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



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Non-canonical amino acids bearing thiophene and bithiophene: synthesis by an Ugi multicomponent reaction and studies on ion recognition ability
Cátia I. C. Esteves, M. Manuela M. Raposo and Susana P. G. Costa

Centre of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal

Abstract: Novel thienyl and bithienyl amino acids with different substituents were obtained by a multicomponent Ugi reaction between a heterocyclic aldehyde, an amine, an acid and an isocyanide. Due to the presence of the sulphur heterocycle at the side chain, these unnatural amino acids are highly emissive and bear extra electron donating atoms so they were tested for their ability to act as fluorescent probes and chemosensors in the recognition of biomedically relevant ions in acetonitrile and acetonitrile/water solutions. The results obtained from spectrophotometric/spectrofluorimeric titrations in the presence of organic and inorganic anions, and alkaline, alkaline-earth and transition metal cations indicated that the bithienyl amino acid bearing a methoxy group is a selective colorimetric chemosensor for Cu2+, while the other (bi)thienyl amino acids act as fluorimetric chemosensors with high sensitivity towards Fe3+ and Cu2+ in a metal–ligand complex with 1:2 stoichiometry. The photophysical and ion sensing properties of these amino acids confirm their potential as fluorescent probes suitable for incorporation into peptidic frameworks with chemosensory ability.

Introduction

Non-canonical amino acids of synthetic origin are useful for the preparation of functional peptides with tailored properties for varied applications such as increased fluorescence, conformational rigidity, and metal complexation ability, among other properties. Recent reports include the application of such amino acids in studies of molecular flexibility and protein folding, substrate binding activity of proteins, antigenicity or enzymatic activity, targeting peptides for molecular imaging, peptidomimetics biological activity and protein engineering (Kajihara et al. 2006; Hennig et al. 2007; Katritzki and Narindoshvili 2009; Lee et al. 2010; Wang et al 2012; Pless and Ahern 2013; Niu and Guo, 2013; Liu et al. 2015, Zhou et al 2016).

Many biochemical processes rely on the coordinating ability that amino acids and peptides display towards metal ions because they possess electron donor atoms like nitrogen, oxygen and sulphur at the main and side chains (Zheng et al. 2003; Shimazaki et al. 2009). Therefore, the insertion of suitable heterocycles at the side chain of natural amino acids, along increasing the number of binding sites, can provide increased UV absorption and fluorescence, which can be valuable for biochemistry, cellular biology and cellular imaging applications. Fluorescent probes are indispensable tools for monitoring ions and biomolecules with high sensitivity in cells and tissues, as they present distinct advantages in fluorescence detection in terms of sensitivity, selectivity, response time and local observation, etc. There are various examples of fluorescent unnatural amino acids, displaying better photophysical properties than tryptophan, that have been inserted in peptide and protein frameworks in order to afford fluorescently labelled entities (Katritzki and Narindoshvili 2009; Cheruku et al. 2015).

Thiophene and its derivatives exhibit interesting optical properties that have led to their application as sensors and fluorescent reporters (Capobianco et al. 2012). Oligomers of thiophene present improved luminescent properties are more readily soluble in organic solvents, improves absorption efficiency and thermal stability of the resultant molecule without reducing fluorescence (Pina et al. 2010).

Selective recognition of anions is also a very dynamic topic due to their importance in medicinal and environmental areas. Especially, the development of colorimetric and fluorimetric chemosensors for anions has been widely investigated due to the relevance of several anions in biological processes (Veale and Gunnlaugsson 2010; Moragues et al. 2011; Santos-Figueroa et al. 2013).

