Fluorescence derivatisation of amino acids in short and long-wavelengths

Article abstract of DOI:10.1016/j.tetlet.2007.03.070

3-(Naphthalen-1-ylamino)propanoic acid was coupled to the amino group of the main and lateral chains of various amino acids in order to evaluate its applicability as a fluorescent derivatising reagent. The resulting amino acid derivatives are strongly flu

Full text of DOI:10.1016/j.tetlet.2007.03.070

Tetrahedron Letters 48 (2007) 3403–3407
Fluorescence derivatisation of amino acids in short and
long-wavelengths
*
´
Vaˆnia H. J. Frade, Sıria A. Barros, Joao C. V. P. Moura and M. Sameiro T. Gonc¸alves
˜
Centro de Quı´mica, Universidade do Minho, Gualtar, 4710-057 Braga, Portugal
Received 31 January 2007; revised 6 March 2007; accepted 9 March 2007
Available online 15 March 2007
Abstract—3-(Naphthalen-1-ylamino)propanoic acid was coupled to the amino group of the main and lateral chains of various
amino acids in order to evaluate its applicability as a fluorescent derivatising reagent. The resulting amino acid derivatives are
strongly fluorescent with a maximum emission of about 415 nm. Condensation of these derivatives with 5-ethylamino-4-methyl-
2-nitrosophenol hydrochloride resulted in the corresponding blue benzo[a]phenoxazinium conjugates, also revealing strong fluores-
cence in ethanol and water at physiological pH and good quantum yields, but with emission wavelengths between 644 and 657 nm,
which was preferable in biological assays.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Amino acids are an important class of compounds
found in a large number of samples, namely, tissues, bio-
logical fluids, food and industrial products. Most of
them are small aliphatic molecules with neither auto-
fluorescence nor strong absorbance in the ultraviolet/
visible region. Thus, derivatisation is a needed to en-
hance both the selectivity and the sensitivity of amino
acid analysis in biological samples. Many fluoro-
phores have been tested to produce highly fluorescent
compounds with amino acids.1–4 Among them are naph-
thalene reagents, such as naphthalene-2,3-dicarboxalde-
hyde (NDA)5–7 or dansyl chloride,8,9 which form
conjugates with absorption and emission maxima in
wavelengths below 500 nm in the electromagnetic
spectrum.
Bearing this in mind, as well as our actual research inter-
ests,10–12 we decided to use the 3-(naphthalen-1-yl-
amino)propanoic acid in the derivatisation of several
a-amino acids, that have no (or low) intrinsic fluores-
cence or that are not easily detectable by ultraviolet
absorption, thus excluding tryptophan and tyrosine, as
representative models of biomolecules. Furthermore,
these amino acid derivatives were reacted with 5-ethyl-
amino-4-methyl-2-nitrosophenol hydrochloride to allo-
cate their transformation into the corresponding
benzo[a]phenoxazinium conjugates. Evaluation of
absorption and emission properties of all compounds
synthesised were performed in ethanol and water at
physiological pH (when soluble).
The synthesis of 3-(naphthalen-1-ylamino)propanoic
acid 1a10 was undertaken by the alkylation of 1-naph-
thylamine with methyl-3-bromopropionate, followed
by hydrolysis (NaOH/1,4-dioxane) of the isolated ester
intermediate methyl 3-(naphthalen-1-ylamino)propa-
noate (1b).13
Despite the importance of short-wavelength fluoro-
phores, the derivatisation and analysis of amino acids
and other molecules related with them are sometimes
limited in bioapplications, where long-wavelength
light-emitting (600–1000 nm) molecules are required be-
cause of the minimum interference from the absorption
scattering and the intrinsic fluorescence of biological
materials.
In order to investigate the possibility of using naph-
thalene carboxylic acid 1a in the derivatisation of
biomolecules, the covalent linkage between this com-
pound and several a-amino acids, as representative
models, was studied. Compound 1a was bonded to the
a-amine group of L-amino acid methyl esters, namely,
alanine, glycine, phenylalanine and glutamic acid
(protected at its side-chain as methyl ester) (2a–d), by
Keywords: Fluorescent derivatisation; Naphthalene derivatives; Long-
wavelength dyes; Benzo[a]phenoxazine; Fluorescent label.
