A naphto[2,1-b]furan as a new fluorescent label

Table 4. Logarithm of association constants (log K_ass) for the interaction of (bi)thienyl amino acids 2b-f,h-j with several cations in acetonitrile (ligand:metal stoichiometry 2:1).

Cation Cpd	Cu^2+	Fe^3+	Hg^2+	Pd^2+
2b	7.232 ± 0.006	7.233 ± 0.007	---	7.247 ± 0.005
2c	12.29 ± 0.31	11.33 ± 0.29	---	11.09 ± 0.07
2d	11.24 ± 0.23	11.35 ± 0.24	---	11.07 ± 0.08
2e	12.54 ± 0.04	13.38 ± 0.05	---	12.15 ± 0.06
2f	12.80 ± 0.07	12.00 ± 0.06	---	---
2h	11.42 ± 0.20	11.40 ± 0.19	11.07 ± 0.08	11.11 ± 0.05
2i	13.33 ± 0.10	13.80 ± 0.15	11.97 ± 0.07	12.20 ± 0.07
2j	13.21 ± 0.05	13.32 ± 0.05	---	12.79 ± 0.06

Conclusions

New heterocyclic amino acids 2 containing thiophene and bithiophene units as side chain were synthesised and evaluated as fluorescent chemosensors based on an amino acid core for a series of biomedically relevant ions. From the spectrofluorimetric titrations in acetonitrile, it was found that the (bi)thienyl amino acids were more sensitive towards Fe³⁺ and Cu²⁺, when compared to the other tested ions, as a very low number of metal equivalents was enough to obtain a complete fluorescence quenching. The results indicated that there is a strong interaction with Fe³⁺ and Cu²⁺ through the donor N, O and S atoms at the side chain of the various amino acids and they can act as fluorimetric chemosensors. Interestingly, the methoxybithienyl amino acid 2i was found to be a very sensitive and selective colorimetric chemosensor for Cu²⁺ as it displayed a marked colour change from pale yellow to pink.

Due to their emissive properties and their recognition ability, these heterocyclic amino acids could find application as fluorescent building blocks for the preparation of peptides with chemosensory ability. Further studies will be undertaken in order to clarify aspects which are important for the pratical biomedical application of the synthesized compounds, such as the behaviour of these probes in the complex mixture of compounds in biological systems and their performance in the presence of ions and probes at physiological concentrations.
Acknowledgments

Thanks are due to Fundação para a Ciência e Tecnologia (FCT-Portugal) and FEDER-COMPETE for financial support through Centro de Química [PEst-C/QUI/UI0686/2013 (F-COMP-01-0124-FEDER-037302)] and a PhD grant to C.I.C. Esteves (SFRH/BD/68360/2010). The NMR spectrometer Bruker Avance III 400 is part of the National NMR Network and was purchased with funds from FCT and FEDER.

