In Silico Design of Short Peptides as Sensing Elements for Phenolic

Jan 11, 2016 - illycaffè S.p.A., Via Flavia 110, 34147 Trieste, Italy. ACS Sens. , 2016, 1 (3), pp 279–286. DOI: 10.1021/acssensors.5b00225. Public...
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In silico design of short peptides as sensing elements for phenolic compounds Michele Del Carlo, Denise Capoferri, Ivan Gladich, Filomena Guida, Cristina Forzato, Luciano Navarini, Dario Compagnone, Alessandro Laio, and Federico Berti ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.5b00225 • Publication Date (Web): 11 Jan 2016 Downloaded from http://pubs.acs.org on January 19, 2016

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In silico design of short peptides as sensing elements for phenolic compounds Michele Del Carlo^,¤, Denise Capoferri^,¤, Ivan Gladich§,¤, Filomena Guida#,¤, Cristina Forzato#, Luciano Navarini+, Dario Compagnone^, Alessandro Laio§ and Federico Berti#,* #

Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Trieste, via Giorgieri 1, 34127 Trieste, § ^ Italy. SISSA – ISAS, via Bonomea 265, 34136 Trieste, Italy. Faculty of Biosciences and Technology for Food, Agricul+ ture and Environment, University of Teramo, Via Lerici 1, 64023, Teramo, Italy. illycaffè S.p.A. Via Flavia 110, 34147, Trieste, Italy. KEYWORDS: peptides, molecular dynamics, biosensors, chlorogenic acid. ABSTRACT: We exploit a recently developed computational approach [I. Gladich et al, J. Chem. Phys. B, DOI: 10.1021/acs.jpcb.5b06227 (2015)] to design cyclic peptides capable to recognize chlorogenic acid and related phenolic compound. A peptide designed by this procedure was synthesized and characterized by circular dichroism and fluorescence spectroscopy, cyclic voltammetry and differential pulse voltammetry. We found that the peptide is selective for chlorogenic acid against other ortho-diphenols such as caffeic acid, and mono-phenols as ferulic and coumaric acid. Indeed, when chlorogenic or caffeic acid are bound to the cyclic peptide, the ortho-diphenol moiety capable to undergo oxidation is not available to electrode surface due to diffusion limitation and steric hindrance. This phenomenon did not occur for cumaric and ferulic acid possibly because of limited complex formation with the cyclic peptide. In an electrochemical sensing system the peptide can therefore discriminate ortho-diphenols in a mixture of phenols.

Among the different biological recognition elements that can constitute the receptor part of a biosensor, peptides offer an invaluable opportunity for developing a cheap device1-4. Indeed, the number of different properties (affinity towards a target, selectivity, etc.) that can be obtained by combining the natural aminoacids is potentially enormous, making conceivable designing peptides capable of recognizing any molecule. Moreover short peptides can be easily synthesized in large quantities, making the scale-up of the production of the device economically affordable. The opportunity of exploiting short peptides as sensing units has indeed been recognized since a long time. Recently, we have introduced an algorithm capable of designing peptides of 10 residues that bind Efavirenz, a drug used in HIV treatment5. In this work we exploit a version of the algorithm based on the same ideas but with several important innovations6. First, the conformational search was carried out by finite temperature molecular dynamics, exploiting a state-of-the-art force field which properly describes the flexibility of both the peptide and ligand. Second, with the scope of reducing the entropic contribution, we have now designed cyclic peptides with disulfide bridge between the first and the last residue. Finally, we have carefully benchmarked the design by long finite temperature molecular dynamics with an explicit atomistic description of the solvent. The algorithm has been exploited to design a cyclic peptide for 5-O-caffeoylquinic acid (known as chlorogenic acid, CGA). Such phenolic food target has been identified because there is a continuous interest in the development

