Comparative study of polyaromatic and polyheteroaromatic fluorescent photocleavable protecting groups

Article abstract of DOI:10.1016/j.tet.2008.01.032

Fluorescent conjugates of N-benzyloxycarbonyl protected γ-aminobutyric acid were prepared by coupling to its C-terminus several polyheteroaromatic, based on the oxobenzopyran skeleton (trivially known as coumarin) and polyaromatic labels, such as naphthalene and pyrene. Photophysical properties were evaluated, as well as their behaviour towards photocleavage by irradiation in MeOH/HEPES buffer solution (80:20), in a photochemical reactor at different wavelengths (254, 300, 350 and 419 nm), followed by HPLC/UV monitoring.

Full text of DOI:10.1016/j.tet.2008.01.032

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 3032e3038
www.elsevier.com/locate/tet
Comparative study of polyaromatic and polyheteroaromatic
fluorescent photocleavable protecting groups
*
Maria J.G. Fernandes, M. Sameiro T. Gonc¸alves, Susana P.G. Costa
Centro de Qu´ımica, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
Received 21 November 2007; received in revised form 20 December 2007; accepted 9 January 2008
Available online 12 January 2008
Abstract
Fluorescent conjugates of N-benzyloxycarbonyl protected g-aminobutyric acid were prepared by coupling to its C-terminus several polyhe-
teroaromatic, based on the oxobenzopyran skeleton (trivially known as coumarin) and polyaromatic labels, such as naphthalene and pyrene.
Photophysical properties were evaluated, as well as their behaviour towards photocleavage by irradiation in MeOH/HEPES buffer solution
(80:20), in a photochemical reactor at different wavelengths (254, 300, 350 and 419 nm), followed by HPLC/UV monitoring.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Oxobenzopyran; Oxobenzobenzopyran; Coumarin; g-Aminobutyric acid (GABA); Photocleavable protecting groups
1. Introduction
aldehydes and ketones. Within this last class of heterocycles,
7-methoxycoumarin-4-yl derivatives are some of the most
widely used photoreleasable groups in cell biology and biophys-
ics.23 These compoundsare fluorophores, whichare moreconve-
nient as phototriggers than non-fluorescent protecting groups,
since they may be useful in monitoring the course of reaction
and thus allow tracing of the location of caged molecules inside
living cells by fluorescent techniques as well as the visualisation
of processes during in situ synthesis of oligonucleotides and the
functioning of peptides.24e26 Although fluorescence deactiva-
tion may be an inconvenience in some photochemical processes,
in recent years, the direction of improvement on photoreleasable
groups has been towards the development and application of
polycyclic structures (both benzene and heterocycle derived),
which are fluorophores in most cases. These compounds have
been reported as having improved properties as photolabile
protecting groups.
g-Aminobutyric acid (GABA), one of the main transmitters
in the central nervous system, has been widely used in photore-
lease applications in neurological sciences, for studying the
chemical mechanisms and the kinetics of synaptic transmis-
sion.27,28 Bearing in mind these facts in connection with our
current research interests in the development of new fluorescent
heterocyclic compounds and their applications as labels and as
Initially reported by Barltrop and Schofield in 1962,1 photo-
labile protecting groups have found extensive application in
both synthetic and biological chemistry, being responsible for
recent advances in various areas and technologies, such as in
organic synthesis, particularly that involving polyfunctional
molecules,2,3 photoactive calcium chelators,4,5 photoactive pre-
cursors of neurotransmitters6e8 and in time-resolved studies for
a wide range of subjects, varying from cell biology9 to X-ray
crystallography.10
Light-sensitive molecules, including groups like 2-nitro-
benzyl,11 benzyl,12 benzoin,13 phenacyl,14 cinnamyl,15 vinylsi-
lane16 and their derivatives, have been developed and used as
photoreleasable groups. Polycyclic aromatics, namely anthra-
quinon-2-ylmethoxycarbonyl,17 anthraquinon-2-ylethyl-10,20-
diol,18 pyren-1-ylmethyl,19,20 pyren-1-ylmethoxycarbonyl,17
phenanthren-9-ylmethoxycarbonyl,17 anthracene-9-methanol,21
and oxobenzopyrans (coumarins)22 have also been applied in the
protection of alcohols, amines, phosphates, carboxylic acids,
* Corresponding author. Tel.: þ351 253 604054; fax: þ351 253 604382.
E-mail address: spc@quimica.uminho.pt (S.P.G. Costa).
0040-4020/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.01.032

M.J.G. Fernandes et al. / Tetrahedron 64 (2008) 3032e3038
3033
photoreleasable protecting groups,29 we now report the use of
different recognised and new fluorophores of aromatic, namely
naphthalene and pyrene, and heteroaromatic nature, such as
oxobenzopyran derivatives, in the preparation of fluorescent
conjugates of GABA, with the aim of undertaking a comparative
study of their performance as photolabile groups.
Derivatisation at the C-terminus of N-benzyloxycarbonyl-
protected GABA with labels 1aee was carried out in DMF, at
room temperature, with the aid of N,N0-dicyclohexylcarbodi-
imide (DCC) assisted by 1-hydroxybenzotriazole (HOBt) under
standard conditions32 (for 1a,d) or by using potassium fluoride33
(for 1b,c,e), yielding fluorescent GABA conjugates 2aee
(Scheme 1, Table 1). All conjugates were characterised by IR,
1H and 13C NMR spectroscopy and elemental analyses or high
resolution mass spectrometry.
2. Results and discussion
The UV/vis absorption and emission spectra of degassed
10ꢀ5e10ꢀ6 M solutions in absolute ethanol of compounds
2aee were measured, absorption and emission maxima, molar
absorptivities and fluorescence quantum yields are also reported
(Table 1, Fig. 2). Fluorescence quantum yields were calculated
using 9,10-diphenylanthracene as standard (FF¼0.95 in etha-
nol).34 Labelled GABA 2aee exhibited moderate to excellent
quantum yields (0.15<FF<0.76), and Stokes’ shift from 33 to
158 nm, the highest values being associated with the heteroaro-
matic moieties.
