Oxobenzo[f]benzopyrans as new fluorescent photolabile protecting groups for the carboxylic function

Article abstract of DOI:10.1016/j.tet.2006.09.085

The properties of three oxobenzo[f]benzopyrans as new fluorogenic photolabile protecting groups for the carboxylic function of?amino acids were studied. Fluorescent amino acid conjugates were efficiently prepared and characterised. Photodeprotection of these compounds was carried out by irradiation at 300, 350 and 419 nm, the most suitable wavelength being 350 nm, on account of short irradiation times and good deprotection yields.

Full text of DOI:10.1016/j.tet.2006.09.085

Tetrahedron 62 (2006) 11955–11962
Oxobenzo[f]benzopyrans as new fluorescent photolabile
protecting groups for the carboxylic function
*
Ana M. Piloto, Daniel Rovira, Susana P. G. Costa and M. Sameiro T. Gonc¸alves
´
Centro de Quımica, Universidade do Minho, Campus de Gualtar, P-4710-057 Braga, Portugal
Received 1 August 2006; revised 20 September 2006; accepted 21 September 2006
Available online 27 October 2006
Abstract—The properties of three oxobenzo[f]benzopyrans as new fluorogenic photolabile protecting groups for the carboxylic function
of amino acids were studied. Fluorescent amino acid conjugates were efficiently prepared and characterised. Photodeprotection of these
compounds was carried out by irradiation at 300, 350 and 419 nm, the most suitable wavelength being 350 nm, on account of short irradiation
times and good deprotection yields.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
derivatives have been reported as food additives, in cos-
metics, as optical brightening agents, disperse fluorescent
and laser dyes.10–12 In addition, these compounds have
also been suggested as photolabile protecting groups for
biomolecules,13–15 as well as other polycyclic aromatics
such as anthraquinone, phenanthrene and pyrene.16,17
In organic synthesis protecting groups are many times a
laborious necessity, as a convenient and efficient synthesis,
chemical stability towards different reagents and selective
removal is required.1 Photochemically releaseable groups
have become an important tool in organic synthesis, biotech-
nology and cell biology, because cleavage only requires
light, which is a very mild deprotection strategy that is usu-
ally orthogonal to chemical conditions, allowing the removal
of protecting groups in sensitive molecules, otherwise in-
compatible with acidic or basic treatment.2
Taking these facts into consideration together with our re-
search work related to the area of fluorescent heterocycles
synthesis and also amino acid labelling,18,19 we decided to
investigate the possibility of using oxobenzo[f]benzopyrans
as new fluorescent photocleavable protecting groups for the
carboxylic function of organic molecules. Using amino
acids as models, the synthesis and characterisation of
new fluorescent amino acid conjugates were carried out.
Absorption and emission properties of all compounds were
measured and the results showed that these conjugates ex-
hibited moderate to excellent fluorescence quantum yields
and Stokes’ shifts.
Fluorescent labelling allows easy and reliable detection of
target compounds, both qualitatively and quantitatively
with improved sensitivity and selectivity, and its application
is well reported in many areas including amino acid and pep-
tide chemistry.3 Fluorescent photolabile protecting groups
have advantages over other photolabile groups, because
they can act as temporary fluorescent labels, allowing the
visualisation of non-fluorescent systems, like most amino
acid residues, during the course of organic reactions.
Photocleavage of these fully protected amino acids was
achieved by using radiation of 300, 350 and 419 nm. The
consumption of starting materials as well as the formation
of the released amino acid was monitored by RP-HPLC
and kinetic data were also obtained.
2-Oxobenzopyrans, trivially named as coumarins, represent
one of the most widespread and interesting class of het-
erocyclic compounds. These oxygen heterocycles are the
structural units of natural products and many exhibit di-
verse biological acivity with applications in pharmaceuti-
cals, agrochemicals and insecticides.4–9 2-Oxobenzopyran
2. Results and discussion
Chloromethyl oxobenzo[ f ]benzopyrans 1a–c were prepared
through a Pechmann reaction of the corresponding 2-naph-
thol and its derivatives, and ethyl 4-chloroacetoacetate cata-
lysed by sulfuric acid, at room temperature in good yields.20
The fluorophores will be designated in this report by a three
letter code for simplicity of naming the various amino acid
Keywords: Benzopyrans; Photocleavable groups; Protecting groups; Tem-
porary labels; Fluorophores.
* Corresponding author. Tel.: +351 253 604 386; fax: +351 253 678 986;
e-mail: msameiro@quimica.uminho.pt
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.09.085

11956
A. M. Piloto et al. / Tetrahedron 62 (2006) 11955–11962
Table 1. Synthesis of compounds 3a–g
Compound Yield (%)
Z-Phe-Obb
fluorescent derivatives (1-methylene-3-oxo-3H-benzo[f]-
benzopyran, Obb, 9-hydroxy-1-methylene-3-oxo-3H-benzo-
[f]benzopyran, Obh and 9-methoxy-1-methylene-3-oxo-
3H-benzo[f]benzopyran, Obm).
Mp (ꢁC)
3a
3b
3c
3d
3e
3f
96
81
71
86
83
94
82
127.8–129.8
189.6–190.7
180.8–182.1
181.6–184.0
132.8–134.0
122.6–124.0
184.8–186.6
Z-Phe-Obh
Z-Phe-Obm
Z-Gly-Obm
Z-Ala-Obm
Z-Val-Obm
Tos-Phe-Obm
In order to investigate the linkage of compounds 1a–c to the
carboxylic function of a-amino acids by an ester bond and
to compare the influence of the substituent at the oxo-
benzo[f]benzopyrans, N-benzyloxycarbonyl-L-phenylala-
nine, Z-Phe-OH (2a) was chosen as model. Derivatisation
at the C-terminus of 2a with heterocycles 1a–c was carried
out with potassium fluoride, in DMF, at room temperature
(Scheme 1), yielding derivatives 3a–c.
3g
wavelength absorption maxima of all compounds were
located between 345 and 361 nm, with molar absorptivity
ranging from 10,174 to 14,125 Mꢀ1 cmꢀ1. The wavelengths
of maximum absorption obtained for these compounds
showed a bathochromic shiftwhencompared withotherpoly-
cyclic aromatic photocleavable groups, namely 7-methoxy-
coumarin-4-ylmethoxycarbonyl (Mmoc, lmax 343 nm),22
anthraquinon-2-ylmethoxycarbonyl(Aqmoc,lmax 327 nm),16
pyren-1-ylmethoxycarbonyl (Pmoc, lmax 323 nm)17 and phe-
nanthren-9-ylmethoxycarbonyl (Phmoc, lmax 297 nm).16
The wavelengths of maximum emission were found between
411 and 478 nm. Emission of compound 3c was batho-
chromically shifted when compared to 3a,b, the difference
being 67 (3c/3a) and 22 nm (3c/3b) due to the substituent.
Although there was not a significant variation in the maxi-
mum wavelengths of emission of compounds 1a–c in their
isolated or conjugated forms, they displayed low FF in their
isolated form (the highest value was 0.08, 1a), which
By comparing fluorescence data obtained for these deriva-
tives, which will be discussed later, it was concluded that
compound 1c was the most fluorogenic reagent. Thus, using
the same method reported above, heterocycle 1c was reacted
with N-benzyloxycarbonyl derivatives of glycine (2b),
alanine (2c) and valine (2d) and also to N-p-toluenosulfonyl-
phenylalanine (2e). After dry chromatography on silica gel,
the corresponding fluorescent derivatives 3a–g were ob-
tained as solid materials in good to excellent yields (71–
96%) (Table 1) and were characterised by elemental analysis
or high resolution mass spectrometry, IR, 1H and 13C NMR
spectroscopies.
