Light-induced cleavage of model phenylalanine conjugates based on coumarins and quinolones

Article abstract of DOI:10.1007/s00726-010-0492-8

In order to evaluate the application of quinolone as a new photocleavable protecting group, in comparison with coumarin, a series of model phenylalanine conjugates were prepared by reaction with chloromethylated O and N heterocycles. The photophysical pro

Full text of DOI:10.1007/s00726-010-0492-8

Amino Acids (2010) 39:699–712
DOI 10.1007/s00726-010-0492-8
ORIGINAL ARTICLE
Light-induced cleavage of model phenylalanine conjugates based
on coumarins and quinolones
•
Andrea S. C. Fonseca M. Sameiro T. Gonc¸alves
•
Susana P. G. Costa
Received: 24 November 2009 / Accepted: 19 January 2010 / Published online: 5 February 2010
Ó Springer-Verlag 2010
Abstract In order to evaluate the application of quino-
lone as a new photocleavable protecting group, in com-
parison with coumarin, a series of model phenylalanine
conjugates were prepared by reaction with chloromethy-
lated O and N heterocycles. The photophysical properties
of the resulting ester conjugates were evaluated as well as
the photosensitivity under irradiation at 250, 300, 350, and
419 nm. The results obtained showed that the quinolone
conjugates were readily photolysed, with complete release
of the amino acid in short irradiation times and could be
considered a new addition to the family of photocleavable
protecting groups for the carboxylic acid function of amino
acids.
photorelease process. Additionally, photochemical cleav-
age is a very mild deprotection strategy that is usually
orthogonal to chemical conditions, allowing the removal of
protecting groups in sensitive molecules, otherwise
incompatible with acidic or basic treatment. Light sensitive
groups have become an important tool in organic synthesis,
biotechnology and cell biology, for example in molecular
caging—a strategy in which a bioactive molecule is ren-
dered inactive by a covalent linkage to a photolabile group.
By irradiation of the cage, the caged molecule can be
released in its active form. These properties are appealing
to both basic and applied research fields, including solution
and solid-phase organic synthesis, combinatorial chemis-
try, photolithography, and biochemical and biophysical
research (Guillier et al. 2000; Bochet 2002; Pelliccioli and
Wirz 2002; Corrie et al. 2005; Mayer and Heckel 2006).
The concept of chromatic orthogonality (different phot-
olabile protecting groups that cleave in different wave-
lengths of irradiation) has been reported in the complete
solid-phase synthesis of a peptide using only photodepro-
tection, with both photolabile linker and protecting groups
(Kessler et al. 2003).
Keywords Coumarin ꢀ Quinolone ꢀ Bioconjugates ꢀ
Amino acids ꢀ Photocleavable protecting groups
Introduction
In organic synthesis, and especially in peptide chemistry,
protecting groups are a laborious necessity, as a convenient
and efficient synthesis, chemical stability towards different
reagents and selective removal are required. Photochemi-
cally controllable release is an attractive feature for pro-
tecting groups, since typically requires no chemical
reagents and provides spatial and temporal resolution in the
Recently reported photolabile protecting groups include
coumarin derivatives, which have been successfully stud-
ied and applied in the protection of phosphates (Hagen
et al. 2003, 2005; Furuta et al. 2004; Geissler et al. 2005),
carboxylates (Hagen et al. 2005; Furuta et al. 1999), sul-
fates (Geissler et al. 2005), sulfonates (Furuta et al. 2004),
diols (Lin and Lawrence 2002) and carbonyl compounds
(Lu et al. 2003; Shembekar et al. 2007). Protection of
hydroxyl and amino functional groups has also been car-
ried out through carbonate (Suzuki et al. 2003; Gilbert et al.
2007) or carbamate bonds (Hagen et al. 2005; Takaoka
et al. 2003, 2004). Coumarins have been applied in
the preparation of photoresponsive prodrugs that release
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 parent drug by UV and visible light irradiation
(Skwarczynski et al. 2006; Noguchi et al. 2008).
Experimental section
Quinolones and its derivatives constitute a major class
of antibacterial chemotherapeutic agents, which have a
broad spectrum of activity against bacteria, mycobacteria,
parasites, and other diseases (Gootz and Brighty 1998;
Ma et al. 2009; Reddy et al. 2009; Senthilkumar et al.
2009). Considering the biological potential, this family of
heterocycles is of extreme importance. Although the
structural resemblance between quinolones and couma-
rins, contrary to the latter, the use of quinolones as
photolabile protecting groups has not been reported,
while quinolines have been considered for this applica-
tion with promising results (Fedoryak and Dore 2002;
Zhu et al. 2006).
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. 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
1
Coumarins and quinolones are fluorophores, which are
more convenient than non-fluorescent protecting groups,
since they may be useful in the visualisation of processes
during synthesis as well as monitoring the course of reac-
tion and thus allow tracing of the location of caged mole-
cules inside living cells by fluorescent techniques.
Although fluorescence deactivation may be an inconve-
nience in some photochemical processes, considering the
body of work in this area published in the last years, the
direction of improvement on photoreleasable groups has
been towards the development and application of polycy-
clic structures (both benzene and heterocycle derived)
which are fluorophores in most cases and have been
reported as having improved properties as photolabile
protecting groups.
for H 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 com-
parison of chemical shifts, peak multiplicities and J values
and were supported by spin decoupling-double resonance
and bidimensional heteronuclear HMBC and HMQC
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 UV/vis Shimadzu
SPD-GAV detector and a Shimadzu C-RGA Chromatopac
register. LC/MS analyses of photolised samples were car-
ried out in a Finnigan LXQ mass detector coupled to a
Surveyor Plus LC system, and mass spectra were obtained
by ESI technique. All reagents were used as received.
Considering these facts, we decided to evaluate the
behaviour of series of quinolones as photocleavable pro-
tecting groups of the carboxylic acid function, compara-
tively to related coumarins. Compounds possessing the
carboxylic group are of great interest in organic chemistry
and commonly need to be protected in sequential synthesis
against various reagents including reactive nucleophiles,
reducing and oxidant agents. Among the most appealing
carboxylic compounds are amino acids, which play central
roles as building blocks of peptides, proteins and as inter-
mediates in metabolism.
Synthesis of 4-chloromethyl-7-methylcoumarin 1a
Therefore, we synthesised the above-mentioned het-
erocycles as well as their corresponding conjugates with
phenylalanine, which was chosen as a model bifunctional
molecule given the relevance of amino acids as scaffolds
in organic and peptide synthesis. The obtained bioconju-
gates were studied under different photocleavage condi-
tions, as part of our continuing interest in the investigation
of different heteroaromatic fluorophores as fluorescent
labels and photocleavable protecting groups, applied to
amino acids, including neurotransmitters (Piloto et al.
2005, 2006a, b; Fernandes et al. 2007, 2008a, b; Fonseca
et al. 2007).
3-Methylphenol (1.034 g, 9.6 9 10-3 mol) was mixed
with ethyl 4-chloro-3-oxobutanoate (1.5 eq, 1.94 mL,
1.4 9 10-2 mol) at room temperature and aqueous H2SO4
70% (5 mL) was added. The reaction mixture was stirred
at room temperature for 17 h, poured into ice, the pre-
cipitate formed was filtered, washed with water and dried
at 50°C. Compound 1a was obtained as a white solid
1
(1.785 g, 90%); mp = 209–210°C; H NMR (300 MHz,
DMSO-d6): d = 2.41 (3H, s, CH3), 5.00 (2H, d, J 0.6,
CH2), 6.60 (1H, s, H-3), 7.23 (1H, dd, J 8.4 and 1.2 Hz, H-
6), 7.27 (1H, s, H-8), 7.72 (1H, d, J 8.1 Hz, H-5); 13C
123

Light-induced cleavage of model phenylalanine conjugates
701
NMR (75.4 MHz, DMSO-d6): d = 21.08 (CH3), 41.27
(CH2), 114.31 (C-3), 114.62 (C-4a), 116.83 (C-8), 125.01
(C-5), 125.56 (C-6), 143.35 (C-7), 150.72 (C-4), 153.43
(C-8a), 159.82 (C-2); IR (KBr 1%, cm-1): m = 3,075,
2,850, 1,729, 1,617, 1,555, 1,513, 1,567, 1,445, 1,392,
1,377, 1,276, 1,263, 1,225, 1,193, 1,149, 1,056, 1,037,
1,018, 979, 887, 882, 692; UV/Vis (ethanol, nm): kmax (log
e) = 314 (3.83); MS: m/z (EI) 210 (M? 37Cl, 8), 208 (M?