Considering that the main goal of this research was to com-
pare the performance of compounds 1aee as fluorescent pho-
tocleavable protecting groups, photolysis studies of GABA
conjugates 2aee were carried out. Solutions of the mentioned
compounds in methanol/HEPES buffer 80:20 solution were
irradiated in a Rayonet RPR-100 reactor, at 254, 300, 350
and 419 nm, in order to determine the best cleavage condi-
tions. The course of the photocleavage reaction was followed
by reverse phase HPLC with UV detection.
The plots of peak area of the starting material versus irra-
diation 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
(Table 2).
1-Hydroxymethyl-7-methoxy-3-oxo-3H-benzo[g]benzopyran
1awas synthesised through a Pechmann reaction, between 2,7-di-
hydroxynaphthalene and ethyl acetoacetate, catalysed by sulfuric
acid at room temperature,30 followed by methylation of the result-
ing 1-methyl-7-hydroxy-3-oxo-3H-benzo[g]benzopyran with
methyl iodide. The methyl group was oxidised to the aldehyde,
by reaction with selenium dioxide, which was then reacted with
sodium borohydride, affording the hydroxymethyl group.31 1-
Chloromethyl-9-methoxy-3-oxo-3H-benzo[f]benzopyran 1b and
1-chloromethyl-6-methoxy-3-oxo-3H-benzopyran 1c were ob-
tained by a similar procedure from the reaction of 7-methoxy-2-
naphthol and 3-methoxyphenol with ethyl chloroacetoacetate.29b
Starting from 4-methoxy-naphthaldehyde, 1-hydroxymethyl-4-
methoxy-naphthalene 1d was obtained by reduction of the formyl
group with sodium borohydride. 1-Chloromethylpyrene 1e was
commercially available. Fluorophores 1aee will be designated
inthisreportbyathreelettercodeforsimplicityofnamingthevar-
ious fluorescent conjugates, as indicated in Table 1 and Figure 1.
Our purpose being the investigation of compounds 1aee as
fluorescent photocleavable protecting groups for neurotrans-
mitter amino acids, namely g-aminobutyric acid (GABA), we
synthesised the corresponding conjugates in order to do a com-
parative study of the behaviour in photolysis conditions of the
ester linkage between fluorophores 1aee and the carboxylic
function of GABA.
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 meth-
odology for a straight line.
Concerning the influence of the wavelength of irradiation on
the rate of the photocleavage reactions of GABA conjugates
2aee in methanol/HEPES buffer 80:20 solution, it was found
that the most suitable was 254 nm (compound 2a), 300 nm
(compounds 2cee) and 350 nm (compound 2b). Cleavage at
419 nm lead to a very large increase in the irradiation time
Table 1
Yields, UV/vis and fluorescence data for GABA ester conjugates 2aee in
absolute ethanol
Compound
Yield UV/vis
(%)
Fluorescence
lmax log 3 lmax Stokes’
(nm)
FF
(nm) shift (nm)
2a ZeGABAeOBbl
2b ZeGABAeOBba
2c ZeGABAeOBpm
2d ZeGABAeONpm
27
81
85
17
345 3.91 503 158
345 3.95 472 127
0.21ꢁ0.03
0.76ꢁ0.02
0.27ꢁ0.03
0.20ꢁ0.02
0.15ꢁ0.01
320 4.22 393
295 3.80 339
342 4.61 375
73
44
33
2e ZeGABAeOPym29e 98
Flu =
OCH3
CH2
CH2
O
CH2
CH2
CH2
H3CO
O
O
H3CO
O
O
O
OCH3
Bbl
Bba
Bpm
Npm
Pym
Figure 1. Structure and three letter code of fluorophores 1aee.

3034
M.J.G. Fernandes et al. / Tetrahedron 64 (2008) 3032e3038
O
O
O
OCH2
PhH2CO
N
H
3
O
2b
O
O
H3CO
O
OCH2
O
O
PhH2CO
N
H
3
O
O
b)
2a
OCH2
PhH2CO
N
H
3
2c
O
a)
b)
OCH3
OCH3
Z-GABA-OH + Flu-R
1
b)
a)
O
O
OCH3
OCH2
OCH2
PhH2CO
N
PhH2CO
N
H
3
3
H
2d
O
O
2e
1a R = OH, Flu = (7-methoxy-3-oxo-3H-benzo[g]benzopyran-1-yl)methyl (Bbl)
b R = Cl, Flu = (9-methoxy-3-oxo-3H-benzo[f]benzopyran-1-yl)methyl (Bba)
c R = Cl, Flu = (6-methoxy-3-oxo-3H-benzopyran-1-yl)methyl (Bpm)
d R = OH, Flu = (4-methoxy-naphthalen-1-yl)methyl (Npm)
e R = Cl, Flu = (pyren-1-yl)methyl (Pym)
Scheme 1. Synthesis of fluorescent GABA ester conjugates 2aee. Reagents and conditions: (a) DCC, HOBt, DMF, rt; (b) KF, DMF, rt.
1.2
for conjugates ZeGABAeOBbl (2a) and ZeGABAeOBba
2d
2c
2b
2a
(2b), in which the label was a linear or an angular 3-oxoben-
zobenzopyran, respectively, the cleavage for the angular deriv-
ative was faster, for all wavelengths of irradiation. Comparison
between ZeGABAeOBpm (2c) with compound 2b, which
differ in the number of fused aromatic rings, showed that the
irradiation times are comparable at 254 and 300 nm, with 2c
cleaving slightly faster. However, at 350 nm, this trend is re-
versed (2b cleaves approximately 12 times faster) probably
due to the proximity of the wavelength of maximum absorp-
tion of 2b to the irradiation wavelength. Regarding the polyar-
omatic or polyheteroaromatic nature of the label, it was found
that the cleavage of compounds 2dee (aromatic) was faster
than that of compounds 2aec (heteroaromatic), at 254 and
300 nm.