of analytical methods able to measure analytes in food samples based both on sensors technology7. In this respect biosensors7 appears to satisfy all industry requests such as high selectivity and specificity, relative low costs of construction and storage, potential for miniaturization, facility of automation8. Chlorogenic acids, which are esters of quinic acid with different cinnamic acids, are receiving a large interest among the scientific community, in fact it is estimated that people ingest between 100 and 1000 mg of CGAs daily, depending on the diet9, since they are largely present in the plant kingdom. Because of this large intake many studies evaluated the biological activity of these compounds and it was found that they may be responsible for the reduced risk of some chronic diseases, may prevent diabetes and cardiovascular diseases10-13. Different studies on the correlation between coffee intake and the biological effects of these compounds have been reported14, since coffee is a major source of chlorogenic acids and it is a very diffuse beverage all around the world. Phenolcarboxylic acids such as 3,4-dihydroxycinnamic acid (caffeic acid, CA), trans-4-hydroxycinnamic acid (pcoumaric acid, CuA) and 4-hydroxy-3-methoxycinnamic acid (ferulic acid, FA) (Figure 1), as well as 5-Ocaffeoylquinic acid (known as chlorogenic acid, CGA), are well known antioxidants that have been associated to different beneficial effects on human health, including prevention of degenerative pathologies15,16. Thus, the aim of this work is to demonstrate the potential of the novel developed algorithm by designing a peptide capable to

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bind to chlorogenic acid and by studying its selectivity towards the above mentioned targets. HO

COOH O

HO

O

O

HO

OH

OH

OH

OH

OH

CGA

CA

O

O HO

HO

OH OCH3

OH CuA

FA

Figure 1: structures of the phenolcarboxylic acids EXPERIMENTAL METHODS

Design of the peptide We have designed high affinity peptides toward CGA using a computational procedure which searches the optimal sequence carrying out single point random mutations on the amino acid sequence and molecular dynamics (MD) simulations at finite temperature to verify their viability. The algorithm is described in detail in ref 66. Contrary to other algorithms in which the generation of possible peptide-ligand configurations is based on a docking protocol17-19, here the thermal stability of the peptideligand complexes is automatically ensured during the design by the use of finite temperature molecular dynamics, an approach that allows describing properly the flexibility of both the peptide and the ligand. Moreover, the design of cyclic peptides with disulphide bridge between the terminal residues is able to reduce the entropic contribution to the binding, which is usually not taken into account in the scoring functions. Briefly, the procedure started with a deca-alanine sequence cyclized by the presence of a disulfide bridge between two N- and C-terminal cysteines. The chlorogenic acid molecule was inserted in the middle of the ring. Starting from this structure we attempted a series of single point mutations according to the following procedure: We randomly selected one amino acid from the peptide and replace it with a different one. In this way, the new peptide sequence at the step i+1, SEQi+1, differs from the sequence at the previous step, SEQi, for only one amino acid at a time. The terminal cysteines were never chosen for mutation, in order to preserve the cyclic geometry of the peptide. In order to avoid close contacts and to explore favourable peptide-ligand spatial arrangements, we performed 500000 steps of steepest descent energy minimization, followed by 1 ns MD simulations at 350 K, in order to enhance the conformational sampling. In all the minimizations and MD runs we have used the AMBER 99SD

