Wavelength-selective cleavage of o-nitrobenzyl and polyheteroaromatic benzyl protecting groups

Article abstract of DOI:10.1016/j.tet.2013.11.100

Evaluation of the wavelength-selective cleavage of five photolabile protecting groups from two different families has been performed. Alanine, as a model bifunctional target molecule was masked at the amino terminal with o-nitrobenzyl group and at the carboxylic terminal with benzyl-type nitrogen and oxygen polyheteroaromatics, namely acridine, (thioxo)benzocoumarin and a coumarin built on the julolidine nucleus. The photosensitivity of the corresponding alanine conjugates was studied at selected wavelengths with HPLC/UV and ¹H NMR monitoring. The release of the fully deprotected molecule could be achieved by sequential irradiation in variable irradiation times, which were dependent on the heteroaromatic group used.

Full text of DOI:10.1016/j.tet.2013.11.100

Tetrahedron 70 (2014) 650e657
Contents lists available at ScienceDirect
Tetrahedron
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Wavelength-selective cleavage of o-nitrobenzyl and
polyheteroaromatic benzyl protecting groups
*
Ana M. Piloto, Susana P.G. Costa, M. Sameiro T. Gonc¸ alves
Centre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Evaluation of the wavelength-selective cleavage of five photolabile protecting groups from two different
families has been performed. Alanine, as a model bifunctional target molecule was masked at the amino
terminal with o-nitrobenzyl group and at the carboxylic terminal with benzyl-type nitrogen and oxygen
polyheteroaromatics, namely acridine, (thioxo)benzocoumarin and a coumarin built on the julolidine
nucleus. The photosensitivity of the corresponding alanine conjugates was studied at selected wave-
lengths with HPLC/UV and 1H NMR monitoring. The release of the fully deprotected molecule could be
achieved by sequential irradiation in variable irradiation times, which were dependent on the hetero-
aromatic group used.
Received 26 September 2013
Received in revised form 27 November 2013
Accepted 28 November 2013
Available online 4 December 2013
Keywords:
Photolabile protecting groups
Selective cleavage
o-Nitrobenzyl group
Acridine
Ó 2013 Elsevier Ltd. All rights reserved.
Coumarin
1. Introduction
because it provides an alternative deactivation pathway to the
intended photoreaction.
Photoremovable protecting groups (PPGs) defined as systems
that contain a chromophore, which is sensitive to light, but rela-
tively stable to most chemical reagents, have been used to mask
various functional groups. Their importance has been proved by
their potential applications in synthetic organic chemistry,1e3 bio-
chemistry4e6 and materials science.4,7
In the last two decades, the intense development of fluorescence
techniques along with fluorescent reagents and strategies for var-
ious purposes, including bioapplications, has also evolved the re-
search concerning PPGs to include fluorescent molecules.
Fluorescent photoremovable protecting groups present advantages
over non-fluorescent groups, since in addition to releasing mole-
cules of interest at the desired location for a specific period of time,
they also allow the visualisation, quantification and monitoring of
the spatial distribution, localisation and depletion of the released
molecules.8 Such groups are particularly useful if the released
molecule has higher fluorescence in its free form when compared
to the conjugated form, providing a means to monitor the photo-
release process. Depending on the application, the use of fluores-
cent protecting groups can sometimes difficult the photoactivation
The above strategy of using fluorescent PPGs has been suc-
cessfully employed on the temporally and spatially controlled de-
livery of bioactive molecules in the study of numerous processes in
biological and medical research fields. Also, in organic synthetic
methodologies, the use of fluorescent PPGs is important, since it
can facilitate the follow up of experimental procedures involved in
synthesis and deprotection reaction steps.
Among the fluorescent PPGs, polyaromatics including (anthra-
cen-9-yl)methyl,9 (pyren-1-yl)methyl,10e12 (perylen-3-yl)methyl13
and (phenanthren-9-yl)methyl10 have been introduced. Hetero-
aromatics that can also be considered analogues of the benzyl
protecting group have emerged in the last years, following the re-
port on (coumarin-4-yl)methyl group photosensitivity in the re-
lease of phosphate ester by Givens in 1984,14 and includes
quinolines,15,16
quinolones,17,18
thiocoumarins
and
thio-
quinolones,18 fused oxazoles19,20 and acridine.21,22
Structural modifications at the coumarin ring by the in-
troduction of substituents, particularly at C-6 and C-7 resulted in an
improvement of photochemical and photophysical properties in-
creasing the range of applications of this class of protecting
groups.23 An extension of the aromatic ring of coumarins, resulting
in polyaromatic analogues has led to the enhancement of fluoro-
phoric properties.24e27 Also the exchange of the carbonyl function
to thiocarbonyl affording the corresponding thioxo(benzo)couma-
rin resulted in an increase in the photosensitivity.18,28
* Corresponding author. E-mail address: msameiro@quimica.uminho.pt (M.S.T.
Gonc¸ alves).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.11.100

A.M. Piloto et al. / Tetrahedron 70 (2014) 650e657
651
The advantages of fluorescent protecting groups mentioned do
not diminish the importance of the remaining groups, including
the most well-known o-nitrobenzyl group and its derivatives,
which have been widely used in the caging of different molecules.3
The variety of PPGs, working by different mechanisms and
possessing different types of chromophores/fluorophores, allows
the possibility for wavelength-selective deprotection, a type of or-
thogonality referred to as chromatic orthogonality. This concept
has been considered in certain research works, including in peptide
solid phase synthesis with selective release using the nitroveratryl/
pivaloylglycol pair.29,30 Also, in bioapplications, some examples
have been reported, involving amino acids,31,32 peptides33 and
(poli)nucleotides,34,35 using different pairs of protecting groups,
such as coumarins, nitrophenethyl, 6-nitroveratryl and p-methoxy-
phenacyl groups. These studies involved photolysis of mixtures of
the caged molecules bearing in each case only one photolabile
protecting group, whereas a single report33 refers to the use of two
different groups in the same molecule.
