Photocleavage studies of fluorescent amino acid conjugates bearing different types of linkages

Article abstract of DOI:10.1016/j.tet.2006.11.082

The synthesis and photochemistry of 3-oxo-3H-benzopyran derivatives linked through ester, anhydride, urethane and carbonate bonds to representative l-amino acids, at the amino and carboxylic acid groups at the main chain or the hydroxyl group at the side

Full text of DOI:10.1016/j.tet.2006.11.082

Tetrahedron 63 (2007) 1353–1359
Photocleavage studies of fluorescent amino acid conjugates
bearing different types of linkages
*
Andrea S. C. Fonseca, M. Sameiro T. Gonc¸alves and Susana P. G. Costa
Centro de Quꢀımica, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
Received 31 October 2006; revised 24 November 2006; accepted 30 November 2006
Available online 20 December 2006
Abstract—The synthesis and photochemistry of 3-oxo-3H-benzopyran derivatives linked through ester, anhydride, urethane and carbonate
bonds to representative L-amino acids, at the amino and carboxylic acid groups at the main chain or the hydroxyl group at the side chain, were
carried out. The stability to photolysis of the resulting conjugates was studied at different wavelengths of irradiation (254, 300 and 350 nm),
the anhydride and ester linkages being the most sensitive in the studied conditions.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
ketones, acids and amines,8–11 and in biological applications
as caging groups for nucleotides and amino acid neurotrans-
mitters.12–14
The fluorescent derivatisation of biomolecules with organic
fluorophores for analytical applications represents a steadily
growing field of research, especially through the develop-
ment of new fluorescent ligands. Fluorescence provides
higher sensitivity than the corresponding UV techniques,
thus overcoming problems of low detection limits and allow-
ing the easy monitoring of the course of organic reactions.
Examples are found in many diverse areas such as cell biol-
ogy and biochemistry, as fluorescent probes to investigate the
functionalityandactionofbiomoleculesinsidelivingcells,1,2
in combinatorial chemistry, to allow the rapid screening of
large libraries of compounds,3,4 and in analytical chemis-
try.5,6 In amino acid and peptide chemistry, appropriate fluo-
rophoric moieties are also used, with 3-oxo-3H-benzopyrans
(trivially named as coumarins) representing one of the most
popular fluorogenic reagents, due to their extended spectral
range, high fluorescence quantum yields, good photostability
and solubility in common solvents.7
Bearing in mind our recent research in the synthesis and
application of fluorescent heterocycles, namely their use
in amino acid chemistry as markers and as protecting
groups,15–17 in this work we investigated the possibility of
using 6-hydroxy and 6-methoxy-1-methylene-3-oxo-3H-
benzopyrans as versatile fluorescent photolabile protecting
groups for the amino, carboxyl and hydroxyl functions of
organic molecules through linkages of different nature.
Using amino acids as models, the synthesis and characterisa-
tion 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
exhibited moderate to high fluorescence quantum yields and
Stokes’ shifts.
Photocleavage of these fully protected amino acids was
achieved by using radiation of 254, 300 and 350 nm. The
course of the photolysis was followed by RP-HPLC and
kinetic data were also obtained.
Photolabile protecting groups are advantageous over con-
ventional blocking groups, because the deprotection strategy
only involves radiation and can be orthogonal to chemical
conditions. In addition to this quality, the use of fluorescent
moieties results in an increase in their importance specially
when used for the protection of colourless or nonfluorescent
systems. In recent reports, 3-oxo-3H-benzopyran derivatives
have been proposed as photolabile protecting groups for
a variety of functional groups such as alcohols, aldehydes,
2. Results and discussion
6-Hydroxy and 6-methoxy-1-chloromethyl-3-oxo-3H-benzo-
pyrans1a,bweresynthesisedbyaPechmannreaction, starting
from 1,3-dihydroxybenzene and 3-methoxyphenol, respec-
tively, and ethyl chloroacetoacetate, catalysed by sulfuric
acid at room temperature in good yields, by a known proce-
dure.18 6-Methoxy-1-hydroxymethyl-3-oxo-3H-benzopyran
1c was obtained by a similar procedure by the reaction of
3-methoxyphenol with ethyl acetoacetate. The methyl group
Keywords: 3-Oxo-3H-benzopyrans; Photocleavable protecting groups;
Photocleavage; Amino acids; Fluorophores.
* Corresponding author. Tel: +351 253 604054; fax: +351 253 678986;
e-mail: spc@quimica.uminho.pt
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.11.082

1354
A. S. C. Fonseca et al. / Tetrahedron 63 (2007) 1353–1359
CH2Ph
was oxidised to aldehyde, by reaction with selenium dioxide,
which was then reacted with sodium borohydride, affording
the hydroxymethyl group.19
O
Boc
O
O
2b
2c
2d
4a
N
H
Flu
Flu
O
O
O
Our purpose being to study the linkage of chloromethyl-
3-oxo-3H-benzopyrans 1a,b to the carboxylic function of
a-amino acids by an ester bond and to compare the influence
of the substituent at the oxobenzopyran, N-tert-butyloxy-
carbonyl-L-valine, Boc–Val–OH (2a) and N-tert-butyloxy-
carbonyl-L-phenylalanine, Boc–Phe–OH (2b) were chosen
as models. Derivatisation at the C-terminus of 2a,b with
heterocycles 1a,b was carried out with potassium fluoride,20
in DMF, at room temperature, yielding fluorescent conju-
gates 3a–d (Scheme 1, Table 1).