*
Corresponding author. Fax: +351 253 678 986; e-mail: msameiro@
quimica.uminho.pt
0040-4039/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.03.070

3404
V. H. J. Frade et al. / Tetrahedron Letters 48 (2007) 3403–3407
H
H
R
O
R
N
DCC / HOBt
rt
N
CH2CH2CO2H
H2N C CO2CH3
C N C CO2CH3
CH2CH2
+
H
2
H H
1a
3
2, 3 a R = CH3
b R = H
c R = CH2-Ph
d R = CH2CH2CO2CH3
H
O
N
DCC / HOBt
rt
CH2CH2
C
NH (CH2)4CHCO2CH3
NHCOCH3
1a
+
H2N-(CH2)4-CH-CO2CH3
NHCOCH3
5
4
Scheme 1.
coupling them with the aid of N,N0-dicyclohexyl-
carbodiimide (DCC) assisted by 1-hydroxybenzotriazole
(HOBt) under standard conditions (Scheme 1).14
the expected bands for the carbonyl groups at 1627–
1651 cmꢀ1 (amide) and 1726–1750 cmꢀ1 (ester).
As was expected, and according to the photophysic
studies which will be discussed later, derivatives 3a–d
and 5 showed maximum absorption in the ultraviolet
range (ꢁ330 nm) and a strong emission at the beginning
of the visible region (ꢁ415 nm).
In addition of derivatising amino acids at their N-termi-
nus, the alternative acylation at a lysine x-amine group
was also performed. Thus, the methyl ester of N-acetyl-
lysine (4) was reacted with 1a under the conditions re-
ported above, to give the expected fluorescent derivative
5 (Scheme 1).
Fluorescent derivatisation is one of the most commonly
used methodologies for analysis purposes15 and labelled
biomolecules, revealing fluorescence outside the region
where the biological material fluoresces, are desirable
for the analytic practice. Considering these facts and
with the purpose of red shifting the maximum absor-
bance and emission of fluorescent amino acid derivatives
3a–d and 5, which were previously prepared, we studied
the possibility of generating the corresponding polycy-
clic systems of benzo[a]phenoxazinium. Thus, condensa-
tion of 5-ethylamino-4-methyl-2-nitrosophenol hydro-
chloride 6, synthesised according to the known
procedure involving treatment of the 3-ethylamino-4-
After purification by dry chromatography on silica gel,
derivatives 3a–d and 5 were obtained in yields ranging
from 85% to 98% (Table 1).
The structure of compounds 3a–d and 5 was confirmed
by high resolution mass spectrometry, IR and NMR (1H
and 13C) spectroscopy. In these compounds, the 13C
NMR spectra showed signals at d 170.24–172.17 ppm
and from 171.89 to 173.44 ppm due to the presence of
the carbonyl carbon of the amide and ester type, respec-
tively. The IR of compounds 3a–d and 5 also presented
Table 1. Yields and UV/vis data in ethanol and water (pH 7.4) for compounds 1a, 3a–d, 5, 7a–d and 8
Compound
Yield [%]
kabs [nm] (e)a
kabs [nm] (e)b
1a
3a
3b
3c
3d
5
85c/47d
87
90
85
95
98
57
67
330 (7000)
331 (10,682)
330 (5960)
320 (3596)
330 (7442)
330 (7000)
630 (23,558)
630 (32,283)
630 (40,945)
630 (30,500)
630 (42,857)
—
—
—
—
—
—
—
7a
7b
7c
7d
8
625 (14,970)
625 (21,778)
625 (24,500)
630 (27,439)
76
68
88
a Spectra were measured in absolute ethanol.
b Spectra were measured in water (pH 7.4).
c Yield from compound 1b.
d Yield from 1-naphthylamine.

