European Journal of Medicinal Chemistry 70 (2013) 225e232

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

New derivative of carnosine for nanoparticle assemblies Francesco Bellia a, *, Valentina Oliveri a, b,1, Enrico Rizzarelli b,1, Graziella Vecchio b,1 a b

Institute of Biostructure and Bioimaging, CNR, viale A. Doria 6, 95125 Catania, Italy Department of Chemical Sciences, University of Catania, viale A. Doria 6, 95125 Catania, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 July 2013 Received in revised form 21 September 2013 Accepted 2 October 2013 Available online 10 October 2013

Carnosine (b-alanyl-L-histidine) is an endogenous dipeptide, extensively studied owing to its multifunctional activity exhibited in tissues of several animal species. This natural compound may act as a physiological buffer, ion-chelating agent (especially for copper(II) and zinc(II)), antioxidant and antiglycating agent. The main limit for the therapeutical uses of carnosine is the rapid hydrolysis mostly in human plasma by carnosinase. The chemical derivatization of carnosine is a promising strategy to improve the bioavailability of the dipeptide and facilitating the site-specific transport to different tissues. On this basis, a new carnosine derivative with biotin was synthesized and structurally characterized by NMR and MS measurements, with aim of exploiting the avidinebiotin technology that offers a universal system for selective delivery of any biotinylated agent. The stability of the new carnosine derivative towards the hydrolytic action of serum carnosinase as well as the copper(II) binding ability of the carnosineebiotin conjugate were also assessed. The binding affinity of the new molecular entity to avidin and streptavidin, investigated by a spectrophotometric assay, was exploited to functionalize avidine and streptavidinegold nanoparticles with the carnosineebiotin conjugate. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Carnosine Nanoparticles Copper Biotin Carnosinase

1. Introduction Among all the dipeptides nowadays studied in scientific research, carnosine (b-alanyl-L-histidine, Car) occupies an important position because its increasingly ascertained biological properties are an intriguing stimulus for trying to understand its still unknown physiological functions. Carnosine is the first peptide ever isolated from natural material [1] and reaches very high concentrations (up to 20 mM) in muscle and nervous tissues of several animal species [2]. The massive presence of carnosine could be ascribed to numerous functions, such as physiological buffer, wound healing promoter, ionchelating agent, especially for CuII and ZnII, antioxidant and antiglycating agent [3,4], all being important in physiological and pathological conditions [5]. It has been shown that carnosine retards cancer growth in animal models [6] and protect against alcohol-induced oxidative stress [7] as well as ethanol-induced chronic liver damage [8]. The neuroprotective action of carnosine in oxidative driven diseases has been recently reviewed [9]. All these properties make this natural

* Corresponding author. Tel.: þ39 095 738 5047; fax: þ39 095 337678. E-mail address: [email protected] (F. Bellia). 1 Tel.: þ39 095 738 5064; fax: þ39 095 337678. 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.10.002

dipeptide an interesting compound for several application in biomedical field [10]. The peptidic nature of carnosine compromises its therapeutical uses mainly for the breakdown by specific dipeptidases. The carnosine concentration in the animal species is regulated by the activity of the metalloprotease carnosinases. In mammals, two dipeptidases have been characterized: the serum-circulating form (‘serum carnosinase’, CN1), secreted by brain cells into the cerebrospinal fluid [11,12] and the cytosolic isoform (‘tissue carnosinase’, CN2), a non-specific dipeptidase distributed in several human tissues and in the rodent brain [13,14]. Recently, several carnosine derivatives with saccharides [15], such as b-cyclodextrin [16,17] and trehalose [18], have been synthesized. All these compounds are able to scavenge hydroxyl radicals and their copper(II) complexes exhibit SOD (superoxide dismutase) activity [19,20]. Furthermore, they are resistant to the hydrolysis of the carnosinase [21,18] and have an antioxidant efficacy at concentrations 10e20 times lower than that reported for other synthetic derivatives [22]. An important physiological role of the conjugating moiety in the carnosine derivatization is enhancing the bioavailability of the dipeptide by facilitating the site-specific transport to different tissues. One of the largely used molecules for selective delivery is vitamin H, better known as biotin (Bio). The extensive biomedical investigation of biotin both in vitro and in vivo is mainly due to its specific