The IR spectra of labelled amino acids showed bands due to
stretching vibrations of the carbonyl groups from 1757 to
1
1619 cmꢀ1. H NMR spectra showed signals of the amino
acid residues, such as a multiplet (d 4.29–4.82 ppm) or a dou-
blet (d 4.18 ppm, 3d) for the a-CH, in addition to the protons
of the heterocyclic moiety. In 13C NMR signals of the car-
bonyl function were found at d 155.58–156.90 ppm for the
carbamate, at d 159.84–160.10 ppm for C-3 of the hetero-
cycle and at d 169.4–172.31 ppm for the ester.
Table 2. UV–vis and fluorescence data of compounds 1aꢀc and 3aꢀg
Compound
UV
Fluorescence
Stokes’
shifta
a
lmax (3)b
a
lem
FF
1a Obb-Cl
1b Obh-Cl
352 (11,449) 418
361 (12,190) 462
354 (12,826) 472
345 (14,125) 411
360 (10,174) 456
347 (12,075) 478
347 (11,436) 471
348 (11,640) 477
348 (11,830) 478
0.08ꢂ0.01
66
0.02ꢂ0.002 101
Electronic absorption and emission spectra of 10ꢀ5–10ꢀ6 M
solutions of compounds 1a–c and 3a–g in degassed absolute
ethanol were measured; absorption and emission maxima,
molar absorptivities and fluorescence quantum yields (FF)
are also reported (Table 2). The FF were calculated using
9,10-diphenylanthracene asstandard(FF¼0.95 inethanol).21
For the FF determination, 9,10-diphenylanthracene was
excited at the wavelengths of maximum absorption found
for each one of the compounds to be tested. The longest
1c
Obm-Cl
0.03ꢂ0.004 118
3a Z-Phe-Obb
3b Z-Phe-Obh
3c Z-Phe-Obm
3d Z-Gly-Obm
0.42ꢂ0.01
0.13ꢂ0.01
0.59ꢂ0.02
0.70ꢂ0.01
0.66ꢂ0.01
0.58ꢂ0.02
0.53ꢂ0.01
66
96
131
124
129
130
128
3e
3f
Z-Ala-Obm
Z-Val-Obm
3g Tos-Phe-Obm 347 (12,883) 475
a
Unit: nm.
Unit: Mꢀ1 cmꢀ1
.
b
R
R
1
CH2-O-CO-CHR2-NHR
CH2-Cl
KF/DMF
rt
R1HN-CHR2-CO2H
+
2
O
O
O
3
O
1
a R = H, R1 = Z, R2 = CH2Ph
b R = OH, R1 = Z, R2 = CH2Ph
c R = OCH3, R1 = Z, R2 = CH2Ph
d R = OCH3, R1 = Z, R2 = H
a R1 = Z, R2 = CH2Ph
b R1 = Z, R2 = H
a R = H
b R = OH
c R1 = Z, R2 = CH3
c R = OCH3
d R1 = Z, R2 = CH(CH3)2
e R1 = Tos, R2 = CH2Ph
e R = OCH3, R1 = Z, R2 = CH3
f R = OCH3, R1 = Z, R2 = CH(CH3)2
g R = OCH3, R1 = Tos, R2 = CH2Ph
Z = Benzyloxycarbonyl
Tos = p-Toluenesulphonyl (tosyl)
Scheme 1. Synthesis of fluorescent conjugates 3a–g.

A. M. Piloto et al. / Tetrahedron 62 (2006) 11955–11962
11957
increased upon reaction with the amino acids. All labelled
amino acids 3a–g exhibited moderate to excellent quantum
yields (0.13<FF<0.70), compound 3b having the lowest
value probably due to the presence of H-bonds to the solvent,
and Stokes’ shift from 66 to 131 nm. By comparison of
FF of labelled N-benzyloxycarbonylphenylalanine 3a–c, it
was possible to see that derivative 3c exhibited the highest
value (0.59) and also the larger Stokes’shift (131 nm), which
may be related to the higher electron-donating character of
the methoxy substituent on the oxobenzopyran moiety 1c.
Considering these results, oxobenzo[f]benzopyrans are
promising candidates for fluorescent labelling. In Figure 1,
the fluorescence spectra of amino acid conjugates 3a, 3b,
3c and 3g are shown.
areas were determined by HPLC and were the average of
three runs.
When compounds 3a–c and 3g were irradiated at 350 nm,
the time necessary for the consumption of the starting
materials, until less than 5% of the initial area was detected,
varied from 4 to 22 min (Table 3).
At this wavelength, the time cause of the reaction for com-
pound 3b was similar to that of 350 nm, whereas compounds
3a and 3g were photolysed about three times and two times
faster, respectively; compound 3c showed a slower cleavage.
As expected, irradiation at 419 nm resulted in much longer
irradiation times for all compounds, with compound 3a
requiring a 29 h photolysis for the consumption of more
than 95% of the starting material.
Since our main purpose is the investigation of the potential
application of these fluorophores as photocleavable protect-
ing groups in organic synthesis, we decided to evaluate the
behaviour of the ester linkage between the fluorescent
heterocycles synthesised and the amino acids to photocleav-
age conditions.
At the same time, the study of the stability of Z-Phe-OH
(2a) and Tos-Phe-OH (2e) was carried out under the above
reported photolysis conditions. HPLC studies showed that
both N-blocking groups were stable to the tested conditions,
no cleavage being detected. These results supported the fact
that the disappearance of the starting materials (3a, 3b, 3c
and 3g) was associated with the cleavage of the ester linkage
between the fluorophore and the C-terminus of the amino
acid, as expected.
Fully protected phenylalanine derivatives, Z-Phe-Obb (3a),
Z-Phe-Obh (3b), Z-Phe-Obm (3c) and Tos-Phe-Obm (3g)
were used as representative models (Scheme 2). Solutions of
the mentioned compounds in acetonitrile (ca. 1ꢃ10ꢀ5 M)
were irradiated in a Rayonet RPR-100 reactor, at different
wavelengths. As it is desirable to have short irradiation times
at the highest irradiation wavelength possible if future bio-
applications are to be considered, photolysis was carried
out at 300, 350 and 419 nm, in order to determine the best
cleavage conditions. The cleavage at different wavelengths
was followed by reverse phase HPLC–UV detection.
The formation of N-protected phenylalanine, as the expected
photolysis product, was also followed by RP-HPLC. The
yield of formation of compounds 2a or 2e was calculated
on the basis of a calibration curve (concentration versus
peak area), which was plotted with solutions of these phenyl-
alanine derivatives of known concentration in acetonitrile.
In case of compound 3g, the photorelease yield of the
expected product was 73% (300 and 419 nm) and 82%
(350 nm). N-Protected phenylalanine 2a was obtained from
the photocleavage of compounds 3a–c in yields ranging
The plots of peak area versus irradiation time were obtained
for each compound, at the considered wavelengths. Peak
1.2
Table 3. Photolysis data of compounds 3a–c and 3g
1.0
3c
3a
Compound
300 nm
Releasec Irr
350 nm
Releasec Irr
419 nm
Releasec
0.8
Irr
0.6
3b
timea
timea
timeb
3g
0.4
3a Z-Phe-Obb
3b Z-Phe-Obh
3c Z-Phe-Obm
3g Tos-Phe-Obm
7
4
12
4
79
82
75
73
22
4
8
80
90
81
82
29
2
8
78
92
86
73
0.2
0.0
350
8
9
400
450
500
550
600
a
Irradiation time (min).
