Spectroscopic characterization of a coumarin-labelled therapeutic tetrapeptide

The present work describes the synthesis of the tetrapeptide H-Ala-Ala-Pro-Val-NH2 (AAPV), an important inhibitor of the enzyme human neutrophil elastase, labelled at the C-terminus with a 7-methoxycoumarinyl moiety. Photophysical studies were made with spectroscopic characterization by UV-vis and fluorescence spectroscopy in different organic solvents and mixtures with aqueous HEPES solution, in order to simulate physiological conditions.


Introduction
Coumarins (the trivial designation of 3-oxo-3H-benzopyrans) are an important family of compounds that have been reported as fluorescent labels and probes [1].Coumarins are well known fluorophores with an extended spectral range, high fluorescence quantum yields, good photostability and solubility in common solvents [1][2][3].As most amino acids are poor UV-absorbing, fluorescence labelling is often employed in peptides and, for example, a peptide or protein bound to a fluorescent moiety may be an important tool for conformational studies of protein-protein and ligand-receptor interactions.
Coumarins have also been applied in the synthesis of photolabile protecting groups to release relevant biomolecules by UV and visible light irradiation [4,5] and can thus be used in the preparation of photoactive prodrugs.
The tetrapeptide H-Ala-Ala-Pro-Val-NH 2 (AAPV) is an important inhibitor of the enzyme human neutrophil elastase, HNE), which is present in high levels in a variety of inflammatory disorders, such as psoriasis.The inhibition of HNE by this hydrophobic sequence occurs when the peptide fits the P-P 1 subsites of elastase and competitively inhibits HNE [6,7].Some studies have been conducted for the use of this peptide as a therapeutic agent for transdermal delivery [6].Therefore, in the present communication the tetrapeptide AAPV was synthesized by the stepwise coupling of the corresponding amino acids in solution and labelled at the C-terminus with a 7-methoxycoumarinyl moiety.This peptide conjugate was submitted to photophysical studies for its spectroscopic characterization by UV-vis and fluorescence spectroscopy in different organic solvents and mixtures with aqueous HEPES solution, in order to simulate physiological conditions.

Coupling to Boc-Pro-OH and deprotection of the dipeptide
N-tert-butyloxycarbonyl-L-proline (Boc-Pro-OH) (0.163 g, 7.57 x 10 -4 mol) was dissolved in acetonitrile (10 mL), 1-hydroxybenzotriazole (HOBt) (0.1023 g, 7.57 x 10 -4 mol, 1 eq) was added and the reaction mixture was stirred at room temperature for 30 minutes.Then N,N'-dicyclohexylcarbodiimide (DCC) (0.1562 g, 7.57 x 10 -4 mol, 1 eq) was added and the mixture was stirred at room temperature for 2 hours.Separately, the previously synthesized valine-coumarin conjugate (0.2312 g, 7.57 x 10 -4 mol) was dissolved in acetonitrile (5 mL) and then added to the reaction mixture containing Boc-Pro-OH and the mixture heated at reflux for 20 hours.The formed solid (the corresponding urea) was filtered and the solvent was evaporated in a rotary evaporator.The residue was dissolved in ethyl acetate (25 mL) and extracted with hydrochloric acid 1M (25 mL) and then with sodium hydrogencarbonate 10% (25 mL).Anhydrous magnesium sulphate was added to the organic layer, filtered and the solvent was evaporated under reduced pressure.The dipeptide-coumarin conjugate obtained was an orange oil (0.2417 g, 64%).