Bearing the above facts in mind, there is a practical interest on the design of unnatural amino acids and our research group has been engaged on the synthesis of heterocyclic amino acids and their application as fluorescent markers and fluorimetric probes for metal ions (Batista et al. 2012; Costa et al. 2007, 2008a, 2008b; Esteves et al. 2009, 2010, 2011, 2016; Oliveira et al. 2011). We now report the synthesis and characterization of novel non-canonical amino acids bearing thiophene and bithiophene moieties, by an Ugi multicomponent reaction between a heterocyclic aldehyde, an amine, an acid and an isocyanide. This reaction is a straightforward method for the synthesis of α- and α,αsubstituted glycines that allows the introduction of a variety of groups and functionalities at the side chain (Dömling 2006). The thiophene coordinating/reporting unit was linked with different substituents to tune the photophysical properties of the new probes and optimize the recognition of target analytes through greater fluorescence sensitivity. The recognition ability of these non-canonical amino acids toward different ions of analytical and biological relevance was evaluated by UV–vis absorption and fluorescence spectroscopy. Spectrophotometric and spectrofluorimetric titrations were made to assess their potential to act as fluorescent probes suitable for incorporation into peptidic frameworks with chemosensory ability.



Experimental Section
General

All melting points were measured on a Stuart SMP3 melting point apparatus. TLC analyses were carried out on 0.25 mm thick precoated silica plates (Merck Fertigplatten Kieselgel 60F254) and spots were visualised under UV light. Chromatography on silica gel was carried out on Merck Kieselgel (230-240 mesh). IR spectra were determined on a BOMEM MB 104 spectrophotometer. NMR spectra were obtained on a Varian Unity Plus Spectrometer at an operating frequency of 300 MHz for 1H and 75.4 MHz for 13C or a Bruker Avance III 400 at an operating frequency of 400 MHz for 1H and 100.6 MHz for 13C using the solvent peak as internal reference at 25 ºC. All chemical shifts are given in ppm using tetramethylsilane as reference and J values are given in Hz. Assignments were supported by spin decoupling-double resonance and bidimensional heteronuclear correlation techniques. Low and high resolution mass spectrometry analyses were performed at the “C.A.C.T.I. - Unidad de Espectrometria de Masas”, at University of Vigo, Spain. Fluorescence spectra were collected using a FluoroMax-4 spectrofluorometer. UV-visible absorption spectra (200 – 600 nm) were obtained using a Shimadzu UV/2501PC spectrophotometer. All reagents were commercially available and used as received.


General procedure for the synthesis of thienyl amino acid derivatives 2a-j by an Ugi multicomponent reaction

The appropriate aldehyde 1a-j (1 equiv) and 4-methoxybenzylamine (1 equiv) were dissolved in dry methanol (5 mL/mmol of aldehyde) and stirred for 1 hour at 50 ºC, to form the corresponding imine. Acetic acid (1 equiv) was added to the previous mixture and stirred for 15 minutes at room temperature. Then, cyclohexyl isocyanide was added (1 equiv) and the mixture was left stirring at room temperature for 24 h. The solvent was evaporated and the crude was chromatographed through a silica gel column with dichloromethane-hexane (2:1) (to elute any unreacted isocyanide), followed by dichloromethane (to elute any unreacted aldehyde) and dichloromethane-methanol (90:1) (to elute the desired product). The fractions containing the product were evaporated to dryness in a rotary evaporator.