H
N
4b
CO2CH3
CDI, DMF
rt
Flu-OH
1c
CH2Ph
CO2CH3
Boc
4c
O
O
N
H
Flu
O
CH2-
O
Flu =
2 b R1 = CH2Ph, R2 = Boc, R3 = OH
c R1 = CH2Ph, R2 = H, R3 = OCH3
CO-R3
d R1 = CH2OH, R2 = Boc, R3 = OCH3
R1
R2-HN
H3CO
O
R1
O
Scheme 2. Synthesis of fluorescent conjugates 4a–c.
NH-Boc
CH2Cl
R1
O
KF, DMF
CO2H
rt
+
carbonyl groups of the linkage fluorophore–amino acid
from 1739 to 1756 cmꢀ1 (esters 3a–d), at 1815 and
1750 cmꢀ1 (anhydride 4a), at 1720–1742 cmꢀ1 (urethane
4b) and 1682 cmꢀ1 (carbonate 4c). The spectra also showed
the carbonyl bands of N-tert-butyloxycarbonyl (1673–
1721 cmꢀ1) and methyl ester (1720 cmꢀ1) protecting groups,
as well as the oxo group at the pyran ring (1644–1690 cmꢀ1).
Boc-HN
R
O
O
R
O
O
1 a R = OH
b R = OCH3
2 a R1 = CH(CH3)2
b R1 = CH2Ph
3 a R = OH, R1 = CH(CH3)2
b R = OH, R1 = CH2Ph
c R = OCH3, R1 = CH(CH3)2
d R = OCH3, R1 = CH2Ph
Scheme 1. Synthesis of fluorescent conjugates 3a–d.
1H NMR spectra showed signals of the amino acid residues,
such as the multiplets for the a-CH (d 4.24–4.75 ppm), as
well as the characteristic protons of the heterocyclic moiety
at d 6.12–6.37 ppm for the H-2 and d 5.20–5.39 ppm for the
methylene group. The confirmation of the presence of the
newly formed linkages was also supported by 13C NMR
spectral signals of the carbonyl group, which were found
at about d 171 ppm for the ester (3a–d), at d 171.45 and
175.33 ppm for the anhydride (4a), at d 155.21 ppm for
the urethane (4b) and d 173.43 ppm for the carbonate (4c),
as well as the signals of the oxo group of the heterocycle
(d 160.54 to 161.16 ppm) and the carbonyl of the Boc (at
about d 155 ppm) and methyl ester (d 169.75 ppm) protect-
ing groups.
In order to explore the versatility of benzopyran in establish-
ing different types of covalent bonds to diverse functional
groups of organic molecules and also their stability to pho-
tolysis conditions, amino acid conjugates bearing the fluoro-
phore connected through linkages other than ester bonds
were prepared.
6-Methoxy-1-hydroxymethyl-3-oxo-3H-benzopyran 1c was
linked to Boc–Phe–OH (2b) and L-phenylalanine methyl
ester, H–Phe–OMe (2c), at the main chain carboxyl and
amino functions, and to N-tert-butyloxycarbonyl-L-serine
methyl ester, Boc–Ser–OMe (2d), at the side chain hydroxyl
group. The corresponding fluorescent conjugates 4a–c were
obtained by a 1,10-carbonyldiimidazole (CDI) carbonyl
transfer reaction21 with fluorophore 1c linked to the amino
acid through a mixed carbonic anhydride (4a), urethane
(4b) and carbonate (4c) bond (Scheme 2, Table 1).
Electronic absorption and emission spectra of 10ꢀ5–10ꢀ6 m
solutions of compounds 3a–d and 4a–c in degassed absolute
ethanol were measured; absorption and emission maxima,
molar absorptivities and fluorescence quantum yields (FF)
are also reported (Table 2). The FF was calculated using
9,10-diphenylanthracene asstandard(FF¼0.95in ethanol).22
For 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 wavelength
After column chromatography on silica gel, the correspond-
ing fluorescent derivatives were obtained in good to excel-
lent yields (62–97%), in the case of compounds 3a–d, and
in lower yields (24–36%) for compounds 4a–c (Table 1)
and were characterised by high-resolution mass spectro-
metry, IR, 1H and 13C NMR spectroscopy.
The IR spectra of labelled amino acids 3a–d and 4a–c
showed bands due to stretching vibrations of the various
Table 2. UV–vis and fluorescence data for compounds 3a–d and 4a–c
Compound
UV–vis
lmax (nm)
Fluorescence
Stokes’
shift (nm)
Table 1. Synthesis of fluorescent amino acid conjugates 3a–d and 4a–c
a
log 3
lem (nm)
FF
Entry
Label
Amino acid
Product
Yield (%)
3a
3b
3c
3d
4a
4b
4c
4.12
4.15
4.14
4.11
3.99
3.67
3.70
325
324
323
323
323
322
323
398
397
392
388
390
386
389
0.61
0.62
0.28
0.27
0.20
0.26
0.22
73
73
69
65
67
64
66
1
2
3
4
5
6
7
1a
1a
1b
1b
1c
1c
1c
2a
2b
2a
2b
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
86
97
78
62
24
36
25
a
Mean deviation from 0.02 to 0.04.

