Synthesis of short and long-wavelength functionalised probes: amino acids' labelling and photophysical studies

Article abstract of DOI:10.1016/j.tet.2007.09.042

Fluorescent labelling of α-amino acids at their N or C terminals in the main and lateral chains at short and long wavelengths was carried out in different ways. The N-[3-(naphthalen-1-ylamino)propanoyl]amino acid methyl esters synthesised showed strong fluorescence in the visible region (～415 nm) of the electromagnetic spectrum. Condensation of these compounds with 5-diethylamino-2-nitrosophenol or 5-ethylamino-4-methyl-2-nitrosophenol produced the benzo[a]phenoxazine derivatives, with maximum emission wavelengths shifted to values higher than 644 nm. The synthesis of novel functionalised 5,9-diaminobenzo[a]phenoxazinium salts, by reaction of 5-ethylamino-4-methyl-2-nitrosophenol and N-substituted 1-naphthylamine and their use in the covalent labelling of the N or C terminals of valine, produced derivatives with long-wavelength emissions (644-653 nm). Photophysical studies using the synthesised compounds both in different solvents and in controlled pH were undertaken. Preliminary evaluation of photostability of the cationic polycyclic heterocycles in ethanol and water at physiological pH was also performed.

Full text of DOI:10.1016/j.tet.2007.09.042

Tetrahedron 63 (2007) 12405–12418
Synthesis of short and long-wavelength functionalised probes:
amino acids’ labelling and photophysical studies
a
a
a
b
V ^a nia H. J. Frade, S ꢀı ria A. Barros, Jo ~a o C. V. P. Moura, Paulo J. G. Coutinho and
a,
*
M. Sameiro T. Gon c¸ alves
a
Centro de Qu ꢀı mica, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
b
Centro de F ꢀı sica, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
Received 1 August 2007; revised 11 September 2007; accepted 19 September 2007
Available online 23 September 2007
Abstract—Fluorescent labelling of a-amino acids at their N or C terminals in the main and lateral chains at short and long wavelengths was
carried out in different ways. The N-[3-(naphthalen-1-ylamino)propanoyl]amino acid methyl esters synthesised showed strong fluorescence in
the visible region (w 415 nm) of the electromagnetic spectrum. Condensation of these compounds with 5-diethylamino-2-nitrosophenol or
5
-ethylamino-4-methyl-2-nitrosophenol produced the benzo[a]phenoxazine derivatives, with maximum emission wavelengths shifted to
values higher than 644 nm. The synthesis of novel functionalised 5,9-diaminobenzo[a]phenoxazinium salts, by reaction of 5-ethylamino-
-methyl-2-nitrosophenol and N-substituted 1-naphthylamine and their use in the covalent labelling of the N or C terminals of valine,
4
produced derivatives with long-wavelength emissions (644–653 nm). Photophysical studies using the synthesised compounds both in differ-
ent solvents and in controlled pH were undertaken. Preliminary evaluation of photostability of the cationic polycyclic heterocycles in ethanol
and water at physiological pH was also performed.
Ó 2007 Elsevier Ltd. All rights reserved.
1
3,14
15,16
1. Introduction
fluorophores (>600 nm) include squaraine,
as well as benzo[a]phenoxazine heterocycles.
cyanine
1
7,18
Fluorescent probes for labelling of biomolecules have been
widely used for analytical purposes in many areas of con-
temporary science. The label molecule may contain a reac-
tive functional group for a covalent linkage to the analyte.
Alternatively, a non-functionalised marker may possess
a strong non-covalent interaction with the biomolecule for
the formation of a stable complex. Despite the fact that the
interactions resulting from the latter methodology are signifi-
cantly less stable than the covalent attachment, it is also
extremely important.
Despite the interest of all these compounds, the use of labels
that absorb and fluoresce in the red (600–700 nm) or near-
infrared (>700 nm) regions of the electromagnetic spectrum
is valuable. Considering the low interference from solvents
and the biological matrix, the far-wavelength detection is
the most advantageous.
Bearing this in mind and in connection with our interest in
the synthesis, characterisation and application of fluorescent
2
,19,20
21
labels,
we have extended our preliminary work
Covalent labelling at short wavelengths (<500 nm) of bio-
molecules has been achieved by using heterocycles, such as
related to the fluorescent derivatisation of biomolecules
through the preparation of N-[3-(naphthalen-1-ylamino)pro-
panoyl]amino acid methyl esters and their conversion into
the corresponding benzo[a]phenoxazinium conjugates
through condensation with 5-diethylamino-2-nitrosophenol
or 5-ethylamino-4-methyl-2-nitrosophenol.
1
2
oxobenzopyranes, oxobenzo[f]benzopyranes, naphtho-
furanes, oligothiophenes, 4,7-phenanthroline-5,6-dione
3
4
5
6
and benzoxadiazoles. The use of naphthalene derivatives,
such as dansyl chloride and naphthalene 2,3-dicarboxalde-
7
,8
9
,10
11
hyde, hasalsobeenreported. Fluorescein andrhodamine
based labels produced fluorescent bioconjugates in the
range of about 500–600 nm. Examples of long-wavelength
1
2
Maximum emission wavelengths were shifted from about
415 nm, in the former fluorescent derivatives, to values
higher than 644 nm in the latter. Another approach for
long-wavelength fluorescent labelling was also performed
through the synthesis of novel functionalised 5,9-diamino-
benzo[a]phenoxazinium salts through the reaction of 5-
ethylamino-4-methyl-2-nitrosophenol with N-substituted
1-naphthylamine, and their use in the covalent labelling of
Keywords: Functionalised probes; Amino acids’ derivatisation; Long-wave-
length fluorescent dyes; Benzo[a]phenoxazine; Fluorescent labelling.
*
Corresponding author. Fax: +351 253604382; e-mail: msameiro@
quimica.uminho.pt
0
040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.09.042

1
2406
V. H. J. Frade et al. / Tetrahedron 63 (2007) 12405–12418
Table 1. Synthesis of labelled amino acid derivatives 3a–e, 5, 8a–f and 9
the N or C terminals of valine. Photophysical characterisa-
tion of the synthesised compounds in different solvents
and in controlled pH was undertaken. The last objective
of this research was to study the behaviour of the cationic
polycyclic heterocycles synthesised when exposed to
ultraviolet and visible radiation. Thus, preliminary evalua-
tion of their photostability at 350 and 419 nm was also car-
ried out.
Entry
Compound
Yield (%)
1
2
3
4
5
6
7
8
9
10
1
12
3a
3b
3c
3d
3e
5
8a
8b
8c
8d
8e
8f
9
Nap-Ala-OMe
Nap-Val-OMe
87
85
90
85
95
98
96
57
78
67
76
68
88
Nap-Gly-OMe
Nap-Phe-OMe
Nap-Glu(OMe)-OMe
Ac-Lys(Nap)-OMe
Bfa-Ala-OMe
Bfe-Ala-OMe
Bfe-Val-OMe
Bfe-Gly-OMe
Bfe-Phe-OMe
2
. Results and discussion
1
1
9
Bfe-Glu(OMe)-OMe
Ac-Lys(Bfe)-OMe
3
-(Naphthalen-1-ylamino)propanoic acid, Nap-OH (1a)
1
3
was synthesised by alkylation of 1-naphthylamine with
methyl-3-bromopropionate, followed by hydrolysis (NaOH/
1
,4-dioxane) of the isolated ester intermediate—methyl 3-
naphthalen-1-ylamino)propanoate (1b). Compound 1a
1
(
was used in the covalent derivatisation of the a-amine group
of L-alanine methyl ester (2a), by coupling with N,N -dicy-
H NMR spectra showed signals of the amino acid residues,
such as a singlet for the methyl ester protons (d 3.69–
3.74 ppm), a multiplet for the a-CH (d 4.50–4.95 ppm,
except for 3c, which was a doublet at 4.03 ppm) and a
doublet or broad singlet (3c) for a-NH (d 6.24–6.78 ppm),
in addition to the protons of the naphthalene moiety.
0
clohexylcarbodiimide (DCC) assisted by 1-hydroxybenzo-
triazole (HOBt) under standard conditions. To extend this
research to other residues, a set of representative a-amino
acid methyl esters, namely valine, glycine, phenylalanine
and glutamic acid (protected at its carboxyl side-chain as
methyl ester) were used in the same conditions (Scheme 1
and Table 1, entries 2–5). In addition to labelling amino acids
at their N-terminus, the alternative acylation at a lysine
u-amine group was also investigated. Thus, the methyl ester
of N-acetyl-L-lysine (4) was reacted with 1a under the condi-
tions reported above to give the expected fluorescent deriva-
tive 5 (Scheme 1 and Table 1, entry 6).
1
3
In C NMR, signals of amide type carbonyl were found
from d 170.24 to 172.17 ppm and those of the ester type
occurred from d 171.89 to 173.44 ppm.
As was expected, and in accordance with the results ob-
tained in the photophysical study, which will be discussed
latter, labelled amino acid derivatives 3a–e and 5 showed
maximum absorbance and fluorescence inferior to 331 nm
and at about 415 nm, respectively.
After purification by chromatography on silica gel, the
corresponding conjugates 3a–e and 5 were obtained as oils
or oily solid (3b) in yields ranging from 85 to 98% (Table 1,
entries 1–6) and were characterised by high resolution
Considering the importance of long-wavelength fluorescent
labelling, we decided to study the possibility of red shifting
the fluorescence of the previously synthesised amino acid
derivatives 3a–e and 5 by using them in the preparation
of the corresponding polycyclic systems based on the
benzo[a]phenoxazine nucleus.
1
13
mass spectrometry, NMR ( H and C) and IR spectroscopies.
The IR spectra of labelled compounds showed bands due to
stretching vibrations of the carbonyl group from 1627 to
ꢀ1
ꢀ1
1
(
651 cm (amide linkage) and from 1726 to 1750 cm
ester group).
From the few synthetic methodologies for the preparation
of benzo[a]phenoxazinium salts, which have been
H
H
O
C
R
R
C
H
2
N
a)
N
CH2CH2CO2H
+
H2N
CO2CH3
CH2CH2
N
H
C
H
CO2CH3
1a
3
2
, 3 a R = CH3
b R = CH(CH3)2
c R = H
d R = CH2-Ph
e R = CH2CH2CO2CH3
H
O
a)
N
1a
+
H2N-(CH2)4-CH-CO2CH3
CH2CH2
C
NH (CH2)4CHCO2CH3
NHCOCH3
NHCOCH3
4
5
Scheme 1. Synthesis of labelled amino acid derivatives 3a–e and 5. Reagents and condition: (a) DCC, HOBt, DMF, rt.