1
0.8
0.6
0.4
0.2
0
300
350
400
450
500
550
600
650
Wavelength (nm)
Figure 2. Normalised emission spectra of GABA conjugates 2aed in absolute
ethanol ([2a]¼1.0ꢂ10ꢀ5 M, lexc¼345 nm; [2b]¼1.0ꢂ10ꢀ5 M, lexc¼345 nm;
[2c]¼5.2ꢂ10ꢀ6 M, lexc¼320 nm; [2d]¼1.0ꢂ10ꢀ5 M, lexc¼295 nm).
As reported before,29c the N-blocking group was stable in the
tested conditions, no cleavage being detected. The photochem-
ical quantum yields were calculated as previously descri-
bed24,29e and are given in Table 2. Although the efficiency of
the photocleavage process was not high, due to fluorescence
deactivation and other photophysical processes, which limit
the overall quantum yield of deprotection, regarding the low
irradiation times, these fluorescent labels can be considered
as suitable photocleavable protecting groups in organic
synthesis.
Table 2
Irradiation times (in min) and photochemical quantum yield (Fphot, ꢂ10ꢀ3) for
the photolysis of compounds 2aee at different wavelengths in MeOH/HEPES
buffer (80:20) solution
Compound
254 nm
300 nm
350 nm
Irr time Fphot Irr time Fphot Irr time Fphot
2a ZeGABAeOBbl
479
0.037 1148
0.095 124
0.024 790
0.112 84
0.007
0.062
0.015
0.192
0.065
2b ZeGABAeOBba 169
2c ZeGABAeOBpm 131
2d ZeGABAeONpm 20
0.418
0.570
0.521
90
7
0.805 986
1.330 4284
0.564 158
2e ZeGABAeOPym
17
19
3. Conclusions
(ca. 68 h in the case of compound 2e, and higher for the other
compounds), which is not useful for practical applications.
Taking into consideration the influence of the structure of
the conjugates on the photocleavage rates, it was found that
Fluorescent g-aminobutyric acid ester conjugates 2aee were
prepared in low to excellent yields by using general synthetic
methods, involving chloromethyl or hydroxymethyl precursors
(oxobenzopyran, naphthalene and pyrene derivatives) and the

M.J.G. Fernandes et al. / Tetrahedron 64 (2008) 3032e3038
3035
C-terminus of N-benzyloxycarbonyl-protected GABA. The
photophysical studies showed that all labels are appropriate
fluorogenic reagents for the derivatisation of non-fluorescent
molecules, being 1-chloromethyl-9-methoxy-3-oxo-3H-ben-
zo[f]benzopyran (1b), together with the other heteroaromatic
moieties, the most interesting fluorophores.
(PymeCl) from TCI and other reagents from Sigma-Aldrich. All
reagents were used as received.
4.2. Synthesis of 1-hydroxymethyl-7-methoxy-3-oxo-3H-
benzo[g]benzopyran, BbleOH (1a)
Regarding the photocleavage studies of the fluorescent con-
jugates, in methanol/HEPES buffer solution (80:20), at 254,
300 and 350 nm, it was possible to conclude that the irradia-
tion time depended on the structure of the label. Depending
on the stability to radiation of the analyte, a choice of the pro-
tecting group can be made based on the wavelength of irradi-
ation, i.e., at 350 nm, benzo[f]benzopyran (1b), at 300 nm
naphthalene (1d) and at 254 nm pyrene (1e). Irradiation times
at 419 nm were too long and not convenient for practical
applications.
In summary, all labels considered lead to conjugates, which
required low irradiation times for photocleavage to occur,
making them appropriate to use as photolabile protecting
groups for organic molecules, including amino acids and other
relevant biomolecules, in addition to their usefulness in fluo-
rescent labelling due to the high Stokes’ shifts and moderate
to excellent fluorescence quantum yields.
4.2.1. 1-Methyl-7-hydroxy-3-oxo-3H-benzo[g]benzopyran
2,7-Dihydroxynaphthalene (0.506 g, 3.16 mmol) and ethyl
acetoacetate (0.60 mL, 4.68 mmol), catalysed by 80% aqueous
sulfuric acid, were stirred at room temperature for 3 days. After
column chromatography using chloroform as eluent, the com-
pound was obtained as a light green solid (0.616 g, 41%).
Mp¼262.3e264.5 ꢃC. [lit. not quoted]. 1H NMR (DMSO-d6):
d¼2.47 (d, J 0.9 Hz, 3H, CH3), 6.31 (d, J 1.2 Hz, 1H, H-2),
7.11 (dd, J 9.0 and 2.4 Hz, 1H, H-8), 7.15 (d, J 2.4 Hz, 1H,
H-6), 7.58 (s, 1H, H-5), 7.92 (d, J 9.0 Hz, 1H, H-9), 8.23 (s,
1H, H-10), 10.23 (s, 1H, OH).