Coupling to Boc-Ala-OH and deprotection of the tripeptide
N-tert-butyloxycarbonyl-L-alanine (Boc-Ala-OH) (0.057 g, 3.01 x 10 -4 mol) was dissolved in acetonitrile (10 mL), HOBt (0.041 g, 3.01 x 10 -4 mol, 1 eq) was added and the reaction mixture was stirred at room temperature for 30 minutes.Then DCC (0.062 g, 3.01 x 10 -4 mol, 1 eq) was added and the mixture was stirred at room temperature for 2 hours.Separately, the previously synthesized dipeptidecoumarin conjugate (0.1207 g, 3.01 x 10 -4 mol) was dissolved in acetonitrile (5 mL) and added to the reaction mixture containing Boc-Ala-OH and heated at reflux for 20 hours.The formed solid was filtered and the solvent was evaporated in a rotary evaporator.The residue was dissolved in ethyl acetate (25 mL) and extracted with hydrochloric acid 1M (25 mL) and then with sodium hydrogencarbonate 10% (25 mL).Anhydrous magnesium sulphate was added to the organic layer, filtered and the solvent was evaporated under reduced pressure.The crude tripeptide-coumarin conjugate was obtained as an orange oil (0.069 g, 40%).

Coupling to Boc-Ala-OH and isolation of the tetrapeptide
Boc-Ala-OH (0.028 g, 1.46 x 10 -4 mol) was dissolved in acetonitrile (10 mL), HOBt (0.020 g, 1.46 x 10 -4 mol, 1 eq) was added and the reaction mixture was stirred at room temperature for 30 minutes.Then DCC (0.030 g, 1.46 x 10 -4 mol, 1 eq) was added and the mixture was stirred at room temperature for 2 hours.Separately, the previously synthesized tripeptide-coumarin conjugate (0.0756 g, 1.46 x 10 -4 mol) was dissolved in acetonitrile (5 mL) and then added the reaction mixture containing Boc-Ala-OH and heated at reflux for 20 hours.The formed solid was filtered and the solvent was evaporated in a rotary evaporator.The residue was dissolved in ethyl acetate (25 mL) and extracted with hydrochloric acid 1M (25 mL) and then with sodium hydrogencarbonate 10% (25 mL).Anhydrous magnesium sulphate was added to the organic layer, filtered and the solvent was evaporated under reduced pressure.The residue was purified by silica gel column chromatography using dichloromethane/methanol (50:1) as eluent, the fractions containing the product were combined and evaporated, and the desired tetrapeptide-coumarin conjugate was obtained as a yellow oil (0.0165 g, 18 %).
This compound bearing a reactive chloromethyl group was used in the derivatization of the carboxylic acid function C-terminal amino acid (valine) of the desired peptide in the presence of potassium fluoride.The protected ester conjugate was obtained in excellent yield (87%) and then the Nprotecting tert-butyloxycarbonyl (Boc) group was removed by acidolysis with trifluoracetic acid, resulting in the deprotected valine-coumarin conjugate (Scheme 2).
The coupling of the remaining amino acids was carried out with a standard DCC/HOBt coupling procedure, followed by the removal of the N-protecting group after each coupling step (Scheme 3).
The coumarin-labelled therapeutic tetrapeptide was obtained in the form of oil in 18% yield and charaterised by The absorption and fluorescence spectra of degassed 10 -5 M solutions were measured and the data collected (absorption and emission maxima, molar absorptivities and fluorescence quantum yields) is presented in the Table 1.The relative fluorescence quantum yields were calculated using 9,10diphenylanthracene as standard (Φ F = 0.95 in ethanol) [9].For the determination of the quantum yields, the absorbance of the solutions did not exceed 0.1 and excitation was made at the wavelength of maximum absorption.By comparison of the absorption and emission maxima in the different solvents, no significant differences were observed, with values in the range 319-325 nm and 393-400 nm, respectively.The Stokes' shift (λ) was between 69 and 80 nm, which is an advantageous property in fluorescence techniques, as it will minimize self-quenching phenomena.
Considering the relative fluorescence quantum yields, it was found that the tetrapetide-coumarin conjugate was more emissive in the aqueous solvent mixture by comparison of the Φ F in the organic solvent (MeOH or ACN) and in the corresponding organic solvent-aqueous HEPES buffer mixture.
Overall, the fluorescence quantum yield was higher in MeOH or MeOH/HEPES (80:20).In Figure 1, the fluorescence spectra of the tetrapetide-coumarin conjugate in the various solvents (ca.

Table 1 -
UV-visible absorption and emission data for the tetrapeptide-coumarin conjugate in different organic solvents and mixtures with aqueous solutions.