-(4-methylene)coumarin-6-yl-alaninyl group will be des-
ignated by the three letter code Mca in the NMR spectra
description.
Taking these facts into consideration, in connection with
our current research interests in the development of new
oxygen and nitrogen heterocycles and their applications as
fluorescent labels and photoreleasable protecting groups
(Piloto et al. 2006; Fonseca et al. 2007, 2010; Fernandes
et al. 2008; Soares et al. 2010a, b), we now report the
synthesis of a functional alanine derivative bearing a
fluorescent coumarin moiety at its side chain, namely
N-acetyl-4-chloromethylcoumarin-6-yl-alanine methyl ester.
The incorporation of a fluorescent heterocycle such as cou-
marin in the amino acid core results in an advantageous fea-
ture for fluorescence-based biological studies and the
molecule shows high chemical stability due to the fact that the
fluorophore is linked by a side-chain carbon–carbon bond. In
addition, given a proper choice of protecting groups that allow
the sequence of its assembly and after deprotection, such an
amino acid has the potential to be incorporated into peptidic
structures by standard coupling procedures through its N- and
C-termini, yielding a functional fluorescent peptide and the
presence of a reactive chloromethyl group at the coumarin
moiety provides an extra coupling site, useful for constriction
strategies.
Synthesis of compounds 3 and 5–11
N-acetyl-L-(4-chloromethyl)coumarin-6-yl-alanine methyl
ester 3
Moreover, in order to evaluate the potential application
of this new amino acid in photoactivable processes, we
decided to synthesize model ester conjugates by reaction
with the carboxylic terminal of N-protected alanine, with
the aim of undertaking a study of the photostability of the
newly formed ester linkage to irradiation at different
wavelengths.
N-acetyl-L-tyrosine methyl ester 1a was dissolved (1 equiv,
0.450 g, 1.90 9 10-3 mol) in aqueous 70 % sulphuric acid
(5 mL), and ethyl 4-chloro-3-oxobutanoate 2 (1.5 equiv,
0.39 mL, 2.85 9 10-3 mol) was added to the mixture. The
reaction mixture was stirred at room temperature for 7 days,
poured into ice, and the precipitate was filtered and dried in
a vacuum oven. The resulting residue was purified by silica
gel column chromatography using chloroform/methanol
(100:1) as eluent. Fractions containing the product were
combined and evaporated, yielding compound 3 as a beige
solid (0.350 g, 55 %); mp = 172.3–173.9 °C; 1H NMR
(400 MHz, DMSO-d6): d = 1.78 (3H, s, CH3 Ac), 2.95
(1H, dd, J 9.6 and 4.4 Hz, b-CH2), 3.11 (1H, dd, J 13.6 and
5.2 Hz, b-CH2), 3.61 (3H, s, OCH3), 4.44–4.52 (1H, m,
a-H), 4.99 (2H, s, CH2), 6.67 (1H, s, H-3), 7.37 (1H, d,
J 8.4 Hz, H-8), 7.51 (1H, dd, J 8.4 and 2.0 Hz, H-7), 7.72
(1H, d, J 2.0 Hz, H-5), 8.35 (1H, d, J 7.6 Hz, NH); 13C
NMR (100.6 MHz, DMSO-d6): d = 22.24 (CH3 Ac), 35.95
(b-CH2), 41.13 (CH2), 51.92 (OCH3), 53.47 (a-C), 115.56
(C-3), 116.62 (C-8), 116.74 (C-4a), 125.58 (C-5), 133.33
Experimental section
General
All melting points were measured on a Stuart SMP3
melting point apparatus. 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 deter-
mined on a BOMEM MB 104 spectrophotometer. UV/Vis
absorption spectra (200–700 nm) were obtained using a