4.2.2. 1-Methyl-7-methoxy-3-oxo-3H-benzo[g]benzopyran
The previous compound (0.450 g, 1.99 mmol) was reacted
with potassium carbonate (1.100 g, 7.96 mmol) and methyl io-
dide (1.51 mL, 9.95 mmol) in DMF (5 mL), at 50e60 ꢃC for 4 h
under a nitrogen atmosphere. Water (15 mL) was added and the
aqueous phase extracted with ethyl acetate (4ꢂ10 mL). The or-
ganic phases were combined and washed with NaCl saturated
solution, dried with magnesium sulfate, filtered and evaporated
under reduced pressure. After column chromatography using
chloroform as eluent, the methoxy derivative was obtained as
a yellow solid (0.360 g, 75%). Mp¼220.6e223.0 ꢃC. 1H
NMR (CDCl3): d¼2.51 (d, J 0.9 Hz, 3H, CH3), 3.95 (s, 3H,
OCH3), 6.27 (d, J 1.2 Hz, 1H, H-2), 7.09 (d, J 2.4 Hz, 1H,
H-6), 7.14 (dd, J 9.0 and 2.7 Hz, 1H, H-8), 7.56 (s, 1H, H-5),
7.80 (d, J 9.0 Hz, 1H, H-9), 7.97 (s, 1H, H-10). 13C NMR
(CDCl3): d¼18.64 (CH3), 55.41 (OCH3), 104.51 (C-6),
111.59 (C-5), 114.53 (C-2), 117.87 (C-10a), 119.42 (C-8),
124.63 (C-10), 125.54 (C-4a), 130.13 (C-9), 136.38 (C-5a),
150.82 (C-9a), 152.11 (C-1), 159.50 (C-7), 160.95 (C-3). IR
(KBr 1%, cmꢀ1): n¼3055, 3005, 2937, 1703, 1625, 1484,
1434, 1400, 1388, 1374, 1313, 1264, 1233, 1209, 1183, 1147,
1134, 1057, 1030, 919, 896, 882, 865, 821, 803, 793, 755,
693. UV/vis (EtOH, nm): lmax (log 3)¼343 (4.16), 286 (4.24).
MS (EI, %): m/z¼240 ([M]þ, 100), 212 (21), 197 (17), 169
(25). HRMS (EI): calcd for C15H12O3 [M]þ 240.0786; found
240.0782.
4. Experimental section
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 Fertigplat-
ten Kieselgel 60F254) and spots were visualised under UV light.
Chromatography on silica gel was carried out on Merck Kiesel-
gel (230e240 mesh). IR spectra were determined on a Perkine
Elmer FTIR-1600 using KBr discs. UV/visible spectra were run
on a Hitachi U-2000 spectrophotometer. 1H NMR spectra were
recorded on a Varian 300 spectrometer in CDCl3 or DMSO-d6
at 300 MHz at 25 ꢃC. 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 instru-
ment 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 de-
coupling-double resonance and bidimensional heteronuclear
HMBC and HMQC correlation techniques. Mass spectrometry
analyses were performed at the ‘C.A.C.T.I.dUnidad de Espec-
trometria de Masas’, at University of Vigo, Spain. Fluorescence
spectra were collected using a Spex Fluorolog 1680 Spectrom-
eter. Photolyses were carried out using a Rayonet RPR-100
chamber reactor equipped with 10 lamps of 254 (35 W), 300
(21 W), 350 (24 W) and 419 (14 W) nm. HPLC analyses
were performed using a Licrospher 100 RP18 (5 mm) column
in a HPLC system composed by a Jasco PU-980 pump, a UV/
vis Shimadzu SPD-GAV detector and a Shimadzu C-RGA
Chromatopac register.
4.2.3. 1-Formyl-7-methoxy-3-oxo-3H-benzo[g]benzopyran
The previous compound (0.300 g, 1.25 mmol) was reacted
with selenium dioxide (0.420 g, 3.75 mmol) in chlorobenzene
(30 mL), at reflux for 48 h. The mixture was filtered hot and
the solvent was removed by rotary evaporation. The crude resi-
due, was used in the next reaction without purification (yellow
1
solid). Mp¼273.8e275.6 ꢃC. H NMR (DMSO-d6): d¼3.90
(s, 3H, OCH3), 6.51 (s, 1H, H-2), 7.17 (dd, J 8.7 and 2.1 Hz,
1H, H-8), 7.37 (d, J 2.1 Hz, 1H, H-6), 7.81 (s, 1H, H-5), 8.02
(d, J 8.7 Hz, 1H, H-9), 9.00 (s, 1H, H-10), 10.20 (s, 1H,
CHO). 13C NMR (DMSO-d6): d¼55.54 (OCH3), 105.01
(C-6), 110.99 (C-2), 111.42 (C-5), 119.17 (C-8), 124.44
N-Benzyloxycarbonyl-L-g-aminobutyric acid (ZeGABAe
OH)waspurchasedfromSennChemicals,1-chloromethylpyrene

3036
M.J.G. Fernandes et al. / Tetrahedron 64 (2008) 3032e3038
(C-10a), 125.07 (C-9a), 126.69 (C-10), 130.66 (C-9), 135.84 (C-
5a), 143.43 (C-1), 151.05 (C-4a), 159.58 (C-7), 160.76 (C-3),
193.70 (CHO). IR (KBr 1% cmꢀ1): n¼1705, 1628, 1617,
1561, 1487, 1401, 1235, 1167, 1131, 1041, 1026, 930, 902,
883, 810, 725. UV/vis (EtOH, nm): lmax (log 3)¼343 (4.16),
285 (4.25). MS (EI, %): m/z¼254 ([M]þ, 21), 137 (18), 121
(17), 95 (19), 81 (57), 69 (100). HRMS (EI): calcd for
C15H10O4 [M]þ 254.0579; found 254.0589.