Considering these facts and taking advantage of our knowledge
in fluorescent PPGs, especially benzyl-based heteroaromatic
groups, the present work aims to evaluate the possibility of
wavelength-selective photolysis between four heteroaromatic
photolabile protecting groups, previously introduced by our re-
search team, and o-nitrobenzyl group. Given our promising earlier
findings with (acridin-9-yl)methyl, (9-methoxy-3-oxo-3H-naphtho
[2,1-b]pyran-1-yl)methyl (trivially named benzocoumarin), [11-
oxo-2,3,5,6,7,11-hexahydro-1H-pyrano[2,3-f]pyrido[3,2,1-ij]quino-
lin-9-yl]methyl (commonly called coumarin, based on the juloli-
dine nucleus) and (9-methoxy-3-thioxo-3H-naphtho[2,1-b]pyran-
1-yl)methyl (trivially designated (thioxo)benzocoumarin) groups,
these systems were selected for the present study. Alanine was
used as model of a bifunctionalised molecule, possessing different
functional groups, an amine and a carboxylic acid. Conjugates were
prepared bearing the o-nitrobenzyl and the heterocyclic group as
protections of the N- and C-terminus through carbamate and ester
linkages, respectively. Photolysis of these conjugates at selected
wavelengths was carried out and the process course was monitored
by HPLC/UV and 1H NMR.
pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-9-yl)methylene (Jul) and (9-
methoxy-3-thioxo-3H-naphtho[2,1-b]pyran-1-yl)methylene (Tba),
as indicated in Schemes 1 and 2 and Tables 1 and 2.
All new compounds were fully characterised by high resolution
mass spectrometry, IR, 1H and 13C NMR spectroscopy. 1H NMR
spectra showed signals of the alanine residue, such as the
3.50e4.60 ppm) and -CH3 1.17e1.54 ppm). The 2-
nitrobenzyloxycarbonyl (conjugates 1, 2, 6e9) and heterocycle
(conjugates 6e9) methylene groups were also visible
5.32e5.60 ppm, and 5.17e6.20 ppm, respectively). The confir-
a-CH (d
b
(d
(
d
d
mation of the presence of the carbamate bond linking the N-ter-
minal of alanine to 2-nitrobenzyl group, as well as the ester linkage
between the C-terminal of N-(2-nitrobenzyloxycarbonyl)-
L-alanine
and the heterocyclic moieties were also supported by 13C NMR
spectra signals at
respectively.
d 154.46e155.55 ppm, and d 172.17e172.69 ppm,
In order to evaluate the possibility of wavelength-selective
photolysis and to obtain the parameters required for monitoring
the processes, fundamental UV/vis photophysical characterisation
was carried out. The UV/vis absorption and emission spectra of
degassed 10ꢀ5 or 10ꢀ6 M solutions in absolute ethanol and in
a methanol/HEPES buffer (80:20) solution of conjugates 2 and 6e9
were measured; absorption and emission maxima, molar absorp-
tion coefficients and relative fluorescence quantum yields are also
reported (Table 1). Relative fluorescence quantum yields were
calculated using naphthalene (FF¼0.23 in cyclohexane),40 9,10-
diphenylanthracene (FF¼0.95 in ethanol),41 or a 0.05 M solution
of quinine in sulfuric acid (FF¼0.54)42 as standards. For the FF
determination, the fluorescence standard was excited at the
wavelengths of maximum absorption found for each one of the
compounds to be tested and in all fluorimetric measurements the
absorbance of the solution did not exceed 0.1.
Regarding the absorption data of conjugates 6e9, in both sol-
vents, it was found that all conjugates displayed two maximum
absorption wavelengths (labs), one of them at about 260 nm, which
is related to the presence of 2-nitrobenzyloxycarbonyl group, as it
was confirmed by the spectra of N-(2-nitrobenzyloxycarbonyl)-L-
alanine 2 (labs about 260 nm), and the other associated to the
heterocycle moiety, which differs accordingly to its structure.
Conjugate 6 bearing the nitrogen heterocycle acridine displayed
absorption maxima at about 360 nm in both solvents. Concerning
conjugate 7, possessing the naphtho[2,1-b]pyran skeleton showed
the lowest value of the set (labs about 347 nm), but the replacement
of the carbonyl group at the pyran ring by a thiocarbonyl (conjugate
9) resulted in a bathochromic shift, tuning absorption maxima to
values in the visible region (labs 400 or 414 nm). In addition, it was
observed that for conjugate 8 with a 3H-benzopyran fused juloli-
dine, the maximum absorption wavelengths were at about 400 nm
in both solvents (labs 395 or 403 nm). For easier comparison, Fig. 1
shows the UV/vis spectra of the photosensitive compounds 2, 6e9
in methanol/HEPES buffer (80:20) solutions.
2. Results and discussion
2.1. Synthesis of alanine conjugates 1,2 and 6e9
(2-Nitrophenyl)methanol was reacted with L-alanine methyl
ester via a N,N0-carbonyldiimidazole (CDI) mediated carbonyl
transfer reaction in N,N-dimethylformamide, at room temperature
to give N-(2-nitrobenzyloxycarbonyl)- -alanine methyl ester 1.
L
Subsequent basic hydrolysis of the methyl ester with aqueous so-
dium hydroxide in 1,4-dioxane, at low temperature gave N-(2-
nitrobenzyloxycarbonyl)- -alanine 2. This carbamate conjugate
L
was used in the reaction with bromo- or chloromethylated het-
erocycles, namely 9-(bromomethyl)acridine 3, 1-chloromethyl-9-
Concerning the fluorescence spectra, in both tested solvents, it
was observed that emission maxima (lem) of conjugates 6e9 oc-
curred in the range (412e496 nm), being the conjugate possessing
3H-benzopyran fused julolidine 8 associated to the longer wave-
methoxy-3-oxo-3H-naphtho[2,1-b]pyran36
4
and
9-(chlor-
omethyl)-2,3,6,7-tetrahydro-1H-pyrano[2,3-f]pyrido[3,2,1-ij]qui-
nolin-11(5H)-one37 5, in the presence of potassium fluoride in N,N-
dimethylformamide, at room temperature (Scheme 1, Table 1),38 to
afford the corresponding conjugates 6e8. In order to exchange the
carbonyl by a thiocarbonyl group at the naphtho[2,1-b]pyran con-
jugate 7, this compound was reacted with Lawesson’s reagent in
toluene, under reflux conditions,39 yielding the corresponding
thionaphthopyran conjugate 9 (Scheme 1, Table 1).