Shimadzu UV/2501PC spectrophotometer. NMR spectra
123

A photoactivable amino acid based on a novel functional coumarin-6-yl-alanine
2331
(C-7), 133.67 (C-6), 150.49 (C-4), 152.15 (C-8a), 159.62
(C-2), 169.40 (C=O amide), 171.95 (C=O ester); FT-IR
(KBr 1 %, cm-1): m = 3,289, 3,080, 2,961, 2,927, 2,855,
1,732, 1,646, 1,572, 1,541, 1,378, 1,357, 1,210, 1,173,
1,135, 1,057, 984, 961, 894, 736; UV/Vis (ethanol, nm):
Compound 6 was obtained as beige oil (0.075 g, 83 %); 1H
NMR (400 MHz, DMSO-d6): d = 1.76 (3H, s, CH3 Ac),
2.88–3.13 (2H, m, b-CH2), 3.64 (2H, s, CH2), 4.35–4.41 (1H,
m, a-H), 7.17 (1H, dd, J 8.4 and 1.6 Hz, H-6), 7.42 (1H, d,
J 0.8 Hz, H-4), 7.44 (1H, d, J 8.4 Hz, H-7), 7.83 (1H, s, H-2),
8.17 (1H, d, J 8.4 Hz, NH); 13C NMR (100.6 MHz, DMSO-
d6): d = 22.37 (CH3 Ac), 28.99 (CH2), 36.75 (b-CH2), 54.13
(a-C), 110.88 (C-7), 113.87 (C-3), 120.26 (C-4), 125.66
(C-6), 127.80 (C-3a), 132.08 (C-5), 143.71 (C-2), 153.52
(C-7a), 169.43 (C=O amide), 171.92 (C=O acid), 173.23
(C=O acid); FT-IR (KBr 1 %, cm-1): m = 3,301, 2,928,
1,723, 1,651, 1,615, 1,557, 1,539, 1,475, 1,446, 1,379, 1,338,
1,279, 1,256, 1,230, 1,217, 1,187, 1,161, 1,124, 1,091, 1,023,
971, 954, 943, 884, 871, 752, 508; UV/Vis (ethanol, nm):
k
max (log e) = 320 (3.67); MS: m/z (ESI) 340 (M??1 37Cl,
29), 338 (M??1 35Cl, 100), HRMS: m/z (ESI) calc. for
C16H17NO357Cl 340.07603, found 340.07593; calc. for
C16H17NO355Cl 338.07898, found 338.07882.
Coumarin-6-yl-alanine ester conjugate 5
Compound 3 (1 equiv, 0.050 g, 1.48 9 10-4 mol) was dis-
solved in dry N,N-dimethylformamide (3 mL). N-t-Butyl-
oxycarbonyl)-L-alanine 4a (1 equiv, 0.025 g, 1.48 9 10-4
mol) and potassium fluoride (3 equiv, 0.028 g, 4.45 9 10-4
mol) were added to the mixture. The reaction mixture was
stirred at room temperature for 3 days. The mixture was
filtered and the solvent was removed by rotary evaporation.
The crude solid was recrystallized from methanol and diethyl
ether and conjugate 5 was obtained as a light yellow solid
(0.067 g, 95 %); mp = 174.0–175.3 °C; 1H NMR
(400 MHz, DMSO-d6): d = 1.31 (3H, d, J 7.5 Hz, b-CH3
Ala), 1.38 (9H, s, (C(CH3)3), 1.77 (3H, s, CH3 Ac), 2.92 (1H,
dd, J 9.6 and 3.9 Hz, b-CH2 Mca), 3.12 (1H, dd, J 14.1 and
5.4 Hz, b-CH2 Mca), 3.61 (3H, s, OCH3), 4.17–4.02 (1H, m,
a-H Ala), 4.48–4.53 (1H, m, a-H Mca), 5.42 (2H, s, CH2),
6.48 (1H, s, H-3), 7.36 (1H, d, J 8.7 Hz, H-8), 7.48–7.50 (2H,
m, H-7 and NH Ala), 7.62 (1H, d, J 1.6 Hz, H-5), 8.35 (1H, d,
J 7.8 Hz, NH Mca); 13C NMR (100.6 MHz, DMSO-d6):
d = 16.69 (b-CH3 Ala), 22.19 (CH3 Ac), 28.11 (C(CH3)3)
35.84 (b-CH2 Mca), 49.15 (a-C Ala), 51.87 (OCH3), 53.39
(a-C Mca), 61.39 (CH2), 78.41 (C(CH3)3), 111.75 (C-3),
116.31 (C-4a), 116.43 (C-8), 125.08 (C-5), 133.16 (C-7),
133.69 (C-6), 149.98 (C-4), 151.73 (C-8a), 155.44 (C=O
urethane), 159.50 (C-2), 169.36 (C=O amide), 172.00 (C=O
methyl ester), 172.64 (C=O ester); FT-IR (KBr 1 %, cm-1):
m = 3,315, 3,295, 3,084, 2,996, 2,985, 2,938, 1,755, 1,731,
1,688, 1,666, 1,575, 1,551, 1,534, 1,436, 1,372, 1,350, 1,316,
1,287, 1,235, 1,174, 1,160, 1,133, 1,070, 1,013, 965, 834;
UV/Vis (ethanol, nm): kmax (log e) = 318 (3.58); MS:
m/z (ESI) 491 (M??1, 27); HRMS: m/z (ESI) calc. for
C24H31N2O9 491.20241, found 491.20289.