A. S. C. Fonseca et al. / Tetrahedron 63 (2007) 1353–1359
1355
absorption maxima of all compounds were at about 325 nm,
with log 3 ranging from 3.67 to 4.15. The wavelengths of
maximum emission were found between 386 and 398 nm.
The nature of the amino acid and of the substituent present
at the fluorophore did not appear to influence the position of
the wavelengths of maximum absorption and emission. All
labelled amino acids 3a–d and 4a–c exhibited good to
excellent quantum yields (0.20<FF<0.62) and Stokes’shifts
ranging from 64 to 73 nm. It was possible to see that deriv-
atives 3a,b exhibited the highest value of fluorescence quan-
tum yield (w0.62) and also the larger Stokes’ shift (73 nm),
which may be related to the electron-donating character of
the hydroxy substituent at the oxobenzopyran moiety.
character of the solvent on FF was observed, as lower values
of FF seemed to be associated with low polarity solvents,
such as n-hexane and diethyl ether (entries 1 and 2).
Our main purpose being the investigation of the potential
application of fluorophore 1c as a versatile photocleavable
protecting group in organic synthesis, and in order to assess
the sensitivity of the various types of linkages between the
fluorescent oxobenzopyrans and the amino acids, photo-
cleavage studies of phenylalanine conjugates 3b,d and
4a,b and serine conjugate 4c were carried out. Solutions
of the mentioned compounds in acetonitrile (1ꢁ10ꢀ5 m)
were irradiated in a Rayonet RPR-100 reactor, at 254, 300
and 350 nm, in order to determine the best cleavage con-
ditions. The course of the photocleavage reaction was fol-
lowed by reverse phase HPLC with UV detection.
Furthermore, the absorption and emission spectra of com-
pound 3b, which had the highest value of fluorescence quan-
tum yield in ethanol, were measured in 10 more solvents
of different polarities and proton donor abilities, such as
n-hexane, diethyl ether, 1,4-dioxane, ethyl acetate, tetrahy-
drofuran (THF), acetonitrile (ACN), chloroform, dichloro-
methane (DCM), N,N-dimethylformamide (DMF) and
dimethyl sulfoxide (DMSO). The wavelengths of maximum
absorption and emission and FF for this compound are listed
in Table 3.
The plots of peak area of the starting material versus irradia-
tion time were obtained for each compound, at the consid-
ered wavelengths. Peak areas were determined by HPLC,
which revealed a gradual decrease with time, and were the
average of three runs. The determined irradiation time repre-
sents the time necessary for the consumption of the starting
materials until less than 5% of the initial area was detected,
at 254 and 300 nm. When the photolysis was carried out at
350 nm, the reactions were followed until less than 10% of
the initial area could be detected.
From the results, it can be seen that the polarity and the pro-
ton donor ability of the solvent did not influence significantly
the position of the band of absorption, with the wavelength
of maximum absorption ranging from 319 to 323 nm. In
the case of the wavelength of maximum emission, the
same behaviour was observed (from 383 to 395 nm), except
for n-hexane (entry 1) in which the lowest value of lem was
obtained (350 nm). However, a slight influence of the
Based on HPLC data, the kinetic study of the photocleavage
reactions was also carried out. For each compound, the plot
of ln Aversus irradiation time showed a linear correlation for
the disappearance of the starting material, which suggested
a first order reaction. The rate constant (k) values were
obtained by the linear least squares methodology for
a straight line (Table 4). Figure 1 summarises the behaviour
of phenylalanine conjugates 3b,d and 4a,b at 300 nm.
Table 3. UV–vis and fluorescence data for compound 3b in different
solvents
Concerning the influence of the wavelength of irradiation on
the rate of the photocleavage reactions of compounds 3b,d
and 4a–c in acetonitrile it can be seen that at 350 nm the
cleavage occurs very slowly (1090–5310 min for 90% of
starting material consumption) when compared to 254 and
300 nm that require similar irradiation times (30–175 and
40–130 min, respectively) to achieve the cleavage of at least
95% of the starting material.
Entry Solvent
UV–vis
Fluorescence
Stokes’
shift (nm)
a
lmax (nm) lem (nm) FF
1
2
3
4
5
6
7
8
9
10
n-Hexane
Diethyl ether 319
1,4-Dioxane 321
Ethyl acetate 320
320
350
383
392
386
388
390
394
395
393
392
0.003 30
0.054 64
0.15
0.14
0.10
0.19
0.51
0.23
0.47
0.36
71
66
68
70
71
73
71
69
THF
320
320
323
322
322
323
Acetonitrile
Chloroform
DCM
DMF
DMSO
The presence of a hydroxyl substituent at the oxobenzopyran
moiety appeared to have a positive effect on the cleavage
rate, as compound 3b cleaved twice as fast than compound
3d at 300 nm and 2.5 times faster at 350 nm. At 254 nm
both compounds showed similar cleavage rates.
a
Mean deviation from 0.01 to 0.04, except for entries 1 (0.001) and
2 (0.003).