Irradiation time (h).
Yield (%) of the released amino acid as determined by HPLC.
Wavelength (nm)
b
c
Figure 1. Normalised fluorescence spectra of compounds 3a–c and 3g.
H2C
R
O
C
C
H
NHR1
CH2
H2C
O
hν
R1HN
C
H
CO2H
O
+ side products
acetonitrile
O
3a R = H, R1 = Z, b R = OH, R1 = Z
c R = OMe, R1 = Z, g R = OCH3, R1 = Tos
2 a R1 = Z
e R1 = Tos
Scheme 2. Photocleavage of compounds 3a–c and 3g.

11958
A. M. Piloto et al. / Tetrahedron 62 (2006) 11955–11962
12
11
10
9
from 75 to 92%, the highest value in the case of compound 3b
at 419 nm.
3a
3b
3c
3g
From the obtained data, it was possible to see that the most
suitable wavelength of irradiation was 350 nm for com-
pounds 3b, 3c and 3g. Although in the case of compound
3a the irradiation time was longer at this wavelength, it is
preferable to avoid the use of shorter wavelengths of irradi-
ation. The results also indicated an influence of the substitu-
ent at position 9 of the heterocycle, as the substituted
compounds 3b, 3c and 3g required shorter irradiation times
for equal percentage of consumption of the starting material,
which was more evident when the wavelength of irradiation
was 350 or 419 nm.
8
7
6
0
5
10
15
20
25
Irradiation time (min)
Figure 2. Plot of ln Aversus irradiation time at 350 nm for compounds 3a–c
and 3g.
Based on HPLC data, the kinetic study of the photocleavage
reactions was also carried out. For each compound, the plot
of ln A versus irradiation time showed a linear correlation
for the decrease of the starting material, which suggested
a first order reaction. The observed values were calculated
by the linear least squares methodology for a straight line
(Table 4). Figure 2 summarises the behaviour of conjugates
3a–c and 3g at 350 nm.
Table 5. Stability tests/chemical cleavage of Tos-Phe-Obm (3g)
Cleavage
Time (h)
Yield (%)
Tos-Phe-Obm Tos-Phe-OH
(3g)
(2e)
Pd/C/1,4-cyclohexadiene
TFA (rt)
TFA (reflux)
6 M HCl (rt)
6 M HCl (reflux)
aq HBr (rt)
aq HBr (reflux)
HBr/CH3CO2H (rt)c
HBr/CH3CO2H (reflux)c
Mg/MeOH
6
2.5
8.5
4.5
17
4.5
5
4.5
5
3
36
100
100
100
83
100
—
90
11a,b
—
—
—
—
—
From these results, it was possible to confirm that the wave-
length of irradiation influenced the rate of the photocleav-
age, 350 nm being the selected wavelength on account of
the short irradiation times and the potentially less damaging
effect on biological systems. The substituent at the 9-posi-
tion of the fluorophore was also important in this process,
the presence of a hydroxyl group leading to a reduction of
the irradiation time.
57a
—
—
—
—
68a
56a
100
1 M NaOH
9
a
Yield of isolated product by dry chromatography.
Obm-H (4) was also obtained (25%).
HBr/CH3CO2H (45% m/v).
b
c
Although the main purpose of this work was to study the
suitability of oxobenzo[f]benzopyrans as photocleavable
groups for the carboxylic acid function of bifunctional
molecules such as amino acids, we also considered their
behaviour towards classical chemical cleavage. Therefore,
stability tests were carried out using fluorescent N-p-tolue-
nosulfonylphenylalanine, Tos-Phe-Obm (3g) as model.
Compound 3g was submitted to similar conditions to those
usually required for cleavage of protecting groups during
peptide synthesis, such us catalytic hydrogenation (Pd/C/
1,4-cyclohexadiene), acidolysis at room temperature and
reflux (TFA, 6 M HCl, aqueous HBr and HBr in acetic
acid),23 reduction with metals (Mg/MeOH)24 and alkaline
hydrolysis (1 M NaOH) (Table 5).
1-methyl-9-methoxy-3-oxo-3H-benzo[f]benzopyran (Obm-
H) (4) being isolated in low yields.
Compound 3g was stable under acidolysis conditions, at
room temperature, and was quantitatively recovered (TFA,
6 M HCl and aqueous HBr) or in 90% yield (HBr in acetic
acid). Considering acidolysis at reflux, the fully protected
fluorescent conjugate (3g) was stable in TFA (9 h, 100% re-
covery) and 6 M HCl (17 h, 83% recovery). However, in
HBr at reflux (5 h), cleavage of the ester linkage between
the fluorophore and the amino acid occurred, the expected
product 2e being isolated in moderate yields (57%, aqueous
HBr and 68%, HBr in acetic acid).
The results showed that under catalytic hydrogenation con-
jugate 3g was cleaved, compounds Tos-Phe-OH (2e) and
Reaction of compound 3g with magnesium gave Tos-Phe-
OH (2e) in 56%. The stability of tosyl group to this metal
was verified with studies of the behaviour of Tos-Phe-OH
(2e) in the same experimental conditions, which confirmed
that it was stable, being recovered quantitatively. As ex-
pected, cleavage of the ester bond was achieved by treatment
with base (1 M NaOH), resulting in quantitative isolation of
compound 2e.
Table 4. Kinetic data of the photolysis studies of compounds 3a–c and 3g
Compound
300 nm
350 nm
419 nm
ka
Rc
ka
Rc
kb
Rc
3a Z-Phe-Obb
3b Z-Phe-Obh
3c Z-Phe-Obm
0.4587 0.9965 0.1175 0.9556 0.1063 0.9965
0.9416 0.9983 0.8310 0.9940 1.6883 0.9893
0.2569 0.9998 0.3684 0.9771 0.3841 0.9993
From the results obtained in these chemical cleavage tests, it
was possible to conclude that fluorophore 1c had the appro-
priate behaviour to be considered also a suitable conven-
tional protecting group for the carboxylic function of
amino acids, in peptide synthesis. All labelled compounds
were stable in prolonged storage at room temperature.
3g Tos-Phe-Obm 0.8159 0.9980 0.4092 0.9992 0.3353 0.9995
Rate constant (minꢀ1).
a
Rate constant (hꢀ1).
Correlation coefficient.
b
c

A. M. Piloto et al. / Tetrahedron 62 (2006) 11955–11962
11959
3. Conclusions
spectrometry analyses were performed at the C.A.C.T.I.—
Unidad de Espectrometria de Masas of the University of
Vigo, Spain, on a Hewlett Packard 5989 A spectrometer
for low resolution spectra and a Autospec M spectrometer
for high resolution mass spectra. Elemental analyses were
carried out on a Leco CHNS 932 instrument. Fluorescence
spectra were collected using a Spex Fluorolog 1680
Spectrometer.
A series of fluorescent amino acid conjugates were synthes-
ised in excellent yields by a straightforward procedure, be-
tween a chloromethylated fluorophore and the C-terminus
of several N-protected amino acids, through an ester bond.
The photophysical properties of these conjugates showed
that oxobenzo[f]benzopyrans are good fluorogenic reagents
for amino acid and peptide chemistry.
4.1.1. N-(Benzyloxycarbonyl) phenylalanine (3-oxo-3H-
benzo[f]benzopyran-1-yl) methyl ester, Z-Phe-Obb (3a).