V. H. J. Frade et al. / Tetrahedron 63 (2007) 12405–12418
12407
R1
R2
R1
NO
N
a)
+
3a-e
R2
H
N
OH
N+
O
N
O
C
R
C
R3
R3 Cl
-
CH CH
N
H
2 3
CO CH
7
2
2
8
a R1 = H, R2 = R = CH2CH3
3
H
1
2
3
b R = CH3, R = H, R = CH2CH3
a R = CH3, R = H, R2 = R = CH2CH3
1
3
1
2
3
b R = CH3, R = CH3, R = H, R = CH2CH3
1
2
3
c R = CH(CH3)2, R = CH3, R = H, R = CH2CH3
1
2
3
d R = H, R = CH3, R = H, R = CH2CH3
1
2
3
e R = CH2Ph, R = CH3, R = H, R = CH2CH3
f R = (CH2)2CO2CH3, R = CH3, R = H, R = CH2CH3
1
2
3
R1
N
O
a)
7b
+
5
H
H
+
-
N
N
O
H3CH2C
Cl
CH2CH2
C N (CH2)4CH CO2CH3
9
H
NHCOCH3
+
Scheme 2. Synthesis of benzo[a]phenoxazinium chlorides 8a–f and 9. Reagents and condition: (a) H /methanol, reflux.
2
2,17
1
reported,
themostappliedsyntheticprotocoliscondensa-
H NMR spectra showed signals of the naphthalene ring and
tion, in the presence of a strong mineral acid, of nitro-
soanilines or nitrosonaphthylamines with the appropriate
the amino acid residues (coming from derivatives 3a–e and
5), as well as the aliphatic and aromatic protons resulting
from the introduction of the new unity. Thus, the protons
1
-naphthylamine, 3-aminophenol or 2-naphthol. Following
this methodology, compounds 3a–e and 5 were reacted with
-diethylamino-2-nitrosophenol (7a) or 5-ethylamino-4-
1
of methyl groups (R ) appeared as a singlet (d 2.25–
2.40 ppm) and the protons of the ethyl groups (R and R ),
2
3
5
methyl-2-nitrosophenol(7b), usingconcentratedhydrochloric
acid (Scheme 2). The required 5-alkylamino-2-nitrosophenol
hydrochloride 7a and 7b was synthesised using the usual
procedure involving treatment of the corresponding 3-alkyl-
aminophenol with sodium nitrite in an acid solution.
namely CH as a multiplet (8a, 8e and 9), broad singlet
3
(8b) or triplet (8c, 8d and 8f) (d 1.23–1.60 ppm) and CH2
as a multiplet or a broad singlet (8b) (d 3.0–3.80 ppm).
The aromatic protons, such as H-10 occurred as a double
doublet (d 6.97 ppm) (8a), H-8 as a singlet or doublet
2
3
(
doublet (8a) (d 7.36–7.77 ppm).
J¼2.4 Hz) (d 6.18–6.62 ppm) and H-11 as a singlet or
The fluorophores are designated in this report as a letter co-
de so as to simplify the naming of the various amino acid
fluorescent derivatives. For example: N-ethyl-N-{5-[3-
(1-methoxy-1-oxopropan-2-ylamino)-3-oxopropylamino]-9H-
benzo[a]phenoxazin-9-ylidene}ethanaminium chloride (8a)
is Bfa-Ala-OMe.
1
3
In C NMR, we highlighted the signals of the aliphatic
carbons of the heterocyclic, namely the methyl (d 17.64–
18.36 ppm) and ethyl groups (CH d 12.61–14.16 ppm and
3
CH d 37.54–45.89 ppm), as well as the aromatic signals
2
of C-6 (d 92.81–94.03 ppm), C-8 (d 93.24–95.60 ppm) and
C-11 (d 131.06–132.58 ppm). Signals of the carbonyl group
were found from d 170.65 to 171.97 ppm (amide type) and
from d 171.87 to 173.51 ppm (ester bond).
Reaction of the nitrosophenol 7a with the alanine derivative
3a (reflux in methanol) produced conjugate Bfa-Ala-OMe
8a). Starting from the nitroso compound 7b and using
(
derivative 3a once again, Bfe-Ala-OMe (8b) was obtained.
Through the comparison of fluorescent data obtained for
these derivatives, which will be discussed later, it was
concluded that 5-ethylamino-4-methyl-2-nitrosophenol (7b)
produced the conjugate with superior fluorescence proper-
ties. As a result, precursor 7b was used as the nitroso
component in the cyclisation reactions with derivatives
The design of new probes is determined by the spectral
properties of the material to be marked. To obtain fluoro-
phores with the adequate characteristics for the non-covalent
or covalent labelling of biological material, as was purpose
of this work, one of the following possibilities must be
considered. The first is the use of labels with high lifetimes
(>100 ns). In this case, the detection is initiated after
a certain time of decay so as to eliminate the intrinsic fluo-
rescence of the biological material. The other possibility
is labelling using fluorophores possessing wavelengths of
excitation and emission, which are considerably superior
to that of the background. The auto-fluorescence of the
biological material is particularly high at wavelengths infe-
rior to 500 nm and reduced at wavelengths higher than
600 nm.
3
b–e and 5 (Scheme 2).
After purification by dry chromatography, compounds 8a–f
and 9 were isolated as blue solids in yields ranging from 57
to 96% (Table 1, entries 7–13) and were fully characterised
using the usual analytical techniques.
The IR spectra of labelled compounds showed bands due to
the stretching vibrations of the carbonyl group from 1633 to
ꢀ
1
ꢀ1
1
(
659 cm (amide linkage) and from 1720 to 1746 cm
ester group), as happened in the case of compounds 3a–e
and 5.
On the other hand, the number of fluorescent probes, which
absorb and fluoresce in the region of 600–1000 nm is low

12408
V. H. J. Frade et al. / Tetrahedron 63 (2007) 12405–12418
H3C
NO
OH
H3C
N
a)
+
H
H
H
N
H
N
O
N+
N
H3CH2C
CH2CH2R
H CH C
-
2 2
CH CH R
3
2
Cl
7b
1
10
a R = CO2H
c R = OH
d R = NH2
a R = CO2H
b R = OH
c R = NH2
+
ꢁ
Scheme 3. Synthesis of benzo[a]phenoxazinium chlorides 10a–c. Reagents and conditions: (a) H /DMF (70 C, compound 10a) or methanol (reflux,
compounds 10b,c).
and it is even lower in a suitable functional group for cova-
lent linkage of the analyte.
(d 1.36–1.43 ppm) and CH as doublet (2ꢂd) (10a), broad
2
singlet (10b) ormultiplet(10c) (d 3.51–3.78 ppm), and NCH -
2
CH as a triplet or a broad singlet (10b) (d 2.71–4.08 ppm).
2
Considering these facts, we decided to synthesise new func-
tionalised benzo[a]phenoxazinium salts analogous to Nile
Blue as well as to study the possibility of covalent bonding
of these fluorophores with organic molecules of biological
interest, using L-valine as a model.
The highlighted aromatic protons are H-8 (singlet, d 6.42–
7.02 ppm), H-6 (singlet, d 6.70–7.17 ppm) and H-11 (singlet
or multiplet (10c), d 7.19–7.96 ppm).
1
3
The C NMR spectra of compounds 10a–c also confirmed
the presence of the methyl group and the N-substituents,
with signals assigned to the methyl groups (d 17.77–
Benzo[a]phenoxazinium chlorides 10a–c were synthesised
by condensation of 5-ethylamino-2-nitrosophenol hydro-
chloride 7b with N-alkylated-naphthylamines 1a and 1c,d
in the presence of hydrochloric acid; heating occurred at
18.47 ppm), ethyl groups (CH , d 14.17–14.51 ppm and
3
CH , d 39.70 and 48.03 ppm) and NCH CH (d 34.93–
2
2
2
ꢁ
19
7
3
1
0 C (10a) or reflux in ethanol (10b and 10c) (Scheme
). Intermediates 1c and 1d were prepared by alkylation of
-naphthylamine with the appropriate chloro-derivative, 2-
60.98 ppm). Concerning the aromatic carbons, signals of
C-8 (d 93.92–94.60 ppm), C-6 (d 94.40–95.17 ppm) and
C-11 (d 132.60–133.71 ppm) were highlighted. In the case
of compound 10a, a signal at d 164.30 ppm was assigned
to the carbonyl function.
chloroethanol and 2-chloroethylamine monohydrochloride,
respectively. Preparation of intermediate 1a was previously
described.
Compounds 10a–c, functionalised charged heterocycles,
have the potential ability for covalent labelling applications,
in addition to their non-covalent labelling capacity. In this
work, the possibility of using these compounds in covalent
labelling of biomolecules was carried out, using L-valine
as a model.
After dry chromatography purification compounds 1c and
1
d were obtained as an oil (1c, 24%) or as a solid (1d,
1%), and they were characterised by high resolution mass
7
1
13
spectrometry, NMR ( H and C) and IR spectroscopies.
The IR spectra showed strong stretching vibration bands
for the hydroxyl and amine groups between 3055 and
ꢀ1
3
389 cm
.
Dyes 10a–c were linked to the a-amine group of the L-valine
methyl ester, H-Val-OMe (2b) or the carboxylic acid of
N-tert-butyloxycarbonyl-L-valine Boc-Val-OH (11), by cou-
1
13
In the H and C NMR spectra, signals of N-substituent
protons occurred at d 3.38–4.02 ppm and d 39.62–
0
pling with the aid of N,N -dicyclohexylcarbodiimide (DCC)
61.13 ppm, respectively, in addition to the presence of naph-
thalene signals.
assisted by 1-hydroxybenzotriazole (HOBt), under standard
conditions. Thus, reaction of the carboxylic acid dye Bfe-
OH (10a) with H-Val-OMe (2b) produced the amide deriva-
tive Bfe-Val-OMe (8c), which was also obtained using the
first labelling methodology described. When Boc-Val-OH
(11) was coupled to the hydroxyl dye, Bfh-H (10b), the ester
conjugate Boc-Val-OBfh (12) was isolated. Derivatisation of
L-valine (Boc-Val-OH, 11) with the amino fluorophore
Bfn-H (10c) resulted in the fluorescent derivative Boc-Val-
OBfn (13) (Scheme 4).
Dry chromatography purification or successive washes with
solvents (10c) produced benzo[a]phenoxazinium chlorides
1
0a–c as blue solids in yields ranging from 45 to 98% (Table
). These polycyclic functionalised heterocycles were fully
2
characterised by the usual analytical techniques.
1
H NMR spectra showed signals of the aliphatic protons of
methyl groups, such as a singlet (d 2.16–2.44 ppm) and ethyl
groups, namely CH as a triplet or a broad singlet (10b)
3
After dry chromatography purification, the L-valine conju-
gates 8c, 12 and 13 were obtained as blue solids in yields
ranging from 89 to 94% (Table 2) and were characterised
by high resolution mass spectrometry, NMR ( H and C)
and IR spectroscopies. Data related to compound 8c were
previously discussed.
Table 2. Synthesis of benzo[a]phenoxazinium chlorides 10a–c and labelled
amino acid derivatives 8c, 12 and 13
1
13
Entry
Compound
Yield (%)
1
2
3
4
5
6
10a
10b
10c
8c
12
13
Bfe-OH
Bfh-H
Bfn-H
Bfe-Val-OMe
Boc-Val-OBfh
Boc-Val-OBfn
45
95
98
92
94
89
The IR spectra of conjugates 12 and 13 showed bands due to
the stretching vibrations of the carbonyl group of the ester
ꢀ1
and the amide functions at 1743 cm
1
and at about
ꢀ
1
643 cm , respectively.

V. H. J. Frade et al. / Tetrahedron 63 (2007) 12405–12418
12409
CH(CH3)2
H3C
N
O
a)
10a
+
H2N
C
H
CO CH
2
3
H
H
O
C
CH(CH3)2
C CO2CH3
H
N+
Cl
N
H3CH2C
CH2CH2
N
H
-
2b
8c
H3C
N
10b
H
H
CH(CH3)2
N+
O
12
N
N
H3CH2C
-
C
CH2CH2
O
C
O
N
Boc
Cl
CH(CH3)2 a)
H
H
Boc
N
H
C
H
CO2H
1
1
H3C
N
10c
H
H
CH(CH3)2
N+
Cl-
O
H3CH2C
CH2CH2
N
H
C C
N
Boc
13
O H
H
Boc - tert-butyloxycarbonyl
Scheme 4. Synthesis of labelled amino acid derivatives 8c, 12 and 13. Reagents and condition: (a) DCC, HOBt, DMF, rt.
1
H NMR spectra of these compounds showed signals of the
valine amino acid, such as a singlet for the methyl protons of
the Boc group (d 1.37 ppm), a multiplet (d 3.95–4.10 ppm,
the presence of the amino acid residues did not produce al-
teration in their value or was negligible (1a, 330 nm/3d,
320 nm).
1
2) or a doublet (d 3.90 ppm, 13) for the a-CH, as well as
1
3
the protons of the label moiety. In C NMR, signals of the
carbonyl were found at about d 158 ppm (urethane type),
d 173.98 ppm (ester bond, 12) and d 176.36 ppm (amide
type, 13).
It is known that the photophysics of naphthylamines in aque-
2
4
ous solutions is strongly pH-dependent, through acid–base
equilibria both in the excited and ground states. In order to
see if the amino acid substitution had some effect on the
corresponding pK values, some preliminary studies were
a
ꢀ
5
ꢀ6
The electronic absorption spectra of 10 to 10 M solu-
tions of label molecules, and the corresponding conjugates
synthesised (1a, 3a–e, 5, 8a–f, 9, 10a–c, 12 and 13) in
degassed absolute ethanol, were measured. Summarised
data of this study are presented in Table 3. Regarding com-
pounds 1a, 3a–e and 5, which absorbed in the ultraviolet
region of the electromagnetic spectrum, as was expected,
the longest wavelength of maximum absorption (lmax) being
w330 or 320 nm (3d, entry 5), with molar absorptivities
undertaken with compound 3a (solutions of methanol/
buffers, 0.02:0.98). From the normalised fluorescence spec-
tra, in Figure 1, it can be seen that only at pH¼3 the proton-
ated form started to appear through a structured emission to
the blue side of the spectrum. In DMAN (N,N-dimethyl-1-
aminonaphthalene) at pH¼3, the fluorescence spectrum
2
4
was dominated by the acidic form. Thus, the pK in the ex-
a
cited state of 3a was much lower than the reported value of
2.5 for DMAN. This can be partly explained by the nearby
carbonyl group, which, through hydrogen bond interaction,
probably forms a six-membered ring with the amino group.
ꢀ
1
ꢀ1
between 3139 (3b, entry 3) and 10,682 M cm (3a).
Comparison of lmax values of derivatives 3a–e (entries 2–
6
) and 5 (entry 7) with compound 1a (entry 1) showed that
The transformation of derivatised amino acids 3a–e and 5,
by condensation with the appropriate nitrosophenol, into
Table 3. UV/visible data for compounds 1a, 3a–e, 5, 8a–f, 9, 10a–c, 12 and
3 in ethanol and water (pH 7.4)
1
Entry
Compound
Ethanol
abs [nm] (3)
Water (pH 7.4)
labs [nm] (3)
l
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
1a
3a
3b
3c
3d
3e
5
8a
8b
8c
8d
8e
8f
Nap-OH
330 (7000)
331 (10,682)
330 (3139)
330 (5960)
320 (3596)
—
—
—
—
—
—
—
Nap-Ala-OMe
Nap-Val-OMe
Nap-Gly-OMe
Nap-Phe-OMe
Nap-Glu(OMe)-OMe 330 (7442)
Ac-Lys(Nap)-OMe
Bfa-Ala-OMe
Bfe-Ala-OMe
Bfe-Val-OMe
Bfe-Gly-OMe
Bfe-Phe-OMe
330 (7000)
640 (22,115) 650 (11,299)
630 (23,558)
630 (39,815) 625 (34,184)
630 (32,283) 625 (14,970)
630 (40,945) 625 (21,778)
—
0
1
2
3
4
5
6
7
8
9
Bfe-Glu(OMe)-OMe 630 (30,500) 625 (24,500)
Ac-Lys(Bfe)-OMe
9
630 (42,857) 630 (27,439)
625 (26,364) 625 (12,945)
625 (26,479) 619 (21,667)
617 (22,400) 620 (17,389)
629 (22,646) 626 (20,175)
630 (27,917) 627 (15,252)
10a Bfe-OH
10b Bfh-H
10c Bfn-H
12
13
Boc-Val-OBfh
Boc-Val-OBfn
Figure 1. Normalised fluorescence intensity spectra of compound 3a at
different pH values.