The synthetic details and characterization for compounds 2a and 2h, considered as models for the thienyl and bithienyl set of amino acids, respectively, are given below. The synthetic details and characterization for compounds 2b-g,i-j are given in the Supplementary Material.
N-Cyclohexyl-2-(N-(4’-methoxybenzyl)acetamido)-2-(thiophen-2-yl)acetamide 2a. Starting from 2-formylthiophene 1a (0.156 g, 1.39 × 10-3 mol), 4-methoxybenzylamine (0.18 mL, 1.39 × 10-3 mol), acetic acid (0.08 mL, 1.39 × 10-3 mol) and cyclohexyl isocyanide (0.17 mL, 1.39 × 10-3 mol), compound 2a was obtained as an orange oil (0.184 g, 4.6 × 10-4 mol, 33%). 1H NMR (400 MHz, CDCl3): = 1.05-1.13 (m, 3H, 3 × H-cHex), 1.24-1.33 (m, 2H, 2 × H-cHex), 1.50-1.62 (m, 3H, 3 × H-cHex), 1.77-1.86 (m, 2H, 3 × H-cHex), 2.01 (s, 3H, CH3CO), 3.70 (s, 4H, OCH3 and H1-cHex), 4.58 (s, 2H, NCH2), 6.07 (s, 1H, -H), 6.23 (d, J 8.0 Hz, 1H, NH), 6.72 (d, J 8.4 Hz, 2H, H3’ and H5’), 6.87 (dd, J 3.4 and 5.0 Hz, 1H, H4), 6.95 (d, J 8.4 Hz, 2H, H2’ and H6’), 7.05 (d, J 3.4 Hz, 1H, H3), 7.21 (d, J 5.0 Hz, 1H, H5). 13C NMR (100.6 MHz, CDCl3): = 22.15 (CH3CO), 24.43 (C-cHex), 24.48 (C-cHex), 25.24 (C-cHex), 32.38 (2 × C-cHex), 48.32 (C1-cHex), 50.07 (NCH2), 54.96 (OCH3), 57.52 (-CH), 113.63 (C3’ and C5’), 126.26 (C4), 127.14 (C5), 127.31 (C2’ and C6’), 129.02 (C3), 129.30 (C1’), 136.91 (C2), 158.42 (C4’), 167.62 (C=O amide), 171.95 (CH3CO); IR (liquid film, cm–1): ν = 3306, 3070, 2933, 2855, 1633, 1586, 1542, 1513, 1464, 1451, 1409, 1364, 1350, 1289, 1247, 1207, 1176, 1111, 1093, 1036, 979, 912, 892, 840, 811, 735, 699, 665, 543. UV/Vis (ethanol, nm): max (log ) = 275 (4.19). MS: m/z (ESI, %) 401 (M+, 100). HMRS: m/z (ESI) calc. for C22H29N2O3S 401.18934, found 401.18900.

2-([2,2’-Bithiophen]-5-yl)-N-cyclohexyl-2-(N-(4’’-methoxybenzyl)acetamido)acetamide 2h. Starting from 5-formyl-2,2’-bithiophene 1h (0.210 g, 1.08 × 10-3 mol), 4-methoxybenzylamine (0.14 mL, 1.08 × 10-3 mol), acetic acid (0.06 mL, 1.08 × 10-3 mol) and cyclohexyl isocyanide (0.13 mL, 1.08 × 10-3 mol), compound 2h was obtained as an orange oil (0.215 g, 4.45 × 10-4 mol, 41%). 1H NMR (400 MHz, CDCl3): = 1.06-1.17 (m, 3H, 3 × H-cHex), 1.27-1.36 (m, 2H, 2 × H-cHex), 1.54-1.66 (m, 3H, 3 × H-cHex), 1.80-1.92 (m, 2H, 2 × H-cHex), 2.08 (s, 3H, CH3CO), 3.73 (s, 4H, OCH3 and H1-cHex), 4.56-4.67 (m, 2H, NCH2), 5.91 (s, 1H, -H), 6.23 (d, J 7.2 Hz, 1H, NH), 6.77 (d, J 8.8 Hz, 2H, H3’’ and H5’’), 6.92 (d, J 3.6 Hz, 1H, H3), 6.95 (d, J 3.6 Hz, 1H, H4), 6.96-6.98 (m, 1H, H4’), 7.06 (d, J 8.8 Hz, 2H, H2’’ and H6’’), 7.12 (d, J 2.8 Hz, 1H, H5’), 7.18 (d, J 5.2 Hz, 1H, H3’). 13C NMR (100.6 MHz, CDCl3): = 22.21 (CH3CO), 24.53 (C-cHex), 24.60 (C-cHex), 25.32 (C-cHex), 32.48 (C-cHex), 32.53 (C-cHex), 48.51 (C1-cHex), 50.49 (NCH2), 55.08 (OCH3), 58.25 (-CH), 113.81 (C3’’ and C5’’), 122.60 (C4), 123.75 (C5’), 124.50 (C3’), 127.59 (C2’’ and C6’’), 127.69 (C4’), 128.83 (C1’’), 129.92 (C3), 135.73 (C2), 135.73 (C2’), 139.28 (C5), 158.65 (C4’’), 167.41 (C=O amide), 172.01 (CH3CO). IR (liquid film, cm-1):  = 3297, 3069, 2932, 2854, 1654, 1586, 1542, 1513, 1451, 1409, 1351, 1303, 1248, 1209, 1177, 1111, 1093, 1035, 979, 956, 918, 892, 840, 811, 735, 699, 665, 543. UV/Vis (ethanol, nm): max (log ) = 310 (4.23). MS: m/z (ESI, %) 483 (M+, 100). HMRS: m/z (ESI) calc. for C26H31N2O3S2 483.17706, found 483.17668.
Spectrophotometric titrations and chemosensing studies for thienyl amino acids 2a-j