4.4. N-Benzyloxycarbonyl-L-g-aminobutyric acid (7-methoxy-
3-oxo-3H-benzo[g]benzopyran-1-yl)methyl ester, ZeGABAe
OBbl (2a)
N-Benzyloxycarbonyl-L-g-aminobutyric acid, ZeGABAe
OH (0.100 g; 0.42 mmol) was reacted with 1-hydroxymethyl-
7-methoxy-3-oxo-3H-benzo[g]benzopyran, BbleOH (1a)
(0.050 g; 0.197 mmol) in DMF (3 mL) using a standard
DCC/HOBt coupling. The solvent was removed by rotary
evaporation under reduced pressure and the crude residue
was purified by column chromatography on silica gel (chloro-
form/methanol, 100:1). Compound 2a was obtained as a yellow
solid (0.025 g, 27%). Mp¼194.0e195.6 ꢃC. 1H NMR (CDCl3):
d¼1.92e1.96 (m, 2H, b-CH2), 2.56 (t, J 7.2 Hz, 2H, a-CH2),
3.28e3.35 (m, 2H, g-CH2), 3.97 (s, 3H, OCH3), 4.90 (br s,
1H, NH), 5.11 (s, 2H, CH2 Bbl), 5.41 (s, 2H, CH2 Z), 6.49
(s, 1H, H-2), 7.13 (d, J 2.4 Hz, 1H, H-6), 7.17 (dd, J 9.3 and
2.7 Hz, 1H, H-8), 7.31e7.36 (m, 5H, 5ꢂPheH), 7.64 (s, 1H,
H-5), 7.81 (d, J 9.0 Hz, 1H, H-9), 7.91 (s, 1H, H-10). 13C
NMR (CDCl3): d¼25.17 (b-CH2), 31.18 (a-CH2), 40.23
(g-CH2), 55.49 (OCH3), 61.22 (CH2 Z), 66.77 (CH2 Bbl),
104.61 (C-6), 112.06 (C-5), 112.66 (C-2), 114.81 (C-10a),
119.85 (C-8), 123.59 (C-10), 125.55 (C-4a), 128.14
(2ꢂPheC), 128.51 (3ꢂPheC), 130.23 (C-9), 136.48 (C-5a),
136.51 (C1 Ph), 148.76 (C-1), 150.71 (C-9a), 156.46 (C]O
urethane), 159.83 (C-7), 160.61 (C-3), 172.41 (C]O ester).
IR (KBr 1%, cmꢀ1): n¼3327, 2928, 2851, 1732, 1721, 1688,
1627, 1575, 1486, 1455, 1312, 1262, 1232, 1171, 1089,
1028. UV/vis (MeOH/HEPES, 80:20, nm): lmax (log 3)¼345
(3.89). MS (FAB, %): m/z¼476 ([MþH]þ, 3), 475 (Mþ, 2),
307 (12), 226 (16), 225 (100), 155 (15), 154 (50). HRMS
(FAB): calcd for C27H26NO7 [MþH]þ 476.1709; found
476.1711.
The previous compound (0.252 g, 0.991 mmol) was reacted
with sodium borohydride (0.026 g, 0.69 mmol) in ethanol
(5 mL) for 3 days at room temperature, affording BbleOH
(1a) as a light pink solid (0.087 g, 34%). Mp¼202.5e
1
206.0 ꢃC. H NMR (DMSO-d6): d¼3.89 (s, 3H, OCH3), 4.86
(d, J 5.4 Hz, 2H, CH2), 5.44 (s, 1H, H-2), 5.70 (t, J 5.4 Hz,
1H, OH), 7.16 (dd, J 9.0 and 2.4 Hz, 1H, H-8), 7.35 (d, J
2.7 Hz, 1H, H-6), 7.70 (s, 1H, H-5), 7.93 (d, J 9.0 Hz, 1H, H-
9), 8.22 (s, 1H, H-10). 13C NMR (DMSO-d6): d¼60.55
(OCH3), 64.20 (CH2), 110.06 (C-6), 115.25 (C-2), 116.10 (C-
5), 120.15 (C-10a), 124.22 (C-8), 129.54 (C-10), 130.30 (C-
9a), 135.57 (C-9), 141.12 (C-5a), 155.52 (C-4a), 161.56 (C-1),
164.25 (C-7), 165.42 (C-3). IR (KBr 1%, cmꢀ1): n¼3380,
2977, 1707, 1630, 1483, 1458, 1444, 1435, 1408, 1333, 1235,
1181, 1147, 1129, 1083, 1024, 980, 972, 939, 899, 879, 859,
842, 808. UV/vis (EtOH, nm): lmax (log 3)¼346 (4.02), 288
(4.05). MS (EI, %): m/z¼256 ([M]þ, 50), 254 (32), 288 (31),
227 (27), 240 (100), 212 (26), 211 (18), 199 (20), 198 (17),
197 (21), 183 (16), 169 (36). HRMS (EI): calcd for C15H12O4
[M]þ 256.0736; found 256.0736.
4.3. Synthesis of 1-hydroxymethyl-4-methoxy-naphthalene,
NpmeOH (1d)
4-Methoxy-1-naphthaldehyde (0.500 g, 2.69 mmol) was
dissolved in ethanol (5 mL) and sodium borohydride
(0.071 g, 1.88 mmol), dissolved previously in ethanol
(6 mL), was added dropwise while stirring in an ice bath.
The reaction mixture was stirred at room temperature for 3
days. The solvent was evaporated to dryness and the residue
submitted to column chromatography (eluent: ethyl acetate/
hexane, 1:3). Compound 1d was obtained as a colourless solid
(0.481 g, 95%). Mp¼65.4e68.0 ꢃC. 1H NMR (CDCl3):
d¼4.02 (s, 3H, OCH3), 5.04 (s, 2H, CH2), 6.74 (d, J 8.1 Hz,
1H, H-3), 7.39 (d, J 7.8 Hz, 1H, H-2), 7.52 (dt, J 6.9 and
1.2 Hz, 1H, H-6), 7.58 (dt, J 6.9 and 1.2 Hz, 1H, H-7), 8.13
(dd, J 8.4 and 1.5 Hz, 1H, H-8), 8.33 (dd, J 8.4 and 1.5 Hz,
1H, H-5). 13C NMR (CDCl3): d¼55.46 (OCH3), 63.61
(CH2), 102.85 (C-3), 122.51 (C-5), 123.61 (C-8), 125.19
(C-6), 125.92 (C-4a), 126.14 (C-2), 126.83 (C-7), 128.45
(C-1), 132.27 (C-8a), 155.66 (C-4). IR (KBr 1%, cmꢀ1):
n¼3375, 3299, 3079, 3014, 2995, 2965, 2934, 2872, 2840,
1583, 1514, 1463, 1450, 1426, 1391, 1338, 1305, 1277,
1249, 1225, 1156, 1091, 1064, 1026, 1010, 999, 822, 767,
738, 616, 510. UV/vis (EtOH, nm): lmax (log 3)¼296 (3.70).