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Supplementary material


1. Synthetic details and characterization for compounds 2b-g,i-j
N-Cyclohexyl-2-(N-(4’’-methoxybenzyl)acetamido)-2-(5-phenylthiophen-2-yl)acetamide 2b. Starting from 2-(4’-formylphenyl)thiophene 1b (0.132 g, 7.03 × 10^-4 mol), 4-methoxybenzylamine (0.09 mL, 7.03 × 10^-4 mol), acetic acid (0.04 mL, 7.03 × 10^-4 mol) and cyclohexyl isocyanide (0.09 mL, 7.03 × 10^-4 mol), compound 2b was obtained as an orange oil (0.201 g, 4.22 × 10^-4 mol, 60%). ¹H NMR (400 MHz, CDCl₃):  = 1.10-1.19 (m, 3H, 3 × H-cHex), 1.28-1.38 (m, 2H, 2 × H-cHex), 1.56-1.69 (m, 3H, 3 × H-cHex), 1.83-1.92 (m, 2H, 2 × H-cHex), 2.11 (s, 3H, CH₃CO), 3.75 (s, 4H, OCH₃ and H1-cHex), 4.63 (d, J 8.0Hz, 2H, NCH₂), 5.89 (s, 1H, -H), 6.01 (d, J 7.6 Hz, 1H, NH), 6.79 (d, J 8.8 Hz, 2H, H3’’ and H5’’), 7.02 (d, J 3.6 Hz, 1H, H3), 7.08 (d, J 8.6 Hz, 2H, H2’’ and H6’’), 7.11 (d, J 3.6 Hz, 1H, H4), 7.29 (d, J 7.2 Hz, 1H, H4’), 7.36 (t, J 7.2 Hz, 2H, H3’ and H5’), 7.56 (d, J 7.2Hz, 2H, H2’ and H6’). ¹³C NMR (100.6 MHz, CDCl₃):  = 22.37 (CH₃CO), 24.68 (C-cHex), 24.74 (C-cHex), 25.48 (C-cHex), 32.69 (C-cHex), 32.73 (C-cHex), 48.68 (C1-cHex), 50.78 (NCH₂), 55.26 (OCH₃), 58.73 (-CH), 113.97 (C3’’ and C5’’), 122.30 (C4), 125.79 (C2’ and C6’), 127.70 (C2’’,C4’ and C-6’’), 128.90 (C3’ and C5’), 129.25 (C1’’), 130.42 (C3), 134.02 (C1’), 136.28 (C2), 146.36 (C5), 158.82 (C4’’), 167.65 (C=O amide), 172.17 (CH₃CO). IR (liquid film, cm^-1):  = 3301, 3060, 2931, 2854, 1649, 1585, 1544, 1513, 1462, 1450, 1407, 1364, 1350, 1303, 1289, 1247, 1209, 1176, 1110, 1093, 1074, 1033, 978, 956, 917, 892, 813, 757, 701, 665, 543. UV/Vis (ethanol, nm): _max (log ) = 286 (4.01). MS: m/z (ESI, %) 477 (M⁺, 100). HMRS: m/z (ESI) calc. for C₂₈H₃₃N₂O₃S 477.22064, found 477.22014.
N-Cyclohexyl-2-(N-(4’’-methoxybenzyl)acetamido)-2-(5-(4’-methoxyphenyl)thiophen-2-yl)acetamide 2c. Starting from 2-formyl-5-(4’-methoxyphenyl)thiophene 1c (0.086 g, 3.95 × 10^-4 mol), 4-methoxybenzylamine (0.05 mL, 3.95 × 10^-4 mol), acetic acid (0.02 mL, 3.95 × 10^-4 mol) and cyclohexyl isocyanide (0.05 mL, 3.95 × 10^-4 mol), compound 2c was obtained as an orange oil (0.050 g, 9.87 × 10^-5 mol, 25%). ¹H NMR (400 MHz, CDCl₃):  = 1.07-1.18 (m, 3H, 3 × H-cHex), 1.26-1.38 (m, 2H, 2 × H-cHex), 1.55-1.70 (m, 3H, 3 × H-cHex), 1.82-1.94 (m, 