35Cl, 25), 173 (28), 146 (25), 145 (100), 115 (26); HRMS:
m/z (EI) calc. for C11H9O235Cl 208.0296, found 208.0291;
calc. for C11H9O237Cl 210.0271, found 210.0262.
CDCl3): d = 18.95 (CH3), 29.21 (NCH3), 114.39 (C-8),
121.13 (C-3), 121.43 (C-4a), 121.87 (C-6), 125.19 (C-5),
130.42 (C-7), 139.81 (C-8a), 146.34 (C-4), 162.11 (C-2);
IR (KBr 1%, cm-1): m = 2,919, 2,886, 2,850, 1,652, 1,615,
1,592, 1,562, 1,503, 1,455, 1,409, 1,387, 1,371, 1,322,
1,268, 1,162, 1,139, 1,113, 1,066, 1,041, 924, 874, 863,
843, 769, 753; UV/Vis (ethanol, nm): kmax (log e) = 329
(3.62); MS: m/z (EI) 174 (M??1, 21), 173 (M?, 100), 145
(21), 144 (89), 130 (52); HRMS: m/z (EI) calc. for
C11H11NO 173.0847, found 173.0841.
General procedure for the synthesis of 4-methylquinolin-
2(1H)-ones 3b,c
Synthesis of 4-chloromethyl-7-methyl-6-nitrocoumarin 1b
Compound 1a (0.060 g, 2.9 9 10-4 mol) was dissolved in
concentrated H2SO4 (0.5 mL), in a ice bath and HNO3 65%
(0.1 mL) was added. The reaction mixture was stirred at
room temperature for 30 min and then poured into ice, to
give a precipitate, which was filtered and dried. Compound
1b was obtained as an off-white solid (0.067 g, 92%);
mp = 133–135°C; 1H NMR (400 MHz, DMSO-d6):
d = 2.62 (3H, s, CH3), 5.09 (2H, s, CH2), 6.79 (1H, s,
H-3), 7.60 (1H, s, H-8), 8.50 (1H, s, H-5); 13C NMR
(100.6 MHz, DMSO-d6): d = 19.86 (CH3), 40.88 (CH2),
115.58 (C-4a), 116.60 (C-3), 120.50 (C-8), 122.29 (C-5),
137.89 (C-6), 144.88 (C-7), 149.60 (C-4), 155.26 (C-8a),
158.74 (C-2); IR (KBr 1%, cm-1): m = 3,086, 3,066,
2,841, 1,747, 1,625, 1,551, 1,526, 1,494, 1,457, 1,448,
1,420, 1,376, 1,351, 1,324, 1,286, 1,261, 1,214, 1,197,
1,166, 1,086, 1,030, 1,003, 927, 898, 850, 775; UV/Vis
(ethanol, nm): kmax (log e) = 319 (3.61); MS: m/z (EI) 255
(M? 37Cl, 5), 253 (M? 35Cl, 17), 238 (21), 236 (78), 202
(100), 174 (29), 146 (27), 144 (24), 117 (28), 116 (29), 115
(76), 91 (23); HRMS: m/z (EI) calc. for C11H8NO435Cl
253.0153, found 253.0142; calc. for C11H8NO237Cl
255.0112, found 255.0112.
Compounds 3b,c were prepared by reaction of the corre-
sponding N-methylated aniline 2b,c (1 equiv) with ethyl
3-oxobutanoate (2 equiv), by stirring at 180°C for 45 min.
After evaporation of the excess ethyl 3-oxobutanoate under
vacuum, the residue was stirred with aqueous H2SO4 70%
(5 mL) at 95°C for 45 min. After cooling to room tem-
perature, water (10 mL) was added to the mixture, fol-
lowed by extraction with ethyl acetate (3 9 15 mL). The
organic layer was dried with anhydrous MgSO4, filtered,
and evaporated under reduced pressure. The resulting solid
was purified by silica gel column chromatography using
chloroform/methanol (50:1) as eluent. Fractions containing
the product were combined and evaporated, yielding
compounds 3b,c as solids.
1,4,6-Trimethylquinolin-2(1H)-one 3b
Starting from N-methyl-4-methylaniline 2b (0.31 mL,
2.5 9 10-3 mol) and ethyl 3-oxobutanoate (0.63 mL,
5.0 9 10-3 mol), compound 3b was obtained as an off-
1
white solid (0.346 g, 75%); mp = 105–107°C; H NMR
(300 MHz, CDCl3): d = 2.45 (6H, s, 2 9 CH3), 3.69
(3H, s, NCH3), 6.58 (1H, d, J 1.2 Hz, H-3), 7.26 (1H, d, J
8.7 Hz, H-8), 7.39 (1H, dd, J 8.7 and 1.8 Hz, H-7), 7.48
(1H, br s, H-5); 13C NMR (75.4 MHz, CDCl3): d = 18.93
(CH3), 20.78 (CH3), 29.18 (NCH3), 114.28 (C-8), 121.02
(C-3), 121.31 (C-4a), 125.07 (C-5), 131.33 (C-6), 131.54
(C-7), 137.73 (C-8a), 146.13 (C-4), 162.01 (C-2); IR (KBr
1%, cm-1): m = 2,850, 1,671, 1,640, 1,572, 1,490, 1,460,
1,375, 1,200, 879, 817, 722; UV/Vis (ethanol, nm): kmax
(log e) = 337 (3.49); MS: m/z (EI) 187 (M?, 100), 158
(54), 144 (49); HRMS: m/z (EI) calc. for C12H13NO
187.0997, found 187.0990.
Synthesis of 1,4-dimethylquinolin-2(1H)-one 3a
N-Methylaniline 2a (2 mL, 1.9 9 10-2 mol) and ethyl
3-oxobutanoate (4.67 mL, 3.8 9 10-2 mol) were heated at
reflux in acetic acid (10 mL) for 48 h. The solvent was
evaporated under reduced pressure and the residue was
heated in H2SO4 96% (8 mL) at 100°C for 1 h. After
cooling to room temperature, the mixture was neutralised
with aqueous NaOH 6 M and a precipitate formed, which
was filtered, washed with water and dried. Compound 3a
was obtained as a white solid (0.212 g, 66%); mp = 126–
1
127°C; H NMR (300 MHz, CDCl3): d = 2.48 (3H, d, J
1,4-Dimethyl-6-methoxyquinolin-2(1H)-one 3c
0.9 Hz, CH3), 3.72 (3H, s, NCH3), 6.62 (1H, d, J 0.9 Hz,
H-3), 7.27 (1H, dt, J 8.7 and 1.2 Hz, H-6), 7.39 (1H, d, J
8.7 Hz, H-8), 7.59 (1H, dt, J 8.7 and 1.2 Hz, H-7), 7.72
(1H, dd, J 7.8 and 1.2 Hz, H-5); 13C NMR (75.4 MHz,
Starting from N-methyl-4-methoxyaniline 2c (0.25 mL,
1.8 9 10-3 mol) and ethyl 3-oxobutanoate (0.46 mL,
3.6 9 10-3 mol), compound 3c was obtained as an
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702
A. S. C. Fonseca et al.
off-white solid (0.263 g, 71%); mp = 122–124°C; 1H
NMR (300 MHz, CDCl3): d = 2.43 (3H, d, J 0.9 Hz,
CH3), 3.68 (3H, s, NCH3), 3.88 (3H, s, OCH3), 6.60 (1H, d,
J 0.9 Hz, H-3), 7.11 (1H, d, J 2.7 Hz, H-5), 7.18 (1H, dd, J
9.3 and 2.7 Hz, H-7), 7.30 (1H, d, J 9.3 Hz, H-8); 13C
NMR (75.4 MHz, CDCl3): d = 18.99 (CH3), 29.27
(NCH3), 55.66 (OCH3), 107.91 (C-5), 115.55 (C-8), 118.12
(C-7), 121.62 (C-3), 122.17 (C-4a), 134.27 (C-8a), 145.61
(C-4), 154.49 (C-6), 161.61 (C-2); IR (KBr 1%, cm-1):
m = 3,003, 2,950, 2,831, 1,650, 1,624, 1,591, 1,570, 1,505,
1,465, 1,429, 1,417, 1,371, 1,314, 1,276, 1,244, 1,201,
1,163, 1,092, 1,033, 910, 886, 844, 806; UV/Vis (ethanol,
nm): kmax (log e) = 351 (3.90); MS: m/z (EI) 203 (M?, 75),
188 (100), 160 (24); HRMS: m/z (EI) calc. for C12H13NO2
203.0949, found 203.0946.