For simplicity of naming the various conjugates in this report,
the photocleavable protecting groups will be designated by a two-
or three-letter code, as follows: 2-nitrobenzyloxycarbonyl (NB),
acridin-9-yl methylene (Acm), (9-methoxy-3-oxo-3H-naphtho[2,1-
b]pyran-1-yl)methylene (Bba), (11-oxo-2,3,5,6,7,11-hexahydro-1H-
lengths, and the N-(2-nitrobenzyloxycarbonyl)- -alanine 2 dis-
L
playing the lowest value (lem at 304 nm), as expected due to its less
conjugated structure. As for the fluorescence quantum yields,
naphtho[2,1-b]pyran 7 was the most emissive (FF 0.44 and 0.22,
depending on the solvent). The emission of thiocarbonyl conjugate
9 was bathochromically shifted in ethanol with lower fluorescent
quantum yields, in comparison with the corresponding carbonyl
precursor (compound 7). Stokes’ shifts (Dl) occurred in a broad
range (41e130 nm), being the lowest values associated to conjugate
2, and the highest values corresponding to conjugates 7 and 8,
bearing coumarin moieties.

652
A.M. Piloto et al. / Tetrahedron 70 (2014) 650e657
Scheme 1. Synthesis of photosensitive conjugates 2 and 6e9.
Table 1
Yields, UV/vis and fluorescence data for conjugates 2, 6e9 in absolute ethanol and methanol/HEPES buffer (80:20) solution
Compound
Yield/%
Ethanol
Methanol/Hepes (80:20)
labs/nm
log
3
lem/nm
FF
Dl/nm
labs/nm
log
3
lem/nm
FF
Dl/nm
2
6
NB-Ala-OH
92
32
257
254
354
259
347
254
395
250
307
400
3.91
5.61
4.46
4.65
4.59
4.68
4.50
4.48
3.98
3.96
304
0.064
47
263
254
362
257
350
259
403
261
309
414
3.78
5.42
4.60
4.96
4.92
4.66
4.67
4.65
4.11
4.09
304
0.073
41
NB-Ala-OAcm
NB-Ala-OBba
NB-Ala-OJul
NB-Ala-OTba
412
466
486
0.113
0.439
0.184
58
119
91
413
480
496
0.157
0.222
0.207
51
130
93
7
8
9
55
44
28
475
0.012
75
480
0.003
66
2.2. Photolysis studies of alanine conjugates 2 and 6e9
and at its C-terminal with benzyl-type nitrogen and oxygen poly-
heteroaromatics, namely acridine, (thioxo)benzocoumarin and
a coumarin built on the julolidine nucleus, through an ester bond.
The later groups were selected from the collection of photo-
removable protecting groups developed by our research group
taking into consideration the photophysical properties, namely the
wavelengths of maximum absorption displayed when linked to
The possibility of selective deprotection of either terminus of
a bifunctionalised molecule by choosing the appropriate wave-
length was the main goal of the present work. As mentioned before,
alanine was chosen as model target, and protected at its N-terminal
with the well-known o-nitrobenzyl group via a carbamate linkage,

A.M. Piloto et al. / Tetrahedron 70 (2014) 650e657
653
NB-Ala-OAcm
6
HO-Acm
10
properties of the selected groups and the o-nitrobenzyl group to-
gether in the same molecule.
Overall, owing to the wavelengths of maximum absorption of
conjugates 6e9 in MeOH/HEPES buffer (80:20), it was found that
photolysis at 350 nm would probably be suitable for the depro-
tection of the C-terminal of alanine in conjugates 6 and 7, whereas
irradiation at 419 nm would be adequate in the case of conjugates 8
and 9. In order to check this and also to verify the stability to other
nearby wavelengths, solutions of conjugates 2 and 6e9 were irra-
diated at different wavelengths (254, 300, 350 and 419 nm) in
a Rayonet RPR-100 reactor (Scheme 2, Table 2). The photolysis re-
action was monitored by reverse phase HPLC with UV detection,
peak areas of the starting material (A, average of three runs for each
compound) revealing a gradual decrease with time and plots of A
versus irradiation time were obtained for each compound, at the
considered wavelengths. The determined irradiation time repre-
sents the time necessary for the consumption of the starting ma-
terials until less than 5% of the initial area was detected (Table 2).
For each compound and based on HPLC data, the plot of ln A versus
irradiation time showed a linear correlation for the disappearance
of the starting material, which suggested a first order reaction,
obtained by the linear least squares methodology for a straight line.