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forcefield for the peptide and solvent molecules, while intramolecular and non-bonded interactions were calculated with the GAFF forcefield. Relevant peptide-ligand complexes were selected from the last 800 ps of the MD trajectory according to a cluster analysis20. Statistically relevant configurations were identified as the centres of clusters with at least 10 configurations. For these structures, the peptide-ligand binding affinity are estimated by the Vina21 scoring function, storing the peptide-ligand coordinates, CNFi+1, of the configuration with lowest score, Ei+1. The new peptide sequence SEQi+1 was accepted or rejected according to the Metropolis criterion, with a probability min(1,exp[(Ei+1-Ei)/Te]) controlling the acceptance rate by an efficacious temperature, Te. In the case of acceptance, a new mutation was attempted from the new state (CNFi+1,SEQi+1,Ei+1), otherwise from the previous one (CNFi, SEQi, Ei). This mutation cycle was iterated until the estimated binding affinity reached a plateau. In order to further enhance the exploration of the sequence space, five simultaneously and independent mutation cycles at five efficacious temperatures Te=0.2, 0.4, 0.6, 0.8, 1.0 kcal/mol were performed, trying to swap two replicas, r and r′, at the end of each mutation step. A swap between the states of replica r at Tr, (CNFr,SEQr,Er) and the state of replica r′ at Tr', (CNFr′,SEQr′,Er′) was accepted with probability min(1,exp[(Er-Er')(1/Tr-1/Tr')]). The computational setup of the MD simulations was the same as the one described in ref. 5. For reasons of computational efficiency we here performed the design in vacuum, and not in explicit solvent. To mimic a water-like solvation environment we applied a cut-off of 0.5 nm on the Coulombic interactions. This cut-off value is (roughly) the space required between two charge groups to host a water molecule. Following ref 5, the best structures and sequences obtained in the design were then selected to benchmark their thermal stability over 250 ns constant pressure (NPT) simulations at 300 K in explicit water. This ensures that the artifacts produced by the design in vacuum are recognized and excluded from experimental validation. Peptide synthesis The cyclic peptide was synthesized in the solid phase using Fluorenyl-methyloxycarbonyl (Fmoc) chemistry on a Biotage automated microwave peptide synthesizer. The synthesis scale was 0.1 mmol using the 2-chlorotrityl resin (substitution 0.22 mmol/g). The resin was manually loaded with a 4-fold molar excess of Fmoc-aa-OH with 4 eq. of Diisopropyl ethyl amine (DIPEA) in dichloromethane (DCM). For subsequent couplings, Fmoc- and side-chain protected aminoacids, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBop) activator and DIPEA were added in a 5-fold excess using N-Methyl-pyrrolidone (NMP) as solvent. The coupling temperature was set to 45°C. The Fmoc deprotection was obtained by using 20% piperidine in N,NDimethylformamide (DMF). The peptide was cleaved

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from the resin using a cocktail of trifluoroacetic acid (TFA), 3,6-dioxa-1,8-octane-dithiol (DODT), thioanisole, phenol, water, tri-isopropylsilane (TIPS) (85%, 6%, 3%, 2%, 2%, 2% v/v), and precipitated in tert-butylmethyl ether (-20°C). The quality of the crude, cysteine reduced, linear peptide was found to be sufficiently high to undergo the folding procedure directly, without previous purification. Folding was carried out in oxidizing conditions by dissolving the crude peptide in an aqueous buffer consisting of 0.1M ammonium acetate, 2 mM EDTA and 0.5 M guanidinium chloride, at a 7.5-8 final pH, under nitrogen, in order to obtain a final peptide concentration of about 40 µM. Cysteine (100 fold excess) and cystine (10 fold excess) were also added immediately prior to use, to catalyze disulfide exchange and facilitate the correct connectivity. The folding reaction was carried out at room temperature for 24h and was monitored by analytical RPHPLC (Kinetex C18, 3µm, 100 A, 50 x 4.6 mm column from Phenomenex, USA). The folding solution was then subjected directly to preparative RP-HPLC on a Phenomenex column (Jupiter™, C18,10 µm, 90 Å, 250x21,20 mm) using a 5-35% CH3CN in 50 min gradient with a 8 ml/min flow, and the peptide was then lyophilized. ESI-MS showed that the peptide was correctly folded and of high purity. Fluorescence spectroscopy Steady-state fluorescence spectra were acquired at 25 °C on a CARY Eclipse (Varian) spectrofluorimeter. The concentration of the peptide was maintained at 5 µM in 350 µL phosphate buffer, whereas the concentration of the tested ligands were gradually increased from 1nM to 300 µM by adding aliquots of 100 times concentrated mother solutions in dimethyl sulfoxide (DMSO). The amount of DMSO in the final solutions was always kept under 1%, and we have verified that such amount of solvent does not affect the emission of the peptide. After the addition of each ligand, the fluorescence intensity at the maximum emission wavelength and the drift of such maximums were measured after equilibrium had been reached (15 min). The measurements were carried out in a 5 mm optical path cell. Quenching data were corrected by inner filter effects due to the absorption of the added ligands22. The correction turned out significant only at ligand concentrations exceeding 30 µM. All the experiments were repeated three times, and the quenching data analyses were carried out on the average values. CD spectroscopy Circular Dichroism spectra were obtained on a Jasco J710 spectropolarimeter. Spectra were recorded from 250 to 195 nm in a 1 cm quartz cuvette at 25 °C. Data were collected with a data pitch of 0.05 nm, a scanning speed of 50 nm/min and a band width of 1 nm. Eight scans were averaged and smoothed to improve signal – to – noise ratio. The background spectrum was subtracted for each analysis. The spectra were recorded at a 10 µM solution of peptide in phosphate buffer at pH 7.3, and at increasing concentrations of CGA in the 10 – 50 µM range, by addition of aliquots from a solution of CGA dissolved in meth-