2H, 2x × H-cHex), 2.10 (s, 3H, CH₃CO), 3.75 (s, 4H, 4’’-OCH₃ and H1-cHex), 3.83 (s, 3H, 4’-OCH₃), 4.57-4.68 (m, 2H, NCH₂), 5.87 (s, 1H, -H), 6.04 (d, J 8.0 Hz, 1H, NH), 6.78 (d, J 8.4 Hz, 2H, H3’’ and H5’’), 6.89 (d, J 8.8 Hz, 2H, H3’ and H5’), 6.99 (s, 2H, H3 and H4), 7.08 (d, J 8.4 Hz, 2H, H2’’ and H6’’), 7.48 (d, J 8.8 Hz, 2H, H2’ and H6’). ¹³C NMR (100.6 MHz, CDCl₃):  = 22.31 (CH₃CO), 24.63 (C-cHex), 24.69 (C-cHex), 25.42 (C-cHex), 32.62 (C-cHex), 32.66 (C-cHex), 48.59 (C1-cHex), 50.67 (NCH₂), 55.19 (4’’-OCH₃), 55.29 (4’-OCH₃), 58.69 (-CH), 113.89 (C3’’ and C5’’), 114.22 (C3’ and C5’), 121.18 (C4), 126.81 (C1’), 127.00 (C2’ and C6’), 127.70 (C2’’ and C6’’), 129.01 (C1’’), 130.32 (C3), 135.05 (C2), 146.24 (C5), 158.72 (C4’’), 159.35 (C4’), 167.67 (C=O amide), 172.08 (CH₃CO). IR (liquid film, cm^-1):  = 3410, 3297, 3066, 3001, 2932, 2854, 1650, 1632, 1612, 1586, 1544, 1513, 1463, 1409, 1364, 1351, 1287, 1249, 1177, 1111, 1090, 1033, 979, 957, 917, 892, 830, 805. UV/Vis (ethanol, nm): _max (log ) = 299 (4.21). MS: m/z (ESI, %) 507 (M⁺, 100). HMRS: m/z (ESI) calc. for C₂₉H₃₅N₂O₄S 507.23120, found 507.23085.
N-Cyclohexyl-2-(N-(4’’-methoxybenzyl)acetamido)-2-(5-(4’-ethoxyphenyl)thiophen-2-yl)acetamide 2d. Starting from 2-formyl-5-(4’-ethoxyphenyl)thiophene 1d (0.187 g, 8.05 × 10^-4 mol), 4-methoxybenzylamine (0.11 mL, 8.05 × 10^-4 mol), acetic acid (0.05 mL, 8.05 × 10^-4 mol) and cyclohexyl isocyanide (0.10 mL, 8.05 × 10^-4 mol), compound 2d was obtained as an orange oil (0.109 g, 2.09 × 10^-4 mol, 26%). ¹H NMR (400 MHz, CDCl₃):  = 1.08-1.17 (m, 3H, 3 × H-cHex), 1.26-1.37 (m, 2H, 2 × H-cHex), 1.40 (t, J 6.8 Hz, 3H, OCH₂CH₃), 1.54-1.67 (m, 3H, 3 × H-cHex), 1.81-1.93 (m, 2H, 2 × H-cHex), 2.07 (s, 3H, CH₃CO), 3.72 (s, 4H, OCH₃ and H1-cHex), 4.02 (q, J 6.8 Hz, 2H, OCH₂CH₃), 4.56-4.67 (m, 2H, NCH₂), 5.90 (s, 1H, -H), 6.18 (d, J 2.4 Hz, 1H, NH), 6.79 (d, J 8.6 Hz, 2H, H3’’ and H5’’), 6.86 (d, J 8.8 Hz, 2H, H3’ and H5’), 6.97 (s, 2H, H3 and H4), 7.06 (d, J 8.6 Hz, 2H, H2’’ and H6’’), 7.44 (d, J 8.8Hz, 2H, H2’ and H6’). ¹³C NMR (100.6 MHz, CDCl₃):  = 14.64 (OCH₂CH₃), 22.23 (CH₃CO), 24.55 (C-cHex), 24.61 (C-cHex), 25.50 (C-cHex), 32.50 (C-cHex), 32.53 (C-cHex), 48.50 (C1-cHex), 50.52 (NCH₂), 55.07 (OCH₃), 58.48 (-CH), 63.39 (OCH₂CH₃), 113.78 (C3’’ and C5’’), 114.67 (C3’ and C5’), 121.03 (C4), 126.86 (C2’ and C6’), 