The photochemical quantum yields (FPhot) were calculated based
NB-Ala-OBba
7
HO-Bba
11
h
ν
(350 or 419 nm)
+
NB-Ala-OH
MeOH/HEPES 80:20
2
NB-Ala-OJul
8
HO-Jul
12
hν (254 nm)
MeOH/HEPES
80:20
or
or
NB-Ala-OTba
9
HO-Tba
13
OHC
+
H-Ala-OH
ON
14
O
O
S
O
O
Acm =
Bba =
Jul =
Tba =
O
N
N
O
O
Scheme 2. Photocleavage reactions of conjugates 2 and 6e9.
on half-lives (t1/2), molar extinction coefficients ( ) and the incident
3
photon flux (I0), which was determined by potassium ferrioxalate
actinometry.43
Conjugates 6 and 7 were exposed to light at 350 nm and results
revealed that cleavage of the ester bond with release of N-(2-
nitrobenzyloxycarbonyl)- -alanine 2 occurred with an irradiation
L
Table 2
Irradiation times, tirr, and photochemical quantum yields, FPhot, for the photolysis of conjugates 2, 6e9 at 254, 300, 350 and 419 nm in methanol/HEPES buffer (80:20) solution
Compound
254 nm
300 nm
350 nm
419 nm
tirr/min
FPhot
tirr/min
FPhot
tirr/min
FPhot
tirr/min
FPhot
2
6
7
8
9
NB-Ala-OH
153
25
48
12
38
1.17ꢁ10ꢀ4
2.28ꢁ10ꢀ4
3.53ꢁ10ꢀ5
1.94ꢁ10ꢀ4
1.09ꢁ10ꢀ4
256
35
98
13
41
3.33ꢁ10ꢀ5
1.04ꢁ10ꢀ4
6.38ꢁ10ꢀ6
8.53ꢁ10ꢀ5
3.63ꢁ10ꢀ5
469
18
60
21
51
1.72ꢁ10ꢀ5
7.90ꢁ10ꢀ5
1.01ꢁ10ꢀ5
5.20ꢁ10ꢀ5
6.89ꢁ10ꢀ5
647
108
243
17
1.29ꢁ10ꢀ5
1.32ꢁ10ꢀ4
2.51ꢁ10ꢀ6
6.25ꢁ10ꢀ5
1.11ꢁ10ꢀ5
NB-Ala-OAcm
NB-Ala-OBba
NB-Ala-OJul
NB-Ala-OTba
31
time (tirr) of 18 and 60 min, respectively. On the other hand, irra-
diation at 419 nm of conjugates 8 and 9 cleaved the carboxylic
protecting group in 17 and 31 min, respectively (Scheme 2, Table 2).
Conjugates 6e9 possess benzyl-type nitrogen and oxygen hetero-
cycles connected through an ester bond, which have been found to
cleave by formation of an ion pair that can recombine or proceed to
the products after nucleophilic attack by the solvent molecules.3 In
this case, the formation of the corresponding heterocyclic alcohols
10e13 as by-products, respectively, was expected.
7
6
8
9
1.0
0.8
0.6
0.4
0.2
0.0
In order to estimate the stability of 2-nitrobenzyloxycarbonyl
group in the above conditions, N-(2-nitrobenzyloxycarbonyl)-L-al-
2
anine 2 was irradiated at 350 and 419 nm. The results showed that
2-nitrobenzyloxycarbonyl group cleaved in 11% (after 18 min), 31%
(after 60 min) at 350 nm, and in 9% (after 17 min),15% (after 31 min)
at 419 nm. Considering these results, the tested protecting groups
cannot be considered totally orthogonal but rather quasi-
orthogonal since selective cleavage can only be achieved in a par-
ticular order of irradiation: in order to minimise simultaneous
cleavage at both terminals, the molecule should always be irradi-
ated first at the longer wavelength of irradiation.
250
300
350
400
450
500
Wavelength (nm)
Fig. 1. Normalised UV/vis absorption spectra of conjugates 2, 6e9 in methanol/HEPES
buffer (80:20).
amino acid residues or butyric acid,17e21,24e28,36,37 and also the
promising photolytic results especially at longer wavelengths,
which provided potential orthogonal behaviour to the o-nitro-
benzyl group. Nevertheless, the fundamental photophysical studies
of conjugates 2 and 6e9 were obviously essential to ascertain the
Considering that after deprotection of the C-terminal, cleavage
of protecting group of N-terminal is required, N-(2-
nitrobenzyloxycarbonyl)-L-alanine 2 was exposed to light of

654
A.M. Piloto et al. / Tetrahedron 70 (2014) 650e657
254 nm, and it was found that cleavage of the carbamate linkage
occurred in 153 min. The photolytic process performed at 300, 350
and 419 nm with conjugate 2, revealed that the irradiation times
increased considerably (tirr between 256 and 647 min), thus con-
firming the expected behaviour, with 254 nm being the most ad-
equate wavelength for the cleavage of o-nitrobenzyl group. This
group is known to cleave by an intramolecular proton abstraction
mechanism yielding the released product (in the present case in-
volving a carbamate linkage, accompanied by spontaneous de-
carboxylation to the free amine) and a nitroso compound 14.3
Bearing in mind the above findings, the release of the fully
deprotected alanine residue by sequential irradiation was attemp-
ted, by irradiating first at the longer wavelength of irradiation (350
or 419 nm, depending on the heteroaromatic group) followed by
the shorter wavelength of irradiation (254 nm). It was found that
the release was possible in irradiation times very similar to the sum
of the irradiation times presented in Table 2 (relative to the cleav-
age of each individual group), revealing that the by-products of the
first photolysis (which remained in the solution being irradiated)
did not influence the overall outcome of the second photolysis.
Additionally to monitoring the photolysis process by HPLC/UV
detection, the photolysis of conjugates 2 (NB-Ala-OH) and 8 (NB-
Ala-OJul), as representative examples of the tested set of com-
pounds, was also followed by 1H NMR in a methanol-d4/D2O
(80:20) solution. In the case of conjugate 2, upon irradiation at
heterocyclic C-terminal protecting group (the coumarin built on the
julolidine nucleus). The alanine -CH and -CH3 signals in the
conjugated form, at about 4.35 and 1.50 ppm, were replaced by
the new set of signals corresponding to the released compound, N-
(2-nitrobenzyloxycarbonyl)- -alanine 2, at about 4.20 and
a
b
d
L
d
1.45 ppm, respectively (Fig. 3). The multiplets corresponding to the
benzylic CH2 of the N- and C-termini protecting groups of conjugate
8, between
d 5.30e5.50 ppm, were substituted by a singlet due to
the remaining N-(2-nitrobenzyloxycarbonyl) group. After irradia-
tion at 419 nm for 11 h, ensuring almost complete conversion of
conjugate 8 to conjugate 2, the same solution was further irradiated
at 254 nm to cleave the N-terminal nitrobenzyl protecting group.