anol. The spectra were corrected by subtracting also the spectra of pure CGA at the same concentrations. Electrochemistry All the electrochemical measurements were carried out in phosphate buffer, pH 7.0, in 50 mM KCl as supporting electrolyte using carbon-based screen printed electrode as sensing probes. 100 µl of the working solution were added to the electrode surface to close the electrochemical cell circuit. The electrochemical behaviour of both the cyclic peptide and CGA, CA, CuA and FA was evaluated by cyclic voltammetry and differential pulse voltammetry. Cyclic Voltammetry was run from -400 mV to +900 mV for CGA, CA and FA and from -400 mV to +1200 mV for CuA vs a Ag/AgCl pseudo-reference electrode with a scan rate of 100 mV sec-1, using a 300 µM concentration of phenolic acid (PA) in all cases. Differential pulse voltammetry conditions were as follows: step potential 9 mV; modulation amplitude 50 mV; scan rate 50 mV/s. A calibration curve was built for all the tested analytes in the concentration interval 0.25-62.50 µM; measurements were carried out in triplicate. Binding of cyclic peptide and PAs was evaluated incubating the solution of the peptide and the ligand at room temperature for 30 minutes, and transferring 100 µL of the mixture on the sensor surface for the electrochemical measurement. 5 µM solutions of the tested PA (CGA, CA, CuA and FA) were mixed with 100 µM cyclic peptide solution. As a control 5 µM solution of the tested PA was measured on the same sensor surface before and after the measurement. The decrease (%) of the PA anodic peak was used as analytical signal. In a following experiment a wider interval of cyclic peptide (7.50-375 µM) concentrations were tested with respect to a fixed concentration of CGA (3.75 µM). Also in this case the analytical signal was the decrease (%) of the anodic peak of CGA measured by DPV. RESULTS AND DISCUSSION Design and identification of the peptide According to the procedure reported in the previous section, we selected for experimental test the peptide of sequence CWWEVITFFKEC. We have verified on previous tests that a 12 aminoacid sequence is optimal for the interaction with a small molecule of the size of CGA. The binding energy becomes in fact more favourable upon enhancing the peptide length up to 12 residues, and then it remains basically unchanged if the chain is further elongated. Figure 2a shows the outcome of the mutations runs carried out at different temperatures, and the plateau in the binding energy that is reached after about 100 mutation steps. The binding affinity of the peptide, estimated as the time average over the last 200 ns of a molecular dynamics in explicit solvent, is of approximately -9 kcal/mol (Figure 2b). Figure 2c shows the peptide backbone positions around CGA, further proving the stability of the peptide-ligand interaction. After about 100 ns a slight change in the backbone conformation occurs,

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leading to a more close interaction with CGA, and the ligand never leaves out the binding site up to the end of the simulation, without any further change in the overall conformation of the complex.