k
HRMS: m/z (ESI) calc. for C15H16NO6 306.09721, found
306.09697.
max (log e) = 314 (3.72); MS: m/z (ESI) 306 (M??1, 35);
N-acetyl-L-alaninyl-L-tyrosine methyl ester 7
N-acetyl-L-alanine 4b (1 equiv, 0.200 g, 1.53 9 10-3 mol)
was dissolved in dry N,N-dimethylformamide (3 mL).
Anhydrous 1-hydroxybenzotriazole (1.1 equiv, 0.206 g,
1.68 9 10-3 mol), N,N0-dicyclohexylcarbodiimide (1 equiv,
0.315 g, 1.53 9 10-3 mol) and L-tyrosine methyl ester 1b (1
equiv, 0.354 g, 1.53 9 10-3 mol), were added to the mixture
with intervals of 10 min. The reaction mixture was stirred at
room temperature for 1 day. The mixture was filtered and the
solvent was removed by rotary evaporation. The crude res-
idue was dissolved in acetone and left in the freezer for some
days. The mixture was filtered and the solvent was removed
by rotary evaporation. The solid mixture was used in the next
1
reaction without further purification. H NMR (300 MHz,
DMSO-d6): d = 1.11–1.14 (3H, m, b-CH3 Ala), 1.79 (3H, s,
CH3 Ac), 3.80–3.20 (2H, m, b-CH2 Tyr), 3.56 (3H, s, OCH3),
4.20–4.40 (2H, m, a-H Ala and Tyr), 6.64 (2H, d, J 8.4 Hz,
H-3 and H-5), 6.97 (2H, d, J 8.4 Hz, H-2 and H-6), 7.97 (1H,
d, J 8.1 Hz, NH Ala), 8.17 (1H, d, J 7.2 Hz, NH Tyr), 9.20
(1H, broad s, OH).
N-acetyl-L-alaninyl-L-(4-chloromethyl)-coumarin-6-yl-
alanine methyl ester 8
Compound 7 (1 equiv, 0.470 g, 1.53 9 10-3 mol) was
dissolved in aqueous 70 % sulphuric acid (5 mL) and ethyl
4-chloro-3-oxobutanoate 2 (2 equiv, 0.45 mL, 3.05 9 10-3
mol) was added to the mixture. The reaction mixture was
stirred at room temperature for 3 weeks, poured into ice,
and the precipitate formed was filtered and dried in a vac-
uum oven. Recrystallization from methanol and diethyl
ether afforded dipeptide 8 as a brown solid (0.100 g, 16 %);
mp = 182.0–183.4 °C; 1H NMR (400 MHz, MeOH-d4):
N-acetyl-L-(3-carboxymethyl)benzofuran-5-yl-alanine 6
Compound 3 (0.100 g, 2.96 9 10-4 mol) was dissolved in
1 M NaOH (5 mL) and the mixture was stirred by heating at
reflux for 17 h. The reaction mixture was acidified with a few
drops of 6 M HCl, and the aqueous phase was extracted with
ethyl acetate (3 9 10 mL). The organic layers were com-
bined and the solvent was removed by rotary evaporation.
123

2332
A. S. C. Fonseca et al.
d = 1.31 (3H, d, J 7.2 Hz, b-CH3 Ala), 1.94 (3H, s, CH3
Ac), 3.10–3.32 (2H, m, b-CH2 Mca), 4.30–4.35 (1H, m, a-H
Ala), 4.77–4.81 (1H, m, a-H Mca), 4.96 (2H, s, CH2), 6.64
(1H, s, H-3), 7.34 (1H, d, J 8.8 Hz, H-8), 7.52 (1H, dd, J 8.8
and 2.0 Hz, H-7), 7.70 (1H, d, J 1.6 Hz, H-5); 13C NMR
(100.6 MHz, MeOH-d4): d = 17.84 (b-CH3 Ala), 22.21
(CH3 Ac), 37.65 (b-CH2 Mca), 42.15 (CH2), 50.36 (a-C
Ala), 52.90 (OCH3), 54.71 (a-H Mca), 116.11 (C-3), 118.07
(C-8), 118.59 (C-4a), 126.51 (C-5), 134.74 (C-7), 134.91
(C-6), 152.68 (C-4), 154.04 (C-8a), 162.39 (C-2), 172.87
(C=O ester), 173.07 (C=O amide Ac), 175.05 (C=O amide);
FT-IR (KBr 1 %, cm-1): m = 3,288, 3,084, 2,937, 1,737,
1,648, 1,572, 1,550, 1,493, 1,439, 1,384, 1,352, 1,285,
1,251, 1,176, 1,058, 960, 907, 830, 735, 709, 661, 581, 514,
523; UV/vis (ethanol, nm): kmax (log e): 320 (3.62) MS:
m/z (ESI): 411 (M??1 37Cl, 23), 409 (M??1 35Cl, 70);
HRMS: m/z (ESI) calc. for C19H22N2O6 37Cl 411.11341,
found 411.11377; calc. for C19H22N2O6 35Cl 409.11609,
found 409.11589.