12410
V. H. J. Frade et al. / Tetrahedron 63 (2007) 12405–12418
the corresponding benzo[a]phenoxazine polycyclic deriva-
tives 8a–f and 9 shifted the maximum absorbance to 630
and 640 nm (8a), with molar absorptivities between
1
10c
ꢀ1 ꢀ1
0.8
2
2,115 and 42,857 M cm .
10b
0
0
0
.6
.4
.2
0
The comparison of the maximum absorption wavelengths of
the two groups of labelled amino acids 3a–e and 5 (entries 2–
13
1
2
7
)/8a–f and 9 (entries 8–14), in Table 3, reveals that a batho-
chromic shift from 299 nm (3a/8b alanine) to 310 nm (3d/8e
phenylalanine) has occurred. Thus, for example, it was
possible to obtain derivatives of phenylalanine with absorp-
tion at 320 and 630 nm. Functionalised benzo[a]phenoxazi-
nium salts 10a–c also presented high maximum absorption
wavelengths (617 nm—10c and 625 nm), as well as molar
400
500
600
700
Wavelength (nm)
ꢀ1
ꢀ1
absorptivities from 22,400 (10c, entry 17)to 26,479 M cm
10b, entry 16).
Figure 2. Normalised absorbance spectra of compounds 10b,c, 12 and 13 in
water (pH 7.4).
(
When compared to 10a–c (entry 2, entries 15–17), the
labelled derivatives of L-valine (8c, 12 and 13, entries 10,
18 and 19) absorb at slightly longer wavelengths, the batho-
chromic shift is between 4 and 13 nm, the most accentuated
being for compounds 10c/13 (617/630 nm).
variation (10a) or only a slight hypsochromic (10b, 12 and
13) or bathochromic shift (10c) occurred. Figure 2 shows
normalised absorption spectra of compounds 10b,c, 12 and
13 in water. The shoulder seen in the blue region was due
to H-aggregate formation in water solutions. It was possi-
ble to conclude that this aggregation increased from 10b to
12 but decreased from 10c to 13.
1
9
In order to be closer to the biological environment, the
absorption properties of all the above compounds (8a–f, 9,
1
pH (pH 7.4, adjusted with HCl and NaOH). As was ex-
0a–c, 12 and 13) were also studied in water at physiological
Studies of the fluorescent properties of compounds 1a, 3a–e,
5, 8a–f, 9, 10a–c, 12 and 13 were also carried out in ethanol
and water (pH 7.4). Maximum excitation (l ) and emission
pected, compounds 1a, 3a–e and 5 were not soluble in water
pH 7.4) (Table 3).
(
ex
(
(F ) for all compounds were obtained and are presented in
lem) wavelengths, as well as fluorescence quantum yields
Considering compounds 8a–f and 9 (entries 8–14) in ethanol
and water (pH 7.4), lmax values were equal (9) or only a slight
alteration occurred (5 or 10 nm). Despite the fact that a red
shift tendency was observed for compound 8a (640 nm, in
ethanol/650 nm, in water), a blue shift predominated in the
other compounds (8c–f).
F
Table 4. For the determination of F , 9,10-diphenylanthra-
F
2
5
cene (F ¼0.95 in ethanol, 1a, 3a–e and 5) or Oxazine 1
F
2
6
were used as standards (F ¼0.11 in ethanol, 8a–f, 9,
F
10a–c, 12 and 13) and they were excited at the l of each
ex
of the compounds tested. Compounds 1a, 3a–e and 5 (entries
1–7) exhibited emission maxima in the range of 414–
417 nm, with high Stokes’ shifts (84–95 nm) and FF
between 0.54 and 0.66, in ethanol.
When compared to ethanol, in aqueous solution, the lmax of
compounds 10a–c, 12 and 13 (entries 15–19) showed no
Table 4. Fluorescence data for compounds 1a, 3a–e, 5, 8a–f, 9, 10a–c, 12 and 13 in ethanol and water (pH 7.4)
Entry
Compound
Ethanol
Water (pH 7.4)
a
ex
a
b
DS
a
ex
a
b
l
l
em
F
F
l
l
em
F
F
DS
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
1a
3a
3b
3c
3d
3e
5
8a
8b
8c
8d
8e
8f
Nap-OH
330
331
330
330
330
330
330
600
590
590
600
580
600
590
590
590
590
590
590
417
415
415
417
415
414
417
672
644
644
644
644
644
645
644
643
647
652
653
0.54
0.66
0.55
0.55
0.55
0.57
0.54
0.18
0.39
0.40
0.37
0.45
0.48
0.32
0.44
0.49
0.27
0.32
0.35
87
84
85
87
95
84
87
32
14
14
14
14
14
15
19
18
30
23
23
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
34
—
28
27
32
27
24
27
32
27
26
26
Nap-Ala-OMe
Nap-Val-OMe
Nap-Gly-OMe
Nap-Phe-OMe
Nap-Glu(OMe)-OMe
Ac-Lys(Nap)-OMe
Bfa-Ala-OMe
Bfe-Ala-OMe
Bfe-Val-OMe
Bfe-Gly-OMe
Bfe-Phe-OMe
Bfe-Glu(OMe)-OMe
Ac-Lys(Bfe)-OMe
Bfe-OH
580
—
684
—
0.13
—
0
1
2
3
4
5
6
7
8
9
580
590
590
600
590
580
590
590
590
590
653
652
657
652
654
652
651
647
652
653
0.26
0.45
0.40
0.16
0.46
0.28
0.36
0.18
0.26
0.48
9
10a
10b
10c
12
13
Bfh-H
Bfn-H
Boc-Val-OBfh
Boc-Val-OBfn
a
Units in nm.
Stokes’ shift (nm).
b

V. H. J. Frade et al. / Tetrahedron 63 (2007) 12405–12418
12411
Comparison of lem and F values of derivatives 3a–e and
F
5 with compound 1a showed that the presence of the
amino acid residues did not produce alteration or
1.0
10c
increased the F value, the highest being for derivative 3a
F
0.8
10b
(
F ¼0.66).
F
0
0
0
0
.6
.4
.2
.0
Despite the importance of fluorescent derivatisation in the
UV/visible region, the possibility of labelling at long wave-
lengths (>600 nm) is extremely interesting, mainly in bio-
logical applications. The fluorescent amino acid derivatives
8a–f and 9 are included in the latter category, revealing lem
between 644 and 684 nm in ethanol and water (pH 7.4).
13
600
650
700
750
800
When compared to ethanol, in aqueous solutions at physio-
logical pH, the lem showed a red shift (8–13 nm) for all
compounds. Despite their low Stokes’ shifts, the best values
occurred in aqueous solution (34 nm, 8a). The highest lem of
the all dyes studied either in ethanol or water is related to
compound 8a (672 nm ethanol, 684 nm water).
Wavelength (nm)
Figure 3. Normalised fluorescence intensity spectra of compounds 10b,c
and 13 in water (pH 7.4).
In order to further elucidate the photophysical alterations of
these types of compounds upon coupling to amino acids,
studies in various aqueous buffers at different pH as well
as in various organic solvents were conducted. As in our pre-
vious study, with the increase of pH, a slight increase of H-
aggregate formation was observed as well as a tendency for
the decrease of the maximum molar absorptivity (Table 5).
While compound 10c retained its pH-dependency upon ami-
no acid linkage, the same behaviour was not observed for
compound 10b. For all compounds tested the fluorescence
quantum yields did not vary significantly with pH.
Compounds 8a–f and 9 exhibited moderate to high F in
F
both solvents (0.18<F <0.48, ethanol and 0.13<F <0.46,
F
F
2
0
water).
Comparison of the maximum emission wavelengths of the
two groups of labelled amino acids 3a–e and 5/8a–f and 9
reveals that a bathochromic shift from 173 nm (5/9 lysine)
to 257 nm (3a/8a alanine) has occurred. Thus, for example,
it was possible to obtain alanine derivatives with emission at
4
15 and 672 nm.
Table 5. Variation of photophysical properties with pH of compounds 10b,c,
1
2 and 13
Regarding fluorophores 10a–c and the labelled L-valine 8c,
2 and 13, in ethanol and water (pH 7.4), lem was located be-
tween 643 and 653 nm with low Stokes’ shifts (14–32 nm),
but superior in water (with the exception of compound
1
pH
10b
F
10c
F
12
13
a
/3
b
F
a
3/3
b
F
a
3/3
b
F
a
3/3
b
3
F
/F
F
/F
F
F
/F
F F
F /F
1
0c). When compared to ethanol, in aqueous solutions at
physiological pH, lem showed no variation (10c, 12 and
3) or a slight red shift (8–9 nm, 8c, 10a and 10b) for all
3
5
7
8
1
1
1
1
1
1
1
1
0.898 1.013
0.791 1.080
0.697 1.082
0.778 1.020
0.723 1.131
0.604 1.201
0.845 0.919
0.780 0.974
0.656 1.035
1.309 0.933
1.097 1.042
1.019 1.026
1
compounds.
a
3
value at pH 3.
F value at pH 3.
F
b
Through the comparison of lmax values of labelled valine
and fluorophores 10a–c, it was verified that the presence
of the amino acid residue did not produce a considerable
alteration in their values, showing a bathochromic shift in
pairs 10b/12 (643/652 nm) and 10c/13 (647/653 nm), in
ethanol or in both solvents, respectively. Figure 3 shows
the normalised fluorescence spectra of compounds 10b,c
and 13 in water (pH 7.4).
1
9
In a previous study, we reported that, in organic solvents,
the benzo[a]phenoxazine compounds are sensitive to the
proton acceptance capability of the solvent through a depro-
tonation process. In relation to the cationic acidic form, the
absorption and the fluorescence of the basic form appeared
shifted to the blue approximately 100 and 50 nm, respec-
tively, with a 10-fold decrease in quantum yield. The ratio
of acid and basic forms thus depended on the solvent, but
also on the substituent groups at the benzo[a]phenoxazine.
The same behaviour was observed in compounds 10b,c, 12
and 13. Comparing 10c and 13, a very similar ratio of acidic
and basic forms was detected in the studied solvents with
compound 12 showing a much larger fraction of the basic
form when compared to 10b in the same solvent. As an ex-
ample, in Figure 4, the absorption spectra in acetone are
shown.
Regarding the F values of compounds 10a–c, 8c, 12 and 13
F
in the solvents used (0.18<F <0.49), it was possible to con-
F
clude that the highest values were found in ethanol (with the
exception of compound 13, 0.35 ethanol/0.48 water).
By comparison of F values of labelled derivatives 8c, 12
F
and 13 with compounds 10a–c in both solvents, it was veri-
fied that there was a decrease in pairs 10a/8c and 10b/12, the
most important being in the last case and in ethanol (10b/12,
0
.49/0.32); in pair 10c/13 an increase occurred, which was
higher in water (10c/13, 0.18/0.48). The higher quantum
yield of compound 13 can be explained by the previously
mentioned decrease in the formation of H-aggregates, which
are non-fluorescent.
In order to confirm that the magnitude of the slight red shift
of the normal cationic form absorption with the polarity
of the solvent was maintained upon amino acid linkage,
2
7
Lippert–Mataga plots were constructed for the absorption