To a solution of 1-chloromethyl-3-oxo-3H-benzo[f]benzo-
pyran, Obb-Cl (1a) (0.106 g, 4.30ꢃ10ꢀ4 mol) in DMF
(1.5 mL), potassium fluoride (0.076 g, 1.30ꢃ10ꢀ3 mol)
and Z-Phe-OH (2a) were added with stirring at room temper-
ature. The reaction mixture was maintained in these condi-
tions for 25 h and monitored by TLC (ethyl acetate/
n-hexane, 4:6). The precipitate was filtered and the remain-
ing solution was evaporated until dryness. Purification of
the residue by dry chromatography using ethyl acetate/
n-hexane, 3:7 as the eluent, followed by recrystallisation
from ethyl acetate/n-hexane, gave Z-Phe-Obb (3a) as a white
solid (0.209 g, 96%). Mp¼127.8–129.8 ꢁC. TLC (ethyl
acetate/n-hexane, 4:6): Rf¼0.58. 1H NMR (CDCl3,
300 MHz): d¼3.17 (d, J¼6.3 Hz, 2H, b-CH2 Phe), 4.62–
4.82 (m, 1H, a-CH Phe), 5.02–5.20 (m, 2H, CH2 Z), 5.30
(d, J¼7.8 Hz, 1H, a-NH Phe), 5.60–5.76 (m, 2H, CH2),
6.54 (s, 1H, H-2), 7.10–7.18 (m, 2H, 2ꢃAr-H Phe), 7.20–
7.30 (m, 3H, 3ꢃAr-H Phe), 7.32–7.40 (m, 5H, 5ꢃAr-H Z),
7.49 (d, J¼8.7 Hz, 1H, H-5), 7.59 (t, J¼6.9 Hz, 1H, H-8),
7.67 (dt, J¼8.3 and 1.5 Hz, 1H, H-9), 7.94 (dd, J¼8.0 and
1.2 Hz, 1H, H-7), 8.01 (d, J¼9.0 Hz, 1H, H-6), 8.08 (d,
J¼8.4 Hz, 1H, H-10). 13C NMR (CDCl3, 75.4 MHz):
dC¼38.10 (b-CH2 Phe), 55.17 (a-CH Phe), 64.94 (CH2),
67.22 (CH2 Z), 112.44 (C-4b), 113.78 (C-2), 117.78 (C-5),
124.54 (C-10), 125.74 (C-8), 127.44 (C-4 Phe), 128.18
(C-4 Z), 128.23 (C-3 and C-5 Phe), 128.49 (C-2 and C-6
Z), 128.61 (C-9), 128.80 (C-3 and C-5 Z), 128.97 (C-6b),
129.04 (C-2 and C-6 Phe), 129.92 (C-7), 131.24 (C-6a),
134.11 (C-6), 135.11 (C-1 Phe), 135.95 (C-1 Z), 149.85
(C-1), 154.86 (C-4a), 155.69 (CONH), 159.84 (C-3),
171.10 (CO2CH3). IR (KBr 1%, cmꢀ1): n¼3287, 3028,
2963, 2918, 2848, 1740, 1728, 1686, 1553, 1532, 1496,
1455, 1413, 1340, 1290, 1258, 1207, 1198, 1166, 1052,
1019, 822, 803. UV–vis (ethanol, nm): lmax (3)¼345
(14,125 Mꢀ1 cmꢀ1). Anal. Calcd for C31H25NO6 (507.52):
C, 73.36; H, 4.97; N, 2.76. Found: C, 73.28; H, 4.98; N, 2.85.
The photocleavage studies of the fluorescent phenylalanine
derivatives with the three oxobenzo[f]benzopyrans showed
that the rate of cleavage of the ester bond depended on the
wavelength of irradiation as well as on the substituent at
the 9-position of the heterocycle. The most suitable wave-
length was 350 nm, allowing short irradiation times, which
are convenient for possible future biological applications,
for example, as caging groups.
In the absence of radiation, we also confirmed that these
groups were efficiently chemically cleavable by soft alkaline
hydrolysis or in moderate yields by acidolysis with HBr at
reflux or with magnesium.
The results obtained, mainly their photophysical properties,
allowed us to conclude that the considered functionalised
heterocycles are potential candidates as fluorescent labels
for biomolecules. Considering the efficient derivatisation
reactions and also the good results of the photocleavage pro-
cess, 1-chloromethyl-3-oxo-3H-benzo[f]benzopyrans 1a–c
could be used as fluorescent photocleavable protecting
groups in organic synthesis. In addition, they can also be
used as conventional protecting groups, quantitatively cleav-
able by basic hydrolysis (1 M NaOH, room temperature).
Bearing in mind the properties displayed by these com-
pounds, further investigations into their applicability in bio-
logical systems, for example, as caging groups, will be
carried out in the near future.
4. Experimental
4.1. General
All melting points were measured on a Gallenkamp melting
point apparatus and were 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 de-
termined on a Perkin Elmer FTIR-1600 using KBr discs or
Nujol. UV–vis spectra were run on a Hitachi U-2000 spec-
trophotometer. 1H NMR spectra were recorded on a Varian
300 spectrometer in CDCl3 or DMSO-d6 at 300 MHz at
25 ꢁC. All chemical shifts are given in parts per million using
dH Me4Si¼0 ppm as reference and J values are given in
hertz. 13C NMR spectra were run in the same instrument
at 75.4 MHz using the solvent peak as internal reference.
Assignments were made by comparison of chemical shifts,
peak multiplicities and J values and were supported by spin
decoupling-double resonance and bidimensional hetero-
nuclear correlation HMBC and HMQC techniques. Mass
4.1.2. N-(Benzyloxycarbonyl) phenylalanine (9-hydroxy-
3-oxo-3H-benzo[ f]benzopyran-1-yl) methyl ester,
Z-Phe-Obh (3b). The product of reaction of 1-chloromethyl-
9-hydroxy-3-oxo-3H-benzo[f]benzopyran, Obh-Cl (1b)
(0.100 g, 3.84ꢃ10ꢀ4 mol), with Z-Phe-OH (0.115 g,
3.84ꢃ10ꢀ4 mol) (2a) was chromatographed using ethyl
acetate/n-hexane, 3:7 as eluent, to give compound Z-Phe-
Obh (3b) as a white solid (0.163 g, 81%). Mp¼189.6–
1
190.7 ꢁC. TLC (acetate/n-hexane, 4:6): Rf¼0.31. H NMR
(CDCl3, 300 MHz): d¼3.00–3.10 (m, 2H, b-CH2 Phe),
4.40–4.54 (m, 1H, a-CH Phe), 4.95 (d, J¼12.0 Hz, 1H,
CH2), 5.10–5.20 (m, 2H, CH2 Z), 5.36 (d, J¼5.1 Hz,
1H, a-NH Phe), 5.48 (d, J¼11.7 Hz, 1H, CH2), 6.26 (s,
1H, H-2), 7.14–7.24 (m, 2H, H-8 and 1ꢃAr-H Phe), 7.28
(d, J¼8.7 Hz, 3H, H-5 and 2ꢃAr-H Phe), 7.32–7.42

11960
A. M. Piloto et al. / Tetrahedron 62 (2006) 11955–11962
(m, 7H, 2ꢃAr-H Phe and 5ꢃAr-H Z), 7.61 (d, J¼1.8 Hz, 1H,
H-10), 7.80 (d, J¼8.7 Hz, 1H, H-7), 7.91 (d, J¼9.0 Hz, 1H,
H-6), 8.44 (s, 1H, OH). 13C NMR (CDCl3, 75.4 MHz):
dC¼37.05 (b-CH2 Phe), 56.26 (a-CH Phe), 66.66 (CH2),
68.26 (CH2 Z), 107.54 (C-10), 112.30 (C-4b), 114.39
(C-5), 117.79 (C-8), 118.68 (C-2), 125.93 (C-6a), 127.87
(1ꢃAr-C Phe), 128.39 (1ꢃAr-C Z), 128.65 (2ꢃAr-C Z),
128.96 (2ꢃAr-C Phe), 129.07 (2ꢃAr-C Z), 130.61 (C-6b),
131.50 (C-7), 134.15 (2ꢃAr-C Phe), 134.61 (C-6), 134.80
(C-1 Phe and C-1 Z), 148.10 (C-1), 155.78 (C-4a), 156.86
(CONH), 157.59 (C-9), 160.07 (C-3), 171.60 (CO2CH2).