127.59 (C2’’ and C6’’), 129.01 (C1’’), 130.19 (C3), 131.86 (C1’), 135.05 (C2), 146.15 (C5), 158.60 (C4’ and C4’’), 167.62 (C=O amide), 172.02 (CH₃CO). IR (liquid film, cm^-1):  = 3410, 3297, 3062, 2979, 2930, 2855, 1652, 1611, 1586, 1572, 1544, 1513, 1463, 1408, 1364, 1351, 1303, 1287, 1248, 1177, 1116, 1090, 1039, 979, 958, 921, 892, 825, 803. UV/Vis (ethanol, nm): _max (log ) = 299 (4.21). MS: m/z (ESI, %) 342 (M⁺-178, 100), 521 (M⁺, 89). HMRS: m/z (ESI) calc. for C₃₀H₃₇N₂O₄S 521.24685, found 521.24652.
N-Cyclohexyl-2-(N-(4’’-methoxybenzyl)acetamido)-2-(5-(4’-phenoxyphenyl)thiophen-2-yl)acetamide 2e. Starting from 2-formyl-5-(4’-phenoxyphenyl)thiophene 1e (0.188 g, 6.70 × 10^-4 mol), 4-methoxybenzylamine (0.09 mL, 6.70 × 10^-4 mol), acetic acid (0.04 mL, 6.70 × 10^-4 mol) and cyclohexyl isocyanide (0.08 mL, 6.70 × 10^-4 mol), compound 2e was obtained as an orange oil (0.080 g, 1.41 × 10^-4 mmol, 21%). ¹H NMR (300 MHz, CDCl₃):  = 1.10-1.19 (m, 3H, 3 × H-cHex), 1.26-1.40 (m, 2H, 2 × H-cHex), 1.55-1.70 (m, 3H, 3 × H-cHex), 1.83-1.95 (m, 2H, 2 × H-cHex), 2.11 (s, 3H, CH₃CO), 3.75 (s, 4H, OCH₃ and H1-cHex), 4.63 (d, J 4.8Hz, 2H, NCH₂), 5.89 (br s, 1H, -H), 6.04 (d, J 7.8 Hz, 1H, NH), 6.79 (d, J 8.7 Hz, 2H, H3’’’ and H5’’’), 7.08 (d, J 8.7 Hz, 2H, H2’’’ and H6’’’), 6.98-7.05 (m, 6H, H3, H4, H2’’, H3’, H5’ and H6’’), 7.13 (dt, J 6.0 and 0.9 Hz, 1H, H4’’), 7.36 (dt, J 6.0 and 0.9 Hz, 2H, H3’’ and H5’’), 7.51 (d, J 8.7 Hz, 2H, H2’ and H6’). ¹³C NMR (75.4 MHz, CDCl₃):  = 22.33 (CH₃CO), 24.63 (C-cHex), 24.69 (C-cHex), 25.42 (C-cHex), 32.63 (C-cHex), 32.68 (C-cHex), 48.61 (C1-cHex), 50.68 (NCH₂), 55.20 (OCH₃), 58.63 (-CH), 113.90 (C3’’’ and C5’’’), 118.98 (C2’’ and C6’’), 119.02 (C3’ and C5’), 121.80 (C4), 123.52 (C4’’), 127.17 (C2’ and C6’), 127.71 (C2’’’ and C6’’’), 129.14 (C1’’’), 129.78 (C3’’ and C5’’), 130.38 (C3), 135.79 (C2), 140.09 (C1’), 145.76 (C5), 156.79 (C1’’), 157.08 (C4’), 158.75 (C4’’’), 167.61 (C=O amide), 172.11 (CH₃CO). IR (liquid film, cm^-1):  = 3301, 3060, 2931, 2854, 1649, 1585, 1544, 1513, 1462, 1450, 1407, 1364, 1350, 1303, 1289, 1247, 1209, 1176, 1110, 1093, 1074, 1033, 978, 956, 917, 892, 813, 757, 701, 665, 543. UV/Vis (ethanol, nm): _max (log ) = 299 (4.15). MS: m/z (ESI, %) 390 (M⁺-178, 100), 569 (M⁺, 96). HMRS: m/z (ESI) calc. for C₃₄H₃₇N₂O₄S 569.24685, found 569.24654.