HRMS: m/z (ESI) calc. for C24H30N3O9 504.19766 found
504.19663.
N-acetyl-L-tyrosinyl-L-alanine methyl ester 10
N-acetyl-L-tyrosine 1c (0.300 g, 1.34 9 10-3 mol) was
dissolved in dry N,N-dimethylformamide (3 mL). Anhy-
drous 1-hydroxybenzotriazole (1.1 equiv, 0.200 g,
1.48 9 10-3 mol), N,N0-dicyclohexylcarbodiimide (1
equiv, 0.277 g, 1.34 9 10-3 mol) and L-alanine methyl ester
4c (1 equiv, 0.188 g, 1.34 9 10-3 mol) were added to the
mixture with intervals of 10 min. The reaction mixture was
stirred at room temperature for 2 days. The mixture was
filtered and the solvent was removed by rotary evaporation.
The crude residue was dissolved in acetone and left in the
freezer for some days. The first precipitate formed was fil-
tered and the solvent volume reduced. A new precipitate was
collected and dipeptide 10 was obtained as a white solid
(0.233 g, 56 %); mp = 100.3–102.2 °C; 1H NMR
(300 MHz, DMSO-d6): d = 1.27 (3H, d, J 7.2 Hz, b-CH3
Ala), 1.73 (3H, s, CH3 Ac), 2.53–2.89 (2H, m, b-CH2 Tyr),
3.60 (3H, s, OCH3), 4.21–4.30 (1H, m, a-H Ala), 4.38–4.46
(1H, m, a-H Tyr), 6.63 (2H, d, J 8.4 Hz, H-3 and H-5), 7.03
(2H, d, J 8.1 Hz, H-2 and H-6), 7.99 (1H, d, J 8.7 Hz, NH
Tyr), 8.42 (1H, d, J 6.9 Hz, NH Ala), 9.18 (1H, broad s, OH);
13C NMR (75.4 MHz, DMSO-d6): 16.86 (b-CH3 Ala), 22.46
(CH3 Ac), 36.86 (b-CH2 Tyr), 47.53 (a-C Ala), 51.85
(OCH3), 53.91 (a-C Tyr), 114.80 (C-3 and C-5), 128.02
(C-4), 130.04 (C-2 and C-6), 155.72 (C-1), 169.03 (C=O
amide Ac), 171.60 (C=O amide), 172.94 (C=O ester); FT-IR
(KBr 1 %, cm-1): m = 3,538, 3,342, 3,271, 3,074, 2,978,
2,928, 1,717, 1,676, 1,652, 1,600, 1,557, 1,519, 1,457, 1,435,
1,374, 1,298, 1,281, 1,245, 1,187, 1,162, 1,107, 1,057, 981,
960, 925, 872, 845, 832, 814, 756, 723, 707; UV/Vis (etha-
nol, nm): kmax (log e) = 303 (3.17); MS: m/z (ESI) 309
(M??1, 100); HRMS: m/z (ESI) calc. for C15H21N2O6
309.14450, found 309.14360.