12412
V. H. J. Frade et al. / Tetrahedron 63 (2007) 12405–12418
basic form contribution. It was concluded that the variation
!
compounds.
!
!
in the dipole moment, D m ¼ m ꢀ m , was the same for all
e
g
Long-wavelength fluorophores with high fluorescence
efficiency and good stability are required in several applica-
tions. Bearing this in mind, our final purpose was the inves-
tigation of the photostability of fluorophores 10a–c, in
comparison with the commercial analogue Nile Blue, under
irradiation in the ultraviolet and visible regions. In this study,
solutions of compounds 10a–c and Nile Blue, in ethanol or
ꢀ
5
water at physiological pH (C¼1ꢂ10 M), were irradiated
at 350 and 419 nm in a Rayonet RPR-100 reactor.
After 2 h of irradiation at 419 nm, in ethanol, the photo-
stabilities of compounds 10a and 10b were similar to each
other, comparable to the Nile Blue value and superior to
that of compound 10c. The values of the residual absorption
were approximately 100% (10a,b and Nile Blue) and 80%
Figure 4. Absorbance spectra of compounds 10b,c, 12 and 13 in acetone.
(
(
10c). Irradiation of the solutions for a long period of time
7 h) did not affect the photostabilities of compounds
maximum wavenumber, ~n abs (Fig. 5). The use of absorption
maxima instead of the usual difference between absorption
and emission can be justified by the fact that the basic
form was more separated from the cationic acidic one in
absorption spectra than the corresponding emission. The
10a,b and Nile Blue, but this lowered the value of the resi-
dual absorption of fluorophore 10c for 70%.
The photostabilities of these fluorophores by irradiation at
419 nm decreased in water (pH 7.4). After 2 h of irradiation,
they presented residual absorption at about 48% (10a), 57%
2
7
expression used is given below:
À
Á
(10b) and 62% (10c). When the experiment was extended to
7 h, it was verified that the photostabilities decreased for all
2
¼ ꢀ m $ m ꢀ m aꢀ3Df þ const
!
!
!
~
n
abs
g
e
g
hc
compounds, compound 10c revealing the lowest value (32%,
1
!
dipole moments, a is the molecular radius and Df is the
!
0c; 43%, 10a,b).
where m and m e are the ground state and excited state
g
Studies of the behaviour of compounds 10a–c, in ethanol
and water (pH 7.4), with irradiation at 350 nm, were also car-
ried out. The results showed that, after 2 h of irradiation, the
photostabilities of compounds 10a and 10b were similar to
each other and, comparable to Nile Blue (ꢃ97%), superior
to that of compound 10c in ethanol. Irradiation of solutions
for 7 h did not significantly change the stability of fluoro-
phores 10a,b and Nile Blue (ꢃ95%), and reduced the value
of residual absorption of compound 10c (66%).
solvent orientation polarizability, which is given by:
2
3
ꢀ 1
n ꢀ 1
Df ¼ 2
3 þ 1 2n þ 1
ꢀ
2
where 3 and n are, respectively, the solvents’ dielectric con-
stant and refractive index. The solvents used were water,
methanol, acetone and dichloromethane in which the acidic
form absorption was well resolved and separated from the
In water, at physiological pH, irradiation at 350 nm showed
a tendency for a decrease in the photostabilities, as previ-
ously occurred. Thus, after 2 h of irradiation, residual
absorption of compounds 10a and 10b was the same (84%)
and superior to that of compound 10c (76%). The irradiation
of solutions during 7 h reduced the photostabilities of all
compounds, compounds 10a and 10b (61%) being similar
to that of Nile Blue (66%) and inferior in the case of
compound 10c (46%).
In fact, by irradiation at 419 and 350 nm, compounds 10a and
1
1
0bpresentedphotostabilitieshigherthanthoseof compound
0c, similar to that of Nile Blue, in both solvents studied.
The results obtained suggest that the photostabilities of the
benzo[a]phenoxazinium salts studied are related with the
solvent (ethanol or water at physiologic pH) as well as
with the wavelength of irradiation.
There was also a relationship detected between the func-
tional group of the lateral chain and the photostabilities of
Figure 5. Lippert–Mataga plot of maximum absorption wavenumbers of
compounds 10b,c, 12 and 13.

V. H. J. Frade et al. / Tetrahedron 63 (2007) 12405–12418
12413
these cationic dyes. The photostability experiments per-
formed were only a preliminary study of the behaviour of
the synthesised compounds. Further studies will be carried
out for a better understanding of this correlation.
spectrometer for low resolution spectra and an Autospec
M spectrometer for high resolution mass spectra. Fluores-
cence spectra were collected using a Spex Fluorolog 1680
spectrometer.
Photostability studies were carried out using a Rayonet
RPR-100 chamber reactor equipped with eight lamps of
wavelengths of 350 and 419 nm (14 W each).
3. Conclusion
In this work, fluorescent conjugates of several a-amino acids
were synthesised with absorption and fluorescence at low
wavelengths (330 nm and 414–417 nm, respectively).
4.2. Synthesis of 3-(naphthalen-1-ylamino)propanoic
acid, Nap-OH (1a)
1
9
ꢀ3
The efficient cyclisation of these compounds with an appro-
priate nitrosophenol generated the corresponding blue
fluorescent derivatives possessing the benzo[a]phenoxazine
nucleus, which presented maximum absorption in the region
between 625 and 650 nm and strong fluorescence, with
maximum emissions between 644 and 684 nm.
To a solution of compound 1b (0.470 g, 2.05ꢂ10 mol)
in 1,4-dioxane (4 mL) 1 M NaOH (3.08 mL, 3.08ꢂ
ꢀ
3
10 mol) was added at low temperature. The solution was
stirred at 0 C for 6 h and acidified to pH 3 with 1 M
ꢁ
KHSO . After extraction with chloroform and evaporation
of the solvent, compound 1a was obtained as a white solid
4
(0.375 g, 85%). Spectroscopic data confirmed the expected
structure and were in accordance with those previously
obtained.
New water-soluble monofunctionalised benzo[a]phen-
oxazine derivatives were also synthesised and characterised.
These were used in the efficient derivatisation of L-valine,
through an ester or amide linkage, producing conjugates
with absorption between 617 and 630 nm and emission
between 644 and 653 nm, respectively.
1
9
4.3. General method for the synthesis of fluorescently
labelled L-amino acids 3a–e and 5
3-(Naphthalen-1-ylamino)propanoic acid, Nap-OH (1a),
Considering the obtained results, 3-(naphthalen-1-ylamino)-
propanoic acid is a potential candidate for low-wavelength
derivatisation, producing derivatives with a red shift in fluo-
rescence for the region above 644 nm, after condensation
with an appropriate nitrosophenol component. On the other
hand, the new functionalised benzo[a]phenoxazine deriva-
tives present the adequate structural and photophysical
characteristics for their use as covalent and non-covalent
labels of biomolecules.
was reacted with an amino acid methyl ester, in DMF, using
a standard DCC/HOBt coupling. After evaporation of the
solvent and chromatography on silica gel, the required
derivative (3a–e and 5) was obtained.
4.3.1. N-[3-(Naphthalen-1-ylamino)propanoyl]alanine
methyl ester, Nap-Ala-OMe (3a). The product of reaction
ꢀ
3
of Nap-OH (1a) (0.224 g, 1.04ꢂ10 mol) with alanine
ꢀ
3
methyl ester hydrochloride (2a) (0.208 g, 1.50ꢂ10 mol)
was chromatographed using chloroform as the eluent to
give compound 3a as a colourless oil (0.270 g, 87%). TLC
1
4
. Experimental
(chloroform/methanol, 5.8:0.2): R ¼0.69.
H
NMR
f
(
CDCl , 300 MHz): d¼1.38 (d, J¼7.2 Hz, 3H, b-CH
3
3
4
.1. General
Ala), 2.67 (t, J¼6.2 Hz, 2H, NHCH CH ), 3.66 (t,
2 2 3
2 2
J¼5.7 Hz, 2H, NHCH CH ), 3.74 (s, 3H, OCH ), 4.56–
All melting points were measured on a Gallenkamp melting
point apparatus and were uncorrected. TLC analyses were
carried out on precoated silica plates, 0.25 mm in thickness
4.67 (m, 1H, a-CH Ala), 5.16 (br s, 1H, NH), 6.24 (d,
J¼6.6 Hz, 1H, a-NH Ala), 6.66 (d, J¼7.5 Hz, 1H, 4-H),
7.27 (d, J¼8.1 Hz, 1H, 2-H), 7.36 (t, J¼7.5 Hz, 1H, 3-H),
7.40–7.50 (m, 2H, 6-H and 7-H), 7.76–7.82 (m, 1H, 5-H),
(
Merck Fertigplatten Kieselgel 60F254) and spots were
1
3
visualised under UV light. Dry 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, Nujol or neat samples. UV/visible spectra
7.84–7.92 (m, 1H, 8-H). C NMR (CDCl , 75.4 MHz):
3
d ¼18.21 (b-CH Ala), 35.04 (NHCH CH ), 40.26
C
3
2
2
(NHCH CH ), 48.03 (a-CH Ala), 52.46 (OCH ), 104.56
2
2
3
(C-4), 117.79 (C-2), 120.18 (C-5), 123.80 (C-4a), 124.79
(C-6), 125.75 (C-7), 126.41 (C-3), 128.48 (C-8), 134.31
(C-8a), 142.85 (C-1), 171.49 (CONH), 173.44 (CO CH ).
1
were run on a Hitachi U-2000 spectrophotometer. H NMR
spectra were recorded on a Varian 300 spectrometer in
2
3
ꢁ
ꢀ1
CDCl or CD OD solution at 300 MHz at 25 C. All chem-
3
IR (KBr 1%, cm ): n¼3393, 3310, 3049, 2929, 2852,
1750, 1641, 1587, 1532, 1490, 1467, 1412, 1351, 1287,
1216, 1120, 983. HRMS (FAB): calcd for C H N O
7 20 2 3
3
ical shifts are given in parts per million using dH Me4-
Si¼0 ppm as a reference and J values are given in Hertz.
1
1
3
+
C NMR spectra were run on the same instrument at
5.4 MHz using the solvent peak as an internal reference.
[M ]: 300.1474; found: 300.1484.
7
Assignments were carried out by comparison of chemical
shifts, peak multiplicities and J values and were supported
by spin decoupling-double resonance and bidimensional
heteronuclear correlation HMBC and HMQC techniques.
Mass spectrometry analyses were performed at the
C.A.C.T.I.—Unidad de Espectrometria de Masas of the
University of Vigo, Spain, on a Hewlett Packard 5989 A
4.3.2. N-[3-(Naphthalen-1-ylamino)propanoyl]valine
methyl ester, Nap-Val-OMe (3b). The product of reaction
ꢀ
4
of Nap-OH (1a) (0.152 g, 7.07ꢂ10 mol) with valine
ꢀ
3
methyl ester hydrochloride (2b) (0.178 g, 1.06ꢂ10 mol)
was chromatographed using chloroform as the eluent to
give compound 3b as brownish oily solid (0.197 g, 85%).
1
TLC (chloroform/methanol, 5.5:0.5): R ¼0.63. H NMR
f