Table 4. Irradiation times and kinetic data for the photolysis of compounds 3b,d and 4a–c at the considered wavelengths, in acetonitrile solution (1ꢁ10ꢀ5 m)
Compound
254 nm
300 nm
350 nm
Irradiation
time (min)
Rate constant,
Irradiation
time (min)
Rate constant,
Irradiation
time (min)
Rate constant,
k (ꢁ10ꢀ2 minꢀ1 a
)
k (ꢁ10ꢀ2 minꢀ1 a
)
k (ꢁ10ꢀ2 minꢀ1 a
)
3b
3d
4a
4b
4c
35
35
30
55
9.03
8.92
10.41
4.97
1.94
40
80
45
65
7.34
3.99
7.21
4.59
2.81
1090
2730
1740
3190
5310
0.21
0.10
0.14
0.07
0.05
175
130
a
Correlation coefficient varied from 0.9786 to 0.9984.

1356
14
A. S. C. Fonseca et al. / Tetrahedron 63 (2007) 1353–1359
suitable and versatile photolabile protecting groups for the
3b
3d
4a
4b
amino, carboxyl and hydroxyl functions of amino acids,
on account of the short irradiation times, which are desirable
in the process of photodeprotection, to prevent side reactions
in other functionalities present in the molecule that is being
cleaved. Although irradiation times at 254 nm are slightly
shorter for all compounds except 4c, the wavelength of
300 nm seems more suitable to perform such deprotection
reactions because the radiation will have a less detrimental
effect on the rest of the molecule. Irradiation at 350 nm
would be less appropriate because of the required long
irradiation periods.
13
12
11
10
0
20
40
60
80
Irradiation time (min)
3. Conclusions
Figure 1. Plot of ln A versus irradiation time for the photolysis of phenyl-
alanine conjugates 3b,d and 4a,b at 300 nm.
A series of fluorescent amino acid conjugates were syn-
thesised in good to excellent yields by a straightforward
procedure, between 6-hydroxy and 6-methoxy-1-chloro-
methyl-3-oxo-3H-benzopyrans and the C-terminus of
N-Boc-protected L-valine and L-phenylalanine, through an
ester bond. Another series of fluorescent amino acid conju-
gates, bearing the same fluorophore (6-methoxy-1-methyl-
ene-3-oxo-3H-benzopyran) connected by different linkages
to the N- and C-terminus of L-phenylalanine (carbonic anhy-
dride and urethane) and to the hydroxyl group at the side
chain of L-serine (carbonate), were also obtained in moder-
ate yields.
Regarding the nature of the linkage of fluorophore to the
amino acid, we found out that when the linkage is done
through a carbonate bond (compound 4c), the cleavage is
slower at all considered wavelengths of irradiation, when
compared to the other types of linkages.
Considering compounds 3d and 4a, in which the same oxo-
benzopyran was linked to the C-terminal of phenylalanine
through different linkages (ester and anhydride, respec-
tively), our results indicated that the anhydride bond cleaved
faster at the considered wavelengths. For compounds 4a
and 4b, in which the same fluorophore was linked to the
C- and N-terminal, through an anhydride and a urethane
bond, respectively, it could be seen that the urethane bond
was more stable to radiation, cleaving in a slower rate.
The photocleavage studies of the fluorescent amino acid
conjugates with 1-methylene-3-oxo-3H-benzopyrans at 254,
300 and 350 nm showed that these heterocycles could
be used as photolabile protecting groups through different
linkages, the most suitable wavelength of irradiation being
300 nm as a result of short irradiation times. The type of
linkage between the heterocycle and the amino acid was
found to be determinant to the time of irradiation required
to cleave the link: the anhydride and ester bonds were more
sensitive to radiation whereas the carbonate bond was the
most stable, at all considered wavelengths of irradiation.
Considering the three types of possible linkages (ester,
anhydride and urethane) between the same fluorophore
(6-methoxy-1-methylene-3-oxo-3H-benzopyran) and the
main chain amino or carboxyl terminal of the amino acid
(compounds 3d, 4a and 4b), the relative sensitivity of bond
cleavage follows the order anhydride>ester>urethane, at
254 and 350 nm, whereas at 300 nm the order becomes
anhydride>urethane>ester.
Bearing in mind the photophysical properties displayed
by these compounds, they can be considered as versatile
fluorogenic reagents as well as photolabile protecting groups
for organic molecules, including amino acids and other
biomolecules.
We also studied the influence of the solvent on the course
of the photocleavage reaction for compound 3b at 300 nm,
by using other solvents like methanol and a THF/H2O mix-
ture, 4:1. The data collected revealed that in both cases the
cleavage occurred faster than that in acetonitrile, revealing
that for this compound protic solvents improved the photo-
cleavage rate, a dramatic increase being obtained in the
case of THF/H2O mixture (Table 5).
4. Experimental
4.1. General
All melting points were measured on a Gallenkamp 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 Perkin Elmer FTIR-1600 using KBr discs.