Alaninyl-coumarin-6-yl-alanine ester conjugate 9
Dipeptide 8 (1 equiv, 0.032 g, 7.83 9 10-4 mol) was
dissolved in dry N,N-dimethylformamide (3 mL). N-
acetyl-L-alanine 4b (1 equiv, 0.011 g, 7.83 9 10-4 mol)
and potassium fluoride (3 equiv, 0.014 g, 2.35 9 10-3
mol) were added to the mixture. The reaction mixture was
stirred at room temperature for 2 days. The mixture was
filtered and the solvent was removed by rotary evapora-
tion. Recrystallization from methanol and diethyl ether
resulted in dipeptide conjugate 9 as a beige solid (0.025 g,
63 %); 1H NMR (400 MHz, DMSO-d6): d = 1.10–1.13
(3H, m, b-CH3 Ala), 1.33–1.35 (3H, m, b-CH3 Ala), 1.74
(3H, s, CH3 Ac), 1.85 (3H, s, CH3 Ac), 2.96–3.16 (2H, m,
b-CH2 Mca), 3.61 (3H, s, OCH3), 4.22–4.28 (1H, m, a-H
Ala), 3.34–4.41 (1H, m, a-H Ala), 4.48–4.57 (1H, m, a-H
Mca), 5.37–5.47 (2H, m, CH2), 6.44 (1H, s, H-3), 7.33
(1H, d, J 8.4 Hz, H-8), 7.48–7.51 (1H, m, H-7), 7.60 (1H,
d, J 1.6 Hz, H-5), 7.92 (1H, d, J 7.6 Hz, NH Ala), 8.29
(1H, d, J 8.0 Hz, NH Mca), 8.43 (1H, d, J 6.8 Hz, NH
Ala); 13C NMR (100.6 MHz, DMSO-d6): d = 16.71
(b-CH3 Ala), 17.96 (b-CH3 Ala), 22.11 (CH3 Ac), 22.30
(CH3 Ac), 35.76 (b-CH2 Mca), 47.71 (a-C Ala), 47.85
(a-C Ala), 51.93 (OCH3), 53.15 (a-C Mca), 61.49 (CH2),
111.90 (C-3), 116.39 (C-8), 125.12 (C-5), 133.27 (C-7),
133.53 (C-6), 150.14 (C-4), 151.75 (C-4a), 159.29 (C-2),
162.29 (C-8a), 168.84 (C=O ester), 169.47 (C=O amide
Ac), 172.27 (C=O amide Ac), 172.60 (C=O ester), 174.26
(C=O amide); FT-IR (KBr 1 %, cm-1): m = 3,290, 3,068,
2,984, 2,934, 1,726, 1,654, 1,634, 1,574, 1,536, 1,445,
1,375, 1,305, 1,264, 1,208, 1,159, 1,042, 1,014, 972, 940,
876, 835, 754, 732, 665, 514; UV/Vis (ethanol, nm): kmax
(log e) = 319 (3.51); MS: m/z (ESI) 504 (M??1, 100);
N-acetyl-L-(4-chloromethyl)-coumarin-6-yl-alaninyl-L-
alanine methyl ester 11
Dipeptide 10 (1 equiv, 0.300 g, 9.73 9 10-4 mol) was
dissolved in aqueous 70 % sulphuric acid (5 mL) and ethyl
4-chloro-3-oxobutanoate 2 (1.5 equiv, 0.2 mL, 1.46 9
10-3 mol) was added to the mixture. The reaction mixture
was stirred at room temperature for 3 weeks, poured into
ice, and the precipitate formed was filtered and dried in a
vacuum oven. Dipeptide 11 was obtained as a dark oil
(0.150 g, 26 %); 1H NMR (400 MHz, MeOH-d4): 1.57
(3H, d, J 7.2 Hz, b-CH3 Ala), 2.01 (3H, s, CH3 Ac),
3.13–3.33 (2H, m, b-CH2 Mca), 3.70 (3H, OCH3), 4.07
(1H, m, a-H Ala), 4.40 (1H, m, a-H Mca), 4.95 (2H, s,
CH2), 6.63 (1H, s, H-3), 7.54 (1H, d, J 8.4 Hz, H-8), 7.58
123

A photoactivable amino acid based on a novel functional coumarin-6-yl-alanine
2333
(1H, dd, J 8.4 and 2.0 Hz, H-7), 7.75 (1H, d, J 2.0 Hz,
H-5); 13C NMR (100.6 MHz, MeOH-d4): 16.24 (b-CH3
Ala), 21.95 (CH3 Ac), 37.62 (b-CH2 Mca), 42.08 (CH2),
54.20 (OCH3), 55.22 (a-C Mca), 55.96 (a-C Ala), 116.08
(C-3), 117.96 (C-8), 118.50 (C-4a), 126.50 (C-5), 134.50
(C-7), 152.44 (C-8a), 152.65 (C-4), 153.99 (C-6), 162.34
(C-2), 171.40 (C=O ester), 172.29 (C=O amide), 173.88
(C=O amide Ac); UV/Vis (ethanol, nm): kmax (log
e) = 312 (3.28); MS: m/z (ESI) 411 (M??1 37Cl, 7), 409
distilled water with HEPES [4-(2-hydroxyethyl)-1-pipera-
zine ethanesulfonic acid] (10 mM), sodium chloride
(120 mM), potassium chloride (3 mM), calcium chloride
(1 mM) and magnesium chloride (1 mM) and the pH
adjusted to 7.2.