The same observations were made concerning the shifts of the al-
anine a-CH and b-CH3 signals, as in the above referred photolysis
for the pure conjugate 2.
254 nm, the signals related to the alanine
conjugated form at about 4.20 and 1.45 ppm, gave rise to a close
set of signals corresponding to alanine in its free form at about
3.75 and 1.50 ppm, respectively (Fig. 2). This conversion was also
accompanied by the decrease with time of the integration of the
signal due to the benzylic CH2 at about 5.5 ppm. In the case of
a-CH and b-CH3 in the
d
d
d
conjugate 8, irradiation was carried out first at 419 nm, to cleave the
Fig. 3. Partial 1H NMR spectra in methanol-d4/D2O (80:20) of the photolysis of con-
jugate 8, NB-Ala-OJul (C¼2ꢁ10ꢀ2 M) at 419 nm: (a) before irradiation; (b) after irra-
diation for 2 h; (c) after irradiation for 10 h; (d) free NB-Ala-OH 2.
The NMR monitoring was carried out with a 2ꢁ10ꢀ2 M solution,
which led to an expected increase in the photolysis time for the
complete release of the molecule, when compared to the irradia-
tion times in Table 2 obtained with dilute solutions.
3. Conclusions
It was investigated the photolysis of four conjugates bearing in
the same molecule two photolabile groups, the o-nitro-
benzyloxycarbonyl and a benzyl-type polyheteroaromatics, namely
acridine, (thioxo)benzocoumarin and a coumarin built on the
julolidine nucleus, and identified the wavelengths where the
chromophores show different sensitivities. It was demonstrated
Fig. 2. Partial 1H NMR spectra in methanol-d4/D2O (80:20) of the photolysis of con-
jugate 2, NB-Ala-OH (C¼2ꢁ10ꢀ2 M) at 254 nm: (a) before irradiation; (b) after irra-
diation for 18 h; (c) after irradiation for 34 h; (d) free H-Ala-OH.

A.M. Piloto et al. / Tetrahedron 70 (2014) 650e657
655
that selective cleavage of the photolabile groups was possible by
choosing the most appropriate wavelength, being for the tested
heterocyclic chromophores 350 and 419 nm, and for the o-nitro-
benzyl group 254 nm. Overall, the results showed that the combi-
nations of photolabile protecting groups used in conjugates 6e9 are
wavelength-selective; however, a specific irradiation sequence is
required, meaning that the heterocyclic chromophore should al-
ways be cleaved before the o-nitrobenzyl group.
The possibility of using other classes of photolabile protecting
group, together with other photolysis conditions, and even the use
of two-photon excitation are possibilities under study in our re-
search group.
1344, 1302, 1250, 1216, 1177, 1151, 1117, 1086, 1071, 980, 859, 816,
791, 733, 703, 672. HRMS (EI): calcd for
282.08522; found: 282.08543.
C
12H14N2O6 [Mþ]:
4.2.2. N-(2-Nitrobenzyloxycarbonyl)- -alanine, 2. To a suspension
L
of compound 1 (0.403 g, 1.44ꢁ10ꢀ3 mol) in 1,4-dioxane (4.0 mL) at
0 ꢂC, 1 M aqueous sodium hydroxide (1.44 mL, 1.44ꢁ10ꢀ3 mol) was
added. The solution was stirred at low temperature for 18 h and
acidified to pH 2e3 with 1 M aqueous potassium hydrogensulfate.
The reaction mixture was reduced to half the initial volume in
a
rotary evaporator and extracted with dichloromethane
(3ꢁ10 mL). The organic extracts were combined, dried with anhy-
drous magnesium sulfate and after solvent evaporation, compound
2 was obtained as a colourless oil (0.355 g, 92%). Rf¼0.39 (ethyl
4. Experimental section
4.1. General
acetate). 1H NMR (DMSO-d6, 400 MHz):
d
¼1.17 (d, J 6.8 Hz, 3H,
b-
CH3), 3.50e3.59 (m,1H,
-NH), 7.58 (dt, J 7.6 and 1.2 Hz, 1H, H-4), 7.68 (dd, 1H, J 7.6 and
0.8 Hz, H-6), 7.77 (dt, J 7.2 and 0.8 Hz, 1H, H-5), 8.08 (dd, J 8.4 and
1.2 Hz, 1H, H-3). 13C NMR (DMSO-d6, 100.6 MHz):
¼19.41 ( -CH3),
51.40 ( -CH), 61.76 (CH2), 124.73 (C-3), 128.74 (C-6), 128.86 (C-4),
133.22 (C-1), 134.20 (C-5), 147.12 (C-2), 154.46 (C]O carbamate),
174.33 (C]O acid). IR (film, cmꢀ1):
a-CH), 5.32 (s, 2H, CH2), 6.64 (d, J 6.0 Hz,1H,
a
All melting points were measured on a Stuart SMP3 melting
point apparatus. TLC analyses were carried out on 0.25 mm thick
precoated silica plates (Merck Fertigplatten Kieselgel 60F254) and
spots were visualised under UV light. Chromatography on silica gel
was carried out on Merck Kieselgel (230e240 mesh). IR spectra
were determined on a BOMEM MB 104 spectrophotometer using
KBr discs. UV/vis absorption spectra (200e700 nm) were obtained
using a Shimadzu UV/2501PC spectrophotometer. NMR spectra
were obtained on a Varian Unity Plus Spectrometer at an operating
frequency of 300 MHz for 1H or a Bruker Avance III 400 at an op-
erating frequency of 400 MHz for 1H and 100.6 MHz for 13C using
the solvent peak as internal reference at 25 ꢂC. All chemical shifts
are given in parts per million using dH Me4Si¼0 ppm as reference
and J values are given in hertz. 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 correlation techniques. Mass spectrometry analyses
were performed at the ‘C.A.C.T.I.dUnidad de Espectrometria de
Masas’, at University of Vigo, Spain. Fluorescence spectra were
collected using a FluoroMax-4 spectrofluorometer. Photolysis was
carried out using a Rayonet RPR-100 chamber reactor equipped
with 10 lamps of 254, 300, 350 and 419ꢃ10 nm. HPLC analyses were
d
b
a
n
¼3369, 2987, 1692, 1614, 1576,
1533, 1451, 1343, 1295, 1264, 1215, 1120, 1067, 1020, 982, 936, 896,
860, 802, 738, 704. HRMS (EI): calcd for C11H12N2O6 [Mþ]:
268.06956; found: 268.06986.