(1H, d, J 0.9 Hz, H-5), 10.15 (1H, s, CHO); 13C NMR
(75.4 MHz, CDCl3): d = 20.88 (CH3), 29.41 (NCH3),
114.28 (C-8), 116.41 (C-4a), 126.28 (C-5), 131.70 (C-3),
132.80 (C-7), 133.12 (C-6), 138.55 (C-8a), 139.86 (C-4),
161.46 (C-2), 192.96 (CHO). IR (KBr 1%, cm-1):
m = 3,039, 2,925, 2,859, 1,702, 1,673, 1,656, 1,585, 1,567,
1,451, 1,413, 1,378, 1,322, 1,303, 1,189, 1,108, 1,055, 939,
902, 813, 758; UV/Vis (ethanol, nm): kmax (log e) = 338
(3.72); MS: m/z (EI) 201 (M?, 100), 172 (48), 144 (49);
HRMS: m/z (EI) calc. for C12H11NO2 201.0790, found
201.0790.
6-Methoxy-1-methylquinolin-2(1H)-one-4-carbaldehyde 4c
Starting from compound 3c (0.170 g, 8.4 9 10-4 mol) and
selenium dioxide (0.376 g, 3.4 9 10-3 mol), compound 4c
was obtained as a yellow solid (0.113 g, 62%); mp = 202–
204°C; 1H NMR (400 MHz, CDCl3): d = 3.77 (3H, s,
NCH3), 3.91 (3H, s, OCH3), 7.19 (1H, s, H-3), 7.27 (1H,
dd, J 6.9 and 2.1 Hz, H-7), 7.39 (1H, d, J 6.9 Hz, H-8),
8.41 (1H, d, J 2.1 Hz, H-5), 10.12 (1H, s, CHO); 13C NMR
(100.6 MHz, CDCl3): d = 30.07 (NCH3), 55.70 (OCH3),
108.06 (C-5), 115.58 (C-8), 117.13 (C-4a), 120.66 (C-7),
132.83 (C-3), 135.19 (C-8a), 139.32 (C-4), 155.66 (C-6),
161.95 (C-2), 193.17 (CHO). IR (KBr 1%, cm-1):
m = 3,020, 2,929, 1,671, 1,659, 1,580, 1,454, 1,433, 1,415,
1,385, 1,331, 1,213, 1,161, 1,055, 936, 898; UV/Vis (eth-
anol, nm): kmax (log e) = 341 (3.69); MS: m/z (EI) 217
(M?, 100), 188 (43); HRMS: m/z (EI) calc. for C12H11NO3
217.0739, found 217.0744.
General procedure for the synthesis of quinolin-2(1H)-one-
4-carbaldehydes 4a–c
Compounds 3a–c (1 equiv) were reacted with selenium
dioxide (4 equiv) in chlorobenzene (30 mL), by heating at
reflux for 2–4 days. The mixture was filtered hot and the
solvent was removed by rotary evaporation. The crude
residue was used in the next reaction without further
purification.
1-Methylquinolin-2(1H)-one-4-carbaldehyde 4a
Starting from compound 3a (0.100 g, 5.8 9 10-4 mol) and
selenium dioxide (0.257 g, 2.3 9 10-3 mol), compound 4a
was obtained as a yellow solid (0.080 g, 74%); mp = 166–
168°C; 1H NMR (300 MHz, CDCl3): d = 3.78 (3H, s,
NCH3), 7.17 (1H, s, H-3), 7.35 (1H, dt, J 8.1 and 1.5 Hz,
H-6), 7.44 (1H, d, J 8.1 Hz, H-8), 7.66 (1H, dt, J 7.5 and
1.5 Hz, H-7), 8.84 (1H, dd, J 8.1 and 1.5 Hz, H-5), 10.15
(1H, s, CHO); 13C NMR (75.4 MHz, CDCl3): d = 29.94
(CH3), 114.43 (C-8), 116.46 (C-4a), 123.35 (C-6), 126.61
(C-5), 131.61 (C-7), 131.66 (C-3), 140.04 (C-4), 140.48
(C-8a), 161.58 (C-2), 192.78 (CHO). IR (KBr 1%, cm-1):
m = 3,023, 2,919, 1,670, 1,664, 1,588, 1,561, 1,454, 1,430,
1,413, 1,391, 1,325, 1,212, 1,160, 1,054, 936, 898, 751;
UV/Vis (ethanol, nm): kmax (log e) = 330 (3.78); MS: m/z
(EI) 187 (M?, 100), 158 (53), 130 (58); HRMS: m/z (EI)
calc. for C11H9NO2 187.0636, found 187.0633.
General procedure for the synthesis
of 4-hydroxymethylquinolin-2(1H)-ones 5a–c
Compounds 4a–c (1 equiv) were reacted with sodium
borohydride (0.7 equiv) in ethanol (10 mL) for 1 day at
room temperature. The solvent was removed under reduced
pressure and after purification by column chromatography
(for 5a and 5c), compounds 5a–c were obtained as solids.
4-Hydroxymethyl-1-methylquinolin-2(1H)-one 5a
Starting from compound 4a (0.807 g, 4.3 9 10-3 mol) and
sodium borohydride (0.114 g, 3.0 9 10-3 mol), and puri-
fication by silica gel column chromatography using
dichloromethane/methanol (25:1) as eluent, compound 5a
was obtained as a light pink solid (0.549 g, 67%);
mp = 182–183°C; 1H NMR (400 MHz, DMSO-d6):
d = 3.60 (3H, s, NCH3), 4.77 (2H, d, J 1.2 Hz, CH2), 5.51
(1H, s, OH), 6.66 (1H, s, H-3), 7.25 (1H, dt, J 8.0 and
1.2 Hz, H-6), 7.55 (1H, dd, J 8.4 and 0.8 Hz, H-8), 7.61
(1H, dt, J 8.4 and 1.2 Hz, H-7), 7.72 (1H, dd, J 8.0
and 1.2 Hz, H-5); 13C NMR (100.6 MHz, DMSO-d6):
1,6-Dimethylquinolin-2(1H)-one-4-carbaldehyde 4b
Starting from compound 3b (0.450 g, 2.4 9 10-3 mol) and
selenium dioxide (1.067 g, 9.6 9 10-3 mol), compound 4b
was obtained as a yellow solid (0.429 g, 89%); mp = 179–
180°C; 1H NMR (300 MHz, CDCl3): d = 2.47 (3H, s,
CH3), 3.76 (3H, s, NCH3), 7.16 (1H, s, H-3), 7.34 (1H, d, J
8.4 Hz, H-7), 7.48 (1H, dd, J 8.7 and 1.8 Hz, H-8), 8.64
123

Light-induced cleavage of model phenylalanine conjugates
703
General procedure for the synthesis
of 4-chloromethylquinolin-2(1H)-ones 6a–c
d = 28.83 (NCH3), 59.40 (CH2), 114.96 (C-8), 116.80
(C-3), 118.31 (C-4a), 121.73 (C-6), 124.19 (C-5), 130.56
(C-7), 139.46 (C-8a), 149.74 (C-4), 161.04 (C-2); IR (KBr
1%, cm-1): m = 3,324, 2,954, 2,924, 2,854, 1,643, 1,575,
1,509, 1,458, 1,442, 1,421, 1,401, 1,377, 1,351, 1,333,
1,302, 1,231, 1,198, 1,140, 1,096, 1,080, 1,050, 1,017, 980,
753; UV/Vis (ethanol, nm): kmax (log e) = 328 (3.72); MS:
m/z (EI) 189 (M?, 100), 160 (26), 144 (39), 130 (22), 118
(36), 117 (20); HRMS: m/z (EI) calc. for C11H11NO2
189.0793, found 189.0790.
Compounds 5a–c (1 equiv) were stirred with thionyl
chloride (20 equiv) in dichloromethane (10 mL) for 1 day
at room temperature. After removal of the solvent under
reduced pressure, compounds 6a–c were obtained as
solids.