UV–vis spectra were run on a Hitachi U-2000 spectropho-
tometer. 1H NMR spectra were recorded on a Varian
300 spectrometer in CDCl3 at 300 MHz at 25 ꢂC. All
chemical shifts are given in parts per million using
From the obtained results, 6-hydroxy and 6-methoxy-1-
methylene-3-oxo-3H-benzopyrans can be considered as
Table 5. Photolysis and kinetic data for compound 3b in different solvents
Solvent
Irradiation
time (min)
Rate constant,
k (ꢁ10-2 minꢀ1 a
)
ACN
MeOH
THF/H2O, 4:1
40
25
10
3.19
4.68
12.31
a
Correlation coefficient varied from 0.9855 to 0.9934.

A. S. C. Fonseca et al. / Tetrahedron 63 (2007) 1353–1359
1357
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
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
Spex Fluorolog 1680 spectrometer. Photolyses were carried
out using a Rayonet RPR-100 chamber reactor equipped
with eight lamps of different wavelengths (254, 300 or
350 nm, 14 W each). HPLC analysis was performed using
a Lichrospher 100 RP18 (5 mm) column in an HPLC system
composed by a Jasco PU-980 pump, a UV–vis Shimadzu
SPD-GAV detector and a Shimadzu C-RGA Chromatopac
register.
DCM/MeOH, 100:1, and DCM/MeOH, 95:5. Compound
3b was obtained as a yellowish solid (0.850 g, 97%).
1
Mp¼125.0–129.3 ꢂC. H NMR (CDCl3): d¼1.43 (s, 9H,
C(CH3)3), 3.11 (d, J¼6.6 Hz, 2H, b-CH2), 4.64–4.68 (m,
1H, a-H), 5.02 (d, J¼7.5 Hz, 1H, NH), 5.20 (s, 2H, CH2),
6.12 (s, 1H, H-2), 6.81 (dd, J¼2.4 and 8.7 Hz, 1H, H-7),
6.86 (d, J¼2.4 Hz, 1H, H-5), 7.11–7.15 (m, 2H, 2ꢁPh–H),
7.24–7.29 (m, 4H, H-8 and 3ꢁPh–H), 9.28 (br s, 1H, OH).
13C NMR (CDCl3): d¼28.19 (C(CH3)3), 38.08 (b-CH2),
54.72 (a-C), 61.92 (CH2), 80.63 (C(CH3)3), 103.46 (C-5),
109.22 (C-2), 109.62 (C-8a), 113.56 (C-7), 124.52 (C-8),
127.29 (C-4 Ph), 128.69 (C-3 and C-5 Ph), 129.02 (C-2
and C-6 Ph), 135.35 (C-1 Ph), 148.60 (C-1), 155.25
(C-4a), 156.00 (C]O urethane), 161.16 (C-3), 161.29
(C-6), 171.38 (C]O ester). IR (KBr 1%, cmꢀ1): n¼3500–
3200 (br, OH), 3347 (NH), 2979, 2931, 1740 (C]O),
1713 (C]O), 1682 (C]O), 1615, 1571, 1525, 1454.
UV–vis (ethanol, nm): lmax (log 3)¼324 (4.15). MS (EI,
ꢃ
%): m/z¼383 ([M]+ ꢀtBu, 35), 366 (15), 322 (20), 248
Compounds 1a–c were synthesised according to published
procedures18,19 and their spectral data compared well with
that of the literature.
(37), 192 (24), 176 (38), 175 (18), 147 (43), 131 (74), 120
(65), 91 (100). HRMS (EI) calcd for C20H17NO7
ꢃ
([M]+ ꢀtBu): 383.1005; found: 383.1003.
4.2. General procedure for the synthesis of compounds
3a–d
4.2.3. N-(tert-Butyloxycarbonyl)-L-valine (6-methoxy-3-
oxo-3H-benzopyran-1-yl) methyl ester (3c). Starting from
1b (0.100 g, 0.45 mmol) and 2a (0.106 g, 0.50 mmol), the
eluent used in the chromatography was CHCl3. Compound
3c was obtained as a yellow solid (0.140 g, 78%). Mp¼
77.8–86.3 ꢂC. 1H NMR (CDCl3): d¼0.92 (d, J¼6.9 Hz,
3H, g-CH3), 1.01 (d, J¼6.9 Hz, 3H, g-CH3), 1.46 (s, 9H,
C(CH3)3), 2.16–2.24 (m, 1H, b-H), 3.89 (s, 3H, OCH3),
4.30–4.36 (m, 1H, a-H), 5.00 (d, J¼9.0 Hz, 1H, NH), 5.33
(s, 2H, CH2), 6.37 (s, 1H, H-2), 6.87 (d, J¼2.7 Hz, 1H,
H-5), 6.88 (dd, J¼2.7 and 8.4 Hz, 1H, H-7), 7.42 (d,
J¼8.4 Hz, 1H, H-8). 13C NMR (CDCl3): d¼17.53
(g-CH3), 19.14 (g-CH3), 28.24 (C(CH3)3), 30.97 (b-C),
55.76 (OCH3), 58.74 (a-C), 61.82 (CH2), 80.14 (C(CH3)3),
101.21 (C-5), 110.43 (C-2), 110.48 (C-8a), 112.64 (C-7),
124.46 (C-8), 148.51 (C-1), 155.53 (C-4a), 155.60 (C]O
urethane), 160.67 (C-3), 162.90 (C-6), 171.85 (C]O ester).