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interaction with avidin (Av) and streptavidin (SAv). These tetrameric proteins have a high-affinity biotin-binding site (Kd z 1015 M1) for each subunit [23] and they selectively binds to specific cells, tissues or organs. Thus avidinebiotin technology offers a universal system for selective delivery of any biotinylated agent to the target using the same affinity carrier. For this reason, a large panel of biomolecules have been biotinylated, usually without significant loss of their biological properties, and they have been investigated for biomedical applications in vitro (cross-linking, staining or targeting biomolecules, etc) [24] and in vivo (drug targeting [25e27], radioimmunoimaging [28], stimulation of immune response [29], etc). Another approach employed in modern scientific era for the selective delivery consists in the use of nanoparticles (NPs). They are attracting considerable and growing interest because of their unique physical and chemical properties. The integration of nanotechnology with biology and medicine has led to further developments of a new emerging research area, nanobiotechnology, which offers opportunities for discovering new materials, processes, and phenomena. Recently, carnosine-coated iron oxide [30] and gold NPs have been obtained [31,32]. These NPs have been investigated as activators of carbonic anhydrase and nickel sensors. As for carnosineNPs as activators of carbonic anhydrase, carnosine has been functionalized with L-lipoic acid in order to synthesize gold NPs through goldesulfur bond formation. The role of carnosine could be related to the presence of histidine, being that the activity of carnosine nanoparticles is very similar to that of histidine nanoparticles. On this basis, a new carnosine derivative with biotin (BioCar) was synthesized (Fig. 1) and structurally characterized by NMR and MS measurements. The stability of BioCar towards the hydrolytic action of serum carnosinase as well as the copper(II) binding ability of the new carnosine derivative were also assessed. The binding affinity of BioCar to avidin and streptavidin, investigated by a spectrophotometric assay, was exploited to functionalize gold nanoparticles with BioCar via Av- and SAv-coated NPs. 2. Materials and methods 2.1. Chemicals In order to synthesize the new carnosine derivative, the following reagents have been used: carnosine (SigmaeAldrich), Biotin succinimidyl ester (Bio-NHS, Carbosynth), N,N-Dimethylformamide (DMF, SigmaeAldrich) and Triethylamine (TEA, Fluka). Phosphate Buffered Saline (PBS) was prepared with the following composition: phosphate buffer (12 mM, pH 7.4), NaCl (137 mM) and KCl (2.7 mM). Thin layer chromatography (TLC) was performed on silica gel (60F-254, 0.20 mm, MachereyeNagel). The compounds not detectable under UV light were revealed with a 1% solution of ninhydrine in acetone and the Pauly’s reagent (10% of Fast Red B salt (Fluka) in deionized water) for detecting primary amino and

O

2

N HN g h,h'

NH

NH f

S

e

O d

β

b c

a

O

NH

A,B 4 X

α

OH

NH O

Fig. 1. Structure of the new carnosineebiotin conjugate. Symbols, letters and numbers are used for the NMR peak assignment.