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 0.4 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 absorption
(318 nm) and the retention time of conjugate 5 was 6.2 min.
(M??1 35Cl, 19); HRMS: m/z (ESI) calc. for C19H
37
21
35
21
ClN2O6 411.11341, found 411.11384; calc. for C19H
ClN2O6 409.11609, found 409.11700.
General photolysis procedure
Results and discussion
A 1 9 10-4 M methanol/HEPES buffer (80:20) solution of
conjugate 5 (5 mL) was 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. HEPES buffer solution was prepared in
Synthesis
N-acetyl-L-(4-chloromethyl)coumarin-6-yl-alanine methyl
ester 3 was prepared in moderate yield by reaction of
Table 1 Synthesis, UV/Vis absorption and emission data for coumarin-6-yl-alanine 3, its derivatives 5, 8, 9, 11, and for benzofuran-5-yl-alanine
6, in absolute ethanol
Compound
Yield (%)
Absorption
Emission
kmax (nm)
log e
kem (nm)
/
F
Stokes’ shift (nm)
3
55
95
83
16
63
26
320
318
314
320
319
312
3.67
3.58
3.72
3.62
3.51
3.28
378
417
386
389
432
372
0.05
0.08
0.08
0.06
0.07
0.06
58
99
5
6
72
8
69
9
113
60
11
O
H
N
O
O
O
O
O
Boc-Ala-OH 4a
O
KF, DMF
O
5
N
H
O
Cl
O
2
O
OH
O
O
Cl
O
O
O
HO
O
70% H2SO4
O
O
N
H
N
H
O
O
O
1a
3
1M NaOH
reflux
O
OH
6
N
H
O
Scheme 1 Synthesis of coumarin-6-yl-alanine 3, its ester conjugate 5 and benzofuran-5-yl-alanine derivative 6
123

2334
A. S. C. Fonseca et al.
N-acetyl-L-tyrosine methyl ester 1a and ethyl 4-chloro-3-
oxobutanoate 2, through a Pechmann reaction, in the
presence of aqueous 70 % sulphuric acid (Fonseca et al.
2010). No evidence was found for the loss of the integrity
of the chiral centre in these reaction conditions. The
structure of the novel amino acid 3 was confirmed by the
signals as multiplets for a-H at d 4.48–4.53 (coumarin-6-yl-
alanine) and at 4.15–4.22 ppm (for the alanine residue), as
well as the characteristic protons of the coumarin ring,
namely H-3 (d 6.48 ppm), H-5 (d 7.62 ppm), H-7 (d
7.50 ppm) and H-8 (d 7.36 ppm), as and its methylene
group at position 4 (d 5.42 ppm). The confirmation of the
presence of the newly formed ester bond was also sup-
ported by the 13C NMR spectrum signal of the carbonyl
group, which was found at d 172.64 ppm.
1
usual spectroscopic techniques, with the H NMR signals
of the coumarin aromatic protons at d 6.67 ppm (singlet for
H-3), d 7.72 ppm (doublet for H-5), d 7.51 ppm (double
doublet for H-7) and d 7.37 ppm (doublet for H-8), as well
as its methylene group at position 4 (singlet at d 4.99 ppm).
The confirmation of the assembly of the coumarin ring at
the amino acid side chain was also possible by the 13C
NMR spectrum signal at d 159.62 ppm corresponding to
the carbonyl group at position 2.
Treatment of coumarin-6-yl-alanine 3 with base yielded
benzofuran-5-yl-alanine derivative 6, accordingly to a
previously reported alkaline ring contraction of a coumarin
bearing a chloromethyl group (Piloto et al. 2006). Its
structure was confirmed by the usual spectroscopic tech-
1
niques, such as the H NMR signals of the benzofuran
Derivatization at the C-terminus of N-t-butyloxycar-
bonyl-L-alanine 4a with the chloromethyl group of
coumarin-6-yl-alanine 3 was carried out in N,N-dimethyl-
formamide, at room temperature, in the presence of
potassium fluoride (Fonseca et al. 2010), yielding fluores-
cent ester conjugate 5 in excellent yield (Table 1;
aromatic protons that appeared at d 7.17 ppm (double
doublet for H-6), d 7.42 ppm (doublet for H-4), d 7.44 ppm
(doublet for H-7) and d 7.83 ppm (singlet for H-2). It was
possible to react both carboxylic acid groups present in the
molecule with L-alanine methyl ester, but the lack of
selectivity in this reaction prevented further application of
the novel benzofuranyl amino acid 6.