4-Chloromethyl-1-methylquinolin-2(1H)-one 6a
Starting from compound 5a (0.068 g, 3.6 9 10-4 mol)
and thionyl chloride (0.5 mL, 7.2 9 10-3 mol), com-
pound 6a was obtained as a white solid (0.078 g, 79%);
mp = 170–171°C; 1H NMR (300 MHz, CDCl3/DMSO-
d6, 1:1): d = 3.56 (3H, s, NCH3), 4.61 (2H, d, J 0.6 Hz,
CH2), 6.67 (1H, s, H-3), 7.16 (1H, dt, J 8.1 and 1.2 Hz,
H-6), 7.28 (1H, d, J 6.9 Hz, H-8), 7.45 (1H, dt, J 8.7 and
1.5 Hz, H-7), 7.66 (1H, dd, J 8.4 and 1.5 Hz, H-5); 13C
NMR (75.4 MHz, CDCl3/DMSO-d6, 1:1): d = 29.03
(CH3), 41.84 (NCH3), 114.44 (C-8), 118.12 (C-4a),
121.29 (C-3), 121.92 (C-6), 124.46 (C-5), 130.67 (C-7),
139.74 (C-8a), 143.91 (C-4), 161.12 (C-2); IR (KBr 1%,
cm-1): m = 3,073, 3,021, 1,674, 1,662, 1,646, 1,593,
1,563, 1,505, 1,453, 1,415, 1,400, 1,325, 1,266, 1,222,
1,169, 1,081, 1,052, 949, 915, 896, 793, 774; UV/Vis
(ethanol, nm): kmax (log e) = 333 (3.78); MS: m/z (EI)
209 (M? 37Cl, 15), 207 (M? 35Cl, 50), 173 (36), 172 (62),
144 (100), 130 (20); HRMS: m/z (EI) calc. for
C11H10NO35Cl 207.0457, found 207.0451; calc. for
C11H10NO37Cl 209.0431, found 209.0421.
1,6-Dimethyl-4-hydroxymethylquinolin-2(1H)-one 5b
Starting from compound 4b (0.221 g, 1.1 9 10-3 mol) and
sodium borohydride (0.029 g, 7.7 9 10-4 mol), compound
5b was obtained as a beige solid (0.188 g, 84%);
mp = 148–150°C; 1H NMR (400 MHz, CDCl3): d = 2.42
(3H, s, CH3), 3.49 (3H, s, NCH3), 4.25 (1H, s, OH), 4.88
(2H, s, CH2), 6.83 (1H, s, H-3), 7.14 (1H, d, J 8.8 Hz, H-8),
7.34 (1H, d, J 8.4 Hz, H-7), 7.48 (1H, s, H-5); 13C NMR
(100.6 MHz, CDCl3): d = 20.76 (CH3), 29.16 (NCH3),
61.32 (CH2), 114.43 (C-8), 118.00 (C-3), 119.06 (C-4a),
124.18 (C-5), 131.63 (C-7), 131.75 (C-6), 137.51 (C-8a),
149.01 (C-4), 162.38 (C-2); IR (KBr 1%, cm-1):
m = 3,356, 2,912, 2,857, 1,660, 1,644, 1,599, 1,573, 1,557,
1,464, 1,417, 1,350, 1,296, 1,279, 1,184, 1,131, 1,095,
1,076, 1,002, 946, 868, 807; UV/Vis (ethanol, nm): kmax
(log e) = 335 (3.51); MS: m/z (EI) 203 (M?, 100), 174
(22), 158 (42), 144 (29), 132 (38); HRMS: m/z (EI) calc.
for C12H13NO2 203.0955, found 203.0946.
4-Hydroxymethyl-6-methoxy-1-methylquinolin-2(1H)-one
5c
4-Chloromethyl-1,6-dimethylquinolin-2(1H)-one 6b
Starting from compound 5b (0.136 g, 6.7 9 10-4 mol) and
thionyl chloride (1.0 mL, 1.3 9 10-2 mol), compound 6b
was obtained as a beige solid (0.122 g, 82%); mp = 153–
155°C; 1H NMR (400 MHz, CDCl3): d = 2.48 (3H, s,
CH3), 3.72 (3H, s NCH3), 4.73 (2H, s, CH2), 6.82 (1H, s,
H-3), 7.32 (1H, d, J 8.4 Hz, H-8), 7.44 (1H, dd, J 8.8 and
1.6 Hz, H-7), 7.59 (1H, s, H-5); 13C NMR (100.6 MHz,
CDCl3): d = 20.88 (CH3), 29.49 (NCH3), 42.34 (CH2),
114.73 (C-8), 118.58 (C-4a), 122.04 (C-3), 124.67 (C-5),
131.89 (C-6), 132.20 (C-7), 138.33 (C-8a), 143.89 (C-4),
161.61 (C-2); IR (KBr 1%, cm-1): m = 3,033, 2,964,
1,656, 1,590, 1,568, 1,505, 1,461, 1,438, 1,418, 1,381,
1,326, 1,262, 1,165, 1,097, 1,052, 1,022, 945, 911, 876,
805; UV/Vis (ethanol, nm): kmax (log e) = 343 (3.48); MS:
m/z (EI) 223 (M? 37Cl, 21), 221 (M? 35Cl, 70), 187 (30),
186 (68), 158 (100), 157 (23), 115 (22); HRMS: m/z (EI)
calc. for C12H12NO35Cl 221.0598, found 221.0607; calc.
for C12H12NO37Cl 223.0583, found 223.0578.
Starting from compound 4c (0.113 g, 5.2 9 10-4 mol) and
sodium borohydride (0.134 g, 3.6 9 10-4 mol), and puri-
fication by silica gel column chromatography using
dichloromethane/methanol (100:1) as eluent, compound 5c
was obtained as a white solid (0.111 g, 97%); mp = 177–
179°C; 1H NMR (300 MHz, CDCl3): d = 3.60 (3H, s,
NCH3), 3.90 (3H, s, OCH3), 3.17 (1H, br s, OH), 4.91 (2H,
s, CH2), 6.87 (1H, s, H-3), 7.13–7.28 (3H, m, H-5, H-7 and
H-8); 13C NMR (75.4 MHz, CDCl3): d = 29.68 (NCH3),
55.73 (OCH3), 61.78 (CH2), 106.94 (C-5), 115.81 (C-8),
118.69 (C-7), 119.04 (C-3), 119.86 (C-4a), 134.33 (C-8a),
147.92 (C-4), 154.68 (C-6), 161.90 (C-2). IR (KBr 1%,
cm-1): m = 3,338, 2,918, 2,849, 1,648, 1,620, 1,573, 1,507,
1,465, 1,430, 1,372, 1,279, 1,241, 1,185, 1,079, 1,034, 864,
809; UV/Vis (ethanol, nm): kmax (log e) = 352 (3.60); MS:
m/z (EI) 219 (M?, 100), 204 (81), 203 (25), 190 (21), 188
(35), 86 (28), 84 (45); HRMS: m/z (EI) calc. for
C12H13NO3 219.0901, found 219.0895.
123

704
A. S. C. Fonseca et al.
4-Chloromethyl-6-methoxy-1-methylquinolin-2(1H)-one 6c
1–3 days. The solvent was removed in a rotary evaporator
and a crude solid was obtained.
Starting from compound 5c (0.100 g, 4.6 9 10-4 mol) and
thionyl chloride (0.7 mL, 9.2 9 10-3 mol), compound 6c
was obtained as a yellowish solid (0.056 g, 52%);
mp = 171–172°C; 1H NMR (300 MHz, CDCl3): d = 3.72
(3H, s, NCH3), 3.91 (3H, s, OCH3), 4.71 (2H, s, CH2), 6.84
(1H, s, H-3), 7.23 (1H, dd, J 8.7 and 2.7 Hz, H-7), 7.25
(1H, s, H-5), 7.36 (1H, d, J 8.7 Hz, H-8); 13C NMR
(300 MHz, CDCl3): d = 29.61 (NCH3), 42.49 (CH2),
55.76 (OCH3), 107.56 (C-5), 116.05 (C-8), 119.06 (C-7),
119.44 (C-4a), 122.67 (C-3), 134.83 (C-8a), 143.46 (C-4),
154.73 (C-6), 161.22 (C-2). IR (KBr 1%, cm-1):
m = 3,081, 2,995, 2,966, 2,942, 2,839, 1,652, 1,622, 1,592,
1,573, 1,508, 1,458, 1,428, 1,415, 1,344, 1,272, 1,246,
1,199, 1,084, 1,043, 929, 865, 808; UV/Vis (ethanol, nm):
N-Benzyloxycarbonyl-L-phenylalanine (7-methyl-
coumarin-4-yl)methyl ester 7a
Starting from compound 1a (0.100 g, 4.79 9 10-4 mol),
Z-Phe-OH (0.143 g, 4.79 9 10-4 mol) and KF (0.083 g,
1.44 9 10-3 mol), the crude solid was recrystallised from
ethyl acetate and petroleum ether 40–60, yielding conju-
gate 7a as a white solid (0.190 g, 84%); mp = 151–152°C;
1H NMR (300 MHz, CDCl3): d = 2.46 (3H, s, CH3), 3.15
(2H, d, J 6.3 Hz, b-CH2 Phe), 4.72–4.79 (1H, m, a-H Phe),
5.12 (2H, s, CH2 Z), 5.24–5.26 (3H, m, CH2 and NH), 6.27
(1H, s, H-3), 7.09–7.35 (13H, m, H-5, H-6, H-8 and
10 9 Ph-H); 13C NMR (75.4 MHz, CDCl3): d = 21.63
(CH3), 38.19 (b-CH2 Phe), 55.06 (a-C Phe), 62.01 (CH2),
67.19 (CH2 Z), 112.76 (C-3), 114.52 (C-4a), 117.52 (C-8),
123.14 (C-5), 125.64 (C-6), 127.43 (C-40 Phe), 128.16 (C-
300 and C-500 Z), 128.25 (C-400 Z), 128.51 (C-200 and C-600
Z), 128.77 (C-30 and C-50 Phe), 129.06 (C-20 and C-60 Phe),
135.14 (C-10 Phe), 135.96 (C-100 Z), 143.46 (C-7), 147.85
(C-4), 153.72 (C-8a), 155.64 (C=O urethane), 160.39
(C-2), 171.06 (C=O ester); IR (KBr 1%, cm-1): m = 3,311,
3,062, 3,027, 1,742, 1,693, 1,620, 1,544, 1,494, 1,456,
1,409, 1,377, 1,272, 1,212, 1,181, 1,149, 1,069, 1,052,
1,016, 962, 863; UV/Vis (ethanol, nm): kmax (log e) = 317
(3.92); MS: m/z (ESI) 472 (M??1, 58), 324 (20), 143 (51),
139 (83); HRMS: m/z (ESI) calc. for C28H26NO6
472.17546, found 472.17537.