4.2.3. N-(2-Nitrobenzyloxycarbonyl)-L-alanine (acridin-9-yl)methyl
ester, 6. To a solution of compound 2 (0.073 g, 2.73ꢁ10ꢀ4 mol) in
dry DMF (3 mL), potassium fluoride (0.047 g, 8.16ꢁ10ꢀ4 mol) and 9-
(bromomethyl)acridine 3 (0.074 g, 2.72ꢁ10ꢀ4 mol) were added. The
reaction mixture was stirred at room temperature for 30 h. Potas-
sium fluoride was removed by filtration, the solvent was removed
by rotary evaporation under reduced pressure and the crude resi-
due was purified by column chromatography using mixtures of
ethyl acetate and light petroleum of increasing polarity as eluent.
Compound 6 was obtained as light brown oil (0.040 g, 32%). Rf¼0.51
(ethyl acetate/light petroleum, 1:1). 1H NMR (CDCl3, 400 MHz):
d
¼1.33 (d, J 7.2 Hz, 3H,
b-CH3), 4.30e4.40 (m, 1H, a-CH), 5.45 (s, 2H,
CH2 NB), 5.58 (d, J 7.6 Hz, 1H, NH), 6.08 (d, J 6.8 Hz, 1H, CH2 OAcm),
6.20 (d, J 6.8 Hz, 1H, CH2 OAcm), 7.35e7.45 (m, 1H, H-40), 7.46e7.55
(m, 2H, H-50and H-60), 7.56e7.62 (m, 2H, H-2 and H-7), 7.73e7.79
(m, 2H, H-3 and H-6), 8.043 (dd, J 6.4 and 1.6 Hz, 1H, H-30),
8.20e8.30 (m, 4H, H-1, H-8, H-4 and H-5). 13C NMR (CDCl3,
performed using a Licrospher 100 RP18 (5 mm) column in a JASCO
HPLC system composed by a PU-2080 pump and a UV-2070 de-
tector with ChromNav software. All reagents were used as received.
4.2. Synthesis of compounds 1, 2 and 6e9
100.6 MHz):
d
¼18.14 (
b-CH3), 49.77 (a-CH), 58.57 (CH2 OAcm),
63.37 (CH2 NB),123.74 (C-1 and C-8),124.83 (C-30), 125.16 (C-8a and
C-9a), 126.82 (C-2 and C-7), 128.34 (C-40), 128.41 (C-50), 129.85 (C-4
and C-5), 130.11 (C-3 and C-6), 132.78 (C-10), 133.65 (C-60), 136.49
(C-9), 147.06 (C-20), 148.32 (C-4a and C-10a), 155.04 (C]O carba-
4.2.1. N-(2-Nitrobenzyloxycarbonyl)-
-alanine methyl ester, 1. N,N0-
L
Carbonyldiimidazole (1.078 g, 6.65ꢁ10ꢀ3 mol) was stirred with (2-
nitrophenyl)methanol (1.018 g, 6.65ꢁ10ꢀ3 mol) in dry DMF (4 mL)
at 0 ꢂC for 30 min.
L-Alanine methyl ester hydrochloride (0.928 g,
mate), 172.69 (C]O ester). IR (film, cmꢀ1):
n
¼3420, 3331, 3056,
6.65ꢁ10ꢀ3 mol), previously treated with triethylamine (1.01 mL,
7.32ꢁ10ꢀ3 mol) in dry DMF (2 mL) was added to the reaction
mixture and the mixture was kept stirring for 18 h. The precipitate
was filtered, the solvent was removed by rotary evaporation under
reduced pressure and the crude residue was purified by column
chromatography using ethyl acetate and light petroleum, with
mixtures of increasing polarity as eluent. Compound 1 was ob-
tained as a pink oily solid (0.430 g, 23%). Rf¼0.48 (ethyl acetate/light
2985, 2941, 2878, 1727, 1630, 1611, 1579, 1526, 1452, 1421, 1344,
1305, 1266, 1209, 1170, 1115, 1068, 966, 910, 897, 859, 819, 790, 738,
704, 674, 642. HRMS (EI): calcd for C25H21N3O6 [Mþ]: 459.14313;
found: 459.14292.