V. H. J. Frade et al. / Tetrahedron Letters 48 (2007) 3403–3407
3405
NO
OH
H3C
H3C
N
O
7
H+/ MeOH
reflux
3
+
H
N
H3CH2C
H
H
N+
Cl -
N
O
C
R
C
H
CH2CH2
H3CH2C
N
H
CO2CH3
6
a R = CH3
b R = H
c R = CH2-Ph
d R = CH2CH2CO2CH3
H3C
H
N
H+/ MeOH
reflux
6
+
5
H
N+
N
O
O
Cl -
CH2CH2
H3CH2C
C
N
H
(CH2)4CH CO2CH3
NHCOCH3
8
Scheme 2.
methylphenol with sodium nitrite in an acid solu-
tion,16,11 with compounds 3a–d and 5 in acidic medium,
followed by dry chromatography purification, gave the
labelled amino acid derivatives 7a–d and 8 as blue mate-
rials in good yields (57–88%) (Scheme 2, Table 1).17
320 nm (3c) with molar absorptivities (e) between 3596
and 10682 Mꢀ1 cmꢀ1. Through the comparison of kabs
of compounds 1a, 3a–d and 5, it was possible to see that
the presence of the amino acid residues did not affect its
value; or was not significant in its variation (330 nm, 1a/
320 nm, 3c). The chemical transformation of derivatives
Compounds 7a–d and 8 were fully characterised by the
usual analytical techniques. As in the case of derivatives
3a–d and 5, the 13C NMR spectra showed signals at d
170.65–171.97 ppm and 171.87–173.51 ppm due to the
presence of the carbonyl carbon of the amide and ester
type, respectively. The IR of these compounds also pre-
sented the expected bands for the carbonyl groups at
1633–1659 cmꢀ1 (amide) and 1720–1746 cmꢀ1 (ester).
3a–d and
5
into the corresponding polycyclic
benzo[a]phenoxazinium conjugates 7a–d and 8, strongly
red shifted the maximum absorption (kabs 630 nm, 7a–d
and 8) with a simultaneously increase of the molar
absorptivities (e 23,558–42,857 Mꢀ1 cmꢀ1).
Comparison of the wavelengths of maxima absorption
of the two series of amino acid derivatives 3a–d and 5,
7a–d and 8, in ethanol, revealed a bathochromic shift
from 299 (3a/7a alanine) to 310 nm (3c/7c
phenylalanine).
Electronic absorption spectra of 10ꢀ6 M solutions of
fluorophore 1a and amino acid derivatives 3a–d, 5, 7a–
d and 8, in degassed absolute ethanol were measured.
Summarised data of this study are presented in Table
1. Compounds 3a–d and 5 showed absorption in the
ultraviolet region, the wavelength of maximum absorp-
tion (kabs) of all compounds being located at 330 or
The absorption properties of compounds 7b–d and 8
were also studied in water at physiological pH (pH
7.4, adjusted with HCl and NaOH); other compounds
were not soluble in water. Comparison of kabs values
Table 2. Fluorescence data in ethanol and water (pH 7.4) for compounds 1a, 3a–d, 5, 7a–d and 8
Compound
Fluorescencea
Stokes’ shift [nm]
Fluorescenceb
Stokes’ shift [nm]
kexc [nm]
kem [nm]
UF
kexc [nm]
kem [nm]
UF
1a
3a
3b
3c
3d
5
330
331
330
330
330
330
590
600
590
600
590
417
415
417
415
414
417
644
644
644
644
645
0.54
0.66
0.55
0.55
0.57
0.54
0.39
0.37
0.45
0.48
0.32
87
84
87
95
84
87
14
14
14
14
15
—
—
—
—
—
—
—
590
590
600
590
—
—
—
—
—
—
—
652
657
652
654
—
—
—
—
—
—
—
0.45
0.40
0.16
0.46
—
—
—
—
—
—
—
27
32
27
24
7a
7b
7c
7d
8
a Spectra were measured in absolute ethanol.
b Spectra were measured in water (pH 7.4).