k
max (log e) = 358 (3.38); MS: m/z (EI) 239 (M? 37Cl, 33),
237 (M? 35Cl, 100), 222 (55), 203 (62), 202 (25), 188 (70),
174 (34), 160 (22), 159 (33), 130 (19); HRMS: m/z (EI)
calc. for C12H12NO235Cl 237.0561, found 237.0557; calc.
for C12H12NO237Cl 239.0517, found 239.0527.
Synthesis of 4-chloromethyl-1,6-dimethyl-5-nitroquinolin-
2(1H)-one 6d
Compound 6b (0.060 g, 2.7 9 10-4 mol) was dissolved
in concentrated H2SO4 (0.5 mL), in an ice bath, and
HNO3 65% (0.05 mL) was added. The mixture was stir-
red for 30 min at room temperature and then poured over
ice. A precipitate was collected by filtration, washed with
water and dried to yield compound 6d as a yellow solid
1
(0.042 g, 58%); H NMR (400 MHz, CDCl3): d = 2.35
N-Benzyloxycarbonyl-L-phenylalanine (7-methyl-6-
nitrocoumarin-4-yl)methyl ester 7b
(3H, s, CH3), 3.76 (3H, s NCH3), 4.65 (2H, d, J 0.8 Hz,
CH2), 7.17 (1H, t, J 0.8 Hz, H-3), 7.51 (2H, m, H-7 and
H-8); 13C NMR (100.6 MHz, CDCl3): d = 17.05 (CH3),
30.25 (NCH3), 41.26 (CH2), 110.68 (C-4a), 116.82 (C-8),
124.23 (C-6), 125.64 (C-3), 133.00 (C-7), 139.57 (C-8a),
140.94 (C-4), 147.90 (C-5), 160.46 (C-2). IR (KBr 1%,
cm-1): m = 2,927, 2,855, 1,663, 1,591, 1,557, 1,530,
1,459, 1,419, 1,376, 1,314, 1,274, 1,107, 980, 869, 817;
UV/Vis (ethanol, nm): kmax (log e) = 343 (3.61); MS: m/z
(EI) 268 (M? 37Cl, 34), 266 (M? 35Cl, 100), 188 (52);
HRMS: m/z (EI) calc. for C12H11N2O335Cl 266.0464,
found 266.0461; C12H11N2O337Cl 268.0438, found
268.0440;
Starting from compound 1b (0.085 g, 3.35 9 10-4 mol),
Z-Phe-OH (0.100 g, 3.35 9 10-4 mol) and KF (0.058 g,
1.00 9 10-3 mol), the crude solid was recrystalised from
ethyl acetate and petroleum ether 40–60, yielding conju-
gate 7b as a white solid (0.122 g, 70%); mp = 140–142°C;
1H NMR (400 MHz, CDCl3): d = 2.73 (3H, s, CH3), 3.13–
3.16 (2H, m, b-CH2 Phe), 4.18–4.20 (1H, m, a-H Phe),
5.11 (2H, s, CH2 Z), 5.25–5.27 (2H, m, CH2), 6.37 (1H, s,
H-3), 7.00–7.12 (5H, m, 5 9 Ph-H), 7.21–7.35 (6H, m, H-8
and 5 9 Ph-H), 8.19 (1H, s, H-5); 13C NMR (100.6 MHz,
CDCl3): d = 21.19 (CH3), 38.21 (b-CH2 Phe), 55.13 (a-C
Phe), 61.44 (CH2), 67.30 (CH2 Z), 115.19 (C-3), 115.37
(C-4a), 121.01 (C-5), 121.19 (C-8), 127.50 (C-40 Phe),
127.81 (C-400 Z), 128.23 (C-300 and C-500 Z), 128.55 (C-200
and C-600 Z), 128.81 (C-30 and C-50 Phe), 129.02 (C-20 and
C-60 Phe), 135.05 (C-10 Phe), 135.94 (C-100 Z), 138.84 (C-
6), 145.09 (C-7), 146.95 (C-4), 155.52 (C-8a), 155.66
(C=O urethane), 158.54 (C-2), 171.07 (C=O ester); IR
(KBr 1%, cm-1): m = 3,325, 2,925, 1,736, 1,698, 1,629,
General procedure for the synthesis of coumarin
conjugates 7a,b and quinolin-2(1H)-one conjugates 8a–d
The corresponding chloromethylcoumarin 1a,b or chlo-
romethylquinolin-2(1H)-one 6a–d (1 equiv) was stirred
with N-benzyloxycarbonyl-L-phenylalanine (1 equiv) and
KF (3 equiv) in DMF (5 mL), at room temperature for
123

Light-induced cleavage of model phenylalanine conjugates
705
1,594, 1,530, 1,496, 1,454, 1,402, 1,382, 1,344, 1,314,
1,260, 1,123, 1,045, 868, 841, 751; UV/Vis (ethanol, nm):
kmax (log e) = 316 (3.58); MS: m/z (ESI) 539
(M? ? 1 ? Na, 83), 474 (53), 374 (27), 338 (38), 323
(20), 322 (100); HRMS: m/z (ESI) calc. for C28H24N2O8Na
539.14135, found 539.14249.
(C-5), 127.26 (C-400 Z), 128.11 (C-200 and C-600 Z), 128.19
(C-40 Phe), 128.50 (C300 and C-500 Z), 128.67 (C-30 and C-50
Phe), 129.12 (C-20 and C-60 Phe), 131.89 (C-6), 132.11 (C-
7), 135.26 (C-10 Phe), 136.08 (C-100 Z), 130.03 (C-8a),
141.98 (C-4), 155.63 (C=O urethane), 161.51 (C-2), 171.17
(C=O ester); IR (KBr 1%, cm-1): m = 3,285, 3,062, 3,031,
2,940, 1,751, 1,720, 1,652, 1,592, 1,570, 1,531, 1,498,
1,455, 1,418, 1,327, 1,260, 1,178, 1,082, 1,062, 880, 809,
749; UV/Vis (ethanol, nm): kmax (log e) = 339 (3.71); MS:
m/z (ESI) 485 (M??1, 100), 486 (M??2, 50); HRMS: m/z
(ESI) calc. for C29H29N2O5 485.20918, found 485.20710.