IR (Nujol, cmꢀ1): n¼3346, 3299, 2954, 2924, 2854, 1744,
1710, 1685, 1622, 1553, 1538, 1463, 1456, 1366, 1333,
1290, 1253, 1232, 1215, 1195, 1163, 1140, 1048, 1014,
987, 963, 883. UV–vis (ethanol, nm): lmax (3)¼360
(10,174 Mꢀ1 cmꢀ1). HRMS (EI): calcd for C31H25NO7
[M+]: 523.1631; found: 523.1611.
1H, H-7), 7.93 (d, J¼8.7 Hz, 1H, H-6). 13C NMR (CDCl3,
75.4 MHz): d¼42.79 (CH2 Gly), 55.47 (OCH3), 64.84
(CH2), 67.36 (CH2 Z), 105.54 (C-10), 111.77 (C-4b),
112.97 (C-2), 115.31 (C-5), 116.71 (C-8), 126.36 (C-6a),
128.17 (Ar-C Z), 128.28 (2ꢃAr-C Z), 128.54 (2ꢃAr-C Z),
130.52 (C-6b), 131.40 (C-7), 133.91 (C-6), 135.95 (C-1
Z), 150.20 (C-1), 155.59 (C-4a), 156.34 (CONH), 159.73
(C-9), 160.13 (C-3), 169.43 (CO2CH2). IR (Nujol, cmꢀ1):
n¼3413, 2954, 2924, 2854, 1757, 1721, 1625, 1553, 1516,
1401, 1368, 1341, 1272, 1232, 1170, 1054, 1016. UV–vis
(ethanol, nm): lmax (3)¼347 (11,436 Mꢀ1 cmꢀ1). Anal.
Calcd for C25H21NO7 (447.43): C, 67.67; H, 5.02; N, 3.03.
Found: C, 67.40; H, 5.01; N, 3.06.
4.1.5. N-(Benzyloxycarbonyl) alanine (9-methoxy-3-oxo-
3H-benzo[f]benzopyran-1-yl) methyl ester, Z-Ala-Obm
(3e). The product of reaction of Obm-Cl (1c) (0.201 g,
7.32ꢃ10ꢀ4 mol), with Z-Ala-OH (2c) (0.183 g, 8.2ꢃ
10ꢀ4 mol) was chromatographed using ethyl acetate/
n-hexane mixtures of increased polarity as the eluent, to
give compound Z-Ala-Obm (3e) as a white solid (0.280 g,
83%). Mp¼132.8–134.0 ꢁC. TLC (acetate/n-hexane, 1:1):
Rf¼0.45. 1H NMR (CDCl3, 300 MHz): d¼1.52 (d,
J¼7.2 Hz, 3H, b-CH3 Ala), 3.97 (s, 3H, OCH3), 4.48–4.62
(m, 1H, a-CH Ala), 5.06–5.20 (m, 2H, CH2 Z), 5.26 (d,
J¼7.2 Hz, 1H, a-NH Ala), 5.66–5.87 (m, 2H, CH2), 6.67
(s, 1H, H-2), 7.24 (dd, J¼9.0 and 2.1 Hz, 1H, H-8), 7.30–
7.41 (m, 6H, H-5 and 5ꢃAr-H), 7.45 (s, 1H, H-10), 7.86
(d, J¼9.0 Hz, 1H, H-7), 7.95 (d, J¼9.0 Hz, 1H, H-6).
13C NMR (CDCl3, 75.4 MHz): dC¼18.14 (b-CH3 Ala),
49.74 (a-CH Ala), 55.42 (OCH3), 64.85 (CH2), 67.10
(CH2 Z), 105.62 (C-10), 111.66 (C-4b), 112.81 (C-2),
115.21 (C-5), 116.52 (C-8), 126.26 (C-6a), 128.12 (1ꢃ
Ar-C Z), 128.17 (1ꢃAr-C Z), 128.46 (1ꢃAr-C Z), 130.44
(C-6b), 131.32 (C-7), 133.79 (C-6), 135.98 (C-1 Z),
150.24 (C-1), 155.48 (C-4a), 155.66 (CONH), 159.63
(C-9), 160.05 (C-3), 172.31 (CO2CH2). IR (Nujol, cmꢀ1):
n¼3422, 3335, 3065, 2958, 2933, 1732, 1719, 1619, 1543,
1518, 1449, 1418, 1355, 1331, 1249, 1230, 1211, 1168,
1105, 1067, 1024. UV–vis (ethanol, nm): lmax (3)¼348
(11,640 Mꢀ1 cmꢀ1). Anal. Calcd for C26H23NO7 (461.45):
C, 67.67; H, 5.02; N, 3.03. Found: C, 67.40; H, 5.01; N, 3.06.
4.1.3. N-(Benzyloxycarbonyl) phenylalanine (9-methoxy-
3-oxo-3H-benzo[ f]benzopyran-1-yl) methyl ester,
Z-Phe-Obm (3c). The product of reaction of 1-chloro-
methyl-9-methoxy-3-oxo-3H-benzo[f]benzopyran, Obm-Cl
(1c) (0.104 g, 3.8ꢃ10ꢀ4 mol), with Z-Phe-OH (2b)
(0.113 g, 3.8ꢃ10ꢀ4 mol) was chromatographed using ethyl
acetate/n-hexane, 3:7 as the eluent, to give compound
Z-Phe-Obm (3c) as a yellow solid (0.145 g, 71%).
Mp¼180.8–182.1 ꢁC. TLC (acetate/n-hexane, 3:7): Rf¼
1
0.45. H NMR (CDCl3, 300 MHz): d¼3.16 (d, J¼6.6 Hz,
2H, b-CH2 Phe), 3.95 (s, 3H, OCH3), 4.72–4.84 (m, 1H,
a-CH Phe), 5.02–5.16 (m, 2H, CH2 Z), 5.28 (d J¼8.1 Hz,
1H, a-NH Phe), 5.66 (s, 2H, CH2), 6.50 (s, 1H, H-2), 7.08–
7.16 (m, 2H, H-3 and H-5 Phe), 7.19–7.28 (m, 3H, H-2, H-
4 and H-6 Phe), 7.30–7.40 (m, 7H, H-5, H-8 and 5ꢃAr-H
Z), 7.42 (s, 1H, H-10), 7.85 (d, J¼9.0 Hz, 1H, H-7), 7.93
(d, J¼9.0 Hz, 1H, H-6). 13C NMR (CDCl3, 75.4 MHz):
dC¼38.21 (b-CH2 Phe), 55.17 (a-CH Phe), 55.44 (OCH3),
64.96 (CH2), 67.25 (CH2 Z), 105.58 (C-10), 111.82 (C-4b),
113.62 (C-2), 115.28 (C-5), 116.69 (C-8), 126.34 (C-6a),
127.45 (C-4 Phe), 128.19 (C-4 Z), 128.26 (C-3 and C-5
Phe), 128.51 (C-2 and C-6 Z), 128.80 (C-3 and C-5 Z),
129.03 (C-2 and C-6 Phe), 130.51 (C-6b), 131.34 (C-7),
133.81 (C-6), 135.07 (C-1 Phe), 135.95 (C-1 Z), 149.64
(C-1), 155.58 (C-4a and CONH), 159.71 (C-9), 160.00
(C-3), 171.16 (CO2CH2). IR (Nujol, cmꢀ1): n¼3285,
2954, 2925, 2854, 1746, 1664, 1630, 1549, 1463, 1409,
1378, 1366, 1275, 1248, 1233, 1201, 1183, 1104,
1086, 1038, 1021. UV–vis (ethanol, nm): lmax (3)¼347
(12,075 Mꢀ1 cmꢀ1). HRMS (EI): calcd for C32H27NO7
[M+]: 537.1788; found: 537.1798.