2a

2b

2c Figure 2: a) the Vina score as a function of the number of mutations of the cyclic peptide sequence during the design. Different colors correspond to different efficacious temperatures, and it can be seen that the score reaches a plateau value after about 100 mutations at any temperature. b) The vina score of the peptide-CGA complex during a 250 ns MD run carried in an explicit solvent box at 300 K. c) RMDS fluctuation of the backbone position with respect to the starting one along the MD run. Insert: snapshots of the peptide-CGA complex during the MD run at 0 ns, 25 ns, 50 ns, 75 ns and 100 ns (blue, purple, green, yellow and red, respectively).

Synthesis and spectroscopic characterization of the peptide The peptide was synthesized by solid-phase, Fmocbased chemistry on a 0.1 mmol scale, and its folding and disulfide formation were carried out in solution by incubation with cysteine for 24 hours. The purity of the peptide after HPLC purification was satisfactory, as assessed by mass spectrometry (see supplementary material). A conformational analysis was carried out first by CD spectroscopy in the far UV region (Figure 3). The peptide shows two negative Cotton effect bands, with minima at 200 nm ([Θ] = -3800 deg residue-1 cm2 dmol-1) and 228 nm ([Θ] = -1500 deg residue-1 cm2 dmol-1). Such minima indi-

cate the presence of β-turn regions (namely, the minimum at 228 nm is typical of turn I structure) as expected in a cyclic peptide, however the minimum at 200 nm or lower region is peculiar of a disordered conformation23. A deconvolution of the spectrum was carried out on the Dichroweb server 24,25. Both the CONTINLL26,27 and the VARSLC28 protocols gave a 52% overall β-turn structure and 48% coil. This is consistent with a Ramachandran analysis of the predicted model structure of the empty and relaxed cyclic peptide, carried out on the RAMPAGE server29, which places four out of the ten residues in the cyclic region of the peptide in the beta allowed region (supplementary figure S5). A similar behaviour has been observed for cyclic, cationic antimicrobial peptides, that

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show a significant fraction of their amino acid content in a random conformation30. Recognition of chlorogenic acid and phenolic compounds A first evidence of interaction between the peptide and CGA was obtained by CD spectroscopy (Figure 3). The dichroism spectrum of the peptide undergoes a relevant change upon increasing the concentration of CGA. The negative ellipticity at 228 nm increases its absolute value by over 100%, the minimum at 200 nm disappears and a novel, strong minimum appears at 205 nm. We have verified that CGA alone has a negligible CD spectrum at the concentrations used in this experiment (supplementary figure S4). A decrease of the random coil amount is consistent with both the disappearance of the minimum at 200 nm and the increased negative Cotton effect at 228 nm, as the typical spectrum of a random coil peptide shows a positive Cotton effect at such wavelength.

We have therefore performed a further, quantitative analysis of the binding of CGA and other phenolic compounds structurally related to CGA by fluorescence spectroscopy. The peptide contains two tryptophan residues. Such amino acid is known to be fluorescent, and if it is excited at around 280 nm, an emission maximum close to 340 nm is often observed; this maximum may vary from 310 nm to 350 nm, depending on the electronic environment of the indole system33. In all the measurements, the concentration of peptide was 1 µM and the ligand concentration was gradually increased during the titration using ligand standard solutions in DMSO. The emission (λexc 280 nm, λem range 300-400 nm) spectra of the peptide alone were recorded at the beginning of any experiments. The emission spectra were monitored after each addition of the ligands. An example of the resulting spectra is reported in figure 4a for CGA. Quenching of the peptide emission was observed (Figure 4b), and after inner filter correction, we analysed the quenching data using the Stern-Volmer equation (equation 1) that describes the quenching process: ࡲ૙