N-Benzyloxycarbonyl-L-phenylalanine (1-methylquinolin-
2(1H)-one-4-yl) methyl ester 8a
Starting from compound 6a (0.080 g, 3.9 9 10-4 mol),
Z-Phe-OH (0.115 g, 3.9 9 10-4 mol) and KF (0.067 g,
1.2 9 10-3 mol), the crude solid was recrystalised from
dichloromethane yielding conjugate 8a as a beige oily solid
(0.165 g, 91%); 1H NMR (400 MHz, DMSO-d6):
d = 2.94–3.11 (2H, m, b-CH2 Phe), 3.62 (3H, s, NCH3),
4.36–4.42 (1H, m, a-H Phe), 4.90–5.00 (2H, m, CH2 Z),
5.36–5.45 (2H, m, CH2), 6.64 (1H, s, H-3), 7.10–7.33
(10H, m, 10 9 Ph-H), 7.56 (1H, d, J 8.4 Hz, H-8), 7.63–
N-Benzyloxycarbonyl-L-phenylalanine (6-methoxy-1-
methylquinolin-2(1H)-one-4-yl) methyl ester 8c
Starting from compound 6c (0.050 g, 2.1 9 10-4 mol),
Z-Phe-OH (0.063 g, 2.1 9 10-4 mol) and KF (0.037 g,
6.3 9 10-4 mol), the crude solid was purified by silica gel
column chromatography using dichloromethane/methanol
(100:1) as eluent, and conjugate 8c was obtained as a light
yellow oily solid (0.065 g, 62%); 1H NMR (400 MHz,
CDCl3): d = 3.13–3.16 (2H, m, b-CH2 Phe), 3.72 (3H, s,
NCH3), 3.90 (3H, s, OCH3), 4.72–4.78 (1H, m, a-H Phe),
5.05–5.12 (2H, m, CH2 Z), 5.27 (1H, d, J 8.0 Hz, NH),
5.33 (2H, s, CH2), 6.75 (1H, s, H-3), 7.02 (1H, d, J 2.4 Hz,
H-5), 7.07–7.09 (2H, m, H-20 and H-60 Phe), 7.21–7.36
(10H, m, H-7, H-8 and 8 9 Ph-H); 13C NMR (100.6 MHz,
CDCl3): d = 29.55 (NCH3), 38.10 (b-CH2 Phe), 54.99
(a-C Phe), 55.73 (OCH3), 63.12 (CH2), 67.08 (CH2 Z),
106.70 (C-5), 115.97 (C-8), 119.13 (C-7), 119.36 (C-4a),
121.28 (C-3), 127.26 (C-40 Phe), 128.09 (C-200 and C-600
Z), 128.18 (C-400 Z), 128.49 (C-300 and C-500 Z), 128.65
(C-30 and C-50 Phe), 129.10 (C-20 and C-60 Phe), 134.57
(C-8a), 135.23 (C-10 Phe), 136.07 (C-100 Z), 141.61 (C-4),
154.80 (C-6), 155.62 (C=O urethane), 161.15 (C-2), 171.18
(C=O ester); IR (KBr 1%, cm-1): m = 3,291, 3,029, 3,006,
2,940, 1,736, 1,652, 1,621, 1,587, 1,571, 1,509, 1,464,
1,456, 1,431, 1,385, 1,313, 1,281, 1,247, 1,205, 1,181,
1,083, 1,041, 876; UV/Vis (ethanol, nm): kmax (log
e) = 354 (3.70); MS: m/z (ESI) 501 (M??1, 63), 407 (21),
204 (100); HRMS: m/z (ESI) calc. for C29H29N2O6
501.20181, found 501.20201.
7.69 (2H, m, H-5 and H-7), 7.95 (1H, d, J 8.0 Hz, NH); 13
C
NMR (100.6 MHz, DMSO-d6): d = 29.03 (CH3), 36.31
(b-CH2 Phe), 55.67 (a-C Phe), 62.48 (CH2), 65.51 (CH2 Z),
115.12 (C-8), 117.88 (C-4a), 119.01 (C-3), 122.03 (C-6),
124.62 (C-5), 126.53 (C-40 Phe), 127.64 (C-200 and C-600
Z), 127.78 (C-400 Z), 128.24 (C-300 and C-500 Z), 128.28
(C-30 and C-50 Phe), 129.08 (C-20 and C-60 Phe), 130.99
(C-7), 136.77 (C-100 Z), 137.19 (C-10 Phe), 139.62 (C-8a),
143.33 (C-4), 156.01 (C=O urethane), 160.54 (C-2), 171.40
(C=O ester); IR (KBr 1%, cm-1): m = 3,031, 2,925, 1,755,
1,720, 1,657, 1,592, 1,498, 1,455, 1,399, 1,325, 1,258,
1,179, 1,082, 1,063, 875, 848, 751; UV/Vis (ethanol, nm):
kmax (log e) = 330 (3.68); MS: m/z (ESI) 471 (M??1,
100); HRMS: m/z (ESI) calc. for C28H27N2O5 471.19184,
found 471.19145.
N-Benzyloxycarbonyl-L-phenylalanine
(1,6-dimethylquinolin-2(1H)-one-4-yl) methyl ester 8b
Starting from compound 6b (0.050 g, 2.3 9 10-4 mol),
Z-Phe-OH (0.068 g, 2.3 9 10-4 mol) and KF (0.039 g,
6.8 9 10-4 mol), the crude solid was purified by silica gel
column chromatography using ethyl acetate/n-hexane (1:2)
as eluent, and conjugate 8b was obtained as a light yellow
1
oily solid (0.061 g, 56%); H NMR (300 MHz, CDCl3):
N-Benzyloxycarbonyl-L-phenylalanine (1,6-dimethyl-5-
nitroquinolin-2(1H)-one-4-yl) methyl ester 8d
d = 2.44 (3H, s, CH3), 3.14–3.17 (2H, m, b-CH2 Phe),
3.72 (3H, s, NCH3), 4.74–4.80 (1H, m, a-H Phe), 5.05–5.15
(2H, m, CH2 Z), 5.26 (1H, d, J 8.1 Hz, NH), 5.34–5.39
(2H, m, CH2), 6.72 (1H, s, H-3), 7.07–7.10 (2H, m, H-20
and H-60 Phe), 7.21–7.45 (12H, m, H-5, H-8 and 10 9 Ph-
H), 7.43 (1H, dd, J 8.7 and 2.1 Hz, H-7); 13C NMR
(75.4 MHz, CDCl3): d = 20.80 (CH3), 29.42 (NCH3),
38.13 (b-CH2 Phe), 54.96 (a-C Phe), 63.05 (CH2), 67.08
(CH2 Z), 114.64 (C-8), 118.48 (C-4a), 120.28 (C-3), 123.99
Starting from compound 6d (0.022 g, 8.3 9 10-5 mol),
Z-Phe-OH (0.025 g, 8.3 9 10-5 mol) and KF (0.015 g,
2.5 9 10-4 mol), the crude solid was purified by silica gel
column chromatography using dichloromethane/methanol
(50:1) as eluent, and conjugate 8d was obtained as a yellow
1
oily solid (0.032 g, 72%); H NMR (300 MHz, CDCl3):
d = 2.35 (3H, s, CH3), 3.06–3.22 (2H, m, b-CH2 Phe),
123

706
A. S. C. Fonseca et al.
3.75 (3H, s, NCH3), 4.70–4.73 (1H, m, a-H Phe), 5.08–5.10
(2H, m, CH2 Z), 5.13 (1H, d, J 8.1 Hz, NH), 5.21–5.26
(2H, m, CH2), 6.83 (1H, s, H-3), 7.09–7.12 (2H, m, H-200
and H-60 Phe), 7.21–7.45 (8H, m, 8 9 Ph-H), 7.46–7.51
(2H, m, H-7 and H-8); 13C NMR (75.4 MHz, CDCl3):
d = 17.13 (CH3), 30.20 (NCH3), 38.04 (b-CH2 Phe), 54.93
(a-C Phe), 62.01 (CH2), 67.07 (CH2 Z), 110.57 (C-4a),
116.78 (C-8), 123.90 (C-3), 124.25 (C-6), 127.19 (C-40
Phe), 128.11 (C-200 and C-600 Z), 128.14 (C-400 Z), 128.48
(C-300 and C-500 Z), 128.64 (C-30 and C-50 Phe), 129.12
(C-20 and C-60 Phe), 133.00 (C-7), 135.40 (C-10 Phe),
136.08 (C-100 Z), 139.38 (C-4), 139.61 (C-8a), 149.00
(C-5), 156.00 (C=O urethane), 160.20 (C-2), 170.57 (C=O
ester); IR (KBr 1%, cm-1): m = 3,303, 3,062, 3,031, 2,921,
2,850, 1,752, 1,720, 1,652, 1,589, 1,558, 1,496, 1,455,
1,418, 1,372, 1,258, 1,210, 1,174, 1,107, 1,083, 1,052, 869,
817, 744; UV/Vis (ethanol, nm): kmax (log e) = 341 (3.86);
MS: m/z (ESI) 530 (M??1, 100), 474 (25); HRMS: m/z
(ESI) calc. for C29H28N3O7 530.19109, found 530.19218.
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,
except for compound 8c (0.6 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 conjugate (retention time: 7a, 6.5; 7b,
6.3; 8a, 4.8; 8b, 5.7; 8c, 6.4; 8d, 5.6 min).