2-(5-(4’-Cyanophenyl)thiophen-2-yl)-N-cyclohexyl-2-(N-(4’’-methoxybenzyl)acetamido)acetamide 2f. Starting from 2-formyl-5-(4’-cyanophenyl)thiophene 1f (0.148 g, 6.97 × 10^-4 mol), 4-methoxybenzylamine (0.09 mL, 6.97 × 10^-4 mol), acetic acid (0.04 mL, 6.97 × 10^-4 mol) and cyclohexyl isocyanide (0.09 mL, 6.97 × 10^-4 mol), compound 2f was obtained as an orange oil (0.091 g, 1.88 × 10^-4 mol, 27%). ¹H NMR (300 MHz, CDCl₃):  = 1.09-1.16 (m, 3H, 3 × H-cHex), 1.24-1.37 (m, 2H, 2 × H-cHex), 1.54-1.67 (m, 3H, 3 × H-cHex), 1.79-1.92 (m, 2H, 2 × H-cHex), 2.11 (s, 3H, CH₃CO), 3.72 (s, 4H, OCH₃ and H1-cHex), 4.61 (d, J 3.0Hz, 2H, NCH₂), 5.97 (s, 1H, -H), 6.22 (d, J 6.9 Hz, 1H, NH), 6.76 (d, J 7.2 Hz, 2H, H3’’ and H5’’), 7.03 (d, J 7.2 Hz, 2H, H2’’ and H6’’), 7.03 (d, J 3.9 Hz, 1H, H3), 7.19 (d, J 3.9 Hz, 1H, H4), 7.58-7.63 (m, 4H, H2’, H3’, H5’ and H6’). ¹³C NMR (75.4 MHz, CDCl₃):  = 22.25 (CH₃CO), 24.54 (C-cHex), 24.59 (C-cHex), 25.30 (C-cHex), 32.49 (C-cHex), 32.54 (C-cHex), 48.61 (C1-cHex), 50.49 (NCH₂), 55.13 (OCH₃), 58.06 (-CH), 110.59 (C4’), 113.85 (C3’’ and C5’’), 118.63 (CN), 124.13 (C4), 125.81 (C2’ and C6’), 127.60 (C2’’ and C6’’), 128.63 (C1’’), 130.51 (C3), 132.58 (C3’ and C5’), 138.19 (C1’), 138.68 (C2), 143.62 (C5), 158.73 (C4’’), 167.23 (C=O amide), 172.15 (CH₃CO). IR (liquid film, cm^-1):  = 3301, 3061, 2999, 2933, 2855, 2226, 1657, 1604 1586, 1538, 1513, 1463, 1451, 1409, 1364, 1350, 1303, 1289, 1248, 1208, 1177, 1111, 1092, 1034, 979, 958, 941, 918, 892, 838, 814, 736, 701, 665, 543, 514. UV/Vis (ethanol, nm): _max (log ) = 317 (4.22). MS: m/z (ESI, %) 502 (M⁺, 100). HMRS: m/z (ESI) calc. for C₂₉H₃₂N₃O₃S 502.21589, found 502.21535.
N-Cyclohexyl-2-(N-(4’’-methoxybenzyl)acetamido)-2-(5-(4’-nitrophenyl)thiophen-2-yl)acetamide 2g. Starting from 2-formyl-5-(4’-nitrophenyl)thiophene 1g (0.186 g, 8.00 × 10^-4 mol), 4-methoxybenzylamine (0.10 mL, 8.00 × 10^-4 mol), acetic acid (0.05 mL, 8.00 × 10^-4 mol) and cyclohexyl isocyanide (0.10 mL, 8.00 × 10^-4 mol), compound 2g was obtained as a yellow oil (0.137 g, 2.72 × 10^-4 mol, 34%). ¹H NMR (400 MHz, CDCl₃):  = 1.09-1.16 (m, 3H, 3 × H-cHex), 1.27-1.32 (m, 2H, 2 × H-cHex), 1.61-1.66 (m, 3H, 3 × H-cHex), 1.82-1.93 (m, 2H, 2 × H-cHex), 2.10 (s, 3H, CH₃CO), 3.71 (s, 4H, OCH₃ and H1-cHex), 4.62 (d, J 8.0Hz, 2H, NCH₂), 6.03 (s, 1H, -H), 6.33 (d, J 7.6 Hz, 1H, NH), 6.75 (d, J 8.8 Hz, 2H, H3’’ and H5’’), 7.03 (d, J 3.6 Hz, 1H, H3), 7.06 (d, J 8.6 Hz, 2H, H2’’ and H6’’), 7.23 (d, J 3.6 Hz, 1H, H4), 7.63 (t, J 7.2 Hz, 2H, H2’ and H6’), 8.14 (d, J 7.2Hz, 2H, H3’ and H5’). ¹³C NMR (100.6 MHz, CDCl₃):  = 22.22 (CH₃CO), 24.52 (C-cHex), 24.57 (C-cHex), 25.29 (C-cHex), 32.45 (C-cHex), 32.52 (C-cHex), 48.59 (C1-cHex), 50.39 (NCH₂), 55.09 (OCH₃), 57.89 (-CH), 113.82 (C3’’ and C5’’), 124.18 (C3’ and C5’), 124.68 (C4), 125.70 (C2’ and C6’), 127.57 (C2’’ and C6’’), 128.64 (C1’’), 130.52 (C3), 139.43 (C2), 140.09 (C1’), 143.00 (C5), 146.51 (C4’), 158.71 (C4’’), 167.14 (C=O amide), 172.11 (CH₃CO). IR (liquid film, cm^-1):  = 3301, 3066, 2931, 2854, 1737, 1648, 1630, 1588, 1543, 1512, 1489, 1463, 1408, 1364, 1350, 1303, 1288, 1243, 1202, 1171, 1108, 1071, 1035, 978, 958, 916, 891, 869, 836, 803, 751, 736, 660, 512, 503. UV/Vis (ethanol, nm): _max (log ) = 349 (4.21). MS: m/z (ESI, %) 522 (M⁺, 100). HMRS: m/z (ESI) calc. for C₂₈H₃₂N₃O₅S 522.20572, found 522.20509.
N-Cyclohexyl-2-(5'-methoxy-[2,2'-bithiophen]-5-yl)-2-(N-(4’’-methoxybenzyl)acetamido)acetamide 2i. Starting from 5-formyl-5’-methoxy-2,2’-bithiophene 1i (0.142 g, 6.34 × 10^-4 mol), 4-methoxybenzylamine (0.08 mL, 6.34 × 10^-4 mol), acetic acid (0.04 mL, 6.34 × 10^-4 mol) and cyclohexyl isocyanide (0.08 mL, 6.34 × 10^-4 mol), compound 2i was obtained as an orange oil (0.131 g, 2.60 × 10^-4 mol, 41%). ¹H NMR (400 MHz, CDCl₃):  = 1.08-1.16 (m, 3H, 3 × H-cHex), 1.26-1.32 (m, 2H, 2 × H-cHex), 1.53-1.66 (m, 3H, 3 × H-cHex), 1.79-1.90 (m, 2H, 2 × H-cHex), 2.06 (s, 3H, CH₃CO), 3.73 (s, 4H, 4’’-OCH₃ and H1-cHex), 3.86 (s, 3H, 4’-OCH₃), 4.54-4.65 (m, 2H, NCH₂), 5.86 (s, 1H, -H), 6.08 (d, J 3.8 Hz, 1H, H4’), 6.21 (br s, 1H, NH), 6.74 (d, J 4.0 Hz, 2H, H3 and H4), 6.77 (d, J 8.4 Hz, 2H, H3’’ and H5’’), 6.87 (d, J 3.8 Hz, 1H, H3’), 7.05 (d, J 8.4 Hz, 2H, H2’’ and H6’’). ¹³C NMR (100.6 MHz, CDCl₃):  = 22.22 (CH₃CO), 24.57 (C-cHex), 24.64 (C-cHex), 25.36 (C-cHex), 32.53 (C-cHex), 32.57 (C-cHex), 48.53 (C1-cHex), 50.58 (NCH₂), 55.12 (4’’-OCH₃), 58.48 (-CH), 60.14 (4’-OCH₃), 104.32 (C4’), 113.84 (C3’’ and C5’’), 121.07 (C4), 121.52 (C3), 123.23 (C5’), 127.65 (C2’’ and C6’’), 128.88 (C1’’), 129.88 (C3’), 134.47 (C2), 140.04 (C5), 158.68 (C4’’), 165.66 (C2’), 167.49 (C=O amide), 172.00 (CH₃CO). IR (liquid film, cm^-1):  = 3411, 3302, 3068, 3007, 2933, 2854, 1650, 1632, 1586, 1532, 1513, 1498, 1462, 1451, 1408, 1350, 1303, 1289, 1249, 1202, 1176, 1111, 1092, 1052, 1036, 993, 911, 892, 873, 803, 770, 732, 646, 510. UV/Vis (ethanol, nm): _max (log ) = 329 (4.20). MS: m/z (ESI, %) 334 (M⁺-178, 100), 513 (M⁺, 49). HMRS: m/z (ESI) calc. for C₂₇H₃₃N₂O₄S₂ 513.18763, found 513.18750.