Eq. 1

ࡲ

ൌ ૚ ൅ ࡷࢗ ࣎૙ ሾࡽሿ ൌ ૚ ൅ ࡷࡿࢂ ሾࡽሿ

The variables F0 and F are the emission intensities before and after the addition of the quencher, respectively, Kq is the bimolecular quenching kinetic constant, i.e. a collisional frequency between freely diffusing molecules, τ0 is the lifetime of the fluorophore (for the tryptophan fluorescence decay τ0 is about 10-8 s)34,35, KSV is the SternVolmer quenching constant and [Q] is the quencher concentration in mol/L; The KSV for the two ligands were determined by linear regression of a plot of F0/F against [Q] (see supplementary material) KSV and Kq (calculated using the equivalence Kq = KSV/τ0) are reported in table 1. Table 1: affinity data from fluorescence spectroscopy. a: from Stern-Volmer analysis; b: from fitting of the fluorescence data to a tetraparametric logistic curve; c: from Hill analysis, n = number of binding sites. Ligand Figure 3: far-UV circular dichroism spectra of a 10 µM solution of the peptide alone and in the presence of increasing concentrations of chlorogenic acid. Dichroism is reported as the average molar ellipticity per aminoacid residue.

The new minimum at 205 nm is conversely found in type III beta turns, while also type I turns exhibit negative ellipticity in this region of the spectrum. All the effects suggest a transition of the random coil regions of the cyclic peptide to an ordered turn upon interaction with CGA. A transition from a disordered to an ordered conformation in cyclic peptides of similar size has been observed, with CD spectral changes very similar to ours, also upon addition of trifluoroethanol31. Divalent metal cations have also shown to induce conformational changes in cyclic peptides structures leading to CD spectra comparable to that of our peptide in the presence of its ligand32.

KSV

a

CGA CA CuA FA

b

Kq -1

Kd -1 -1

L mol

L mol s

47300±2100

4.73x10

4200±380 2200±190 800±200

µmol L

Kd / n -1

12

9.21±0.53

11

103±12

11

199±22

4.20x10 2.20x10

c

µmol L

-1

9.24 / 0.89

10

8.00x10

The bimolecular quenching kinetic constants (Kq) for CGA, CA and CuA are 1-2 orders of magnitude higher than the maximum value for diffusion-limited collisional quenching (2.0 x 1010 L mol-1 s-1)36,37, and the static quenching originating from the association of the fluorophore and quenchers in a bimolecular complex can be estimated as the main contribution to the fluorescence quenching mechanism. Ksv can be thus regarded as the association constant for the formation of the peptide – ligand com-

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plexes. Conversely, the quenching constant for FA is very close to the limit, and in this case the contribution of dynamic quenching cannot be neglected. In any case, the peptide shows selectivity towards its target, and the association constants for the other tested ligands are at least ten time less favorable. We have also evaluated the dissociation constant by fitting the sigmoidal-trending data reported in fig. 4b for CGA, CA and CuA to a tetraparametric logistic function: this analysis yields a 9.21 µM dissociation constant for the CGA complex, being the constants for CA and CuA 103 and 199 µM respectively. A Hill analysis of the data for CGA leads to a similar value and to a number of binding sites per molecule of peptide reasonably close to 1. The affinity value for CGA is approximately consistent with the value predicted by the design procedure. Indeed, the average Vina score observed in a long molecular dynamics run (see Figure 2b) is -8.6 kcal/mol, corresponding to a binding affinity of approximately 1 µM.

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the peptide fluorescence emission upon increasing concentrations of the phenolic compounds.

Electrochemical evaluations Polyphenols, being antioxidants, are electroactive, particularly on carbon electrodes. Depending on the chemical structure of the compounds redox potentials and reversibility is highly variable. Considering the amount and variety of polyphenols present in food, selective detection of a single molecule or a classes of molecules for practical use is very challenging. On the other hand, CGA is electrochemically very interesting belonging to the class of odiphenols that have been reported to have the highest antioxidant activity among polyphenols38. In order to evaluate the possibility of using the cyclic peptide as the recognition element in an electrochemical assay for chlorogenic acid and its derivatives, its electrochemical behaviour has been evaluated both by cyclic voltammetry and differential pulse voltammetry on carbon based screen printed electrodes. In cyclic voltammetry, as observed in figure 5a, there is no evident anodic peak in the region of interest for electrochemical detection of CGA or CA.