12414
V. H. J. Frade et al. / Tetrahedron 63 (2007) 12405–12418
(
0
CDCl , 300 MHz): d¼0.85 (d, J¼6.9 Hz, 3H, g-CH Val),
(C-6), 125.75 (C-7), 126.39 (C-3), 127.02 (C-4 Phe),
128.47 (C-8, C-3 Phe and C-5 Phe), 129.05 (C-2 Phe and
C-6 Phe), 134.30 (C-8a), 135.54 (C-1 Phe), 142.75 (C-1),
3
3
.91 (d, J¼6.9 Hz, 3H, g-CH Val), 2.08–2.20 (m, 1H,
3
b-CH Val), 2.72 (dt, J¼6.0 and 1.8 Hz, 2H, NHCH CH ),
2 2
ꢀ
171.43 (CONH), 171.89 (CO CH ). IR (neat, cm ):
2 3
1
3
4
(
.65 (t, J¼6.0 Hz, 2H, NHCH CH ), 3.72 (s, 3H, OCH ),
2
2
3
.17 (br s, 1H, N–H), 4.54–4.62 (m, 1H, a-CH Val), 6.38
n¼3374, 3324, 3061, 2929, 2851, 1731, 1651, 1582, 1530,
d, J¼8.4 Hz, 1H, a-NH Val), 6.71 (d, J¼7.2 Hz, 1H, 4-H),
1500, 1430, 1410, 1371, 1346, 1283, 1217, 1177, 1121,
1089, 990. HRMS (FAB): calcd for C H N O [M ]:
376.1787; found: 376.1797.
+
7
7
5
7
3
5
1
1
1
.29 (d, J¼9.6 Hz, 1H, 2-H), 7.36 (t, J¼7.5 Hz, 1H, 3-H),
2
3 24 2 3
.40–7.50 (m, 2H, 6-H and 7-H), 7.76–7.84 (m, 1H,
-H), 7.86–7.94 (m, 1H, 8-H).
1
3
C NMR (CDCl3,
5.4 MHz): d ¼17.78 (g-CH Val), 18.90 (g-CH3 Val),
4.3.5. N-[3-(Naphthalen-1-ylamino)propanoyl]glutamic
acid dimethyl ester, Nap-Glu(OMe)-OMe (3e). The prod-
C
3
1.05 (b-CH Val), 35.21 (NHCH CH ), 40.65 (NHCH CH ),
2
2
2
2
ꢀ4
2.19 (OCH ), 57.13 (a-CH Val), 105.05 (C-4), 118.23 (C-2),
3
uct of reaction of Nap-OH (1a) (0.150 g, 6.98ꢂ10 mol)
20.21 (C-8), 123.89 (C-4a), 124.91 (C-6), 125.82 (C-7),
26.38 (C-3), 128.51 (C-5), 134.32 (C-8a), 142.44 (C-1),
71.87 (CONH), 172.54 (CO2 CH ). IR (neat, cm ):
with glutamic acid dimethyl ester hydrochloride (2e)
ꢀ3
(0.222 g, 1.05ꢂ10 mol) was chromatographed using chlo-
roform as the eluent to give compound 3e as a brownish
oil (0.246 g, 95%). TLC (chloroform/methanol, 5.5:0.5):
ꢀ
1
3
n¼3326, 3052, 2954, 2931, 2851, 1736, 1651, 1633, 1583,
1
1
1
486, 1436, 1411, 1373, 1346, 1311, 1283, 1210, 1153,
121, 1089, 1018, 892. HRMS (FAB): calcd for
R ¼0.58. H NMR (CDCl , 300 MHz): d¼0.82–2.00 and
f
3
2.10–2.21 (2ꢂm, 2H, b-CH Glu), 2.30–2.40 (m, 2H,
2
+
C H N O [M ]: 328.1787; found: 328.1796.
1
g-CH Glu), 2.64 (t, J¼6.0 Hz, 2H, NHCH CH ), 3.53–
9
24
2
3
2
2
2
3
(s, 3H, OCH ), 4.60–4.70 (m, 1H, a-CH Glu), 6.62 (d,
.59 (m, 2H, NHCH CH ), 3.60 (s, 3H, OCH ), 3.69
2 2 3
4.3.3. N-[3-(Naphthalen-1-ylamino)propanoyl]glycine
methyl ester, Nap-Gly-OMe (3c). The product of reaction
3
J¼7.2 Hz, 1H, 4-H), 6.78 (d, J¼7.8 Hz, 1H, a-NH Glu),
7.25 (d, J¼8.1 Hz, 1H, 2-H), 7.34 (t, J¼7.5 Hz, 1H, 3-H),
7.40–7.46 (m, 2H, 6-H and 7-H), 7.77 (dd, J¼8.5 and
3.6 Hz, 1H, 5-H), 7.88 (dd, J¼9.0 and 2.7 Hz, 1H, 8-H).
2
ꢀ
4
of Nap-OH (1a) (0.164 g, 7.63ꢂ10 mol) with glycine
ꢀ
3
methyl ester hydrochloride (2c) (0.143 g, 1.14ꢂ10 mol)
was chromatographed using dichloromethane/methanol,
1
3
5
.8:0.2 as the eluent, to give compound 3c as a brownish
oil (0.196 g, 90%). TLC (dichloromethane/methanol,
C
NMR (CDCl , 75.4 MHz): d ¼26.92 (b-CH
2 2
3
C
Glu), 29.91 (g-CH2 Glu), 35.02 (NHCH CH ), 40.15
(NHCH CH ), 51.58 (a-CH Glu), 51.69 (OCH ), 52.41
2 2 3
1
5
.5:0.5): R ¼0.53. H NMR (CDCl , 300 MHz): d¼2.68 (t,
f
3
J¼6.0 Hz, 2H, NHCH CH ), 3.64 (t, J¼6.0 Hz, 2H,
(OCH ), 104.39 (C-4), 117.62 (C-2), 120.16 (C-8), 123.71
3
2
2
NHCH CH ), 3.73 (s, 3H, OCH ), 4.03 (d, J¼5.4 Hz, 2H,
(C-4a), 124.66 (C-7), 125.65 (C-6), 126.37 (C-3), 128.38
(C-5), 134.24 (C-8a), 142.86 (C-1), 171.93 (CO CH ),
2
2
3
CH Gly), 6.45 (br s, 1H, a-NH Gly), 6.65 (d, J¼7.5 Hz,
2
2
3
1
1
H, 4-H), 7.26 (d, J¼7.5 Hz, 1H, 2-H), 7.35 (t, J¼7.5 Hz,
172.17 (CONH), 173.16 (CO CH Glu main chain). IR
2 3
ꢀ
1
H, 3-H), 7.40–7.50 (m, 2H, 6-H and 7-H), 7.72–7.81
(Nujol, cm ): n¼3428, 3314, 2953, 2925, 2854, 1746,
1
3
(
7
4
m, 1H, 5-H), 7.83–7.92 (m, 1H, 8-H). C NMR (CDCl3,
5.4 MHz): d ¼35.96 (NHCH CH ), 40.27 (NHCH CH ),
1726, 1636, 1530, 1463, 1378, 1158. HRMS (FAB): calcd
for C H N O [M ]: 372.1685; found: 372.1692.
+
C
2
2
2
2
20 24 2 5
1.18 (CH Gly), 52.39 (OCH ), 104.63 (C-4), 117.92
2
3
(
(
(
C-2), 120.20 (C-8), 123.82 (C-4a), 124.85 (C-6), 125.79
C-7), 126.42 (C-3), 128.51 (C-5), 134.32 (C-8a), 142.80
C-1), 170.32 (CONH), 172.11 (CO CH ). IR (neat,
4.3.6. N-Acetyl-u-[3-(naphthalen-1-ylamino)propanoyl]-
lysine methyl ester, Ac-Lys(Nap)-OMe (5). The product
ꢀ4
of reaction of Nap-OH (1a) (0.100 g, 4.65ꢂ10 mol) with
2
3
ꢀ1
cm ): n¼3400, 3070, 2952, 2929, 2852, 1747, 1651,
N-acetyl-lysine methyl ester hydrochloride (4) (0.175 g,
7.32ꢂ10 mol) was chromatographed using chloroform
ꢀ
4
1
1
583, 1531, 1486, 1436, 1410, 1372, 1346, 1285, 1214,
181, 1124, 1090, 1039, 984. HRMS (FAB): calcd for
as the eluent to give compound 5 as a brownish oil
(0.182 g, 98%). TLC (chloroform): R ¼0.33. H NMR
+
1
C H N O [M ]: 286.1317; found: 286.1309.
1
6
18
2
3
f
(
CDCl , 300 MHz): d¼1.21–1.35 (m, 2H, g-CH Lys),
3
2
4.3.4. N-[3-(Naphthalen-1-ylamino)propanoyl]phenyl-
alanine methyl ester, Nap-Phe-OMe (3d). The product of
1.40–1.70 (m, 4H, b-CH and d-CH Lys), 1.98 (3H, s,
2 2
CH ), 2.61 (t, J¼6.0 Hz, 2H, NCH CH ), 3.15–3.30 (m,
3
2
2
ꢀ4
reaction of Nap-OH (1a) (0.086 g, 4.04ꢂ10 mol) with
1H, 3-CH Lys), 3.61 (t, J¼6.3 Hz, 1H, NHCH CH ), 3.72
2
2
2
phenylalanine methyl ester hydrochloride (2d) (0.129 g,
(s, 3H, OCH ), 4.50–4.60 (m, 1H, a-CH Lys), 6.15 (br s,
3
ꢀ
4
6
the eluent to give compound 3d as a brownish oil (0.129 g,
8
.0ꢂ10 mol) was chromatographed using chloroform as
1H, NH Lys lateral chain), 6.24 (d, J¼7.8 Hz, 1H, a-NH
Lys), 6.62 (d, J¼7.5 Hz, 1H, 4-H), 7.24 (d, J¼8.1 Hz, 1H,
2-H), 7.28 (t, J¼7.5 Hz, 1H, 3-H), 7.40–7.48 (m, 2H, 6-H
and 7-H), 7.74–7.80 (m, 1H, 5-H), 7.82–7.90 (m, 1H,
1
5%). TLC (chloroform/methanol, 5.8:0.2): R ¼0.75. H
f
NMR (CDCl , 300 MHz): d¼2.61 (t, J¼6.0 Hz, 2H,
3
1
3
NHCH CH ), 3.0–3.20 (m, 2H, b-CH Phe), 3.55–3.65 (m,
2
8-H). C NMR (CDCl , 75.4 MHz): d ¼22.28 (g-CH
2
2
3
C
2
2
2
H, NHCH CH ), 3.71 (s, 3H, OCH ), 4.85–4.95 (m, 2H,
2
Lys), 23.08 (CH ), 28.58 (b-CH Lys), 31.98 (d-CH
3 2
2
3
a-CH Phe and NH), 6.35 (d, J¼7.8 Hz, 1H, a-NH Phe),
Lys), 35.17 (NHCH CH ), 38.80 (3-CH Lys), 40.39
2 2 2
(NHCH CH ), 51.68 (a-CH Lys), 52.40 (OCH ), 104.37
2 2 3
6
6
.62 (d, J¼7.5 Hz, 1H, 4-H), 6.94–7.0 (m, 2H, 2-H and
-H Phe), 7.06–7.16 (m, 3H, 3-H, 4-H and 5-H Phe), 7.28
(C-4), 117.61 (C-2), 120.15 (C-8), 123.72 (C-4a), 124.80
(C-7), 125.78 (C-6), 126.46 (C-3), 128.50 (C-5), 134.28
(C-8a), 143.04 (C-1), 170.24 (COCH ), 172.15 (CONH),
(
7
2
d, J¼8.7 Hz, 1H, 2-H), 7.35 (t, J¼7.5 Hz, 1H, 3-H), 7.41–
.50 (m, 2H, 6-H and 7-H), 7.80 (dd, J¼7.2 and 2.1 Hz,
3
1
3
ꢀ1
H, 5-H and 8-H).
C NMR (CDCl , 75.4 MHz):
3
172.94 (CO CH ). IR (Nujol, cm ): n¼3326, 2954, 2925,
2
3
d ¼35.05 (NHCH CH ), 37.60 (b-CH Phe), 40.16
2854, 1726, 1627, 1463, 1377, 1311, 1260, 1088. HRMS
(EI): calcd for C H N O [M ]: 399.2158; found:
399.2157.
C
2
2
2
+
(
(
NHCH CH ), 52.32 (OCH ), 53.03 (a-CH Phe), 104.57
2
C-4), 117.84 (C-2), 120.15 (C-5), 123.79 (C-4a), 124.81
2
3
22 29 3 4