Results and discussion
Synthesis
4-Chloromethyl-7-methylcoumarin 1a was synthesised
through a Pechmann reaction, between 3-methylphenol and
ethyl 4-chloro-3-oxobutanoate, catalysed by aqueous sul-
phuric acid at room temperature, through a method reported
earlier (Fonseca et al. 2007). This compound was nitrated in
standard conditions with nitric acid and sulphuric
acid, yielding 4-chloromethyl-7-methyl-6-nitrocoumarin 1b
(Scheme 1).
General photolysis procedure
A 1 9 10-4 M methanol/HEPES buffer (80:20) solution of
conjugates 7 and 8 (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. HEPES buffer solution was
prepared in distilled water with HEPES (4-(2-hydroxy-
ethyl)-1-piperazine ethanesulfonic acid) (10 mM), NaCl
(120 mM), KCl (3 mM), CaCl2 (1 mM) and MgCl2
(1 mM) and pH adjusted to 7.2.
1,4-Dimethylquinolin-2(1H)-one 3a, 1,4,6-trimethylquin-
olin-2(1H)-one 3b and 1,4-dimethyl-6-methoxyquinolin-
2(1H)-one 3c were prepared by a modified Knorr synthesis
(Uray et al. 1999) between ethyl 3-oxobutanoate and
N-methylaniline 2a, N,4-dimethylaniline 2b and N-methyl-4-
methoxyaniline2c, respectively. The methylgroup atposition
4 was oxidised to the aldehyde, by reaction with selenium
dioxide,
affording
1-methylquinolin-2(1H)-one-4-
Scheme 1 Synthesis of
coumarins 1a,b and quinolin-
2(1H)-ones 3a–c, 4a–c, 5a–c
and 6a–d
Cl
O
O
Cl
R
O
H2SO4 70%, rt
O
O
OH
1a R = H
1b R = NO2
HNO3 / H2SO4
R
R
CH3
N
R
CHO
N
O
O
R1
R1
R1
SeO2
O
H+, reflux
chlorobenzene,
reflux
O
O
NH
2a-c
3a-c
4a-c
NaBH4
EtOH, rt
R
CH2OH
R
CH2Cl
R1
a R = R1 = H
R1
SOCl2
b R = H, R1 = CH3
c R = H, R1 = OCH3
d R = NO2, R1 = CH3
DCM, rt
HNO3 / H2SO4
N
O
N
O
6a-d
5a-c
123

Light-induced cleavage of model phenylalanine conjugates
707
carbaldehyde 4a, 1,6-dimethylquinolin-2(1H)-one-4-carbal-
dehyde 4b and 6-methoxy-1-methylquinolin-2(1H)-one-4-
carbaldehyde 4c, which were then reacted with sodium
borohydride, giving 4-hydroxymethyl-1-methylquinolin-2
(1H)-one 5a, 1,6-dimethyl-4-hydroxymethylquinolin-2(1H)-
one 5b and 4-hydroxymethyl-6-methoxy-1-methylquinolin-
2(1H)-one 5c. These hydroxymethylated derivatives were
converted through treatment with thionyl chloride to the
chloromethylated counterparts, namely 4-chloromethyl-1-
methylquinolin-2(1H)-one 6a, 4-chloromethyl-1,6-dimeth-
ylquinolin-2(1H)-one 6b and 4-chloromethyl-6-methoxy-1-
methylquinolin-2(1H)-one 6c. Thisstrategywas followed due
to difficulties encountered when the reaction was carried out
directly between the N-methylated amines 2a–c and ethyl 4-
chloro-3-oxobutanoate, with no expected product being
Table 1 Yields, UV–vis and fluorescence data for coumarins 1a,b,
quinolin-2(1H)-ones 6a–d and fluorescent bioconjugates 7a,b and 8a–
d in absolute ethanol
Compound UV–vis
Fluorescence
Yield kmax
log e kem UF
(nm)
Stokes’ shift
(nm)
(%)
(nm)
1a
1b
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
6d
7a
7b
8a
8b
8c
8d
90
92
66
77
71
74
89
62
67
84
97
79
82
52
58
84
70
91
56
62
72
314
320
329
337
351
330
338
341
328
335
352
333
343
358
343
317
316
330
339
354
341
3.83
3.61
3.62
3.49
3.90
3.78
3.72
3.69
3.72
3.51
3.60
3.78
3.48
3.38
3.61
3.92
3.58
3.68
3.71
3.70
3.86
397
384
383
377
400
404
389
395
384
388
410
386
396
408
423
397
401
385
396
418
414
0.03 83
0.02 64
0.13 54
0.12 40
0.05 49
0.11 74
0.06 51
0.02 54
0.14 56
0.13 53
0.06 58
0.05 53
0.05 53
0.01 50
0.02 80
0.03 80
0.02 85
0.10 55
0.07 57
0.05 64
0.01 73
obtained.
4-Chloromethyl-1,6-dimethyl-5-nitroquinolin-2
(1H)-one 6d was obtained by nitration of compound 6b
(Scheme 1). The introduction of the nitro group will later on
allow the design of an alternative bifunctional heterocyclic
analogue of the well-known o-nitrobenzyl photocleavable
protecting group, by subsequent transformation of the adja-
cent methyl group. The chloromethyl derivatives were
obtained in good yields (Table 1) and characterised by IR, 1H
and 13C NMR spectroscopy and mass spectrometry.
Given the need for protecting groups in synthetic strat-
egies involving bifunctional molecules, such as amino
acids, as well as the interest in photocleavable protecting
groups, we evaluated the behaviour of the above hetero-
cycles as photocleavable protecting groups for the car-
boxylic function. Thus, chloromethyl coumarins 1a,b and
quinolones 6a–d were used in the preparation of sev-
eral fluorescent bioconjugates using phenylalanine as a
model, via coupling of N-benzyloxycarbonyl-phenylala-
nine (Z-Phe-OH) in DMF, at room temperature, in the
presence of potassium fluoride (Tjoeng and Heavner 1981).
The corresponding phenylalanine conjugates 7a,b and
8a–d (Scheme 2; Table 1) were obtained in good to
excellent yields (56–91%) and fully characterised by the
usual analytical techniques.
comparison with the starting heterocycles, for the deter-
mination of the parameters needed for monitorisation
during photolysis. As they are novel compounds and for the
sake of comprehensiveness, the UV–vis and fluorescence
data of the synthesized compounds was collected and is
presented in Table 1.
The UV–vis absorption and emission spectra of degas-
sed 1 9 10-5 to 2 9 10-5 M solutions in absolute ethanol
of all tags and precursors (1a,b, 3a–c, 4a–c, 5a–c and
6a–d) and conjugates 7a,b and 8a–d were measured,
absorption and emission maxima, molar absorptivities and
fluorescence quantum yields (UF) are reported (Table 1).
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 fluo-
rescence standard was excited at the wavelengths of max-
imum absorption found for each of the compounds to be
tested and in all fluorimetric measurements, the absorbance
of the solution did not exceed 0.1.
The IR spectra of conjugates 7a,b and 8a–d showed
bands due to stretching vibrations of the ester carbonyl
group of the fluorophore-amino acid linkage from 1,736 to
.
1,755 cm-1 1H NMR spectra showed signals of the
phenylalanine a-CH (d 4.18–4.80 ppm), as well as the
characteristic protons of the fluorophore methylene group
(d 5.21–5.45 ppm). The confirmation of the presence of the
newly formed ester bond was also supported by 13C NMR
spectra signals of the carbonyl group, which were found
between d 170.57 and 171.40 ppm.
By comparison of the data of coumarin conjugates 7a,b
with the corresponding precursors 1a,b, it was found that
the fluorescence quantum yield did not vary after covalent
linkage to the amino acid. Regarding the quinolone
Considering that the present work involves the evalua-
tion of new quinolone derivatives as photocleavable pro-
tecting groups, we carried out the photophysical
characterisation to study the properties of the conjugates in
123

708
A. S. C. Fonseca et al.
Scheme 2 Synthesis of
phenylalanine ester conjugates
7a,b and 8a–d
O
Ph
O
R
O
O
Ph
O
N
H
O
1a,b
7a R = H
7b R = NO2
Ph
O
O
OH
KF
Ph
O
N
H
DMF, rt
O
Ph
6a-d
N
O
O
Ph
O
N
H
O
R
8a R = R1 = H
R1
8b R = H, R1 = CH3
8c R = H, R1 = OCH3
8d R = NO2, R1 = CH3
precursors, the fluorescence quantum yields of the meth-
time represents the time necessary for the consumption of
the starting materials until less than 5% of the initial area
was detected (Table 2).
ylated compounds 3a–c are similar to those of hydroxy-
the
methyl
derivatives
5a–c,
decreasing
for
chloromethylated derivatives 6a–d. The fluorescence
quantum yields for quinolone conjugates 8a–c revealed an
increase in their values, upon linkage of the quinolone
precursor to the amino acid. Considering the Stokes’ shifts
of all compounds, it was found that the data for fluoro-
phores in their free form and their conjugates are compa-
rable, varying from 50 to 85 nm.