4.2.4. N-(2-Nitrobenzyloxycarbonyl)-
3H-naphtho[2,1-b]pyran-1-yl)]methyl ester, 7. To
L
-alanine [(9-methoxy-3-oxo-
solution of
a
compound 2 (0.134 g, 4.99ꢁ10ꢀ4 mol) in dry DMF (3 mL), potassium
fluoride (0.087 g, 1.49ꢁ10ꢀ3 mol) and 1-chloromethyl-9-methoxy-
3-oxo-3H-naphtho[2,1-b]pyran 4 (0.137 g, 4.99ꢁ10ꢀ4 mol) were
added. The reaction mixture was stirred at room temperature for
48 h. Potassium fluoride was removed by filtration, the solvent was
removed by rotary evaporation under reduced pressure and the
crude residue was purified by column chromatography using ethyl
acetate and light petroleum, with mixtures of increasing polarity as
eluent. Compound 7 was obtained as a light yellow solid (0.139 g,
55%); mp 145.0e146.6 ꢂC. Rf¼0.40 (ethyl acetate/light petroleum,
petroleum, 1:1). 1H NMR (CDCl3, 400 MHz):
b
d
¼1.44 (d, J 7.2 Hz, 3H,
-CH3), 3.76 (s, 3H, OCH3), 4.30e4.44 (m, 1H, -CH), 5.49 (broad s,
-NH), 5.53 (s, 2H, CH2), 7.47 (dt, J 7.4 and 1.6 Hz, 1H, H-4), 7.62
a
1H,
a
(dd,1H, J 7.2 and 1.2 Hz, H-6), 7.65 (dt, J 6.8 and 0.8 Hz,1H, H-5), 8.10
(dd, J 8.0 and 0.8 Hz, 1H, H-3). 13C NMR (CDCl3,100.6 MHz):
¼18.54
-CH3), 49.63 ( -CH), 52.49 (OCH3), 63.41 (CH2), 124.92 (C-3),
d
(b
a
128.49 (C-4), 128.56 (C-6), 132.98 (C-1), 133.74 (C-5), 147.22 (C-2),
155.01 (C]O carbamate), 173.32 (C]O ester). IR (KBr 1%, cmꢀ1):
n
¼3345, 3113, 3044, 2991, 2956, 2880, 1729, 1613, 1578, 1526, 1453,

656
A.M. Piloto et al. / Tetrahedron 70 (2014) 650e657
1:1). 1H NMR (CDCl3, 400 MHz):
d
¼1.54 (d, J 7.6 Hz, 3H,
-CH), 5.53 (s, 2H, CH2 NB), 5.55
-NH), 5.60e5.79 (m, 2H, CH2 OBba), 6.64 (s, 1H, H-2),
b
-CH3), 3.96
4b),114.93 (C-5), 117.17 (C-2), 124.97 (C-30), 126.61 (C-6a),127.39 (C-
8), 128.62 (C-40), 128.77 (C-50), 130.23 (C-6b), 131.49 (C-7), 132.68
(C-10), 133.77 (C-60), 134.42 (C-6), 141.34 (C-1), 147.32 (C-20), 155.12
(C]O carbamate), 158.81 (C-4a), 159.97 (C-9), 172.21 (C]O ester),
(s, 3H, OCH3), 4.52e4.58 (m, 1H,
(broad s, 1H,
a
a
7.22 (dd, J 8.8 and 2.0 Hz, 1H, H-8), 7.30 (d, J 9.2 Hz, 1H, H-5),
7.36e7.42 (m, 1H, H-10), 7.45 (dt, J 7.2 and 2.0 Hz, 1H, H-40),
7.55e7.69 (m, 2H, H-50and H-60), 7.82 (d, J 8.8 Hz, 1H, H-7), 7.90 (d, J
8.8 Hz, 1H, H-6), 8.08 (d, J 8.0 Hz, 1H, H-30). 13C NMR (CDCl3,
195.12 (C-3). IR (film, cmꢀ1):
n
¼3338, 3065, 2938, 2852, 1727, 1622,
1591, 1535, 1453, 1343, 1297, 1231, 1174, 1141, 1096, 1069, 1024, 976,
858, 840, 790, 732, 700, 688, 671. HRMS (EI): calcd for C26H22N2O8S
[Mþ]: 522.10976; found: 522.10995.
100.6 MHz):
d
¼18.12 (
b-CH3), 49.86 (a-CH), 55.46 (OCH3), 63.64
(CH2 NB), 64.98 (CH2 OBba), 105.72 (C-10), 111.70 (C-4b), 112.93 (C-
2), 115.23 (C-5), 116.53 (C-8), 124.93 (C-30), 126.32 (C-6a), 128.60 (C-
40), 128.74 (C-50), 130.48 (C-6b), 131.35 (C-7), 132.66 (C-10), 133.75
(C-60), 133.86 (C-6), 147.28 (C-20), 150.25 (C-1), 155.15 (C-4a), 155.55
(C]O carbamate), 159.71 (C-9), 160.07 (C-3), 172.17 (C]O ester). IR
4.3. General photolysis procedure
A 1ꢁ10ꢀ4 M methanol/HEPES (80:20) solution of compounds 2
and 6e9 (5 mL) was placed in a quartz tube and irradiated in
a Rayonet RPR-100 reactor at the desired wavelength. The lamps
used for irradiation were of 254, 300, 350 and 419ꢃ10 nm.
(KBr 1%, cmꢀ1):
n
¼3406, 3057, 2987, 2959, 2942, 1718, 1624, 1579,
1553, 1523,1460,1425, 1345,1232,1214,1171, 1142, 1121,1073,1026,
983, 946, 894, 862, 844, 822, 789, 731, 703, 674, 608. HRMS (EI):
calcd for C26H22N2O9 [Mþ]: 506.13256; found: 506.13268.
Aliquots of 100 mL were taken at regular intervals and analysed
by RP-HPLC. The eluent was acetonitrile/water, 75:25 with 0.1%
trifluoroacetic acid, at a flow rate of 0.8 mL/min, previously filtered
through a Millipore, type HN 0.45 mm filter and degassed by ultra-
4.2.5. N-(2-Nitrobenzyloxycarbonyl)-L-alanine [11-oxo-2,3,5,6,7,11-
hexahydro-1H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-9-yl]methyl es-
ter, 8. To a solution of compound 2 (0.027 g, 1.01ꢁ10ꢀ4 mol) in dry
DMF (3 mL), potassium fluoride (0.017 g, 3.02ꢁ10ꢀ4 mol) and 9-
(chloromethyl)-2,3,6,7-tetrahydro-1H-pyrano[2,3-f]pyrido[3,2,1-ij]
quinolin-11(5H)-one 5 (0.029 g, 1.01ꢁ10ꢀ4 mol) were added. The
reaction mixture was stirred at room temperature for 46 h. Potas-
sium fluoride was removed by filtration, the solvent was removed
by rotary evaporation under reduced pressure and the crude resi-
due was purified by column chromatography using ethyl acetate
and light petroleum, with mixtures of increasing polarity as eluent.