Solutions of compounds 2a-j (1.0 × 10-5 to 1.0 × 10-6 M) and of the ions under study (1.0 × 10-1 to 1.0 × 10-3 M) were prepared in UV-grade acetonitrile (in the form of hydrated tetrafluorborate salts for Cu+, Ag+, Pd2+ and Co2+, hydrated perchlorate salts for K+, Cd2+, Ca2+, Fe3+, Fe2+, Cr3+, Cu2+, Ni2+, Cs+, Na+, Hg2+, Pb2+, Zn2+ and hydrated tetrabutylammonium salts for CH3COO-, F-, I-, ClO4-, CN-, NO3-, BzO-, Cl-, Br- and OH-). Titration of the compounds with the several ions was performed by the sequential addition of ion to the compound solution, in a 10 mm path length quartz cuvette and emission spectra were measured by excitation at the wavelength of maximum absorption for each compound, indicated in Table 2, with a 2 nm slit. The linearity of the absorption versus concentration was checked within the used concentration. The binding stoichiometry of the thienyl amino acids with the ions was determined by Job’s plots. The association constants were obtained with HypSpec program.


Results and Discussion
Synthesis

New non-canonical amino acids bearing thiophene and bithiopene units with substituents of different electronic character as side chains were obtained by an Ugi reaction. This reaction is an isocyanide-based four-component reaction proposed in 1959 by Ivar Ugi as an alternative to the classical methods for amino acid synthesis, by reacting an acid, an amine, an isocyanide and a carbonyl compound. Following the original application of the Ugi reaction, it can be used for the synthesis of α-amino acids (if an aldehyde is used as the carbonyl component) and α,α-dialkylamino acids (if a ketone is used as the carbonyl component) (Dömling 2006; Costa et al. 2003). In this work, acetic acid, 4-methoxybenzylamine and cyclohexyl isocyanide were used, along with a series of thiophene and bithiophene aldehydes 1a-j bearing different substituents. The protected amino acids 2a-j were prepared in fair to moderate yields (15-60%) (Scheme 1, Table 1). These new compounds were fully characterised by the usual spectroscopic techniques.



Scheme 1. Synthesis of (bi)thienyl amino acid derivatives 2a-j.
Table 1. Yields, UV-visible absorption and fluorescence data for amino acids 2a-j in absolute ethanol.

Cpd.