4a

Figure 5: Cyclic voltammograms of 250 µM CP (A), 300 µM CGA (B), 300 µM CA (C), 300 µM CuA (D); 300 µM FA (E) in phosphate buffer pH 7.0, KCl 50 mM.

4b Figure 4: a) emission fluorescence spectra of the cyclic peptide (λexc 280nm) upon additions of increasing concentrations of CGA in the 1nM – 300 mM range; b) quenching of

Further investigation on the electrochemical oxidation of the cyclic peptide was made by differential pulse voltammetry. In these experiments there is no evident oxidation peak in the region of interest for the electrochemical detection of PAs. The PAs have also been characterised by cyclic and differential pulse voltammetry. CGA showed a reversible electrochemical behaviour with an anodic peak at 188±4mV and a cathodic peak at 179±16mV. The peak area of the anodic and cathodic peaks were 1.6x10-5 A and 0.6x10-5 A respectively. CA showed a quasi-reversible electrochemical behaviour with an anodic peak at 307±6mV and a cathodic peak at -40±8mV. The peak area of the anodic and cathodic peaks were 2.6x10-5 and 0.5x10-5 respectively. CuA and FA exhibited a non-reversible electrochemistry with anodic peaks respectively at 737±36mV and 601±21mV.

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Calibration curves were obtained for all the PAs in the concentration range 0.25-62.50 µM.

7.5-375 µM. CGA concentration was 3.75 µM, incubation time 30 minutes in phosphate buffer pH 7.0, KCl 50 mM.

CGA calibration had a linear range between 0.98-62.50 µM, the linear equation was y=187.96x-211.86, R² = 0.999. CA calibration curve was characterized by two well distinct region showing different calibration slope: the former between 0.25 and 1.96 µM, y=7.5392x-2.2168, R² = 0.965 and the latter between 3.91 and 62.5 µM, y=28.313x 128.27, R² = 0.9942.

These data suggest that the CGA or CA binding with the cyclic peptide subtract PAs molecules to the diffusion layer thus reducing the electrochemical signal. No signal at higher potentials indicating complex oxidation was observed. We then hypothesise that when CGA or CA are bound to the cyclic peptide the ortho-diphenols potentially available for the oxidation, are not available to electrode surface due to diffusion limitation and steric hindrance. This phenomenon did not occur for CuA and FA possibly because of limited complex formation with the cyclic peptide. From our results it can be concluded that the ortho-diphenolic moiety appears mandatory in order to obtain the binding of the PAs with the cyclic peptide. An inspection of the predicted structure of the CGApeptide complex shows indeed that the hydroxyl group at position 3 of the aromatic ring (that is present in CGA and CA only) is actually involved in binding of CGA via formation of a network of hydrogen bonds with glutamate 11 (Figure 7).

A similar shape was shown also by the calibration curves of CuA and FA. Lower concentration region of calibration curve was linear in the 0.49-1.96 µM range (y=1.0773x+2.025, R²=0.8331) and in the 3.91-62.50 µM range (y=15.309x-27.617, R² = 0.9983) for CuA. 0.25-3.91 µM (y=2.4438x+5.0363, R² = 0,9922) and 7.82-62.50 µM (y=21.428x-124.58, R² = 0.99) were obtained for PA. Differential pulse voltammetry was used to follow the ability of the cyclic peptide to bind the different PAs under investigation. A preliminary experiment was carried out using a fixed concentration of the PAs (5µM) incubated for 30 minutes with 100 µM of cyclic peptide. CGA and CA showed a significant decrease of the anodic current down to 49.8% and 40.1% respectively. This current reduction can be attributed to the binding ability of the peptide that reduce the electrochemical availability of the phenolic groups of the two PA. As long as CuA and FA (not carrying the ortho-diphenolic moiety), no variation of the anodic peak was observed upon incubation with an excess of the cyclic peptide (see supplementary material). Further investigations on CGA were carried out varying the concentration ratio CGA:CP (cyclic peptide) in the 1:21:100 range using a CGA concentration of 3.75 µM and measuring the reduction of the signal vs the anodic peak signal (%) upon incubation with the cyclic peptide. The semilogaritmic plot (figure 6) show a decrease of 50% of the signal at a cyclic peptide concentration of 65µM that is corresponding to a CGA:CP ratio of 1:17 and confirmed the behaviour observed using 5.00 µM CGA.