IR (KBr 1%, cmꢀ1): n¼3346 (NH), 2963, 2876, 1750
(C]O), 1721 (C]O), 1684 (C]O), 1619, 1559, 1529,
1463, 1450. UV–vis (ethanol, nm): lmax (log 3)¼323
1-Chloromethyl-3-oxo-3H-benzopyrans 1a,b (1 equiv) were
dissolved in dry DMF (2 mL), potassium fluoride (3 equiv)
and Boc–Val–OH, 2a, or Boc–Phe–OH, 2b (1.1 equiv)
were added. The reaction mixture was stirred at room tem-
perature for 3–4 days. The solvent was removed by rotary
evaporation under reduced pressure and the crude residue
was purified by column chromatography.
4.2.1. N-(tert-Butyloxycarbonyl)-L-valine (6-hydroxy-3-
oxo-3H-benzopyran-1-yl) methyl ester (3a). Starting from
1a (0.210 g, 1 mmol) and 2a (0.239 g, 1.1 mmol), the eluent
used in the chromatography was CHCl3/n-hexane, 80:20,
and CHCl3. Compound 3a was obtained as a colourless oil
1
(0.336 g, 86%). H NMR (CDCl3): d¼0.92 (d, J¼6.9 Hz,
3H, g-CH3), 1.01 (d, J¼6.9 Hz, 3H, g-CH3), 1.41 (s, 9H,
C(CH3)3), 2.18–2.24 (m, 1H, b-H), 4.24–4.29 (m, 1H,
a-H), 5.11 (d, J¼8.7 Hz, 1H, NH), 5.25 (d, J¼6.3 Hz,
2H, CH2), 6.25 (s, 1H, H-2), 6.80 (dd, J¼2.4 and
9.0 Hz, 1H, H-7), 6.83 (d, J¼2.4 Hz, 1H, H-5), 7.30 (d,
J¼9.0 Hz, 1H, H-8), 10.01 (br s, 1H, OH). 13C NMR
(CDCl3): d¼17.55 (g-CH3), 19.16 (g-CH3), 28.27
(C(CH3)3), 30.94 (b-C), 58.78 (a-C), 61.89 (CH2), 80.31
(C(CH3)3), 103.62 (C-5), 109.47 (C-2), 109.70 (C-8a),
113.51 (C-7), 124.53 (C-8), 148.77 (C-1), 155.45 (C-4a),
156.00 (C]O urethane), 161.16 (C-3), 161.21 (C-6),
171.83 (C]O ester). IR (KBr 1%, cmꢀ1): n¼3380–3300
(br, OH and NH), 2966, 2931, 2876, 1739 (C]O), 1710
(C]O), 1664 (C]O), 1632, 1573, 1519, 1505, 1455.
UV–vis (ethanol, nm): lmax (log 3)¼325 (4.12). MS (EI,
ꢃ
ꢃ
(4.11). MS (FAB, %): m/z¼406 ([M+H]+ , 15), 405 (M+ ,
65), 332 (39), 262 (19), 190 (100), 189 (16), 161 (29), 116
ꢃ
(63), 72 (79). HRMS (EI) calcd for C21H27NO7 [M]+ :
405.1788; found: 405.1751.
4.2.4. N-(tert-Butyloxycarbonyl)-L-phenylalanine (6-
methoxy-3-oxo-3H-benzopyran-1-yl) methyl ester (3d).
Starting from 1b (0.100 g, 0.45 mmol) and 2b (0.130 g,
0.50 mmol), the eluent used in the chromatography was
CHCl3. Compound 3d was obtained as a yellow solid
1
(0.125 g, 62%). Mp¼118.9–121.2 ꢂC. H NMR (CDCl3):
d¼1.43 (s, 9H, C(CH3)3), 3.11 (d, J¼6.3 Hz, 2H, b-CH2),
3.88 (s, 3H, OCH3), 4.63–4.70 (m, 1H, a-H), 5.02
(d, J¼7.2 Hz, 1H, NH), 5.23 (s, 2H, CH2), 6.18 (s, 1H,
H-2), 6.83–6.85 (m, 2H, H-5 and H-7), 7.12–7.14 (m,
2H, 2ꢁPh–H), 7.25–7.28 (m, 3H, 3ꢁPh–H), 7.32 (d, J¼
9.6 Hz, 1H, H-8). 13C NMR (CDCl3): d¼28.20 (C(CH3)3),
38.26 (b-CH2), 54.69 (a-C), 55.75 (OCH3), 61.92 (CH2),
80.29 (C(CH3)3), 101.13 (C-5), 110.47 (C-8a), 110.52
(C-2), 112.59 (C-7), 124.47 (C-8), 127.28 (C-4 Ph),
ꢃ
%): m/z¼335 ([M]+ ꢀtBu, 37), 176 (41), 147 (20), 116
(18), 72 (100). HRMS (EI) calcd for C16H17NO7
ꢃ
([M]+ ꢀtBu): 335.1005; found: 335.0993.
4.2.2. N-(tert-Butyloxycarbonyl)-L-phenylalanine (6-hy-
droxy-3-oxo-3H-benzopyran-1-yl) methyl ester (3b).