imidazole groups, respectively. Acetonitrile and heptafluorobutyric acid (HFBA) were from Sigma (HPLC grade). Hydrogen tetrachloroaurate (HAuCl4$3H2O), and sodium citrate were purchased from Alfa Aesar. Streptavidin from Streptomyces avidinii and Avidin from egg white were from SigmaeAldrich. Millipore-Q Ultrapure water was used to prepare nanoparticle solutions. Dedicated glassware was cleaned before each reaction with aqua regia and then rinsed with ultra-pure deionized water. All solutions used for nanoparticle preparation were filtered through a 0.45 mm membrane filter (Cellulose Nitrate Membrane Filter). Copper(II) nitrate was prepared from copper(II) basic carbonate by adding a slight excess of HNO3; the concentration of stock solutions was determined by ethylenediaminetetraacetic acid titrations using murexide as the indicator [33]. The HNO3 excess in metal stock solutions was determined by Gran’s method [34,35]. High-purity water (Millipore, Milli-Q Element A 10 ultrapure water) and grade A glassware were employed. 2.2. Synthesis of biotinecarnosine (BioCar) Car (35 mg, 0.15 mmol) was dissolved in water (300 ml) and a solution of Bio-NHS (51 mg, 0.15 mmol) in DMF (1.5 ml) was added drop by drop. 20 ml of TEA (0.15 mmol) were finally added to the mixture and the reaction was carried out at room temperature. After 20 h the solvent was evaporated under vacuum at 40  C. The solid obtained was purified with a Sephadex-DEAE-A25 anion exchange column (HCOe 3 form) using water as the eluent and a linear gradient of NH4HCO3 (0e0.3 M). The fractions collected were analyzed by thin-layer-chromatography (TLC PrOH-H2O-AcOEtNH3 5:3:1:2), and those containing the product were concentrated under vacuum at 40  C. Yield 68%. ESIeMS [BioCar] m/z ¼ 453.1911 (M þ 1). Calculated m/z: 453.1915. 1 H NMR (D2O, 500 MHz) d (ppm): 8.51 (s, 1H, H-2 Im), 7.18 (s, 1H, H-5 Im), 4.52 (dd, 1H, J ¼ 7.5, 5.4 Hz, g Bio), 4.45 (m, 1H, X His), 4.33 (dd, 1H, f Bio, J ¼ 7.9, 4.4 Hz), 3.34 (m, 2H, CH2 Ala), 3.23 (m, 1H, e Bio), 3.16 (dd, 1H, A His, J ¼ 15.3, 5.0 Hz), 3.01 (dd, 1H, B His J ¼ 15.4, 8.1 Hz), 2. 90 (ddd, 1H, h’ Bio, J ¼ 13.0, 4.9, 1.2 Hz), 2.69 (d, h Bio, J ¼ 13.0 Hz), 2.39 (t, 1H, CH2 Ala, J ¼ 6.6 Hz), 2.10 (m, 1H, a Bio), 1.62 (m, 2H, d Bio), 1.50 (m, 2H, b Bio), 1.29 (2H, c Bio). 13 C NMR (D2O, 125 MHz) d (ppm): 62.3 (f Bio), 60.3 (g Bio), 55.7 (e Bio), 53.9 (X His), 39.7 (a Ala), 35.7 (b Ala), 35.4 (a Bio), 35.1 (CH2 His), 27.9 (c Bio), 27.5 (h Bio), 27.4 (d Bio), 25.4 (b Bio). 2.3. Spectroscopic and spectrometric measurements 1 H NMR spectra were recorded at 25  C in D2O with a Varian Unity Plus 500 spectrometer at 499.883 MHz. The 1H NMR spectra were recorded by using the standard pulse programs from the Varian library. In all cases, the length of 90 pulse was ca. 7 ms. The two-dimensional (2D) experiments were acquired using 1 K data points, 256 increments, and a relaxation delay of 1.2 s. 4,4dimethyl-4-silapentane-1-sulfonic acid (DSS) was used as the external standard. Circular dichroism spectra of the ligand and its copper(II) complexes were recorded on a Jasco 810 spectropolarimeter at a scan rate of 50 nm min1 and a resolution of 0.1 nm. The path lengths were 1 or 0.1 cm, in the 190e800 nm range. The spectra were recorded as an average of 10 or 20 scans. Calibration of the instrument was performed with a 0.06% solution of ammonium camphorsulfonate in water (D3 ¼ 2.40 M1 cm1 at 290.5 nm). The 200e800 nm spectral range was covered by using quartz cells of various path lengths. The results are reported as D3 (molar dichroic coefficient) in M1 cm1.