3406
V. H. J. Frade et al. / Tetrahedron Letters 48 (2007) 3403–3407
in ethanol and water showed only a slight hypsochromic
shift (ꢁ5 nm) or were equal (8).
ised, 3-(naphthalen-1-ylamino)propanoic acid. Trans-
formation of the fluorescent derivatives into the
corresponding benzo[a]phenoxazinium based conjugates
following a straightforward procedure was possible in
good yields. The latter amino acid derivatives revealed
a maximum emission of 645 nm (in ethanol) and 652
or 657 nm (in water at physiological pH), which corre-
sponds to a red shift from 227 to 230 nm in relation to
the previously derivatising procedure. Considering the
photophysical properties of the all obtained conjugates,
both possibilities are interesting, depending on the spe-
cific application. However, the last one represents an
example of long-wavelength fluorescent labelling of
biomolecules.
Evaluation of the fluorescent properties of amino acid
derivatives 3a–d, 5, 7a–d and 8 in ethanol and water at
physiological pH (7b–d and 8), using 9,10-diphenylan-
thracene (UF = 0.95 in ethanol,18 3a–d and 5) or oxazine
1 (UF = 0.11 in ethanol,19 7a–d and 8) as standards was
performed (Table 2). In ethanol, the labelled amino
acids 3a–d and 5 exhibited good fluorescence with wave-
lengths of maximum emission (kem) at 414–417 nm,
high Stokes’s shifts (84–95 nm) and quantum yields
ranging from 0.54 to 0.66. Considering these results,
and as in the case of maximum absorption, the presence
of the amino acid residues did not practically affect
the kem of derivatising reagent 1a. However, fluores-
cent quantum yields increased (except for compound
5), the highest value being that of compound 3a (UF
0.66).
Acknowledgment
We thank the Fundac¸ao para a Cieˆncia e Tecnologia
˜
´
(Portugal) for its financial support to the Centro de Quı-
mica (Universidade do Minho).
Regarding blue amino acid derivatives 7a–d and 8 in the
two solvents studied, they fluoresced with wavelengths
of maximum emission in the 644–657 nm region and
showed quantum yields ranging from 0.16 to 0.48 (Table
2). Through the comparison of kem values in ethanol and
water (pH 7.4), it was notorious that a red shift (8 or
13 nm, 7c) occurred for the four derivatives. Despite
the low Stokes’s shifts exhibited for the compounds,
their values are superior in water (being the highest
32 nm, 7c).
References and notes
1. Piloto, A. M.; Costa, S. P. G.; Gonc¸alves, M. S. T.
Tetrahedron Lett. 2005, 46, 4757–4760.
2. Piloto, A. M.; Fonseca, A. S. C.; Costa, S. P. G.;
Gonc¸alves, M. S. T. Tetrahedron 2006, 62, 9258–9267.
3. Nemati, M.; Oveisi, M. R.; Abdollahi, H.; Sabzevari, O. J.
Pharm. Biomed. Anal. 2004, 34, 485–492.
Following the same tendency as the kabs, wavelengths of
maximum emission obtained for two series 3a–d, 5 and
7a–d, 8 presented a separation from 227 to 230 nm in
their values. As a result, for example, it was possible
to obtain alanine derivatives showing maximum fluores-
cence at 415 nm and at 644 nm, in ethanol.
4. Gatti, M.; Gioia, G.; Andreatta, P.; Pentassuglia, G. J.
Pharm. Biomed. Anal. 2004, 35, 339–348.
5. Clarke, G.; O’Mahony, S.; Malone, G.; Dinan, T. G. J.
Neurosci. Methods 2007, 160, 223–230.
6. Wang, C.; Zhao, S.; Juan, H.; Xiao, D. J. Chromatogr. B
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2006, 833, 129–134.
´
7. Gyinesi-Forras, K.; Leitner, A.; Akasaka, K.; Lindner, W.
J. Chromatogr. A. 2005, 1083, 80–88.
Figure 1 shows the comparison between the emission
spectra of compounds 3b in ethanol and 7b in ethanol
and water (pH 7.4).