For each compound, 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 correlation coeffi-
cients varying from 0.9874 to 0.9987 (Fig. 1). The corre-
sponding rate constants were calculated and are presented
in Table 2.
Photolysis studies of phenylalanine conjugates 7a,b
and 8a–d
In the present work, it is intended to assess, in a pre-
liminary approach, the potential for the applicability of
quinolone as a photocleavable protecting group, when
compared to the well-established coumarin. As such, con-
sidering the chromophores presented, definitive conclu-
sions about the influence of the substituents or their
position on the photocleavage properties cannot be drawn.
Taking into consideration the influence of the structure of
The evaluation of heterocycles 1a,b and 6a–d as photo-
cleavable protecting groups was carried out by photolysis
studies of the corresponding ester conjugates 7a,b and
8a–d under irradiation at different wavelengths. Solutions
of the mentioned compounds (1 9 10-4 M) in methanol/
HEPES buffer (80:20) solution were irradiated in a Ray-
onet RPR-100 reactor, at 254, 300, 350, and 419 nm, in
order to determine the most favourable cleavage condi-
tions. The solvent system was chosen based on previous
studies regarding different organic solvents with varying
aqueous content, which revealed that photocleavage was
faster in a mixture of methanol and HEPES buffer
(Fernandes et al. 2007). The course of the photocleavage
reaction was followed by reverse phase HPLC with UV
detection.
Table 2 Irradiation times (in min) and rate constants (k, in min-1
for the photolysis of bioconjugates 7a,b and 8a–d at different
wavelengths in methanol/HEPES buffer (80:20) solution
)
Compound 254 nm
300 nm
350 nm
419 nm
Irr
time
k
Irr
time
k
Irr
time
k
Irr
time
k
7a
7b
8a
8b
8c
8d
34
18
9
0.082 129
0.166 176
0.023 3,293 0.001
0.016 1,317 0.002
–
–
–
–
–
–
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 irradiation
0.321
1.279
8
2
0.384
1.665
69 0.043
2
4
0.754 193 0.015
0.6 9.347 34 0.086
0.035 144 0.021
0.8 3.227
1.2 2.274
25 0.122 85
–
–
123

Light-induced cleavage of model phenylalanine conjugates
709
Fig. 1 Plot of ln A versus
irradiation time for the
photolysis of bioconjugates 7a
(plus sign), 7b (open square), 8a
(open triangle), 8b (times
symbol), 8c (open diamond) and
8d (open circle) at 254 nm in
methanol/HEPES buffer (80:20)
solution
the heterocycle on the photocleavage rates, it was found
that the quinolone conjugates 8a–d displayed shorter irra-
diation times, in all wavelengths of irradiation studied,
when compared to the coumarin conjugates 7a,b. The
presence of a nitro group increased the irradiation times,
for compound 8d in all wavelengths. From this fact and
considering the results in Table 2 for compounds 8b or 8c
and 8d (with a nitro group), one might suggest the feasi-
bility of selective photodeprotection at 300 and 350 nm, if
both the quinolone precursors were used to protect a
bifunctional molecule through ester bonds. At 300 and
350 nm, the irradiation times for the nitro conjugate 8d
were ca. 40 times larger when compared to the irradiation
time for conjugate 8b. The same suggestion could be made
for the combination of a coumarin and a quinolone as
protecting groups, as can be seen by comparison of the
behaviour of coumarin conjugate 7a and quinolone con-
jugate 8b at 300 and 350 nm. Under irradiation at 350 nm,
there was a striking difference between the irradiation
times (the quinolone conjugate cleaved more than 800
times faster than the coumarin conjugate), which could
allow a selective photolysis.
methylquinoline 6b and methoxyquinoline 6c precursors
can be used as photoremovable protecting groups to be used
at 419 nm. These heterocycles, when linked through ester
bonds, could be selectively removed in the presence of other
protecting groups which cleave with much slower rates or
that are not affected by radiation of that wavelength. Pho-
tolysis at 419 nm can be considered the most suitable for
certain practical bioapplications involving caging strategies,
as it avoids cell damage due to short-wavelength light.
As reported before (Piloto et al. 2006b), the N-ben-
zyloxycarbonyl urethane-type blocking group was stable
in the tested conditions, no cleavage being detected at the
various wavelengths of irradiation. Even at 254 nm,
where all the chromophores present at the molecule
absorb, in the reported conditions (using a Rayonet
reactor) no tendency to form side products has been
detected, for example by cleavage of the N-protecting
group or any other rearrangement.
In addition to monitoring of the photolysis process
through HPLC/UV detection in the case of the quinolone
conjugates, the samples collected at different irradiation
times were also analysed by LC/MS. The obtained mass
spectra contained peaks that could be attributed to the
released Z-protected phenylalanine, the hydroxymethylated
quinolone precursor, as well as the methoxymethylated
quinolone. These data are in agreement with a proposed
mechanism (Singh and Khade 2005; Yamaji et al. 2009),
where either by homolytical or by heterolytical cleavage of
the ester bond, the observed cleavage products are
obtained: once the methylene carbocation is formed, it can
be attacked by a proper nucleophile (water or methanol)
yielding the above-mentioned products (Scheme 3).
Regarding irradiation times in the different wavelengths
studied, and accordingly to the lamp power, it was found
for the coumarin conjugates that shorter values were
obtained at 254 nm. For the quinolone conjugates, irradi-
ation at 254 and 300 nm resulted in similar irradiation
times for compounds 8a–c (0.8–9 min), with compounds
8b and 8c showing short irradiation times at 350 nm (0.6
and 4 min, respectively). Conjugate 8d showed the largest
irradiation times in all wavelengths of irradiation studied.
Considering the short irradiation time obtained for com-
pounds 8b and 8c at the studied wavelengths, photocleavage
at 419 nm was also tested. For compound 8b, a large
increase in the irradiation time was observed (193 min, ca.
3.5 h) and for compound 8c, the increase in the irradiation
time was moderate (34 min, ca 0.5 h). Once again, consid-
ering the possibility of selective photodeprotection, these
interesting results suggest that the corresponding
Furthermore, the release of Z-protected phenylalanine,
as the expected photolysis product, was also followed by
1H NMR in a methanol-d4/D2O (80:20) solution of con-
jugates 7 and 8 in a concentration from 5.4 9 10-3 to
9.1 9 10-3 M (ca. 54–91 times larger than the concen-
tration used of the above described experiments), which led
to the expected increase in the photolysis time for the
123

710
A. S. C. Fonseca et al.
homolytical
cleavage
(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-Phe-OH,
thus confirming the quantitative release of the amino acid.
Moreover, signals due to by-products related to the fluo-
CH2-Het
CH2-Het
Z-Phe-O
+
hυ
electron transfer
Z-Phe-OCH2-Het
heterolytical
cleavage
Z-Phe-O
+
1
rophore were also detected in the H NMR spectra.
H2O
MeOH
Het = quinolone
HO-CH2-Het
MeO-CH2-Het Z-Phe-OH
Conclusions
Scheme 3 Proposed mechanism for the formation of photolysis
products from quinolin-2(1H)-one conjugates 8a–d
Phenylalanine ester bioconjugates 7 and 8 were prepared in
good to excellent yields by using a simple synthetic method
involving chloromethylated coumarin 1 and quinolone 6
precursors and the carboxylic acid group of N-benzylox-
ycarbonyl-protected phenylalanine as model of bifunc-
tional compounds.
complete release of the amino acid. Upon irradiation at the
usual wavelengths, a new set of signals appeared, related to
phenylalanine a–CH and b–CH2, at d 4.44 and 2.95 ppm,
respectively, and a decrease of the benzylic-type CH2 of
the fluorophore was visible at d 5.44 ppm (see Fig. 2 for
conjugate 8b). After
a variable irradiation period
Fig. 2 1H NMR spectra in methanol-d4/D2O (80:20) of the photolysis of conjugate 8b (C = 8.8 9 10-3 M) at 350 nm: a before irradiation; b
after irradiation for 15 min; c after irradiation for 95 min; d Z-Phe-OH
123

Light-induced cleavage of model phenylalanine conjugates
711
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that can be considered useful for practical applications,
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Acknowledgments Thanks are due to the Foundation for Science
and Technology (FCT-Portugal) for financial support through project
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