Starting from 1a (0.420 g, 2 mmol) and 2b (0.583 g,
2.2 mmol), the eluent used in the chromatography was

1358
A. S. C. Fonseca et al. / Tetrahedron 63 (2007) 1353–1359
128.68 (C-3 and C-5 Ph), 129.07 (C-2 and C-6 Ph), 135.45
(C-1 Ph), 148.08 (C-1), 155.06 (C]O urethane), 155.50
(C-4a), 160.54 (C-3), 162.83 (C-6), 171.44 (C]O ester).
IR (KBr 1%, cmꢀ1): n¼3349 (NH), 3087, 3063, 3025,
2978, 2934, 2872, 2840, 2631, 2419, 2048, 1942, 1756
(C]O), 1717 (C]O), 1690 (C]O), 1614, 1560, 1532,
1497, 1455. UV–vis (ethanol, nm): lmax (log 3)¼323
(m, 3H, H-5, H-7 and 1ꢁPh–H), 7.20–7.35 (m, 4H, 4ꢁPh–
H), 7.43 (d, J¼8.5 Hz, 1H, H-8). 13C NMR (CDCl3):
d¼38.01 (b-CH2), 52.80 (OCH3 ester), 52.90 (a-C), 55.61
(OCH3), 62.38 (CH2), 102.02 (C-5), 111.10 (C-8a), 111.20
(C-2), 112.91 (C-7), 124.59 (C-8), 127.16 (C-4 Ph), 127.99
(C-5 Ph), 128.43 (C-3 Ph), 128.95 (C-2 Ph), 129.50 (C-6
Ph), 135.90 (C-1 Ph), 146.87 (C-1), 155.21 (C]O urethane),
155.50 (C-4a), 161.02 (C-3), 162.80 (C-6), 169.75 (C]O
ester). IR (KBr 1%, cmꢀ1): n¼3354 (NH), 3029, 2950,
2853, 1742–1720 (C]O), 1632 (C]O), 1614, 1562, 1515,
1497, 1454, 1441. UV–vis (ethanol, nm): lmax (log 3)¼322
ꢃ
(4.14). MS (EI, %): m/z¼453 ([M]+ , 3), 398 (25), 397
(100), 380 (34), 379 (15), 337 (20), 336 (46), 262 (59), 206
(56), 190 (69), 189 (20), 161 (27), 131 (68), 120 (59), 91
ꢃ
(68). HRMS (EI) calcd for C25H27NO7 [M]+ : 453.1788;
found: 453.1798.
ꢃ
(3.67). HRMS (EI) calcd for C22H21NO7 [M]+ : 411.1318;
found: 411.1326.
4.2.5. 2-(tert-Butyloxycarbonylamino)-3-phenylpro-
panoic [(6-methoxy-3-oxo-3H-benzopyran-1-yl) methyl
carbonic] anhydride (4a). 1-Hydroxymethyl-6-methoxy-
3-oxo-3H-benzopyran 1c (0.150 g, 0.728 mmol), Boc–Phe–
OH, 2b (0.386 g, 1.46 mmol) and CDI (0.236 g, 1.46 mmol)
were dissolved in dry DMF (4 mL). The reaction mixture
was stirred for 10 min and then triethylamine (0.5 mL)
was added. The reaction mixture was stirred at room temper-
ature for 24 h and the solvent was removed by rotary evapo-
ration under reduced pressure. The crude residue was
purified by column chromatography, using CHCl3 and
CHCl3/MeOH, 100:1, as eluent. Compound 4a was obtained
as a light yellow solid (0.086 g, 24%). Mp¼71.0–73.5 ꢂC
4.2.7. N-(tert-Butyloxycarbonyl)-O-{[(6-methoxy-3-oxo-
3H-benzopyran-1-yl) methoxy]carbonyl}-L-serine
methyl ester (4c). 1-Hydroxymethyl-6-methoxy-3-oxo-
3H-benzopyran 1c (0.177 g, 0.859 mmol), CDI (0.154 g,
0.948 mmol) and Boc–Ser–OMe, 2d (0.350 g, 1.724 mmol)
were dissolved in dry DMF (5 mL). The reaction mixture
was stirred at room temperature for 66 h, triethylamine
(0.5 mL) was added and the mixture was stirred for another
6 h. The solvent was removed by rotary evaporation under
reduced pressure and the crude brown oil was purified by
column chromatography, using CHCl3 and CHCl3/MeOH,
100:1, as eluent. Compound 4c was obtained as a light
yellow oily solid (0.097 g, 25%). 1H NMR (CDCl3):
d¼1.40 (s, 9H, C(CH3)3), 3.71 (s, 3H, OCH3 ester), 3.85
(s, 3H, OCH3), 4.01–4.04 (m, 2H, b-CH2), 4.63–4.67 (m,
1H, a-H), 5.24 (m, 1H, NH), 5.39 (s, 2H, CH2), 6.18 (s,
1H, H-2), 6.75–6.82 (m, 2H, H-5 and H-7), 7.43 (d, J¼
8.5 Hz, 1H, H-8). 13C NMR (CDCl3): d¼28.22 (C(CH3)3),
39.13 (b-CH2), 53.90 (a-C), 52.40 (OCH3 ester), 55.76
(OCH3), 61.82 (CH2), 80.15 (C(CH3)3), 101.18 (C-5),
110.47 (C-8a), 110.49 (C-2), 112.63 (C-7), 124.48 (C-8),
148.59 (C-1), 155.20 (C]O urethane), 155.52 (C-4a),
160.66 (C-3), 162.91 (C-6), 171.35 (C]O ester), 173.43
(C]O carbonate). IR (KBr 1%, cmꢀ1): n¼3295 (NH),
1682 (C]O), 1673 (C]O), 1651 (C]O), 1645 (C]O).
