1
782
E. Oliveira et al.
chromatography with silica gel (eluent: CH Cl /MeOH
2
Elemental analysis (found: C, 57.4; H, 5.6; N, 12.5 %
CHNS requires: C, 57.1; H, 5.9; N, 12.2). IR (KBr win-
2
100:1). The fractions were combined and the product L1
was obtained as a yellow solid.
-
1
-1
dows) cm : m (NH st) (cm ) = 3,030; m (alkyl-CH)
-
1
-1
Yellow powder (yield: 0.202 g, 70 %), C H NO .
5
(cm ) = 2,923, 2,847; m (C=O lactone) (cm ) = 1,712;
1
4
13
-
1
FW = 275.26.
(C=O st carboxylic acid) (cm ) = 1,681; m (C=C ben-
-
1
Elemental analysis (found: C, 61.3; H, 4.9; N, 4.7 %
CHNS requires: C, 61.0; H, 4.8; N, 5.0). IR (KBr win-
zene) (cm ) = 1,622, 1,446, 1,392, 1,286; m (N–C=O st)
-
1
-1
(cm ) = 1,564; m (C–O–C cyclic ethers) (cm ) = 1,214.
NMR spectrum: d (C H O, 400 MHz) ppm: 1.52–1.54 (d,
-
1
-1
dows) cm : m (NH st) (cm ) = 3,347; m (alkyl-CH)
(
H
3 6
-
1
-1
cm ) = 2,937, 2,947; m (C=O lactone) (cm ) = 1,720;
6H, 2 9 b-CH3 Ala), 2.78 (s, 6H, -CH3 acridine),
4.65–4.68 (m, 1H, a-H Ala), 7.39–7.49 (m, 4H, H6-H7,
-
1
(
C=O st carboxylic acid) (cm ) = 1,711; m (C=C ben-
-
1
0
0
0
0
zene) (cm ) = 1,648, 1,449, 1,362, 1,286; m (N–C=O st)
H6 -H7 ), 7.52–7.56 (m, 4H, H5, H8, H5 , H8 ), 7.70–7.72
-
1
-1
(
cm ) = 1,565; m (C–O–C cyclic ethers) (cm ) = 1,221,
(d, J = 8 Hz, 2H, 29 NH acridine), 7.77–7.81 (m, 3H,
0
0
00
00
00
00
1
,202. NMR spectrum: dH (CDCl , 400 MHz) ppm:
H6 , H10 , H8 ), 7.92–7.98 (m, 5H, H3 ? H13 , 29 NH
0
3
1
.49–1.52 (d, J = 12 Hz, 3H, b-CH Ala), 3.74 (s, 3H,
coumarin), 8.91 (s, 2H, H3, H3 ). d (C H O, 100 MHz)
3
C
3 6
OCH ), 4.70–4.73 (m, 1H, a-H Ala), 7.32–7.38 (m, 2H,
H6, H7), 7.60–7.65 (m, 2H, H5, H8), 8.84 (s, 1H, H3),
ppm: 17.55 (b-CH Ala), 33.44 (CH acridine), 48.15 (aC
Ala), 109.35 (C8 ), 116.24 (C6, C6 ), 118.72 (C6 ), 119.10
3
3
3
00 0 00
00 0 0 0
(C10 ), 124.38 (C7, C7 ), 125.15 (C5, C5 ), 127.18 (C8, C8 ),
9
.17–9.19 (d, J = 8 Hz, 1H, NH Ala). dC (CDCl3,
0
0
00
0
1
00 MHz) ppm: 18.00 (C–b-CH ), 48.63 (aC), 52.45
130.20 (C3 ), 134.15 (C13 ), 148.11 (C3, C3 ). UV–vis in
absolute ethanol (k nm): bands at 295 nm (loge = 4.45),
330 nm (loge = 4.20), and 462 nm (loge = 3.28). Emission
3
(
(
OCH ), 116.64 (C6), 125.25 (C7), 129.80 (C5), 134.16
3
C8), 148.56 (C3), 154.48 (C=O amide carbonyl). UV–vis
in absolute ethanol (k nm), bands at 295 nm (loge = 4.0),
30 nm (loge = 3.75). Emission spectra in absolute etha-
nol (kexc = 330 nm, k = 410 nm). ESI-TOF calc.
spectra in absolute ethanol (kexc1 = 330 nm, kexc2
=
3
462 nm, kemis1 = 410 nm, k = 492 nm). ESI-TOF
emis2
?
calc. (found) %: [L2] H 724.5 (724.7) 100 %.
emis
?
(
found) %: [L1]H , 276.3 (276.5) 100 % m/z.
L3: acridine yellow
L2: coumarin-alanine-acridine-alanine-coumarin
Acridine yellow G was a commercial product from Aldrich
and used without any further purification.
-
4
Coumarin-alanine (L1), (0.060 g, 2.18 9 10 mol) was
dissolved in 1,4-dioxane (1 ml) in an ice bath, and aqueous
sodium hydroxide 1 M solution (0.32 ml, 1.5 eq., 3.27 9
-
4
1
0
M) was added drop wise. The mixture was stirred at
Results and discussion
room temperature for 3 h. The pH was adjusted to 2–3 by
adding aqueous KHSO 1 M solution and extracting it with
4
Synthesis
ethyl acetate (3 9 10 ml). After drying it with anhydrous
sodium sulphate and the evaporation of the solvent, the
residue was triturated with diethyl ether and a white solid
Compound L1 was obtained by a standard DCC/HOBt
coupling reaction between H-Ala-OMe and coumarin-3-
carboxylic acid (A). L1 was purified by gel chromatogra-
phy yielding a yellow powder. The synthesis of compound
L2 was obtained by a reaction between compound
B (coumarin-Ala-OH) and commercial acridine yellow G
(L3), which was stirred for 1 h at 0 °C and 1 h at room
temperature. The final product was purified by precipitation
yielding an orange powder (Scheme 1).
(
B, coumarin-alanine-OH) was obtained.
-
4
Compound B (0.054 g, 2.06 9 10 mol) was dissolved
in distilled DMF (2 ml), cooled in an ice bath, followed by
-
the addition of HOBt (0.03 g, 2.06 9 10 mol) and DCC
4
-
0.05 g, 2.06 9 10 mol). The mixture was stirred in an
4
(
ice bath for 30 min.
Following the next step, acridine yellow G (L3)
-
0.056 g, 2.06 9 10 mol) was added to the solution
4
(
Both compounds were characterized by elemental
1
13
containing compound B and the final mixture was stirred
for 1 h in an ice bath and 1 h at room temperature. The
solvent was evaporated under reduced pressure and the
residue was treated with cooled acetone, to remove N-
acylurea (DCU) through filtration. The solvent was again
evaporated and the residue purified by precipitation.
Product L2 was obtained as an orange solid.
analysis, H and C NMR, infrared spectroscopy (KBr
discs), melting point, ESI-TOF (electrospray ionization-
time of flight-mass), UV–vis absorption and emission
spectroscopy, fluorescence quantum yields and lifetimes.
1
The H NMR spectra of compounds L1 and L2 present
the characteristic signals of the amino acid backbone NH
and a-H and side chain b-CH (for Ala). Also, the signals,
3
Orange powder (yield: 0.070 g, 50 %), C H N O
1 33 5 8
due to the heterocyclic rings, presented in coumarin and
4
6
H O, FW = 831.8.
2
acridine moieties were visible between 7.32 and 8.91 ppm.
1
23