MS (EI, %): m/z¼188 ([M]þ, 95), 187 (18), 172 (75), 171
(80), 156 (18), 144 (39), 129 (68), 128 (100), 127 (46), 115
(48). HRMS (EI): calcd for C12H12O2 [M]þ 188.0837; found
188.0840.
4.5. N-Benzyloxycarbonyl-L-g-aminobutyric acid (4-methoxy-
naphthalen-1-yl)methyl ester, ZeGABAeONpm (2d)
Starting from ZeGABAeOH (0.134 g, 0.567 mmol) and
1-hydroxymethyl-4-methoxy-naphthalene NpmeOH (1d)
(0.050 g, 0.266 mmol), and following a similar procedure as
described for 2a, compound 2d was obtained as a yellow oil
(0.057 g, 52%). 1H NMR (CDCl3): d¼1.79e1.85 (m, 2H,
b-CH2), 2.38 (t, J 7.5 Hz, 2H, a-CH2), 3.18e3.25 (m, 2H,
g-CH2), 4.01 (s, 3H, OCH3), 4.92 (br s, 1H, NH), 5.08 (s, 2H,
CH2 Z), 5.50 (s, 2H, CH2 Npm), 6.76 (d, J 8.1 Hz, 1H, H-3),
7.31e7.38 (m, 5H, 5ꢂPheH), 7.45 (d, J 7.8 Hz, 1H, H-2),
7.51 (dt, J 6.9 and 1.2 Hz, 1H, H-6), 7.57 (dt, J 6.9 and
1.2 Hz, 1H, H-7), 7.95 (dd, J 8.1 and 1.8 Hz, 1H, H-8), 8.33
(dd, J 8.1 and 1.8 Hz, 1H, H-5). 13C NMR (CDCl3): d¼25.06
(b-CH2), 31.47 (a-CH2), 40.28 (g-CH2), 55.48 (OCH3), 64.79
(CH2 Npm), 66.58 (CH2 Z), 102.83 (C-3), 122.62 (C-5),
123.34 (C-8), 125.25 (C-6), 125.81 (C-4a), 127.06 (C-7),
128.02 (2ꢂPheC), 128.44 (3ꢂPheC), 128.45 (C-1), 128.59
(C-2), 132.62 (C-8a), 136.45 (C1 Ph), 156.23 (C]O urethane),
156.37 (C-4), 173.18 (C]O ester). IR (KBr 1%, cmꢀ1):
n¼3418, 3033, 2942, 1726, 1626, 1585, 1515, 1464, 1455,
1394, 1250, 1164, 1093, 818, 768. UV/vis (MeOH/HEPES,

M.J.G. Fernandes et al. / Tetrahedron 64 (2008) 3032e3038
3037
80:20, nm): lmax (log 3)¼295 (3.75). MS (FAB, %): m/z¼408
([MþH]þ, 34), 407 (Mþ, 12), 307 (100), 155 (18), 154 (44).
HRMS (FAB): calcd for C24H26NO5 [MþH]þ 408.1813; found
408.1805.
(C1 Ph), 149.05 (C-1), 155.48 (C-4a), 156.43 (C]O urethane),
160.79 (C-3), 162.85 (C-6), 172.30 (C]O ester). IR (KBr 1%,
cmꢀ1): n¼3311, 3083, 3004, 2948, 1716, 1689, 1622, 1513,
1435, 1411, 1328, 1295, 1207, 1152, 1104, 1063, 1035, 998,
894, 846, 807, 742, 696. UV/vis (MeOH/HEPES, 80:20, nm):
lmax (log 3)¼325 (4.10). MS (FAB, %): m/z¼426 ([MþH]þ,
54), 455 (Mþ, 8), 307 (32), 289 (15), 155 (31), 154 (100).
HRMS (FAB): calcd for C23H24NO7 [MþH]þ: 426.1553;
found: 426.1555.
4.6. N-Benzyloxycarbonyl-L-g-aminobutyric acid (9-methoxy-
3-oxo-3H-benzo[f]benzopyran-1-yl)methyl ester, ZeGABAe
OBba (2b)
ZeGABAeOH (0.095 g; 0.40 mmol) was reacted with 1-
chloromethyl-9-methoxy-3-oxo-3H-benzo[f]benzopyran, Bba-
Cl (1b) (0.110 g; 0.40 mmol) and potassium fluoride (0.070 g,
1.2 mmol) in dry DMF (3 mL). The reaction mixture was stirred
at room temperature for 3 days. The solvent was removed by ro-
tary evaporation under reduced pressure and the crude residue
was purified by column chromatography with chloroform. Com-
pound 2b was obtained as a light brown solid (0.154 g, 81%).
4.8. General photolysis procedure
A 1ꢂ10ꢀ4 M MeOH/HEPES buffer (80:20) solution of
compounds 2aee (5 mL) were placed in a quartz tube and
irradiated in a Rayonet RPR-100 reactor at the desired wave-
length. The lamps used for irradiation were of 254, 300, 350
and 419ꢁ10 nm.