V. H. J. Frade et al. / Tetrahedron 63 (2007) 12405–12418
12415
4
.4. General method for the synthesis of the fluorescently
125.31 (C-4), 127.22 (C-10), 129.98 (C-3), 130.18 (Ar-C),
130.54 (Ar-C), 131.11 (C-11), 131.67 (C-2), 133.42
L-amino acids 8a–f and 9
(Ar-C), 146.92 (Ar-C), 150.51 (Ar-C), 154.29 (C-9),
157.24 (C-5), 170.65 (CONH), 173.17 (CO CH ). IR
To a cold solution (ice bath) of 5-(alkylamino)-2-nitrosophe-
nol hydrochloride 7a,b in methanol, compounds 3a–e or 5
2
3
ꢀ
1
(Nujol, cm ): n¼3400, 2954, 2924, 2854, 1726, 1651,
ꢀ
2
and concentrated hydrochloric acid (5.0ꢂ10 mL) were
added. The mixture was refluxed during the time given
below and monitored by TLC (chloroform/methanol). After
evaporation of the solvent and dry chromatography on silica
gel, the required derivative (8a–f and 9) was obtained as
a blue solid.
1633, 1587, 1556, 1504, 1463, 1377, 1311. HRMS (FAB):
calcd for C H N O [M ]: 461.2189; found: 461.2203.
+
2
6 29 4 4
4.4.3. N-{5-[3-(1-Methoxy-3-methyl-1-oxobutan-2-
ylamino)-3-oxopropylamino]-10-methyl-9H-benzo[a]-
phenoxazin-9-ylidene}ethanaminium chloride, Bfe-Val-
OMe (8c). The product of the reaction of 7b (0.0423 g,
ꢀ
4
ꢀ4
4
.4.1. N-Ethyl-N-{5-[3-(1-methoxy-1-oxopropan-2-yl-
2.35ꢂ10 mol) with 3b (0.077 g, 2.35ꢂ10 mol) (reflux
time 11 h) was chromatographed with dichloromethane/
methanol, 6.6:0.4 as the eluent, to give Bfe-Val-OMe (8c)
amino)-3-oxopropylamino]-9H-benzo[a]phenoxazin-9-
ylidene}ethanaminium chloride, Bfa-Ala-OMe (8a). The
ꢀ
4
ꢁ
product of the reaction of 7a (0.087 g, 3.33ꢂ10 mol)
(0.090 g, 78%). Mp¼204.0–206.1 C. TLC (dichlorome-
ꢀ
4
1
with 3a (0.100 g, 3.33ꢂ10 mol) (reflux time 6 h) was
thane/methanol, 5.5:0.5): R ¼0.88. H NMR (CDCl ,
f
3
chromatographed with chloroform/methanol, 5.3:0.7 as the
eluent, to give Bfa-Ala-OMe (8a) (0.152 g, 96%). Mp¼
300 MHz): d¼0.94 (d, J¼6.9 Hz, 3H, g-CH Val), 0.99 (d,
3
J¼6.9 Hz, 3H, g-CH Val), 1.42 (t, J¼7.2 Hz, 3H,
3
ꢁ
1
01.2–103.2 C. TLC (chloroform/methanol, 5.3:0.7):
NHCH CH ), 2.12–2.26 (m, 1H, b-CH Val), 2.40 (s, 3H,
3
2
1
R ¼0.44. H NMR (CDCl , 300 MHz): d¼1.23–1.45 (m,
CH ), 3.05–3.16 (m, 2H, NHCH CH ), 3.20–3.45 (m, 2H,
3 2 2
NHCH CH ), 3.57 (s, 3H, OCH ), 3.72–3.85 (m, 2H,
2 3 3
NHCH CH ), 4.38–4.46 (m, 1H, a-CH Val), 6.26 (br s,
2 2
f
3
9
NHCH CH ), 3.55 (s, 3H, OCH ), 3.56–3.65 (m, 6H,
H, 2ꢂNCH CH and b-CH Ala), 3.05–3.22 (m, 2H,
2
3
3
2
2
3
2
4
6
7
ꢂNCH CH ), 4.11 (t, J¼6.6 Hz, 2H, NHCH CH ), 4.40–
1H, NH), 6.27 (s, 1H, 8-H), 6.46 (s, 1H, 6-H), 7.51 (s, 1H,
11-H), 7.87 (t, J¼8.7 Hz, 3H, 2-H, 3-H and a-NH Val),
8.78–8.90 (m, 1H, 1-H), 9.19 (br s, 1H, 4-H), 10.63 (br s,
2
3
2
2
.52 (m, 1H, a-CH Ala), 6.62 (d, J¼2.4 Hz, 1H, 8-H),
.83 (s, 1H, 6-H), 6.97 (dd, J¼9.3 and 2.4 Hz, 1H, 10-H),
.77 (d, J¼9.3 Hz, 1H, 11-H), 7.78–7.88 (m, 2H, 2-H and
-H), 8.22 (d, J¼7.2 Hz, 1H, a-NH Ala), 8.79 (dd, J¼7.5
1
3
1H, N–H). C NMR (CDCl , 75.4 MHz): d ¼13.83
3
C
3
(NHCH CH ), 18.09 (CH ), 18.29 (g-CH Val), 19.11 (g-
2 3 3 3
1
3
and 2.4 Hz, 1H, 1-H), 9.24 (d, J¼7.2 Hz, 1H, 4-H).
C
CH Val), 30.51 (b-CH Val), 34.28 (NHCH CH ), 38.76
3 2 2
(NHCH CH ), 42.13 (NHCH CH ), 51.88 (OCH ), 58.21
2 3 2 2 3
NMR (CDCl , 75.4 MHz): d ¼12.61 (2ꢂNCH CH ),
3
C
2
3
1
7.27 (b-CH Ala), 34.67 (NHCH CH ), 42.20 (NHCH -
3
(a-CH Val), 92.81 (C-6), 93.24 (C-8), 124.0 (C-1), 125.51
(C-4), 127.41 (C-10), 129.87 (C-3), 130.25 (2ꢂAr-C),
130.55 (Ar-C), 131.06 (C-11), 131.66 (C-2), 133.29 (Ar-
C), 146.92 (Ar-C), 150.40 (Ar-C), 154.34 (C-9), 157.16
(C-5), 171.10 (CONH), 172.09 (CO CH ). IR (Nujol,
2
2
2
CH ), 45.89 (2ꢂNHCH CH ), 48.34 (a-CH Ala), 52.02
2
2
3
(
(
(
(
(
OCH ), 93.69 (C-6), 95.60 (C-8), 114.06 (C-10), 123.83
3
C-1), 124.08 (Ar-C), 125.31 (C-4), 128.80 (Ar-C), 130.11
C-3), 130.41 (Ar-C), 131.53 (C-2), 132.38 (C-11), 134.74
Ar-C), 147.33 (Ar-C), 151.0 (Ar-C), 153.01 (C-9), 158.38
C-5), 170.77 (CONH), 173.12 (CO CH ). IR (KBr 1%,
2
3
ꢀ1
cm ): n¼3193, 2954, 2923, 2854, 1737, 1659, 1642,
1588, 1563, 1538, 1519, 1455, 1377, 1311, 1281, 1258,
1183, 1160, 1132, 1085, 1054. HRMS (FAB): calcd for
2
3
ꢀ
1
cm ): n¼3437, 3224, 3055, 2963, 2927, 2854, 1740,
+
1
1
640, 1588, 1547, 1497, 1455, 1438, 1384, 1329, 1277,
258, 1197, 1165, 1127, 1074, 1013, 946. HRMS (FAB):
C H N O [M ]: 489.2502; found: 489.2523.
8 33 4 4
2
+
calcd for C H N O [M ]: 475.2345; found: 475.2338.
27 31 4 4
4.4.4. N-{5-[3-(2-Methoxy-2-oxoethylamino)-3-oxopro-
pylamino]-10-methyl-9H-benzo[a]phenoxazin-9-ylide-
ne}ethanaminium chloride, Bfe-Gly-OMe (8d). The
4
3
9
.4.2. N-{5-[3-(1-Methoxy-1-oxopropan-2-ylamino)-
-oxopropylamino]-10-methyl-9H-benzo[a]phenoxazin-
-ylidene}ethanaminium chloride, Bfe-Ala-OMe (8b).
ꢀ
4
product of the reaction of 7b (0.100 g, 5.59ꢂ10 mol)
ꢀ
4
with 3c (0.160 g, 5.59ꢂ10 mol) (reflux time 1 h) was
chromatographed with chloroform/methanol, 5.5:0.5 as the
eluent, to give Bfe-Gly-OMe (8d) (0.167 g, 67%). Mp¼
The product of the reaction of 7b (0.0918 g, 5.1ꢂ
ꢀ
4
ꢀ4
1
0
mol) with 3a (0.300 g, 5.1ꢂ10 mol) (reflux time
ꢁ
1
thane/methanol, 5.4:0.6 as the eluent, to give Bfe-Ala-OMe
h and 30 min) was chromatographed with dichlorome-
above 300 C. TLC (dichloromethane/methanol, 5.5:0.5):
1
R ¼0.34. H NMR (CD OD, 300 MHz): d¼1.40 (t,
f
3
ꢁ
(
8b) (0.133 g, 57%). Mp¼above 300 C. TLC (dichlorome-
J¼7.2 Hz, 3H, NHCH CH ), 2.25 (s, 3H, CH ), 2.89 (br s,
2
3
3
1
thane/methanol, 5.5:0.5): R ¼0.30. H NMR (CD OD,
2H, NHCH CH ), 3.40–3.56 (m, 2H, NHCH CH ), 3.73
2 2 2 3
f
3
3
00 MHz): d¼1.41 (br s, 3H, NCH CH ), 1.48 (d,
(s, 3H, OCH ), 3.93 (br s, 2H, NHCH CH ), 4.04 (s, 2H,
3 2 2
CH Gly), 6.63 (s, 1H, 8-H), 6.78 (s, 1H, 6-H), 7.40 (s,
2
2
3
J¼7.2 Hz, 3H, b-CH Ala), 2.39 (s, 3H, CH ), 3.05 (br s,
3
3
2H, NCH CH ), 3.28 (br s, 2H, NCH CH ), 3.68 (s, 3H,
OCH ), 3.65–3.82 (m, 2H, NCH CH ), 4.42–4.56 (m, 1H,
1H, 11-H), 7.62–7.84 (m, 2H, 2-H and 3-H), 8.16 (br s,
1H, 1-H), 8.50–8.68 (m, 1H, 4-H). C NMR (CD OD,
2
2
2
3
1
3
3
2
2
3
a-CH Ala), 6.18 (s, 1H, 8-H), 6.35 (s, 1H, 6-H), 6.74 (br s,
H, a-NH Ala), 7.37 (s, 1H, 11-H), 7.82 (br s, 2H, 2-H
and 3-H), 8.32 (d, J¼6.6 Hz, 1H, NH), 8.75 (br s, 1H, 1-
75.4 MHz): d ¼14.16 (NHCH CH ), 18.36 (CH ), 35.01
C
2
3
3
1
(NHCH CH ), 39.81 (NHCH CH ), 41.80 (NHCH CH ),
2 2 2 3 2 2
41.93 (CH Gly), 52.70 (OCH ), 94.03 (C-6), 94.48 (C-8),
2
3
1
3
H), 9.04 (br s, 1H, 4-H), 10.16 (br s, 1H, NH). C NMR
CDCl , 75.4 MHz): d ¼13.87 (NCH CH ), 17.42 (b-CH
123.70 (Ar-C), 124.52 (C-1), 125.35 (C-4), 128.97 (Ar-C),
130.65 (C-3), 132.23 (C-10), 132.26 (Ar-C), 132.58 (C-
11), 132.77 (C-2), 133.59 (Ar-C), 149.21 (Ar-C), 152.48
(Ar-C), 156.78 (C-9), 158.17 (C-5), 171.97 (CONH),
(
Ala), 18.05 (CH ), 34.21 (NCH CH ), 38.79 (NCH CH ),
3
C
2
3
3
3
2
2
2
3
4
9
1.98 (NCH CH ), 48.46 (a-CH Ala), 52.21 (OCH ),
2 2 3
2.83 (C-6), 93.27 (C-8), 123.4 (Ar-C), 124.03 (C-1),
ꢀ1
173.51 (CO CH ). IR (Nujol, cm ): n¼3366, 2954, 2924,
2
3