1
Scheme 1). The H NMR spectra of conjugate 5 showed
Scheme 2 Synthesis of model dipeptides 8, 9 and 11, bearing a coumarin-6-yl-alanine by post-coupling assembly of the coumarin ring
123

A photoactivable amino acid based on a novel functional coumarin-6-yl-alanine
2335
Having proven the possibility of assembling the cou-
marin ring by modification at the side chain of free tyro-
sine, the same idea was envisaged for tyrosine in a
dipeptide, through a post-coupling assembly of the
coumarin ring. Thus, L-tyrosine methyl ester 1b or
N-acetyl-L-tyrosine 1c was coupled to N-acetyl-L-alanine
4b or L-alanine methyl ester 4c, by a standard cou-
pling protocol with N,N0-dicyclohexylcarbodiimide and
1-hydroxybenzotriazole in N,N-dimethylformamide, yield-
ing the corresponding model dipeptides 7 and 10. These
compounds, in the presence of ethyl 4-chloro-3-oxobut-
anoate 2, in the same reaction conditions as mentioned
earlier, afforded the dipeptides bearing a chloromethylated
coumarin-6-yl-alanine at the C- and N-terminus, dipeptides
8 and 11, respectively (Scheme 2). The low isolated yields
of these compounds can be explained by the relatively
harsh reaction conditions that cause loss of the N-and
C-termini blocking groups, leading to deprotected by-
products. For the alaninyl–coumarinyl–alaninyl dipeptide
8, further reaction at the chloromethyl group was carried
out with N-acetyl-L-alanine 4b and potassium fluoride in
N,N-dimethylformamide, as previously mentioned. The
dipeptide ester conjugate 9 was obtained in good yield
(63 %) (Scheme 2). All the dipeptides were fully charac-
terised by the usual spectroscopic techniques and the
obtained data were in accordance with that obtained for the
free coumarin-6-yl-alanine.
Coumarin-6-yl-alanine 3, its ester conjugates 5 and 9
and the dipeptides 8 and 11 displayed fair fluorescence
quantum yields. The fact that the absorption and emission
maxima for coumarin-6-yl-alanine 3 and its derivatives 5,
8, 9 and 11 occurs at longer wavelengths than those of the
natural amino acids that exhibit fluorescence, namely
tryptophan (kmax = 278 nm, kem = 352 nm), tyrosine
(kmax = 274 nm, kem = 303 nm) and phenylalanine
(kmax = 257 nm, kem = 282 nm), confirms that coumarin-
6-yl-alanine 3 could be used as an intrinsic fluorescent
label when incorporated into biologically active peptides
and proteins. All compounds displayed large Stokes’ shifts
(58–113 nm), being dipeptide ester conjugate 9 associated
with the largest shift (113 nm), which is an advantageous
property in fluorescence techniques as it will minimise self-
quenching phenomena. From the data in Table 1, it can be
seen that upon reaction of the chloromethyl group to yield
the ester linkage to the amino acid in conjugates 5 and 9, a
40 nm bathochromic shift of the emission band occurred,
when compared to 3 and 8, respectively (Fig. 1).
Photolysis studies of coumarin-6-yl-alanine ester
conjugate 5
One of the goals of this work was to assess the photosta-
bility of the linkage between coumarin-6-yl-alanine and
alanine, as a model amino acid, in ester conjugates, having
in mind a possible application of the novel amino acid in
photoactivable processes for biological studies. Therefore,
photolysis studies were carried out by irradiating solutions
of conjugate 5 in methanol/HEPES buffer (80:20) in a
Rayonet RPR-100 reactor, at 254, 300, 350 and 419 nm.
The course of the photocleavage reaction was followed by
Coumarin and benzofuran derivatives are known for
their fluorescence properties and so the photophysical
properties of all synthesised compounds bearing a cou-
marin (3, 5, 8, 9 and 11) or a benzofuran (6) were evaluated
and the UV/Vis absorption and emission spectra of
degassed 10-5 M solutions in absolute ethanol of these
compounds were measured (Table 1). Relative fluores-
cence quantum yields were calculated using 9,10-diphen-
ylanthracene as standard (/F = 0.95 in ethanol) (Morris
et al. 1976).