UV–vis (ethanol, nm): lmax (log 3)¼323 (3.70). HRMS (EI)
1
(dec). H NMR (CDCl3): d¼1.43 (s, 9H, C(CH3)3), 3.10–
3.12 (m, 2H, b-CH2), 3.88 (s, 3H, OCH3), 4.63–4.70
(m, 1H, a-H), 5.00 (d, J¼7.0 Hz, 1H, NH), 5.24 (s, 2H,
CH2), 6.18 (s, 1H, H-2), 6.80–6.87 (m, 2H, H-5 and H-7),
7.14–7.18 (m, 2H, 2ꢁPh–H), 7.25–7.40 (m, 4H, H-8 and
3ꢁPh–H). 13C NMR (CDCl3): d¼28.21 (C(CH3)3), 38.27
(b-CH2), 54.70 (a-C), 55.76 (OCH3), 61.93 (CH2), 80.30
(C(CH3)3), 101.15 (C-5), 110.49 (C-8a), 110.54 (C-2),
112.61 (C-7), 124.48 (C-8), 127.29 (C-4 Ph), 128.69 (C-3
and C-5 Ph), 129.08 (C-2 and C-6 Ph), 135.46 (C-1 Ph),
148.09 (C-1), 155.10 (C]O urethane), 155.52 (C-4a),
160.56 (C-3), 162.85 (C-6), 171.45 (C]O), 175.33
(C]O). IR (KBr 1%, cmꢀ1): n¼3302 (NH), 1815 (C]O),
1750 (C]O), 1683 (C]O), 1670 (C]O). UV–vis (ethanol,
nm): lmax (log 3)¼323 (3.99). HRMS (EI) calcd for
ꢃ
calcd for C21H25NO10 [M]+ : 451.1478; found: 451.1490.
ꢃ
C26H27NO9 [M]+ : 497.1686; found: 497.1693.
4.3. General photolysis procedure
4.2.6. N-[(6-Methoxy-3-oxo-3H-benzopyran-1-yl)methyl-
oxycarbonyl]-L-phenylalanine methyl ester (4b). 1-Hy-
droxymethyl-6-methoxy-3-oxo-3H-benzopyran 1c (0.150 g,
0.728 mmol) and CDI (0.130 g, 0.80 mmol) were dissolved
in dry DMF (5 mL). The reaction mixture was stirred
for 1 h. L-Phenylalanine methyl ester hydrochloride 2c
(0.345 g, 1.60 mmol) was neutralised with triethylamine
(0.25 mL, 1.80 mmol) in DMF. The resulting solid was
filtered and the filtrate was added to previous reaction mix-
ture, which was then stirred at room temperature for 20 h.
A further addition of triethylamine (1 mL, 7.17 mmol) and
CDI (0.186 g, 0.863 mmol) was done and the mixture was
stirred for another 68 h. The solvent was removed by rotary
evaporation under reduced pressure and the crude oily residue
was purified by column chromatography using CHCl3 and
CHCl3/MeOH, 100:1, as eluent. Compound 4b was obtained
A 1ꢁ10ꢀ5 m solution in acetonitrile of the compound to be
tested (20 mL) was placed in a quartz tube and irradiated in
the reactor at the desired wavelength. Aliquots of 100 mL
were taken at regular intervals and analysed by HPLC.
The eluent was acetonitrile/water, 3:1, at a flow rate of
0.8 mL/min, previously filtered through a Milipore, type
HN 0.45 mm filter and degassed by ultra-sound for 30 min.
The chromatograms were traced by detecting UVabsorption
at the wavelength of maximum absorption for each com-
pound (retention time: 3b, 3.9; 3d, 5.8; 4a, 5.7; 4b, 4.1;
4c, 3.4 min).
Acknowledgements
1
as a yellow oily solid (0.108 g, 36%). H NMR (CDCl3):
~ ^
We thank the Fundac¸ao para a Ciencia e Tecnologia (Portu-
ꢀ
gal) for their financial support to the Centro de Quımica
(Universidade do Minho).
d¼3.06–3.09 (m, 2H, b-CH2), 3.73 (s, 3H, OCH3 ester),
3.90 (s, 3H, OCH3), 4.70–4.75 (m, 1H, a-H), 4.80–4.90 (m,
1H, NH), 5.39 (s, 2H, CH2), 6.20 (s, 1H, H-2), 7.03–7.18

A. S. C. Fonseca et al. / Tetrahedron 63 (2007) 1353–1359
1359
€
12. Schonleber, R. O.; Bendig, J.; Hagen, V.; Giese, B. Bioorg.
Med. Chem. 2002, 10, 97.
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