4.1.6. N-(Benzyloxycarbonyl) valine (9-methoxy-3-oxo-
3H-benzo[f]benzopyran-1-yl) methyl ester, Z-Val-Obm
(3f). The product of reaction of Obm-Cl (1c) (0.104 g,
3.8ꢃ10ꢀ4 mol), with Z-Val-OH (2d) (0.070 g, 2.8ꢃ
10ꢀ4 mol) was chromatographed using ethyl acetate/
n-hexane mixtures of increasing polarity as the eluent, to
give compound Z-Val-Obm (2f) as a white solid (0.129 g,
94%). Mp¼122.6–124.0 ꢁC. TLC (chloroform/methanol,
50:0.5): Rf¼0.48. 1H NMR (CDCl3, 300 MHz): d¼0.95 (d,
J¼7.2 Hz, 3H, g-CH3 Val), 1.04 (d, J¼6.9 Hz, 3H, g-CH3
Val), 2.20–2.35 (m, 1H, b-CH Val), 3.98 (s, 3H, OCH3),
4.40–4.50 (m, 1H, a-CH Val), 5.13 (s, 2H, CH2 Z), 5.25
(d, J¼8.4 Hz, 1H, a-NH Val), 5.76 (d, J¼3.9 Hz, 2H,
CH2), 6.70 (s, 1H, H-2), 7.25 (dd, J¼7.8 and 2.4 Hz, 1H,
H-8), 7.30–7.42 (m, 6H, H-5 and 5ꢃAr-H Z), 7.47 (s, 1H,
H-10), 7.86 (d, J¼9.0 Hz, 1H, H-7), 7.95 (d, J¼9.0 Hz,
1H, H-6). 13C NMR (CDCl3, 75.4 MHz): dC¼17.45
(g-CH3 Val), 19.17 (g-CH3 Val), 30.96 (b-CH Val), 55.43
(OCH3), 59.28 (a-CH Val), 64.81 (CH2), 67.25 (CH2 Z),
105.67 (C-10), 111.75 (C-4b), 113.08 (C-2), 115.25 (C-5),
4.1.4. N-(Benzyloxycarbonyl) glycine (9-methoxy-3-oxo-
3H-benzo[f]benzopyran-1-yl) methyl ester, Z-Gly-Obm
(3d). The product of reaction of Obm-Cl (1c) (0.100 g,
3.64ꢃ10ꢀ4 mol), with Z-Gly-OH (2b) (0.076 g, 3.64ꢃ
10ꢀ4 mol) was chromatographed using ethyl acetate/
n-hexane, 3:7 as the eluent, to give compound Z-Phe-Obm
(3c) as a yellowish solid (0.140 g, 86%). Mp¼181.6–
1
184.0 ꢁC. TLC (acetate/n-hexane, 4:6): Rf¼0.51. H NMR
(CDCl3, 300 MHz): d¼3.97 (s, 3H, OCH3), 4.18 (d,
J¼5.7 Hz, 2H, CH2 Gly), 5.16 (s, 2H, CH2 Z), 5.35 (br s,
1H, a-NH Gly), 5.77 (s, 2H, CH2), 6.66 (s, 1H, H-2), 7.24
(dd, J¼9.0 and 2.4 Hz, 1H, H-8), 7.30–7.40 (m, 6H, H-5
and 5ꢃAr-H Z), 7.42 (s, 1H, H-10), 7.84 (d, J¼9.0 Hz,

A. M. Piloto et al. / Tetrahedron 62 (2006) 11955–11962
11961
116.58 (C-8), 126.31 (C-6a), 128.17 (1ꢃAr-C Z), 128.22
(2ꢃAr-C Z), 128.50 (2ꢃAr-C Z), 130.49 (C-6b), 131.34
(C-7), 133.82 (C-6), 135.99 (C-1 Z), 150.18 (C-1), 155.54
(C-4a), 156.25 (CONH), 159.70 (C-9), 160.07 (C-3),
171.51 (CO2CH2). IR (KBr 1%, cmꢀ1) n¼3391, 2966,
2928, 1731, 1721, 1625, 1553, 1520, 1456, 1426,
1351, 1306, 1275, 1232, 1180, 1164, 1098, 1059, 1025.
UV–vis (ethanol, nm): lmax (3)¼348 (11,838 Mꢀ1 cmꢀ1).
HRMS (EI): calcd for C28H27NO7 [M+]: 489.1788; found:
489.1790.
flow 0.8 mL minꢀ1, tR 3.3 min; 2e, ldet 240 nm, flow
0.8 mL minꢀ1, tR 3.1 min), using eluent A for compounds
3a–c and 3g and eluent B for compounds 2a and 2e.
The yield of photorelease was calculated by comparison of
the HPLC trace (peak area) of the released amino acid
with the corresponding standard calibration curve (concen-
tration vs peak area).