1
Mp¼120.7e121.9 ꢃC. H NMR (CDCl3): d¼1.91e1.96 (m,
Aliquots of 100 mL were taken at regular intervals and an-
alysed by RP-HPLC. The eluent was acetonitrile/water, 3:1, at
a flow rate of 1.0 or 1.2 mL/min (for compound 2e), previ-
ously filtered through a Millipore, type HN 0.45 mm filter
and degassed by ultra-sound for 30 min. The chromatograms
were traced by detecting UV absorption at the wavelength of
maximum absorption for each compound (retention time: 2a,
4.6 min; 2b, 4.4 min; 2c, 3.3 min; 2d, 5.7 min; 2e, 8.8 min).
2H, b-CH2), 2.57 (t, J 7.2 Hz, 2H, a-CH2), 3.28e3.34 (m, 2H,
g-CH2), 3.95 (s, 3H, OCH3), 5.00 (br s, 1H, NH), 5.10 (s, 2H,
CH2 Z), 5.66 (s, 2H, CH2 Bba), 6.65 (s, 1H, H-2), 7.22 (dd, J
8.7 and 2.1 Hz, 1H, H-8), 7.31e7.35 (m, 6H, 5ꢂPheH and
H-5), 7.42 (d, J 2.1 Hz, 1H, H-10), 7.83 (d, J 9.3 Hz, 1H, H-
7), 7.91 (d, J 8.7 Hz, 1H, H-6). 13C NMR (CDCl3): d¼25.04
(b-CH2), 31.12 (a-CH2), 40.16 (g-CH2), 55.43 (OCH3), 64.21
(CH2 Bba), 66.76 (CH2 Z), 105.79 (C-10), 111.88 (C-4b),
112.61 (C-2), 115.26 (C-5), 116.53 (C-8), 126.34 (C-6a),
128.10 (2ꢂPheC), 128.47 (3ꢂPheC), 130.55 (C-6b), 131.31
(C-7), 133.80 (C-6), 136.35 (C1 Ph), 151.02 (C-1), 155.49
(C-4a), 156.54 (C]O urethane), 159.62 (C-9), 160.39 (C-3),
172.33 (C]O ester). IR (KBr 1%, cmꢀ1): n¼3329, 2927,
2851, 1739, 1721, 1691, 1626, 1553, 1456, 1365, 1265, 1229,
1182, 1100, 942, 837. UV/vis (MeOH/HEPES, 80:20, nm):
lmax (log 3)¼350 (3.90). MS (FAB, %): m/z¼476 ([MþH]þ,
22), 475 (Mþ, 5), 432 (10), 314 (28), 276 (14), 240 (10), 230
(14), 193 (12), 192 (61), 175 (22), 174 (42), 154 (25). HRMS
(FAB): calcd for C27H26NO7 [MþH]þ: 476.1709; found:
476.1705.
Acknowledgements
Thanks are due to the Foundation for Science and Technol-
ogy (Portugal) for financial support through project PTDC/
QUI/69607/2006 and a Ph.D. grant to M.J.G.F. (SFRH/BD/
36695/2007).
References and notes
1. Barltrop, J. A.; Schofield, P. Tetrahedron Lett. 1962, 3, 697.
2. (a) Liu, Z.-C.; Shin, D.-S.; Shokouhimehr, M.; Lee, K.-N.; Yoo, B.-W.;
Kim, Y.-K.; Lee, Y.-S. Biosens. Bioelectron. 2007, 22, 2891; (b) Gagey,
N.; Neveu, P.; Benbrahim, C.; Goetz, B.; Aujard, I.; Baudin, J.-B.; Jullien,
L. J. Am. Chem. Soc. 2007, 129, 9986; (c) Gagey, N.; Neveu, P.; Jullien, L.
Angew. Chem., Int. Ed. 2007, 46, 2467; (d) Pelliccioli, A. P.; Wirz, J.
Photochem. Photobiol. Sci. 2002, 1, 441.
4.7. N-Benzyloxycarbonyl-L-g-aminobutyric acid (6-methoxy-
3-oxo-3H-benzopyran-1-yl)methyl ester, ZeGABAe
OBpm (2c)
´
3. Bourgault, S.; Letourneau, M.; Fournier, A. Peptides 2007, 28, 1074.
4. Adams, S. R.; Kao, J. P. Y.; Tsien, R. Y. J. Am. Chem. Soc. 1989, 111,
7957.
Starting from ZeGABAeOH (0.112 g; 0.42 mmol) and
1-chloromethyl-6-methoxy-3-oxo-3H-benzopyran,
Bpm-Cl
5. Ellis-Davies, G. C. R.; Barsotti, R. J. Cell Calcium 2006, 39, 75.
6. Maier, W.; Corrie, J. E. T.; Papageorgiou, G.; Laube, B.; Grewer, C.
J. Neurosci. Methods 2005, 142, 1.
7. Huang, Y. H.; Sinha, S. R.; Fedoryak, O. D.; Ellis-Davies, G. C. R.;
Bergles, D. E. Biochemistry 2005, 44, 3316.
(1c) (0.097 g; 0.42 mmol), and following a similar procedure
as described for 2b, compound 2c was obtained as a off-white
solid (0.152 g, 85%). Mp¼117.8e118.5 ꢃC. 1H NMR
(CDCl3): d¼1.86e1.95 (m, 2H, b-CH2), 2.51 (t, J 7.2 Hz, 2H,
a-CH2), 3.26e3.31 (m, 2H, g-CH2), 3.87 (s, 3H, OCH3), 4.97
(br s, 1H, NH), 5.09 (s, 2H, CH2 Z), 5.24 (s, 2H, CH2 Bpm),
6.31 (s, 1H, H-2), 6.84 (dd, J 7.1 and 2.4 Hz, 1H, H-7), 6.88
(d, J 2.4 Hz, 1H, H-5), 7.27e7.40 (m, 6H, 5ꢂPheH and H-8).