Table 2 Irradiation times (tirr, in min), rate constant (k, in min-1) and
photochemical quantum yield (/phot) for the photolysis of conjugate 5
at different wavelengths in MeOH/HEPES buffer (80:20) solution
Wavelength of irradiation (nm)
254
300
350
419
tirr
k
69
4.2 9 10-3
2.3 9 10-2
79
3.7 9 10-3
1.2 9 10-2
2,910
1.0 9 10-3
0.2 9 10-2
8,542
/
phot
tirr
k
/
phot
tirr
k
/
phot
tirr
k
0.4 9 10-3
Fig. 1 Normalised fluorescence spectra of coumarin-6-yl-alanine 3
and its ester conjugate 5 in absolute ethanol (3, kexc = 320 nm; 5,
kexc = 318 nm)
/
*
phot
* Not determined
123

2336
A. S. C. Fonseca et al.
reverse phase HPLC with UV detection and 1H NMR. The
determined irradiation time represents the time necessary
for the consumption of the starting materials until\5 % of
the initial peak area (A) was detected (Table 2). 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,
with high correlation coefficients, and rate constants were
calculated.
Photochemical quantum yields (/phot) of the photoc-
leavage reaction of conjugate 5 were calculated as previ-
ously described (Muller et al. 2001) and short irradiation
times were obtained for irradiation at 254 and 300 nm.
Irradiation times at 350 and 419 nm were too long to be
useful for practical applications (Table 2). In the tested
conditions, no cleavage of the N-t-butyloxycarbonyl and
N-acetyl groups was detected. In addition to monitoring the
photolysis process by HPLC/UV detection, the release of
Boc-protected alanine, as the expected product of the
Scheme 3 Irradiation of ester conjugate 5 and the corresponding products
Fig. 2 1H NMR spectra in
methanol-d4/D2O (80:20) of the
photolysis of conjugate 5
(C = 7.0 9 10-3 M) at
300 nm: a before irradiation;
b after irradiation for 60 min;
c after irradiation for 120 min
123

A photoactivable amino acid based on a novel functional coumarin-6-yl-alanine
2337
photolysis of conjugate 5, was also followed by 1H NMR in
a methanol-d4/D2O (80:20) solution (Scheme 3).
The incorporation of this amino acid into biologically
active peptides is currently being addressed with the
preparation of cyclic peptides by taking advantage of its
interesting features, namely the possibility of intramolec-
ular crosslinking/cyclization for replacement of disulphide
bonds, the photosensitivity for the preparation of open-
chain derivatives of these peptides, and the intrinsic
labelling with an UV-active moiety, allowing the use of
fluorescence-based techniques.
Upon irradiation at 300 nm, the signals due to the ala-
nine residue in the conjugate form disappeared progres-
sively and were replaced by the corresponding set of
signals of Boc-Ala-OH 4a, with the a-CH and b-CH3
appearing at d 4.10 and 1.40 ppm, respectively. The
decrease of the benzylic-type CH2 at position 4 of the
coumarin ring was visible at d 5.50 ppm, thus confirming
the release of the amino acid. The coumarin-6-yl-alanine
b-CH2 showed a small shift to lower d. Moreover, signals
due to by-products related to the coumarin-6-yl-alanine
Acknowledgments Thanks are due to the Foundation for Science and
Technology (FCT-Portugal) for financial support through projects
PTDC/QUI/69607/2006 (FCOMP-01-0124-FEDER-007449) and PEst-
C/QUI/UI0686/2011 (F-COMP-01-0124-FEDER-022716), FEDER-
COMPETE. A PhD grant to A.S.C.F. (SFRH/BD/32664/2006) is also
acknowledged. The NMR spectrometer Bruker Avance III 400 is part of
the National NMR Network and was purchased with funds from POCI
2010 (FEDER) and FCT.
1
were also detected in the H NMR spectra (Fig. 2).
The mechanism of cleavage is believed to follow the
previously reported mechanism for this type of compounds:
after homolytical (followed by electron transfer) or het-
erolytical cleavage of the ester O–CH2 bond, the methylene
carbocation can be attacked by a nucleophile (in this case,
water or methanol) yielding by-products related to cou-
marinyl-alanine and the free amino acid, by hydrogen
abstraction from the solvent by the amino acid anionic
intermediate (Yamaji et al. 2009; Schmidt et al. 2007).
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