´
´
8. Naval, M. V.; Gomez-Serranillos, M. P.; Carretero, M. E.;
De Arce, C. J. Chromatogr. A. 2006, 1121, 242–247.
9. Kang, X.; Xiao, J.; Huang, X.; Gu, Z. Clin. Chim. Acta
˜
2006, 366, 352–356.
In this work, efficient fluorescent amino acid derivatisa-
tion was achieved in the short wavelength region (kem at
about 414–417 nm) using the naphthalene functional-
10. Frade, V. H. J.; Gonc¸alves, M. S. T.; Coutinho, P. J. G.;
Moura, J. C. V. P. J. Photochem. Photobiol. A 2007, 185,
220–230.
11. Frade, V. H. J.; Gonc¸alves, M. S. T.; Moura, J. C. V. P.
Tetrahedron Lett. 2006, 47, 8567–8570.
12. Frade, V. H. J.; Coutinho, P. J. G.; Moura, J. C. V. P.;
Gonc¸alves, M. S. T. Tetrahedron 2007, 63, 1654–1663.
13. Synthesis of compound 1a: To a suspension of methyl 3-
1.0
3b
7b
(naphthalen-1-ylamino)propanoate
(1b)10
(0.470 g;
7b (water)
2.05 mmol) in 1,4-dioxane (4.0 mL) 1 M NaOH
(3.08 mL; 3.08 mmol) was added. The solution was stirred
at room temperature for 6 hours and acidified to pH 2–3
with 1 M KHSO4. The mixture was extracted with
chloroform (4 · 15 mL) and the organic extracts were
dried (MgSO4) and evaporated to dryness giving 3-
(naphthalen-1-ylamino)propanoic acid 1a as a white solid
(0.375 g, 85%). The spectroscopic data confirmed the
structure of compound 1a and were in accordance with the
previously reported characterisation of this compound
obtained by another procedure.10
0.8
0.6
0.4
0.2
0.0
300
400
500
600
700
800
Wavelength (nm)
14. Typical procedure for the synthesis of compounds 3a–d
and 5 (described for 3a): 3-(Naphthalen-1-ylamino)propa-
noic acid 1a (0.224 g; 1.04 mmol) was reacted with
Figure 1. Normalised fluorescence spectra of compounds 3b and 7b in
ethanol and 7b in water (pH 7.4).

V. H. J. Frade et al. / Tetrahedron Letters 48 (2007) 3403–3407
3407
alanine methyl ester hydrochloride 2a (0.208 g; 1.50 mmol)
in DMF (3 mL) by a standard DCC/HOBt coupling. After
dry chromatography on silica gel (chloroform/methanol,
5.9:0.1), N-[3-(naphthalen-1-ylamino)propanoyl] alanine
methyl ester 3a was obtained as an off-white oil (0.270 g,
87%). Rf = 0.69 (chloroform/methanol, 5.8:0.2). FTIR
(KBr): mmax 3393, 3310, 3049, 2929, 2852, 1750, 1641,
0.51 mmol) and concentrated hydrochloride acid (5.0 ·
10ꢀ2 mL) were added. The mixture was refluxed for 1 h
and 30 min and monitored by TLC (silica: chloroform–
methanol, 5.7:0.3). The solvent was removed under
reduced pressure and the crude mixture was purified by
dry chromatography (silica: dichloromethane–methanol,
5.4:0.6). N-{5-[3-(1-methoxy-1-oxopropan-2-ylamino)-3-
oxopropylamino]-10-methyl-9H-benzo[a]phenoxazin-9-yli-
dene}ethanaminium chloride 7a was obtained as a blue
solid (0.133 g, 57%). Mp above 300 ꢁC. Rf 0.30 (silica:
chloroform–methanol, 5.5:0.5). FTIR (KBr, 1%): mmax
3400, 2954, 2924, 2854, 1726, 1651, 1633, 1587, 1556, 1504,
1587, 1532, 1490, 1467, 1412, 1351, 1287, 1216, 1120,
1
983 cmꢀ1
.