Figure 7: detail of the predicted structure of the CGA – peptide complex. The interaction between the hydroxyl group at position 3 of the ligand aromatic ring and glutamate 11 is likely to favor the binding energy and to enhance the steric protection of the diphenolic system in CGA and CA.

Figure 6: semilogaritmic plot of CGA –CP mix. The I/I0 % signal is the % decrease of the anodic peak upon incubation of CGA with increasing concentration of CP in the interval

The selective binding of the cyclic peptide to CGA and CA observed using electrochemical detection suggest that an electrochemical assay might be designed using the proposed strategy so that the ortho-diphenolic-carboxylic acids may be selectively determined out from a pool of phenolic compounds. Because CGA and CA are among the most important antioxidant found in many food the method could be used to assess the amount of most active antioxidants available as part of the total phenolic fraction. Very recently, a sensing system selective for tea polyphenols has been described by Wang and colleagues39. This very sensitive methodology exploits the peroxidase activity of protein-conjugate gold nanocluster to detect tea polyphenols, mostly including pyrogallol- related compounds as gallic acid and EGCC, with cathechin. The

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system shows a selectivity somewhat complementary to that of our peptide, as it is not sensitive to CGA. In the present, non-optimized format, a test based on our peptide would be less sensitive, by about one order of magnitude, but very selective for ortho-diphenols. However polyphenols in coffee are present in very high concentrations, and the main point is their profile. Our strategy, extended to a sensor array containing few peptides with different binding affinities vs. polyphenols can represent the basis for the development of an e-tongue for the quali/quantitative evaluation of these bioactive compounds in food.

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(8) Luong, J. H.; Groom, C. A.; Male, K. B. The potential role of biosensors in the food and drink industries. Biosens. Bioelectron. 1991, 6, 547-54. (9) Olthof, M. R.; Hollman, P. C. H.; Katan, M. Chlorogenic acid and caffeic acid are absorbed in humans. J. Nutrit. 2001, 131, 6671. (10) Ludwig, I. A.; Clifford, M. N.; Lean, M. E. J.; Ashihara, H.; Crozier, A. Coffee: biochemistry and potential impact on health. Food Funct. 2014, 5, 695-1717. (11) Flanagan, J.; Bily, A.; Rolland, Y.; Roller, M. Lipolytic activity of Svetol, a decaffeinated green coffee bean extract. Phytother. Res. 2014, 28, 946-948.

Mass spectrum of the cyclic peptide, Stern Volmer plots, differential pulse voltammetry of PAs. This material is available free of charge via the Internet at http://pubs.acs.org.”

(12) Cano-Marquina, A.; Tarin, JJ.; Cano, A. The impact of coffee on health. Maturitas 2013, 75, 7-21.

AUTHOR INFORMATION

(13) Frost-Meyer, N. J.; Logomarsino, J. V. Impact of coffee components on inflammatory markers: A review. J. Funct. Foods 2012, 4, 819-830.

Corresponding Author

(14) Stalmach, A.; Williamson, G.; Crozier, A. Impact of dose on the bioavailability of coffee chlorogenic acids in humans. Food Funct. 2014, 5, 1727-1737.

* Federico Berti [email protected]

Author Contributions ¤ These authors equally contributed to the work reported in this paper

Notes The authors declare no conflict of interest.

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