F. Bellia et al. / European Journal of Medicinal Chemistry 70 (2013) 225e232

UVevis spectra were carried out with Varioskan plate reader (Thermo Scientific), Nanodrop 1000 and Agilent 8453 spectrophotometers. For the determination of size distribution and zeta potential of gold nanoparticles, dynamic light-scattering (DLS) measurements were carried out at 25  C using a Zetasizer Nano ZS (Malvern Instruments, UK) at a detection angle of 173 with a 4 mW HeeNe laser (633 nm) as the incident beam. Each DLS measurement was run at least in triplicate using automated, optimal measurement times and laser attenuation settings. The recorded correlation functions and measured particle mobilities were converted into size and zeta potentials, respectively, using Dispersion Technology Software (DTS). For the particle sizing in solution, the software gives multiple aspects and interpretations of the data collected for the sample such as intensity, volume, and number distribution graphs as well as statistical analysis for each. In particular, the transformation from the intensity data into volume is performed using Mie theory and this conversion requires the particle refractive index (n) and absorption (k) values. The optical properties used for the conversion from intensity to volume in this study were 0.2 (n) and 3.32 (k) respectively. The mean particle diameter is calculated by the software from the measured particle distributions, and the polydispersity index (PdI) given is a measure of the size ranges present in the solution. All the ESI-MS measurements were carried out by using a Finnigan LCQ DECA XP PLUS ion trap spectrometer operating in the positive ion mode and equipped with an orthogonal ESI source (Thermo Electron Corporation, USA). Sample water solutions were injected into the ion source without the addition of any other solvent at a flow rate of 5 ml/min, using nitrogen as the drying gas. All the other experimental parameters were the same as described elsewhere [36]. Xcalibur software was used for the elaboration of mass spectra. Each species is indicated in the following with the m/ z value of the first peak of its isotopic cluster. For a more accurate structural assignment, the relative intensity of the peaks in each cluster was compared with that of the peaks in the corresponding simulated spectra. 2.4. Carnosinase assay Enzymatic hydrolysis extent of carnosine and its biotin derivative was assayed by means of a method previously reported [37]. Human plasma (final dilution 1:10) was incubated with Car or BioCar (1 mM) in MOPS buffer (50 mM, pH 7.4) at 37  C for 2 h. The production of L-histidine was evaluated by analytical RPeHPLC analyses, performed by using a Waters 1525 instrument equipped with Waters 2996 photodiode array detector with detection at 222 nm. The chromatographic analysis was performed with solvents A (0.4% HFBA in water) and B (0.4% HFBA in acetonitrile) on a Synergi polar-RP column (Phenomenex) 150  2 mm (100  A pore size, 4 mm particle size) at flow rate of 0.2 mL min1. 2.5. Binding assay of avidin and streptavidin The binding ability of the new carnosine derivative to avidin (Av) or streptavidin (SAv) was assessed by the 2-(4hydroxyphenylazo)benzoic acid (HABA) assay [38]. To a mixture of HABA (100 mM) and Av or SAv (10 mM) in PBS buffer pH 7.4 (100 mL) were added 0.5e1 mL aliquots of biotin (0.33 mM) or BioCar (0.5 mM) in 8 min intervals. The formation of the biotinand BioCar-protein adducts was indicated by a decrease in the absorbance at 495 nm due to the displacement of HABA from the avidin. A preliminary experiment were performed by adding 0.5e 1 ml aliquots of HABA (1 mM) to the avidin solution (10 mM). The formation trend of the HABA complex with Av or SAv, indicated by