H NMR (CDCl3, 300 MHz): d 1.38 (3H, d, J
7.2 Hz, b-CH3 Ala), 2.67 (2H, t, J 6.2 Hz, NCH2CH2),
3.66 (2H, t, J 5.7 Hz, NCH2CH2), 3.74 (3H, s, OMe),
4.56–4.67 (1H, m, a-CH Ala), 5.14–5.18 (1H, br s, NH),
6.24 (1H, d, J 6.6 Hz, a-NH Ala), 6.66 (1H, d, J 7.5 Hz, 4-
H), 7.27 (1H, d, J 8.1 Hz, 2-H), 7.36 (1H, t, J 7.5 Hz, 3-H),
7.40–7.50 (2H, m, 6-H and 7-H), 7.76–7.82 (1H, m, 5-H),
7.84–7.92 (1H, m, 8-H) ppm. 13C NMR (CDCl3,
75.4 MHz): d 18.20 (b-CH3 Ala), 35.04 (NCH2CH2),
40.26 (NCH2CH2), 48.03 (a-CH Ala), 52.46 (OMe),
104.56 (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
(CO2Me). The assignments were supported by HMBC
and HMQC techniques. HRMS: m/z (FAB): calcd for
C17H20N2O3[M+] 300.1474; found 300.1484.
1463, 1377, 1311 cmꢀ1 1H NMR (CDCl3, 300 MHz): d
.
1.41 (3H, br s, NCH2CH3), 1.48 (3H, d, J 7.2 Hz, b-CH3
Ala), 2.39 (3H, s, CH3), 3.05 (2H, br s, NCH2CH2), 3.28
(2H, br s, NCH2CH3), 3.68 (3H, s, OMe), 3.65–3.82 (2H,
m, NCH2CH2), 4.42–4.56 (1H, m, a-CH Ala), 6.18 (1H, s,
8-H), 6.35 (1H, s, 6-H), 6.74 (1H, br s, a-NH Ala), 7.37
(1H, s, 11-H), 7.82 (2H, br s, 2-H and 3-H), 8.32 (1H, d, J
6.6 Hz, NH), 8.75 (1H, br s, 1-H), 9.04 (1H, br s, 4-H),
10.16 (1H, br s, NH) ppm. 13C NMR (CDCl3, 75.4 MHz):
d 13.87 (NCH2CH3), 17.42 (b-CH3 Ala), 18.05 (CH3),
34.21 (NCH2CH2), 38.79 (NCH2CH3), 41.98 (NCH2CH2),
48.46 (a-CH Ala), 52.21 (OMe), 92.83 (C-6), 93.27 (C-8),
123.40 (Ar-C), 124.03 (C-1), 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 (Ar-C), 146.92 (Ar-C), 150.51 (Ar-C),
154.29 (C-9), 157.24 (C-5), 170.65 (CONH), 173.17
(CO2Me) ppm. The assignments were supported by
HMBC and HMQC techniques. HRMS: m/z (FAB):
calcd for C26H29N4O4[M+] 461.2189; found 461.2203.
18. Morris, J. V.; Mahaney, M. A.; Huber, J. R. J. Phys.
Chem. 1976, 80, 969–974.
´
´
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´
´
lova, A.; Zakova, L. J. Chromatogr. B 2004, 808, 75–82.
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bach, P. F.; Parker, R. P. J. Am. Chem. Soc. 1952, 74, 578–
584.
17. Typical procedure for the synthesis of compounds 7a–d
and 8 (described for 7a): To a cold solution (ice bath) of 5-
ethylamino-4-methyl-2-nitrosophenol hydrochloride
6
(0.092 g; 0.51 mmol) in methanol (2 mL), N-[3-(naphthalen-
1-ylamino)propanoyl] alanine methyl ester (3a) (0.300 g;
19. Sens, R.; Drexhage, K. H. J. Lumin. 1981, 24, 709–712.