4.3. Stability tests with Tos-Phe-Obm (3g)
4.1.7. N-(p-Toluenesulfonyl) phenylalanine (9-methoxy-
3-oxo-3H-benzo[f]benzopyran-1-yl) methyl ester, Tos-
Phe-Obm (3g). The product of reaction of Obm-Cl (1c)
(0.060 g, 2.18ꢃ10ꢀ4 mol), with Tos-Phe-OH (2e) (0.069 g,
2.18ꢃ10ꢀ4 mol) was chromatographed using ethyl acetate/
n-hexane, 3:7 as the eluent, to give compound Tos-Phe-
Obm (3g) as a yellow solid (0.100 g, 82%). Mp¼184.8–
4.3.1. Catalytic hydrogenation. A suspension of Tos-Phe-
Obm (3g) (2.90ꢃ10ꢀ2 g, 5.33ꢃ10ꢀ5 mol) in methanol
(1.0 mL) and 1,4-cyclohexadiene (1.35ꢃ10ꢀ2 mL, 1.40ꢃ
10ꢀ4 mol) was mixed with 10% palladium on charcoal cat-
alyst (1.05ꢃ10ꢀ2 g), and refluxed for 6 h with stirring. The
catalyst was filtered off and washed with methanol; the com-
bined liquids were then evaporated under reduced pressure
1
1
186.6 ꢁC. TLC (acetate/n-hexane, 4:6): Rf¼0.45. H NMR
affording the compound as a oily solid (0.185 g, 11%). H
(CDCl3, 300 MHz): d¼2.27 (s, 3H, CH3), 3.08 (d,
J¼6.9 Hz, 2H, b-CH2 Phe), 3.93 (3H, s, OCH3), 4.28–4.40
(m, 1H, a-CH Phe), 5.30–5.53 (m, 3H, CH2 and a-NH
Phe), 6.39 (s, 1H, H-2), 7.02–7.09 (m, 2H, H-3 and H-5
Phe), 7.13–7.19 (m, 5H, H-3 and H-5 Tos, H-2, H-4 and
H-6 Phe), 7.23 (dd, J¼8.7 and 2.4 Hz, 1H, H-8), 7.27–7.32
(m, 2H, H-5 and H-10), 7.61 (d, J¼8.4 Hz, 2H, H-2 and
H-6 Tos), 7.83 (d, J¼8.7 Hz, 1H, H-7), 7.90 (d, J¼9.0 Hz,
1H, H-6). 13C NMR (CDCl3, 75.4 MHz): dC¼21.30 (CH3
Tos), 39.24 (b-CH2 Phe), 55.45 (OCH3), 57.07 (a-CH
Phe), 64.88 (CH2), 105.71 (C-10), 111.64 (C-4b), 113.25
(C-2), 115.20 (C-5), 116.44 (C-8), 126.29 (C-6a), 127.10
(C-2 and C-6 Tos), 127.43 (C-4 Phe), 128.69 (C-3 and
C-5 Phe), 129.13 (C-2 and C-6 Phe), 129.60 (C-3 and
C-5 Tos), 130.35 (C-6b), 131.37 (C-7), 133.82 (C-6),
134.55 (C-1 Phe), 136.51 (C-1 Tos), 143.73 (C-4 Tos),
149.39 (C-1), 155.45 (C-4a), 159.66 (C-9), 159.98 (C-3),
170.55 (CO2CH2). IR (KBr 1%, cmꢀ1): n¼3434, 2921,
2846, 1707, 1625, 1550, 1506, 1443, 1349, 1224, 1161,
1080, 1011. UV–vis (ethanol, nm): lmax (3)¼347
(12,883 Mꢀ1 cmꢀ1). HRMS (EI): calcd for C31H27NO7S
[M+]: 557.1508; found: 557.1519.
NMR was well compared with Tos-Phe-OH (2e). 1-Methyl-
9-methoxy-3-oxo-3H-benzo[f]benzopyran, Obm-H (4)
(3.20ꢃ10ꢀ3 g, 25%) and starting material (3g) (1.07ꢃ
10ꢀ2 g, 36%) were also isolated.
4.3.1.1. 1-Methyl-9-methoxy-3-oxo-3H-benzo[f]benzo-
pyran, Obm-H (4). TLC (ethyl acetate/n-hexane, 6:4):
Rf¼0.63. 1H NMR (CDCl3, 300 MHz): d¼2.95 (s, 3H,
CH3), 3.98 (s, 3H, OCH3), 6.36 (s, 1H, H-2), 7.23 (dd,
J¼8.7 and 2.4 Hz, 1H, H-8), 7.34 (d, J¼8.7 Hz, 1H, H-5),
7.84 (d, J¼8.7 Hz, 1H, H-7), 7.91 (d, J¼9.0 Hz, 1H, H-6),
7.95 (d, J¼2.4 Hz, 1H, H-10). 13C NMR (CDCl3,
75.4 MHz): dC¼26.24 (CH3), 55.42 (OCH3), 106.35 (C-10),
113.77 (C-4b), 115.40 (C-5), 116.09 (C-2), 116.09 (C-8),
126.47 (C-6a), 131.10 (C-7), 131.73 (C-6b), 133.36 (C-6),
154.02 (C-1), 155.37 (C-4a), 159.11 (C-9), 160.47 (C-3). IR
(neat, cmꢀ1): n¼3414, 2918, 2848, 1716, 1623, 1552, 1515,
1455, 1356, 1261, 1228, 1093, 1018, 933 cmꢀ1. HRMS (EI):
calcd for C15H12O3 [M+]: 240.0786; found: 240.0777.
4.3.2. Acidolysis with trifluoracetic acid
(a) To the fully protected amino acid Tos-Phe-Obm (3g)
(3.00ꢃ10ꢀ2 g, 5.39ꢃ10ꢀ5 mol) were added 0.60 mL
of trifluoracetic acid with rapid stirring, at room temper-
ature, over 2.5 h. Evaporation under reduced pressure
gave a yellow solid (3.00ꢃ10ꢀ2 g, 100%). 1H NMR con-
firmed the structure of the starting material.
4.2. General photolysis procedure
A 1ꢃ10ꢀ5 M acetonitrile solution of the compound to be
tested (20 mL) was placed in a quartz tube and irradiated
in a Rayonet RPR-100 chamber reactor with 10 lamps of
different wavelength (300, 350 and 419 nm, 14 W each).
Aliquots were taken at regular intervals and analysed by
reversed phase HPLC using a Licrospher 100 RP18 (5 mm)
column and a system composed by a Jasco PU-980 pump,
a UV–vis Shimadzu SPD-GAV detector and a Shimadzu
C-RGA Chromatopac register. The eluent was acetonitrile/
water, 3:1 (eluent A) or acetonitrile/water, 3:1 with 0.1%
TFA (eluent B), previously filtered through a Milipore,
type HN 0.45 mm filter and degassed by ultra-sound for
30 min.
(b) A solution of Tos-Phe-Obm (3g) (2.80ꢃ10ꢀ2 g,
4.97ꢃ10ꢀ5 mol) in TFA (6 mL) was refluxed for 9 h.
Evaporation under reduced pressure gave a white-
greenish solid (2.80ꢃ10ꢀ2 g, 100%). 1H NMR con-
firmed the structure of the starting material.
4.3.3. Acidolysis with hydrochloric acid
(a) To the fully protected amino acid Tos-Phe-Obm (3g)
(2.10ꢃ10ꢀ2 g, 3.77ꢃ10ꢀ5 mol), was added 6 M HCl
(0.40 mL) under rapid stirring, at room temperature,
over 4.5 h. Evaporation under reduced pressure gave
a white solid (2.10ꢃ10ꢀ2 g; 100%). 1H NMR confirmed
the structure of the starting material.
The chromatograms were traced by detecting UVabsorption
(3a, ldet 347 nm, flow 1.2 mL minꢀ1, retention time—tR
4.9 min; 3b, ldet 360 nm, flow 0.8 mL minꢀ1, tR 5.2 min;
3c, ldet 347 nm, flow 1.2 mL minꢀ1, tR 5.1 min; 3d, ldet
347 nm, flow 1.0 mL minꢀ1, tR 5.5 min; 2a, ldet 240 nm,
(b) A solution of Tos-Phe-Obm (3g) (2.10ꢃ10ꢀ2 g;
3.77ꢃ10ꢀ5 mol) in 6 M HCl (0.40 mL) was refluxed

11962
A. M. Piloto et al. / Tetrahedron 62 (2006) 11955–11962
for 17 h. Evaporation under reduced pressure gave a
of the solvent, Tos-Phe-OH (2g) was obtained as an orange
1
1
white solid (1.74ꢃ10ꢀ3 g, 83%). H NMR confirmed
solid (4.10ꢃ10ꢀ2 g, 100%). H NMR compared well with
the structure of the starting material.
an original sample.
4.3.4. Acidolysis with hydrobromic acid
Acknowledgements
(a) To the fully protected amino acid Tos-Phe-Obm (3g)
(2.20ꢃ10ꢀ2 g, 3.91ꢃ10ꢀ5 mol) was added aqueous
HBr (0.2 mL), under rapid stirring over 4.5 h. Evapo-
ration under reduced pressure gave a white solid
(2.20ꢃ10ꢀ2 g, 100%). 1H NMR confirmed the structure
of the starting material.