12416
V. H. J. Frade et al. / Tetrahedron 63 (2007) 12405–12418
2
1
854, 1739, 1643, 1588, 1557, 1519, 1463, 1455, 1377,
311, 1160, 1133, 1007. HRMS (FAB): calcd for
n¼3417, 2954, 2924, 2854, 1726, 1720, 1651, 1556, 1504,
1463, 1377, 1150, 1000. HRMS (FAB): calcd for
C H N O [M ]: 533.2400; found: 533.2386.
+
+
C H N O [M ]: 447.2032; found: 447.2037.
4
2
5
27
4
29 33 4 6
4
.4.5. N-{5-[3-(1-Methoxy-1-oxo-3-phenylpropan-2-yl-
4.4.7. N-{5-[3-(5-Acetamido-6-methoxy-6-oxohexyl-
amino)-3-oxopropylamino]-10-methyl-9H-benzo[a]phe-
noxazin-9-ylidene}ethanaminium chloride, Ac-Lys(Bfe)-
OMe (9). The product of the reaction of 7b (0.063 g,
amino)-3-oxopropylamino]-10-methyl-9H-benzo[a]phe-
noxazin-9-ylidene}ethanaminium chloride, Bfe-Phe-
OMe (8e). The product of the reaction of 7b (0.144 g,
7.98ꢂ10 mol) with 3d (0.300 g, 7.98ꢂ10 mol) (reflux
time 1 h and 30 min) was chromatographed with chloro-
ꢀ
4
ꢀ4
ꢀ4
ꢀ4
3.50ꢂ10 mol) with 5 (0.140 g, 3.50ꢂ10 mol) (reflux
time 3 h) was chromatographed with dichloromethane/
methanol, 5.3:0.7 as the eluent, to give Ac-Lys(Bfe)-OMe
form/methanol, 5.8:0.2 as the eluent, to give Bfe-Phe-OMe
ꢁ
ꢁ
(
8e) (0.323 g, 76%). Mp¼230.1–232.2 C. TLC (dichloro-
(9) (0.173 g, 88%). Mp¼above 300 C. TLC (chloroform/
1
1
methane/methanol, 5.5:0.5): R ¼0.56. H NMR (CDCl ,
methanol, 5.2:0.8): R ¼0.51. H NMR (CDCl , 300 MHz):
f
3
f
3
3
00 MHz): d¼1.23–1.40 (m, 3H, NHCH CH ), 2.40 (s,
d¼1.20–1.40 (m, 2H, g-CH Lys), 1.41–1.60 (m, 7H,
2
3
2
3
NHCH CH ), 3.22–3.40 (m, 2H, NHCH CH ), 3.53 (s,
H, CH ), 3.0 (br s, 2H, b-CH Phe), 3.02–3.21 (m, 2H,
b-CH Lys, d-CH Lys and NHCH CH ), 1.80 (s, 3H,
2
3
2
2
2
3
COCH ), 2.34 (s, 3H, CH ), 3.0–3.40 (m, 6H, 3-CH Lys,
3 3 2
2
2
2
3
3
H, OCH ), 3.68–3.90 (m, 2H, NHCH CH ), 4.70–4.80
3
NHCH CH and NHCH CH ), 3.70 (s, 3H, OCH ), 3.80–
2 2 2 3 3
2
2
(
1
m, 1H, a-CH Phe), 5.96 (br s, 1H, a-NH Phe), 6.37 (s,
H, 8-H), 6.55 (s, 1H, 6-H), 7.0–7.30 (m, 5H, 5ꢂAr-H
Phe), 7.57 (s, 1H, 11-H), 7.80–7.95 (m, 1H, 2-H), 7.96–
4.0 (m, 2H, NHCH CH ), 4.40–4.50 (m, 1H, a-CH Lys),
2 2
6.19 (br s, 1H, NH Lys lateral chain), 6.34 (s, 1H, 8-H),
6.57 (br s, 1H, 6-H), 7.40–7.50 (m, 2H, 11-H and a-NH
8
4
.05 (m, 1H, 3-H), 8.80–8.90 (m, 1H, 1-H), 9.26 (s, 1H,
-H), 10.73 (s, 1H, N–H). C NMR (CDCl , 75.4 MHz):
Lys), 7.83 (s, 2H, 2-H and 3-H), 8.34 (s, 1H, NH), 8.76 (s,
1H, 1-H), 9.03 (s, 1H, 4-H), 10.25 (br s, 1H, NH).
1
3
13
C
3
d ¼13.93 (NHCH CH ), 17.86 (CH ), 29.68 (b-CH Phe),
NMR (CDCl , 75.4 MHz): d ¼13.97 (NHCH CH ), 17.64
C
2
3
3
2
3
C
2
3
3
4.43 (NHCH CH ), 37.54 (NHCH CH ), 38.77
2
(CH ), 22.53 (d-CH Lys), 28.58 (b-CH Lys), 29.67
3 2 2
2
2
3
(
(
(
NHCH CH ), 52.15 (OCH ), 54.24 (a-CH Phe), 93.08
3
C-6), 93.46 (C-8), 123.66 (Ar-C), 124.11 (C-1), 125.67
C-4), 126.18 (Ar-C), 126.67 (C-10), 126.77 (C-4 Phe),
(g-CH Lys), 30.75 (COCH ), 34.41 (3-CH Lys), 38.39
2 3 2
(NHCH CH ), 39.85 (NHCH CH ), 42.44 (NHCH CH ),
2 3 2 2 2 2
52.16 (OCH ), 52.64 (a-CH Lys), 93.16 (C-6), 93.48
3
2
2
1
1
1
1
28.40 (C-3 and C-5 Phe), 129.19 (C-2 and C-4 Phe),
29.94 (Ar-C), 130.25 (C-3), 130.63 (Ar-C), 131.22 (C-
1), 131.82 (C-2), 134.15 (C-1 Phe), 146.89 (Ar-C),
50.78 (Ar-C), 153.97 (C-9), 157.64 (C-5), 170.83
(C-8), 123.59 (Ar-C), 124.26 (C-1), 124.93 (C-4), 126.31
(C-10), 129.94 (C-3), 130.31 (Ar-C), 130.56 (Ar-C),
131.35 (C-11), 131.87 (C-2), 134.19 (Ar-C), 146.94 (Ar-
C), 150.99 (Ar-C), 153.98 (C-9), 157.92 (C-5), 170.97
(CONH), 171.0 (COCH ), 173.17 (CO CH ). IR (Nujol,
ꢀ
1
(
2
1
CONH), 171.87 (CO CH ). IR (KBr, cm ): n¼3500,
2
3
3
2
3
ꢀ
1
932, 2853, 1720, 1650, 1644, 1586, 1544, 1515, 1451,
316, 1136, 1010. HRMS (FAB): calcd for C H N O
2 33 4 4
cm ): n¼3396, 2954, 2924, 2854, 1746, 1659, 1635,
1592, 1463, 1456, 1377, 1312, 1016. HRMS (FAB): calcd
for C H N O [M ]: 560.2873; found: 560.2858.
3
+
+
[
M ]: 537.2502; found: 537.2501.
3
1 38 5 5
4.4.6. N-{5-[3-(1,5-Dimethoxy-1,5-dioxopentan-2-yl-
amino)-3-oxopropylamino]-10-methyl-9H-benzo[a]phe-
noxazin-9-ylidene}ethanaminium chloride, Bfe-Glu
4.5. Synthesis of the functionalised benzo[a]phenox-
azinium salts 10a–c
(
(
(
OMe)-OMe (8f). The product of the reaction of 7b
0.077 g, 4.30ꢂ10 mol) with 3e (0.160 g, 4.30ꢂ10 mol)
4.5.1. 2-(Naphthalen-1-ylamino)ethanol, Nap-H (1c). To a
ꢀ
4
ꢀ4
ꢀ3
solution of 1-naphthylamine (1.0 g, 6.99ꢂ10 mol) in eth-
ꢀ
3
reflux time 3 h and 30 min) was chromatographed with
anol (2.5 mL), 2-chloroethanol (0.49 mL, 7.34ꢂ10 mol)
was added and the resulting mixture was refluxed for
20 h, and monitored by TLC (dichloromethane/methanol,
5.5:0.5). The solvent was removed under reduced pressure
and the crude mixture was chromatographed with dichloro-
methane/methanol, 5.9:0.1 as the eluent, to give Nap-H (1c)
as a brownish oil (0.309 g, 24%). TLC (chloroform/meth-
chloroform/methanol, 5.3:0.7 as the eluent, to give Bfe-
ꢁ
Glu(OMe)-OMe (8f) (0.156 g, 68%). Mp¼above 300 C.
1
TLC (chloroform/methanol, 5.4:0.6): R ¼0.33. H NMR
f
(
2
CDCl , 300 MHz): d¼1.40 (t, J¼7.2 Hz, 3H, NHCH CH ),
3
2
3
.25 (br s, 2H, b-CH Glu), 2.36 (s, 1H, CH ), 2.42–2.52
2 3
(m, 2H, g-CH Glu), 3.0–3.20 (m, 2H, NHCH CH ), 3.25–
2 2 2
1
3
3
.40 (m, 2H, NHCH CH ), 3.59 (s, 3H, OCH Glu), 3.61 (s,
2
anol, 5.5:0.5): R ¼0.84. H NMR (CDCl , 300 MHz):
3
3
f
3
H, OCH Glu), 3.70–3.80 (m, 2H, NHCH CH ), 4.45–4.60
3
d¼3.50 (t, J¼5.1 Hz, 2H, NHCH CH ), 4.02 (t, J¼5.4 Hz,
2
2
2
2
(
6
m, 1H, a-CH Glu), 5.80 (br s, 1H, NH), 6.20 (s, 1H, 8-H),
.38 (s, 1H, 6-H), 6.75 (br s, 1H, NH), 7.36 (s, 1H, 11-H),
2H, NHCH CH ), 6.68 (d, J¼7.2 Hz, 1H, 4-H), 7.29
2 2
(d, J¼8.4 Hz, 1H, 2-H), 7.36 (t, J¼7.5 Hz, 1H, 3-H),
13
7
NH Glu), 8.71 (br s, 1H, 1-H), 8.98 (br s, 1H, 4-H).
.75–7.85 (m, 2H, 3-H and 2-H), 8.45 (d, J¼7.5 Hz, 1H, a-
7.42–7.50 (m, 2H, 6-H and 7-H), 7.78–7.84 (m, 1H, 8-H),
7.86–7.90 (1H, m, 5-H). C NMR (CDCl , 75.4 MHz):
1
3
C
3
NMR (CDCl , 75.4 MHz): d ¼13.90 (NHCH CH ), 16.57
d ¼46.19 (NHCH CH ), 61.13 (NHCH CH ), 104.78
3
C
2
3
C
2
2
2
2
(
b-CH Glu), 17.87 (CH ), 30.23 (g-CH Glu), 34.31
2
(C-4), 117.97 (C-2), 119.96 (C-5), 123.76 (C-4a), 124.85
(C-7), 125.80 (C-6), 126.46 (C-3), 128.65 (C-8), 134.30
3
2
(NHCH CH ), 38.79 (NHCH CH ), 42.01 (NHCH CH ),
2 2 2 3 2 2
ꢀ
1
5
6
1
1
1
1
1.67 (a-CH Glu), 52.04 (OCH ), 52.28 (OCH ), 92.97 (C-
3
(C-8a), 143.27 (C-1). IR (neat, cm ): n¼3373, 3060,
2963, 2925, 2350, 2283, 1621, 1582, 1514, 1479, 1407,
1378, 1286, 1261, 1089.
3
), 93.38 (C-8), 123.54 (Ar-C), 124.09 (C-1), 124.93 (C-4),
30.10 (Ar-C), 130.17 (C-3), 130.59 (C-10), 131.21 (Ar-C),
31.63 (C-11), 131.75 (C-2), 133.85 (Ar-C), 148.57 (Ar-C),
50.73 (Ar-C), 154.13 (C-9), 157.54 (C-5), 171.13 (CONH),
1
4.5.2. N -(Naphthalen-1-yl)ethane-1,2-diamine, Nan-H
(1d). The product of the reaction of 1-naphthylamine
ꢀ1
71.97 (CO CH ), 173.0 (CO CH ). IR (Nujol, cm ):
3
2
2
3

V. H. J. Frade et al. / Tetrahedron 63 (2007) 12405–12418
12417
ꢀ
3
(
1.0 g, 6.99ꢂ10 mol) with 2-chloroethylamine monohy-
J¼6.3 Hz, 2H, NHCH CH ), 7.02 (s, 1H, 8-H), 7.17 (s, 1H,
2
2
ꢀ
3
drochloride (0.85 g, 7.34ꢂ10 mol) (reflux time 26 h)
was chromatographed with chloroform/methanol, mixtures
of increasing polarity, as the eluent. The oil obtained was
crystallised from dichloromethane to give Nan-H (1d) as
6-H), 7.84–7.96 (m, 2H, 11-H and 3-H), 8.00 (t, J¼7.8 Hz,
1H, 2-H), 8.48 (d, J¼8.4 Hz, 1H, 1-H), 9.07 (d, J¼8.4 Hz,
1
3
1H, 4-H). C NMR (CD OD, 75.4 MHz): d ¼14.51
3
C
(NHCH CH ), 18.47 (CH ), 40.53 (NHCH CH ), 41.95
2
3
3
2
2
ꢁ
a white solid (0.926 g, 71%). Mp¼229.0–231.6 C. TLC
(NHCH CH ), 43.01 (NHCH CH ), 94.60 (C-8), 95.17 (C-
2 2 2 3
1
(
3
(
chloroform/methanol, 6:1): R ¼0.16. H NMR (CD OD,
6), 123.84 (Ar-C), 124.88 (C-1), 125.54 (C-4), 130.48 (C-3),
130.78 (C-10), 132.44 (Ar-C), 133.32 (C-2), 133.48 (Ar-C),
133.52 (Ar-C), 133.71 (C-11), 149.88 (Ar-C), 152.56
f
3
00 MHz): d¼3.38 (t, J¼5.1 Hz, 2H, NHCH CH ), 3.64
2
2
t, J¼6.3 Hz, 2H, NHCH CH ), 6.71 (d, J¼7.2 Hz, 1H,
2 2
ꢀ
1
4
1
1
7
1
1
1
3
1
-H), 7.27 (d, J¼8.1 Hz, 1H, 2-H), 7.35 (t, J¼7.2 Hz,
(Ar-C), 157.83 (C-9), 158.29 (C-5). IR (KBr, cm ):
n¼3452, 2931, 2853, 1642, 1615, 1588, 1557, 1508, 1472,
1438, 1400, 1315, 1393, 1193, 1163, 1135, 1006. HRMS
H, 3-H), 7.40–7.50 (m, 2H, 6-H and 7-H), 7.75–7.82 (m,
H, 8-H), 8.06–8.12 (m, 1H, 5-H). C NMR (CD OD,
1
3
3
+
5.4 MHz): d ¼39.62 (NHCH CH ), 42.23 (NHCH CH ),
(EI): calcd for C H N O [M ]: 347.1872; found: 347.1875.
21 23 4
C
2
2
2
2
05.15 (C-4), 118.90 (C-2), 122.18 (C-5), 125.45 (C-4a),
25.62 (C-7), 126.78 (C-6), 127.44 (C-3), 129.27 (C-8),
4.6. General procedure for the synthesis of the
fluorescently labelled L-valine 8c, 12 and 13
ꢀ
1
35.94 (C-8a), 144.44 (C-1). IR (KBr, cm ): n¼3389,
055, 2983, 2928, 2874, 1585, 1536, 1488, 1452, 1410,
372, 1347, 1282, 1251, 1143, 1025, 948 cm . HRMS
ꢀ
1
The carboxylic derivative (Bfe-OH 10a or Boc-Val-OH 11)
was reacted with L-valine methyl ester hydrochloride H-Val-
OMe (2b), Bfh-H (10b) or Bfn-H (10c) using a standard
DCC/HOBt coupling. After evaporation of the solvent
and dry chromatography, the required derivative Bfe-Val-
OMe (8c), Boc-Val-OBfh (12) or Boc-Val-OBfn (13) was
obtained.
+
(
1
FAB): calcd for C H N [M ]: 186.1157; found:
12 14 2
86.1165.
4
.5.3. N-[5-(2-Hydroxyethylamino)-10-methyl-9H-ben-
zo[a]phenoxazin-9-ylidene]ethanaminium chloride,
Bfh-H (10b). To a cold solution (ice bath) of 5-ethyl-
amino-4-methyl-2-nitrosophenolhydrochloride(7b)(0.173 g,
ꢀ
ꢀ
4
9
9
(
.63ꢂ10 mol) in ethanol (2 mL), compound 1c (0.180 g,
4.6.1. N-{5-[3-(1-Methoxy-3-methyl-1-oxobutan-2-yl-
amino)-3-oxopropylamino]-10-methyl-9H-benzo[a]phen-
oxazin-9-ylidene}ethanaminium chloride, Bfe-Val-
OMe (8c). The product of the reaction of Bfe-OH (10a)
4
.63ꢂ10 mol) and concentrated hydrochloric acid
ꢀ2
5.0ꢂ10 mol) were added. The mixture was refluxed for
3
h and 30 min and monitored by TLC (dichloromethane/
ꢀ
5
methanol, 5.8:0.2). After evaporation of the solvent and
dry chromatography on silica gel with dichloromethane/
methanol, 5.5:0.5 as the eluent, Bfh-H (10b) was obtained
(0.030 g, 7.98ꢂ10 mol) with valine methyl ester
ꢀ4
hydrochloride (2b) (0.020 g, 1.20ꢂ10 mol) was chroma-
tographed with dichloromethane/methanol, 5.5:0.5 as the
eluent, to produce Bfe-Val-OMe (8c) as a blue solid
(0.036 g, 92%). The experimental data confirmed the ex-
pected structure and were in accordance with that previously
described (Section 4.4.3).
ꢁ
as a blue solid (0.317 g, 95%). Mp¼above 300 C. TLC (di-
1
chloromethane/methanol, 5.2:0.8): R ¼0.45. H NMR
f
(
CD OD, 300 MHz): d¼1.36 (br s, 3H, NHCH CH ), 2.16
3
2
3
(
NHCH CH ), 3.97 (br s, 2H, NHCH CH ), 6.42 (s, 1H,
s, 3H, CH ), 3.38 (br s, 2H, NHCH CH ), 3.73 (br s, 2H,
3 2 2
2
3
2
2
8
2
1
-H), 6.70 (s, 1H, 6-H), 7.19 (s, 1H, 11-H), 7.60–7.80 (m,
H, 2-H and 3-H), 8.12 (br s, 1H, 1-H), 8.41 (d, J¼6.9 Hz,
4.6.2. N-{5-[2-(2-(tert-Butoxycarbonylamino)-3-methyl-
butanoyloxy)ethylamino]-10-methyl-9H-benzo[a]phen-
oxazin-9-ylidene}ethanaminium chloride, Boc-Val-OBfh
(12). The product of the reaction of N-tert-butyloxycarbo-
1
3
H, 4-H). C NMR (CD OD, 75.4 MHz): d ¼14.17
3
C
(
(
(
(
NHCH CH ), 17.77 (CH ), 39.77 (NHCH CH ), 48.03
2 3 3 2 2
NHCH CH ), 60.98 (NHCH CH ), 94.30 (C-8), 94.40
2
C-6), 123.72 (C-1), 124.46 (Ar-C), 125.27 (C-4), 128.58
C-10), 130.60 (C-3), 131.79 (Ar-C), 132.02 (Ar-C),
ꢀ
4
nylvaline (0.050 g, 2.30ꢂ10 mol) with Bfn-H (10b)
3
2
2
ꢀ
4
(0.080 g, 2.30ꢂ10 mol) was chromatographed with di-
chloromethane/methanol, 5.6:0.4 as the eluent, to give
Boc-Val-OBfh (12) as a blue solid (0.118 g, 94%).
1
32.54 (C-2), 132.60 (C-11), 133.91 (Ar-C), 148.90
Ar-C), 152.30 (Ar-C), 156.47 (C-9), 158.91 (C-5). IR
ꢁ
(
(
Mp¼110.0–113.0 C. TLC (dichloromethane/methanol,
ꢀ
1
1
KBr, cm ): n¼3500–3118, 2974, 2924, 1641, 1590,
5.2:0.8): R ¼0.67. H NMR (CD OD, 300 MHz): d¼0.90
f
3
1
1
561, 1544, 1521, 1477, 1451, 1434, 1315, 1259, 1185,
163, 1137, 1084, 1056, 1008. HRMS (FAB): calcd for
(t, J¼6.9 Hz, 6H, 2ꢂg-CH Val), 1.37 (s, 9H, C(CH ) ),
3
3 3
1.38–1.50 (m, 3H, NHCH CH ), 2.0–2.15 (m, 1H, b-CH
2 3
+
C H N O [M ]: 348.1712; found: 348.1712.
2
Val), 2.31 (s, 3H, CH ), 3.45–3.55 (m, 2H, NHCH CH ),
3 2 2
1
22
3
2
3.95–4.10 (m, 3H, NHCH CH and a-CH Val), 4.45–4.70
2 3
4
.5.4. N-[5-(2-Aminoethylamino)-10-methyl-9H-ben-
(2ꢂm, 2H, NHCH CH ), 6.74 (s, 1H, 8-H), 6.97 (s, 1H,
2
2
zo[a]phenoxazin-9-ylidene]ethanaminium chloride,
Bfn-H (10c). The product of the reaction of compound 7b
6-H), 7.54 (s, 1H, 11-H), 7.68–7.79 (m, 1H, 3-H), 7.80–
7.90 (m, 1H, 2-H), 8.25 (d, J¼7.5 Hz, 1H, 1-H), 8.75 (d,
ꢀ
4
ꢀ4
13
(
(
0.136 g, 7.53ꢂ10 mol) with 1c (0.140 g, 7.53ꢂ10 mol)
J¼7.8 Hz, 1H, 4-H). C NMR (CD OD, 75.4 MHz):
3
reflux time 1 h and 30 min), following the same procedure
d ¼14.15 (NHCH CH ), 17.78 (CH ), 18.46 (g-CH Val),
C
2
3
3
3
as described for the synthesis of compound 10b, was succes-
sively washed with mixtures of increasing polarity of
chloroform/n-hexane and dichloromethane, to give Bfn-H
19.56 (g-CH Val), 28.65 (C(CH ) ), 31.58 (b-CH Val),
3 3 3
39.85 (NHCH CH ), 44.21 (NHCH CH ), 60.82 (a-CH
2
2
2
3
Val), 63.48 (NHCH CH ), 80.63 (C(CH ) ), 94.12 (C-6),
2
2
3 3
ꢁ
1
(
10c) as a blue solid (0.256 g, 98%). Mp¼above 300 C. H
94.50 (C-8), 123.66 (C-1), 124.44 (Ar-C), 125.38 (C-4),
129.35 (C-10), 130.64 (C-3), 132.28 (Ar-C), 132.59 (C-2),
132.71 (Ar-C), 132.84 (C-11), 133.70 (Ar-C), 149.41
(Ar-C), 152.50 (Ar-C), 157.03 (C-9), 158.26 (CO C(CH ) ),
NMR (CD OD, 300 MHz): d¼1.43 (t, J¼7.2 Hz, 3H,
3
NHCH CH ), 2.44 (s, 3H, CH ), 3.48 (t, J¼6.0 Hz, 2H,
2
3
3
NHCH CH ), 3.68–3.78 (m, 3H, NHCH CH ), 4.08 (t,
3
2
2
2
2
3 3