227

an absorbance increase at 495 nm, allows to calculate the number of binding sites of Av and SAv for HABA and, then, for biotin and BioCar. 2.6. Synthesis of gold nanoparticles Gold NPs were synthesized by citrate reduction of HAuCl4$3H2O according to methods elsewhere described [39]. In brief, 2 mL of trisodium citrate (38.8 mM) were quickly added with vigorous stirring to 20 mL of a boiling solution of HAuCl4$3H2O (1 mM). The color of the solution changed from pale yellow to deep red. A complete reduction of trisodium citrate was obtained after 6e8 min under boiling. The solution was cooled to the room temperature and filtered through a 0.45 mm membrane filter to remove large aggregates. The NP solution was stored at 4  C. UVeVis (l/nm): 520; Volume distribution: 13.7  0.1 nm. 2.7. Synthesis of streptavidin/avidin-coated nanoparticles (Au@SAv) 500 mL of a solution obtained by diluting (1:1) the NPs with H2O was added to 10 mL of a 1 mg mL1 streptavidin solution after adding NaOH (16 mL, 0.1 M). The solution was incubated in ice for 1 h and centrifuged (12,500 g, 30 min). After separation of the supernatant, the protein-coated NPs were resuspended in 90 mL of milli-Q water. As for avidin-coated nanoparticles, the pH of the solution was adjusted at 11 in order to avoid aggregation. Au@SAv. UVeVis (l/nm): 524; Volume distribution: 18.8  0.8 nm. Au@Av. UVeVis (l/nm): 527; Volume distribution: 29.0  0.9 nm. 2.8. Functionalization of protein-coated NPs with biotynilated carnosine Biotinylated carnosine (100 mM in H2O) was incubated for 30 min with protein-coated NPs. After the centrifugation and the removal of the supernatant solution, NPs were suspended in water (pH 7.0 and 11.0 for streptavidin and avidin, respectively). Au@SAv@BioCar. UVeVis (l/nm): 525; Volume distribution: 20.0  0.6 nm. Au@Av@BioCar. UVeVis (l/nm): 528; Volume distribution: 29.5  0.5 nm. 3. Results and discussion 3.1. Synthetic aspect and stability of Biocar The carnosineebiotin conjugate has been synthesized through a convenient one-pot reaction. The carboxyl-activated biotin as succinimidyl ester allows the condensation with the amino group of carnosine and is stable to the hydrolysis in aqueous medium where the dipeptide is soluble. A single purification step is also sufficient to obtain the product in a good yield and purity. BioCar was structurally characterized by ESI-MS and NMR. Looking at the ESI-MS spectra (Fig. S1), the proton adduct M-Hþ (m/ z 453.3) of BioCar is the main detected species, followed by sodium adduct M-Naþ (m/z 475.3). Signals at m/z 905.0 and m/z 927.0 have been attributed to the dimer species M2Hþ and M2Naþ, respectively. Multidimensional fragmentation (MSn), have been also carried out in order to confirm the structure of the new carnosine derivative (Fig. S2). The identity of the new carnosine derivative is also confirmed by NMR spectra (see Supplementary material, Figs. S3eS6). The spectra were assigned by 2D NMR spectroscopy (COSY, TOCSY and

F. Bellia et al. / European Journal of Medicinal Chemistry 70 (2013) 225e232

pH 5.0

53 7. 55 2 5. 2

2

2 5.

51

7. 49

47

5.

2

45

3.

2

228

pH 7.0

Fig. 2. Carnosinase activity in human plasma towards carnosine (dark gray) and its biotin-conjugate (light gray).

HSQC). The 1H NMR spectrum of BioCar displays signals due to the biotin and carnosine moieties. Imidazole protons resonate at 8.51 and 7.18 ppm; the eCH proton of the ABX system of histidine resonates at 4.43 ppm. The methylenic groups of histidine and balanine resonate between 2.3 and 3.4 ppm, whereas the protons of the valerate chain of biotin at higher field (1.2e1.7 ppm). The stability of BioCar to the hydrolysis of the carnosine moiety catalyzed by carnosinase was assessed in human plasma, in which CN1 is the unique peptidase that processes the natural dipeptide. As expected, carnosine is rapidly degraded within 100 min under the experimental conditions employed for this assay (Fig. 2). This result is clearly shown by the simultaneous increase of histidine and decrease of carnosine. Conversely, the BioCar content in human plasma is substantially unchanged within 100 min. Such behavior also exclude any effect of biotinidases on the BioCar stability in human plasma. 3.2. Copper(II) complex with BioCar The interaction between copper(II) ion and BioCar was studied by ESIeMS and CD measurements. The complex species of the CuII-BioCar system detected by ESIe MS are listed in Table 1, whereas representative ESIeMS spectra are shown in Fig. 3. Spectroscopic parameters (CD and UVeVis) are listed in Table 2. ESIeMS, CD and UVeVis measurements were carried out at different pH values, from 5.0 to 9.0. The MS spectra reported in Fig. 3 clearly show the decrease of the proton adduct (m/z 453.3) as well as the increment of the sodium adducts (m/z 475.3 and 497.2) as the pH increases. The formation percentage of almost all the copper(II) species with BioCar also increases from pH 5.0 to 9.0. Fig. 4 shows the Zoom Scan spectra of the main copper(II) species of BioCar detected between pH 5.0 and 9.0. The typical isotopic pattern of natural copper confirms the attribution of these signals to species of the copper(II)-BioCar system. The m/z values of the isotopic cluster showed in the lower