Compound 8 was obtained as brown oil (0.023 g, 44%). Rf¼0.44
(ethyl acetate/light petroleum, 1:1). 1H NMR (CDCl3, 400 MHz):
sound for 30 min. The chromatograms were traced by detecting UV
absorption at the wavelength of maximum absorption for each
compound (retention time: 2, 3.5; 6, 5.0; 7, 6.3; 8, 7.5 and 9
9.5 min).
Acknowledgements
~
^
Thanks are given to the Fundac¸ ao para a Ciencia e Tecnologia
(FCT, Portugal) for financial support to the NMR Portuguese net-
work (PTNMR, Bruker Avance III 400-Univ. Minho), FCT and FEDER
(European Fund for Regional Development)-COMPETE-QREN-EU
for financial support to the research centre CQ/UM [PEst-C/QUI/
UI0686/2011 (FCOMP-01-0124-FEDER-022716)] and for a Ph.D.
grant to A.M.P. (SFRH/BD/61459/2009).
d
¼1.52 (d, J 7.2 Hz, 3H,
2.73e2.78 (m, 2H, H-7), 2.83e2.89 (m, 2H, H-1), 3.20e3.30 (m, 4H,
H-3 and H-5), 4.45e4.55 (m, 1H, -CH), 5.17e5.32 (m, 2H, CH2 Jul),
5.40e5.50 (m, 1H,
b-CH3), 1.90e2.00 (m, 4H, H-2 and H-6),
a
a
-NH), 5.51e5.60 (m, 2H, CH2 NB), 6.06 (s, 1H, H-
10), 6.86 (s, 1H, H-8), 7.47 (dt, J 7.2 and 1.6 Hz, 1H, H-40), 7.59e7.68
References and notes
(m, 2H, H-50and H-60), 8.10 (dd, J 8.4 and 0.8 Hz, 1H, H-30). 13C NMR
1. Pillai, V. N. R. Synthesis 1980, 1e26.
2. Greene, T. W.; Wuts, G. M. Protective Groups in Organic Chemistry, 3rd ed.;
John Wiley: New York, NY, 1999.
(CDCl3, 100.6 MHz):
(C-6), 27.63 (C-7), 49.47 (C-3), 49.82 (
d
¼18.34 (
b
-CH3), 20.36 (C-2), 20.47 (C-1), 21.37
-CH), 49.89 (C-5), 62.49 (CH2
a
Jul), 63.53 (CH2 NB), 105.66 (C-10), 105.90 (C-8a), 106.97 (C-12b),
118.35 (C-7a), 120.48 (C-8), 124.93 (C-30), 128.52 (C-40), 128.57 (C-
50), 132.88 (C-10), 133.81 (C-60), 145.99 (C-7b), 147.19 (C-20), 148.70
(C-9), 151.19 (C-12a), 155.08 (C]O carbamate), 162.01 (C-11), 172.27
3. Klan, P.; Solomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.; Rubina, M.; Popik, V.;
Kostikov, A.; Wirz, J. Chem. Rev. 2013, 113, 119e191.
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London, 1998.
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6. Pelliccioli, A. P.; Wirz, J. Photochem. Photobiol. Sci. 2002, 1, 441e458.
7. San Miguel Arnanz, V.; Bochet, C. G.; del Campo, A. J. Am. Chem. Soc. 2011, 133,
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10133e10139.
(C]O ester). IR (film, cmꢀ1):
n
¼3318, 3060, 2940, 2851, 1717, 1603,
1556,1524,1440, 1381,1343,1312,1255,1206,1177,1122,1071,1015,
960, 885, 858, 820, 790, 732, 700. HRMS (EI): calcd for C27H27N3O8
[Mþ]: 521.17991; found: 521.17989.
4.2.6. N-(2-Nitrobenzyloxycarbonyl)-L-alanine [(9-methoxy-3-
thioxo-3H-naphtho[2,1-b]pyran-1-yl)]methyl ester, 9. Lawesson’s
reagent (0.172 g, 4.26ꢁ10ꢀ4 mol) was added to a solution of com-
pound 7 (0.072 g, 1.42ꢁ10ꢀ4 mol) in toluene (5 mL). The reaction
mixture was refluxed for 9 h and the process was followed by TLC
(ethyl acetate/light petroleum, 1:4). The solvent was removed by
rotary evaporation under reduced pressure and the crude residue
was purified by column chromatography using dichloromethane
and light petroleum, with mixtures of increasing polarity as eluent.
Compound 9 was obtained as an orange oil (0.021 g, 28%). Rf¼0.67
(ethyl acetate/light petroleum, 1:1). 1H NMR (CDCl3, 400 MHz):
12. Fernandes, M. J. G.; Costa, S. P. G.; Gonc¸ alves, M. S. T. New J. Chem. 2013,
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13. Jana, A.; Ikbal, M.; Singh, N. D. P. Tetrahedron 2012, 68, 112811e112836.
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d
¼1.54 (d, J 7.2 Hz, 3H,
b
-CH3), 3.97 (s, 3H, OCH3), 4.50e4.60 (m, 1H,
a
-CH), 5.35e5.45 (m, 1H,
a
-NH), 5.54 (s, 2H, CH2 NB), 5.65e5.80 (m,
2H, CH2 OTba), 7.25 (d, J 2.0 Hz, 1H, H-2), 7.41e7.53 (m, 4H, H-40, H-
10, H-5 and H-8), 7.54e7.70 (m, 2H, H-50and H-60), 7.85 (d, J 8.8 Hz,
1H, H-7), 7.96 (d, J 8.8 Hz, 1H, H-6), 8.03 (d, J 8.0 Hz, 1H, H-30). 13C
NMR (CDCl3, 100.6 MHz):
(OCH3), 63.70 (CH2 NB), 64.79 (CH2 OTba), 106.29 (C-10), 114.17 (C-
d
¼18.21 (
b-CH3), 49.87 (a-CH), 55.55

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