~
We thank the Fundac¸ao para a Ciencia e Tecnologia (Portu-
gal) for its financial support to the Centro de Quımica (Uni-
versidade do Minho).
^
´
(b) A solution of Tos-Phe-Obm (3g) (2.20ꢃ10ꢀ2 g,
3.86ꢃ10ꢀ5 mol) in aqueous HBr (0.2 mL) was refluxed
for 5 h. Evaporation under reduced pressure, followed
by dry chromatography with ethyl acetate/n-hexane
mixtures of increasing polarity, gave Tos-Phe-OH (2e)
References and notes
1. Kocienski, P. J. Protecting Groups; Enders, D., Noyori, R.,
Trost, B. M., Eds.; Georg Thieme: Stuttgart, 1994.
2. Pelliccioli, A. P.; Wizz, J. Photochem. Photobiol. Sci. 2002,
1, 441.
3. Haugland, R. P. Handbook of Fluorescent Probes and Research
Products, 9th ed.; Molecular Probes: Eugene, OR, 2002.
4. Madhavan, G. R.; Balraju, V.; Mallesham, B.; Chaknabarti, R.;
Lohray, V. B. Bioorg. Med. Chem. Lett. 2003, 13, 2547.
5. Cravotto, G.; Nano, G. M.; Palmisano, G.; Tagliapietra, S.
Tetrahedron: Asymmetry 2001, 12, 707.
6. Wang, C. J.; Hsieh, Y. J.; Chu, C. Y.; Lin, Y. L.; Tseng, T. H.
Cancer Lett. 2002, 183, 163.
7. Kirkiacharian, S.; Thuy, D. T.; Sicsic, S.; Bakhchinian, R.;
Kurkjian, R.; Tonnaire, T. Il Farmaco 2002, 57, 703.
8. Oyamada, J.; Jia, C.; Fujiwara, Y.; Kitamura, T. Chem. Lett.
2002, 380.
1
as a yellow oil (6.80ꢃ10ꢀ3 g, 57%). H and 13C NMR
well compared with an original sample.
(c) To the fully protected amino acid Tos-Phe-Obm (3g)
(2.00ꢃ10ꢀ3 g, 3.59ꢃ10ꢀ5 mol), was added a solution
of HBr in CH3CO2H (45% m/v) (0.008 mL), under rapid
stirring over 4.5 h. Evaporation under reduced pressure
gave a yellowish white solid (1.80ꢃ10ꢀ2 g, 90%).
1H NMR confirmed the structure of the starting material.
(d) A solution of Tos-Phe-Obm (3g) 2.00ꢃ10ꢀ2 g;
3.59ꢃ10ꢀ5 mol), in a solution of HBr in CH3CO2H
(45% m/v) (0.008 mL), was refluxed for 5 h. Evapora-
tion under reduced pressure followed by dry chromato-
graphy ethyl acetate/n-hexane, mixtures of increasing
polarity, gave Tos-Phe-OH (2e) (7.80ꢃ10ꢀ3 g, 68%)
9. Murray, R. D. H.; Medez, J.; Brown, S. A. The Natural
Coumarins: Occurrence, Chemistry and Biochemistry; Wiley:
New York, 1982.
1
as a colourless oil. H NMR compared well with an
original sample.
10. Siegrist, A. E.; Hefti, H.; Meyer, H. R.; Schmidt, E. Rev. Prog.
Colour. 1987, 17, 39.
4.3.5. Reduction with Mg/MeOH
11. Valizadeh, H.; Shockravi, A. Tetrahedron Lett. 2005, 46, 3501
and cited references.
12. Christie, R. M.; Lui, C.-H. Dyes Pigments 1999, 42, 85 and
cited references.
13. Lu, M.; Fedoryak, O. D.; Moister, B. R.; Dore, T. M. Org. Lett.
2003, 5, 2119.
14. Susuki, A. Z.; Watanable, T.; Kawamoto, M.; Nishiyama, K.;
Yamashita, H.; Iwamura, M.; Furuta, T. Org. Lett. 2003, 5,
4867.
15. Furuta, T.; Wang, S. S.-H.; Dantzker, J. L.; Dore, T. M.; Bybee,
W. J.; Callaway, E. M.; Denk, W.; Tsien, R. Y. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 1193.
16. Furuta, T.; Hirayama, Y.; Iwamura, M. Org. Lett. 2001, 3, 1809.
17. Iwamura, M.; Hodota, C.; Ishibashi, M. Synlett 1991, 35.
18. Frade, V. H. J.; Gonc¸alves, M. S. T.; Moura, J. C. V. P.
Tetrahedron Lett. 2005, 46, 4949.
19. Piloto, A. M.; Fonseca, A. S. C.; Costa, S. P. G.; Gonc¸alves,
M. S. T. Tetrahedron 2006, 62, 9258.
20. Tao, Z.-F.; Qian, X.; Fan, M. Tetrahedron 1997, 53, 13329.
21. Morris, J. V.; Mahaney, M. A.; Huber, J. R. J. Phys. Chem.
1976, 80, 969.
(a) To a solution of Tos-Phe-Obm (2g) (6.00ꢃ10ꢀ2 g,
1.08ꢃ10ꢀ4 mol), in dry methanol (2 mL) magnesium
powder (3.90ꢃ10ꢀ2 g, 1.62ꢃ10ꢀ3 mol) was added and
the resulting mixture was sonicated for 3 h, at room tem-
perature. The process was monitored by TLC (ethyl
acetate/n-hexane, 6:4) until all compounds were reacted.
The reaction was quenched by addition of saturated
aqueous NH4Cl (4 mL) and extracted with ethyl acetate.
The organic layer was dried with MgSO4, concentrated
to dryness to give Tos-Phe-OH (2e) as an off-white oil
1
(1.20ꢃ10ꢀ2 g, 56%). H NMR compared well with an
original sample.
(b) Starting with Tos-Phe-OH (2e) (5.90ꢃ10ꢀ2 g, 1.88ꢃ
10ꢀ4 mol), in dry methanol (2 mL) magnesium powder
(4.60ꢃ10ꢀ2 g, 1.88ꢃ10ꢀ3 mol) was added and the
resulting mixture was sonicated for 3 h, at room temper-
ature. Treatment by following the procedure described
above gave a white solid (5.00ꢃ10ꢀ2 g, 84%). 1H
NMR confirmed the structure of the compound.
4.3.6. Alkaline hydrolysis. To the fully protected amino
acid Tos-Phe-Obm (3g) (3.10ꢃ10ꢀ2 g, 5.47ꢃ10ꢀ5 mol),
in 1,4-dioxane (2 mL) 1 M NaOH (0.220 mL, 2.19ꢃ
10ꢀ4 mol) was added at low temperature. The solution was
stirred at 0 ꢁC for 9 h and acidified to pH 3 with 1 M
KHSO4. After extraction with ethyl acetate and evaporation
22. Schade, B.; Hagen, V.; Schmidt, R.; Herbrich, R.; Krause, E.;
Eckardt, T.; Bendig, J. J. Org. Chem. 1999, 64, 9109.
23. Bodanszky, M.; Bodanszky, A. The Practice of Peptide
Synthesis; Springer: Berlin, 1984.
~
24. Maia, H. L. S.; Monteiro, L. S.; Sebastiao, J. Eur. J. Org. Chem.
2001, 1967.