Table 1 ESIeMS characterization of all the copper(II) complexes with the new biotinylated derivative of carnosine, BioCar. [Cu2þ] ¼ [ligand] ¼ 1$104 M. Assignment

Theoretical Observed Relative intensity (%) (m/z) (m/z) pH 5.0 pH 6.0 pH 7.0 pH 8.0 pH 9.0

[CuIIL]þHþ [CuIIL]þNaþ [CuIIL]þ [CuIL]Hþ [CuIL]Naþ [CuIL]NaþH2O

257.6 268.6 514.1 515.1 537.1 555.1

257.6 268.5 514.2 515.2 537.2 555.2

15 e 55 100 e e

9 e 48 100 5 e

e 3 51 100 26 23

e 6 83 94 100 59

e e 21 21 29 100

Intensity

3x10 2x10

pH 9.0

1x10 0

400

500

600

700

m/z Fig. 3. Representative ESIeMS spectra of the copper(II)-BioCar system at different pH values (5.0, 7.0 and 9.0).

graph of Fig. 4 differ by 0.5, thus indicating that they have to be attributed to a double-charged species, [CuIIL]þHþ. The protonated form of [CuIIL]þ is detected only at acid pH values, whereas the sodium adduct forms at pH 7.0 and 8.0. However, within the investigated pH interval (5.0e9.0), the single charged form of [CuIIL]þ is the major CuII complex species. The MS spectra also show the formation of an analogous species in which copper is in the þ1 oxidation state. The reduction of copper(II) during ESIeMS experiments has previously been reported [40,37] and could be partially attributed to charge transfer, either because of ligand to metal electron transfer reactions or through a mechanism that happens in the electrospray source, when a high electric field is applied between the capillary and the counter electrode. However, the copper reduction only occurs during the ESIeMS analysis as no reduction of copper(II) complexes was observed in solution. The neutral species [CuIL] is detected as the sodium (m/z 537.1 and 555.1) and proton (m/z 515.1) adducts. The isotopic pattern of the latter partially overlaps with that the of [CuIIL]þ. As observed for the analogous CuII species, the sodiated and protonated forms of [CuIL] increase and decrease, respectively, as the pH increases. CD and UV spectra of the copper(II)-BioCar system are quite different from those of copper(II) complexes with carnosine and other derivatives previously studied. The main reason is the absence of the amino group of the b-alanine residue in BioCar. The binding groups for the copper(II) coordination remain the imidazole and the carboxylate. Moreover, precipitation in solution is observed at basic pH. This event is probably due to the fact the amide deprotonation leads to the formation of a neutral copper(II) species. At pH 5.0, the imidazole nitrogen is the anchoring site for the metal coordination. This statement is confirmed by the CD bands at 302 nm and 235 nm, attributed to the ligand-to-metal charge transfer transitions from p1 and p2 of imidazole nitrogen, respectively. The UVeVis band (l ¼ 807 nm, 3 ¼ 15 M1 cm1) is consistent with the coordination of a nitrogen atom to the metal center [18]. Table 2 Spectroscopic data for the copper(II) complexes with BioCar. pH

UveVis l/nm (3 /M1 cm1)

CD vis l/nm (D3/M-1cm1)

5.0 6.0 7.0

807 (15) 780 (20) 723 (38)

235 (0.91); 302 (0.005) 235 (0.91); 308 (0.022); 718 (0.021) 235 (0.91); 305 (0.026); 740 (0.028)

7.

1

53

5. 51

4

3x10

4

53

0

0

4

54

1x10

0.

53

0

8.

0

7.

6. 51

3

8.0x10

51

2x10

9.

4.

4

0

1

1.6x10 51

Intensity

229

1

F. Bellia et al. / European Journal of Medicinal Chemistry 70 (2013) 225e232

0.0

0 512

514

516

518

520

4

538

540

542

8. 25 4

25

8.