Yield (%)



UV/Vis absorption

Fluorescence

λabs

log ε

λem

Stokes’

shift (cm-1)



Stokes’

shift (nm)



ΦF

2a

33

276

3.60

303

3627

27

0.005

2b

60

286

4.01

353

6636

67

0.089

2c

25

298

3.55

362

5933

64

0.293

2d

26

299

3.54

362

5821

63

0.284

2e

21

299

4.25

358

5512

59

0.221

2f

27

317

4.22

386

5639

69

0.039

2g

34

349

4.21

511

9084

162

0.002

2h

41

310

3.49

375

5591

65

0.420

2i

41

327

4.31

402

5705

75

0.037

2j

15

336

4.21

415

5666

79

0.124

The acid, amine and isocyanide components were chosen considering previous work that ensures straightforward removal of the groups at the N- and C-terminal by acidolysis to afford the free non canonical amino acids for subsequent use in peptide synthesis (Costa et al. 2003; Castro et al. 2016).
Photophysical study of (bi)thienyl amino acid derivatives 2a-j

The electron donor or acceptor character of the substituents envisaged the modulation of the photophysical and the recognition properties of the resulting compounds. Therefore, the absorption and emission spectra of (bi)thienyl amino acids derivatives 2a-j were measured in absolute ethanol (10-6-10-5 m solution) (Table 1). The nature of the substituent had a clear influence on the absorption and emission bands of compounds 2a-j (Figures 1 and 2).


Figure 1. Normalised UV-visible absorption spectra of (bi)thienyl amino acids 2a-j in ACN at T = 298 K.
By comparison to compound 2a, as the parent compound, the presence of a phenyl ring bearing and electron donor group (as in 2c-e) lead to an expected bathochromic shift (ca. 20 nm) of the maximum wavelength of absorption (λabs). The bathochromic shift was more pronounced when electron acceptor groups were present: a 41 nm shift with the cyano group (for 2f) and a 73 nm shift with the nitro group (for 2g). The same trend was seen in the fluorescence spectra, with larger bathochromic shifts (especially for the nitro derivative with a 208 nm shift).

Comparison of the electronic absorption and emission spectra of compound 2b (R = phenyl) with compound 2h (R = thiophene), compound 2c (R = methoxyphenyl), with 2i (R = methoxythiophene), as well as comparison of derivative 2f (R = cyanophenyl), with 2j (R = cyanothiophene) revealed that the substitution of an aryl group by a thiophene caused a red shift of the maximum absorption (between 19-29 nm) and emission (between 22-40 nm) wavelengths. This observation clearly indicates that the incorporation of thiophene units enhances the charge-transfer properties of the overall system and the optical data obtained can be largely explained by the bathochromic effect of sulphur and also the increase of the -overlap between the thiophene units.


Figure 2. Normalised fluorescence spectra of (bi)thienyl amino acids 2a-j in ACN at T = 298 K (λexc = λabs for each compound).
The synthesized compounds showed moderate to large Stokes’ shifts (the lowest being 3009 cm-1 for 2a and the highest 9084 cm-1 for 2g). A large Stokes’ shift is an interesting characteristic for a fluorescent probe, when using fluorescence based techniques, that allows an improved separation of the light inherent to the matrix and the light dispersed by the sample (Holler et al. 2002).

The relative fluorescence quantum yields of the ethanolic solutions of compounds 2a-j were determined using a 10-6 m solution of 9,10-diphenylanthracene in ethanol as standard (ΦF = 0.95) (Morris et al. 1976). It was found that the thienyl amino acids 2c-e (bearing donor groups) and bithienyl amino acids 2h,j were the most emissive (0.124 ≤ F ≤ 0.420). The most fluorescent derivative was compound 2h (bithiophene) and the presence of the nitro group resulted in an expected fluorescence quenching, with compound 2g being practically non-emissive in ethanol.

For the subsequent chemosensing study towards different ions, the absorption and emission spectra of (bi)thienyl amino acids 2a-j were also measured in acetonitrile (10-6-10-5 m solution) and its mixture with water (9:1) (Table 2). It was found that the presence of water did not influence the fluorescence quantum yields but the character of the solvent did, as the quantum yield was lower in ethanol (a protic solvent) when compared to acetonitrile (an aprotic solvent).


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