2-(5'-Cyano-[2,2'-bithiophen]-5-yl)-N-cyclohexyl-2-(N-(4’’-methoxybenzyl)acetamido)acetamide 2j. Starting from 5’-cyano-5-formyl-2,2’-bithiophene 1j (0.175 g, 8.00 × 10^-4 mol), 4-methoxybenzylamine (0.10 mL, 8.00 × 10^-4 mol), acetic acid (0.05 mL, 8.00 × 10^-4 mol) and cyclohexyl isocyanide (0.10 mL, 8.00 × 10^-4 mol), compound 2j was obtained as an orange oil (0.052 g, 1.20 × 10^-4 mol, 15%). ¹H NMR (400 MHz, CDCl₃):  = 1.10-1.19 (m, 3H, 3 × H-cHex), 1.26-1.35 (m, 2H, 2 × H-cHex), 1.57-1.70 (m, 3H, 3 × H-cHex), 1.83-1.93 (m, 2H, 2 × H-cHex), 2.14 (s, 3H, CH₃CO), 3.76 (s, 4H, OCH₃ and H1-cHex), 4.60 (s, 2H, NCH₂), 5.93 (s, 1H, -H), 6.11 (d, J 8.0 Hz, 1H, NH), 6.79 (d, J 8.4 Hz, 2H, H3’’ and H5’’), 6.95 (d, J 3.8 Hz, 1H, H3), 7.04 (d, J 8.4 Hz, 2H, H2’’ and H6’’), 7.06 (d, J 3.8 Hz, 1H, H4), 7.09 (d, J 3.8 Hz, 1H, H3’), 7.51 (d, J 3.8 Hz, 1H, H4’). ¹³C NMR (100.6 MHz, CDCl₃):  = 22.28 (CH₃CO), 24.60 (C-cHex), 24.65 (C-cHex), 25.39 (C-cHex), 32.58 (C-cHex), 32.65 (C-cHex), 48.72 (C1-cHex), 50.62 (NCH₂), 55.25 (OCH₃), 58.10 (-CH), 107.61 (C5’), 113.98 (C3’’ and C5’’), 114.12 (CN), 123.50 (C3’), 124.85 (C4), 127.74 (C2’’ and C6’’), 128.47 (C1’’), 130.33 (C5’), 136.84 (C2’), 138.23 (C4’), 138.48 (C2), 144.29 (C5), 158.92 (C4’’), 167.17 (C=O amide), 172.25 (CH₃CO). IR (liquid film, cm^-1):  = 3302, 3070, 2998, 2932, 2854, 2217, 1652, 1586, 1532, 1513, 1464, 1451, 1436, 1407, 1363, 1351, 1303, 1292, 1248, 1207, 1177, 1112, 1093, 1035, 979, 963, 918, 805, 736, 709, 512. UV/Vis (ethanol, nm): _max (log ) = 337 (4.21). MS: m/z (ESI, %) 508 (M⁺, 100). HMRS: m/z (ESI) calc. for C₂₇H₃₀N₃O₃S₂ 508.17231, found 508.17183.

2. Spectrofluorimetric titrations of compound 2b with all the tested ions (in acetonitrile)


3. Spectrofluorimetric titrations of compound 2h with all the tested ions (in acetonitrile)


4. Spectrofluorimetric titrations of compounds 2a-j with Fe³⁺ (in acetonitrile)
2a

2b

2c

2d

2e

2f

2h

2j


5. Job’s plot for compound 2i with Cu²⁺ (in acetonitrile)
2i

2i + Cu²⁺