13C NMR (CDCl3): d¼25.08 (b-CH2), 31.10 (a-CH2), 40.18
(g-CH2), 55.75 (OCH3), 61.22 (CH2 Z), 66.68 (CH2 Bpm),
101.17 (C-5), 109.99 (C-2), 110.53 (C-8a), 112.60 (C-7),
124.40 (C-8), 128.08 (2ꢂPheC), 128.46 (3ꢂPheC), 136.42
8. Banerjee, A.; Grewer, C.; Ramakrishnan, L.; Jager, J.; Gameiro, A.;
Breitinger, H.-G. A.; Gee, K. R.; Carpenter, B. K.; Hess, G. P. J. Org.
Chem. 2003, 68, 8361.
9. Li, L.-S.; Babendure, J. L.; Sinha, S. C.; Olefsky, J. M.; Lerner, R. A.
Bioorg. Med. Chem. Lett. 2005, 15, 3917.
10. Schlichting, I.; Rapp, G.; John, J.; Wittinghoffer, A.; Pai, E. F.; Goody,
R. S. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 7687.
11. Aujard, I.; Benbrahim, C.; Gouget, M.; Ruel, O.; Baudin, J.-B.; Neveu, P.;
Jullien, L. Chem.dEur. J. 2006, 12, 6865.
12. Pincock, J. A. Acc. Chem. Res. 1997, 30, 43.

3038
M.J.G. Fernandes et al. / Tetrahedron 64 (2008) 3032e3038
´
13. Cameron, J. F.; Willson, C. G.; Frechet, J. M. J. J. Am. Chem. Soc. 1996,
118, 12925.
26. Vasquez, M. E.; Nitz, M.; Stehn, J.; Yaffe, M. B.; Imperiali, B. J. Am.
Chem. Soc. 2003, 125, 10150.
14. Givens, R. S.; Weber, J. F. W.; Conrad, P. G.; Orosz, G.; Donahue, S. L.;
Thayer, S. A. J. Am. Chem. Soc. 2000, 122, 2687.
15. Turner, A. D.; Pizzo, S. V.; Rozakis, G. W.; Porter, N. A. J. Am. Chem.
Soc. 1987, 109, 1274.
27. Canepari, M.; Nelson, L.; Papageorgiou, G.; Corrie, J. E. T.; Ogden, D.
J. Neurosci. Methods 2001, 112, 29.
28. Curten, B.; Kullmann, H. M. P.; Bier, M. E.; Kandler, K.; Schmidt, B. F.
Photochem. Photobiol. 2005, 81, 641.
16. Pirrung, M. C.; Fallon, L.; Zhu, J.; Lee, Y. R. J. Am. Chem. Soc. 2001,
123, 3638.
17. Furuta, T.; Hirayama, Y.; Iwamura, M. Org. Lett. 2001, 3, 1809.
18. Yu, J.-Y.; Tang, W.-J.; Wang, H.-B.; Song, Q.-H. J. Photochem. Photo-
biol., A 2007, 185, 101.
29. (a) Piloto, A. M.; Costa, S. P. G.; Gonc¸alves, M. S. T. Tetrahedron Lett.
2005, 46, 4757; (b) Piloto, A. M.; Fonseca, A. S. C.; Costa, S. P. G.;
Gonc¸alves, M. S. T. Tetrahedron 2006, 62, 9258; (c) Piloto, A. M.;
Rovira, D.; Costa, S. P. G.; Gonc¸alves, M. S. T. Tetrahedron 2006, 62,
11955; (d) Fonseca, A. S. C.; Gonc¸alves, M. S. T.; Costa, S. P. G. Tetra-
hedron 2007, 63, 1353; (e) Fernandes, M. J. G.; Gonc¸alves, M. S. T.;
Costa, S. P. G. Tetrahedron 2007, 63, 10133.
19. Iwamura, M.; Hodota, C.; Ishibashi, M. Synlett 1991, 35.
20. Iwamura, M.; Ishikawa, T.; Koyama, Y.; Sakuma, K.; Iwamura, H.
Tetrahedron Lett. 1987, 28, 679.
30. Tao, Z.-F.; Qian, X.; Fan, M. Tetrahedron 1997, 53, 13329.
31. (a) Buchi, G.; Foulkes, D. M.; Kurono, M.; Mitchell, G. F.; Schneider,
R. S. J. Am. Chem. Soc. 1967, 89, 6745; (b) Furuta, T.; Takeuchi, H.;
Isozaki, M.; Takahashi, Y.; Kahenara, M.; Sugimoto, M.; Watanabe, T.;
Noguchi, K.; Dore, T. M.; Kurahashi, T.; Iwamura, M.; Tsien, R. Y.
ChemBioChem 2004, 5, 1119.
21. Singh, A. K.; Prashant, K. K. Tetrahedron Lett. 2005, 46, 5563.
22. Shembekar, V. R.; Chen, Y.; Carpenter, B. K.; Hess, G. P. Biochemistry
2005, 44, 7107.
23. Hagen, V.; Bendig, J.; Frings, S.; Wiesner, B.; Schade, B.; Helm, S.;
Lorenz, D.; Kaupp, U. B. J. Photochem. Photobiol., B: Biol. 1999,
53, 91.
32. Bodanszky, M.; Bodanszky, A. The Practice of Peptide Synthesis;
Springer: Berlin, 1984.
´
24. Muller, C.; Even, P.; Viriot, M.-L.; Carre, M.-C. Helv. Chim. Acta 2001,
84, 3735.
25. Tang, X. J.; Dmochowski, I. J. Org. Lett. 2005, 7, 279.
33. Tjoeng, F. S.; Heavner, G. A. Synthesis 1981, 897.
34. Morris, J. V.; Mahaney, M. A.; Huber, J. R. J. Phys. Chem. 1976, 80, 969.