12418
V. H. J. Frade et al. / Tetrahedron 63 (2007) 12405–12418
ꢀ
1
1
2
1
58.50 (C-5), 173.98 (CO CH ). IR (KBr, cm ): n¼3250,
References and notes
2
2
974, 2933, 1743, 1708, 1642, 1591, 1561, 1545, 1522,
430, 1317, 1258, 1185, 1164, 1143, 1087, 1011. HRMS
EI): calcd for C H N O [M ]: 547.2920; found:
3
1. Shutes, A.; Der, C. J. Methods 2005, 37, 183.
2. Piloto, A. M.; Rovira, D.; Costa, S. P. G.; Gon c¸ alves, M. S. T.
Tetrahedron 2006, 62, 11955.
+
(
1 39 4 5
5
47.2938.
3
. Piloto, A. M.; Fonseca, A. S. C.; Costa, S. P. G.; Gon c¸ alves,
M. S. T. Tetrahedron 2006, 62, 9258.
4.6.3. N-{5-[2-(2-(tert-Butoxycarbonylamino)-3-methyl-
butanamido)ethylamino]-10-methyl-9H-benzo[a]phen-
4. Barbarella, G.; Zambianchi, M.; Sotgiu, G.; Ventola, A.;
Galeotti, M.; Gigli, G.; Cazzato, A.; Capobianco, M. L.
J. Non-Cryst. Solids 2006, 352, 2465.
5. Gatti, M.; Gioia, M. G.; Andreatta, P.; Pentassuglia, G. J. Pharm.
Biomed. Anal. 2004, 35, 339.
6. Sol ꢀı nov ꢀa , V.; Kasicka, V.; Koval, D.; Barth, T.; Ciencialov ꢀa , A.;
Z ꢀa kov ꢀa , L. J. Chromatogr., B 2004, 808, 75.
7. Naval, M. V.; G ꢀo mez-Serranillos, M. P.; Carretero, M. E.; De
Arce, C. J. Chromatogr., A 2006, 1121, 242.
8. Kang, X.; Xi ~a o, J.; Huang, X.; Gu, Z. Clin. Chim. Acta 2006,
366, 352.
9. Clarke, G.; O’Mahony, S.; Malone, G.; Dian, T. G. J. Neurosci.
Methods 2007, 160, 223.
oxazin-9-ylidene}ethanaminium chloride, Boc-Val-OBfn
(
13). The product of the reaction of N-tert-butyloxycarbo-
nylvaline (0.025 g, 1.15ꢂ10 mol) with Bfn-H (10c)
ꢀ
4
ꢀ
4
(
roform/methanol, 5.7:0.3 as the eluent, to give Boc-Val-
0.040 g, 1.15ꢂ10 mol) was chromatographed with chlo-
OBfn (13) as a blue solid (0.056 g, 89%). Mp¼100.0–
ꢁ
1
03.0 C. TLC (dichloromethane/methanol, 5.5:0.5):
1
R ¼0.47. H NMR (CD OD, 300 MHz): d¼0.96 (t,
f
3
J¼7.2 Hz, 6H, 2ꢂg-CH Val), 1.37 (s, 9H, C(CH ) ),
3
3 3
1
Val), 2.28 (s, 3H, CH ), 3.44–3.55 (m, 2H, NHCH CH ),
.38–1.50 (m, 3H, NHCH CH ), 2.04–2.16 (m, 1H, b-CH
2 3
3
2
2
3
.61–3.80 (m, 4H, NHCH CH and NHCH CH ), 3.90 (d,
2 3 2 2
J¼6.3 Hz, 1H, a-CH Val), 6.66 (s, 1H, 8-H), 6.82 (s, 1H,
10. Wang, C.; Zhao, S.; Yuan, H.; Xi ~a o, D. J. Chromatogr., B 2006,
833, 129.
11. Burchak, O. N.; Mugherli, L.; Chatelain, F.; Balakirev, M. Y.
Bioorg. Med. Chem. 2006, 14, 2559.
12. Meng, Q.; Yu, M.; Zhang, H.; Ren, J.; Huang, D. Dyes
Pigments 2007, 73, 254.
6
8
8
7
-H), 7.33 (s, 1H, 11-H), 7.52 (d, J¼8.1 Hz, 1H, 3-H),
.50 (d, J¼8.4 Hz, 1H, 2-H), 8.18 (d, J¼7.8 Hz, 1H, 1-H),
1
3
.86 (d, J¼7.8 Hz, 1H, 4-H).
C NMR (CD OD,
3
5.4 MHz): d ¼14.19 (NHCH CH ), 17.81 (CH ), 18.46
C
2
3
3
(
g-CH Val), 19.76 (g-CH Val), 28.65 (C(CH ) ), 31.79
3 3 3 3
(
b-CH Val), 39.08 (NHCH CH ), 39.83 (NHCH CH ),
2
5.85 (NHCH CH ), 61.97 (a-CH Val), 80.60 (C(CH ) ),
2 2 3 3
3.88 (C-8), 94.47 (C-6), 123.75 (C-1), 124.31 (Ar-C),
25.33 (C-4), 129.02 (Ar-C), 129.42 (C-10), 130.67 (C-3),
32.12 (Ar-C), 132.55 (C-2), 132.73 (C-11), 133.76
13. Tatarets, A. L.; Fedyunyayeva, I. A.; Dyubko, T. S.; Povrozin,
Y. A.; Doroshenko, A. O.; Terpetschnig, E. A.; Patsenker, L. D.
Anal. Chim. Acta 2006, 570, 214.
14. Volkova, K. D.; Kovalska, V. B.; Tatarets, A. L.; Patsenker,
L. D.; Kryvorotenko, D. V.; Yarmoluk, S. M. Dyes Pigments
2007, 72, 285.
15. Kovalska, V. B.; Volkova, K. D.; Losytskyy, M. Y.; Tolmachev,
O. I.; Balanda, A. O.; Yarmoluk, S. M. Spectrochim. Acta, Part
A 2006, 65, 271.
16. Chen, X.; Peng, X.; Cui, A.; Wang, B.; Wang, L.; Zhang, R.
J. Photochem. Photobiol., A 2006, 181, 79.
3
2
2
4
9
1
1
(
1
Ar-C), 149.16 (Ar-C), 152.37 (Ar-C), 156.79 (C-9),
58.02 (CO C(CH ) ), 158.30 (C-5), 176.36 (CONH). IR
2
3 3
ꢀ1
(
KBr, cm ): n¼3250, 2973, 2933, 1708, 1643, 1591,
1521, 1435, 1315, 1259, 1164, 1142, 1011. HRMS (EI):
calcd for C H N O [M ]: 546.3080; found: 546.3066.
+
3
1 40 5 4
4.7. General procedure for the photofading of
compounds 10a–c
17. Jose, J.; Burgess, K. Tetrahedron 2006, 62, 11021.
18. Ho, N.-H.; Weissleder, R.; Tung, C.-H. Tetrahedron2006, 62,578.
1
9. Frade, V. H. J.; Gon c¸ alves, M. S. T.; Coutinho, P. J. G.; Moura,
J. C. V. P. J. Photochem. Photobiol., A 2007, 185, 220.
Compounds 10a–c were dissolved in ethanol or water (pH
7
ꢀ
5
.4) with concentrations of 1ꢂ10 M. The samples were
20. Frade, V. H. J.; Coutinho, P. J. G.; Moura, J. C. V. P.; Gon c¸ alves,
M. S. T. Tetrahedron 2007, 63, 1654.
21. Frade, V. H. J.; Barros, S. A.; Moura, J. C. V. P.; Gon c¸ alves,
M. S. T. Tetrahedron Lett. 2007, 48, 3403.
22. Kanitz, A.; Hartmann, H. Eur. J. Org. Chem. 1999, 923.
23. Crossley, M. L.; Turner, R. J.; Hofmann, C. M.; Dreisbach,
P. F.; Parker, R. P. J. Am. Chem. Soc. 1952, 74, 578.
irradiated at 350 or 419 nm in a Rayonet RPR-100 chamber
reactor with 10 lamps. The photostabilities were expressed
in terms of their remaining absorption (%) calculated from
the change of absorption intensities at the absorption
maxima before and after irradiation.
2
4. Tajima, S.; Tobita, S.; Shizuka, H. J. Phys. Chem. A 2000,104, 11270.
25. Morris, J. V.; Mahaney, M. A.; Huber, J. R. J. Phys. Chem.
976, 80, 969.
Acknowledgements
1
We thank the Funda c¸ ~a o para a Ci ^e ncia e Tecnologia (Portu-
gal) for its financial support to the Centro de Qu ꢀı mica and
Centro de F ꢀı sica (Universidade do Minho).
26. Sens, R.; Drexhage, K. H. J. Lumin. 1981, 24, 709.
27. Valeur, B. Molecular Fluorescence: Principles and
Applications; Wiley-VCH: Weinheim, 2002.