9

8.

0

2.0x10

25

Intensity

4

25

7.

5

4.0x10

536

0.0 256

257

258

259

260

m/z Fig. 4. Zoom Scan MS spectra of the main copper(II) species of BioCar detected between pH 5.0 and 9.0.

The blue-shift of this band at pH 6.0 and 7.0 could be ascribed to the coordination of the carboxylate group. The involvement of a deprotonated amide to the coordination of copper(II) is excluded by the absence of the CD band at ca. 320 nm, relative to the NeCuII charge transfer transition. The CD bands at pH 6.0 and 7.0 differ to those at pH 5.0 only in terms of intensity, meaning that other nitrogen atoms than that of imidazole ring are not involved in the coordination sphere of copper ion.

or BioCar) concentration. IC50 is the concentration of Bio or BioCar required to displace 50% of HABA from its complex with Av or SAv. The Hill slope constant value (nH) is related to the mechanism by which the ligand interacts with the protein. IC50 and nH values are listed in Table 3. The IC50 value (12.3 mM) of Bio for the interaction with Av is very similar to that reported in literature (5.8 mM) [41], thus confirming

1.5

3.3. Binding of BioCar with Av or SAv

FðtÞ ¼ min þ

max  min 1 þ 10ðlogIC50 xÞnH

where min and max where forced to be 0 and 100%, respectively. The variable x is expressed as decimal logarithm of the ligand (Bio

1.2 Abs

0.9 0.6 0.3 0 320

420

370

470

520

570

Wavelength (nm) 15

I/I 0

It was then tested the ability of BioCar to interact with both avidin and streptavidin and compared to that of the natural cofactor, biotin. The specific interactions between Av and Bio can be a useful tool in designing targeting systems. The very high affinity of Av and SAv for Bio (Kd z 1015 M1) is the strongest noncovalent interaction known between a protein and a ligand. This allows Bio-containing molecules in a complex mixture to be bound to Av or its conjugates. Therefore the affinity of the BioCar conjugate towards Av and SAv has been evaluated by using the HABA spectrophotometric assay [38]. Firstly, the titrations of Av and SAv with HABA were carried out in order to calculate the number of HABA units that bind to the protein. The addition of HABA to avidin produces a yellowe orange complex (lmax ¼ 495 nm) (Fig. 5, upper graph). The discontinued point in Fig. 5 (lower graph) clearly indicates that four equivalents of HABA bind to Av at the Bio binding site. When Bio or BioCar were added to the solutions containing the HABA complex with Av or SAv, HABA is displaced from the Bio binding sites and the decrease of absorbance is measured (Fig. 6). In order to evaluate the binding affinity of BioCar to Av and SAv, the experimental data shown in Fig. 6 were fitted by using the following equation:

10 5 0 0

1

2

3

4

5

6

7

[HABA]/[Avidin] Fig. 5. Absorption spectra of the HABA:avidin complex (upper graph) from 0:1 (lower and thinnest line) to 7:1 (upper and thickest line) ligand-to-protein molar ratio. Titration curve of avidin with HABA at 495 nm (lower graph).

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F. Bellia et al. / European Journal of Medicinal Chemistry 70 (2013) 225e232

70

to Av or SAv, its affinity for a second Bio increases. Finally, the calculated nH values are not significantly different. This clearly means that the mechanisms by which the biotin moiety binds to Av and SAv are the same and they are not affected by the conjugated dipeptide.

50

3.4. BioCar nanoparticles

30

Biotinylated carnosine enables us to explore a new method to obtain carnosine-coated NPs exploiting the high affinity of biotin for avidin and streptavidin proteins (Fig. 7). This supramolecular approach was performed with the following steps: 1) the physisorption of streptavidin or avidin on gold NPs via hydrophobic and electrostatic interactions [39], 2) the non-covalent interaction of biotinylated carnosine with SAv or Av. The NPs were synthesized using the citrate method with a precise ratio between citrate and HAuCl4. This method delivers controllable sizes ranging from a few up to tens of nanometers, so NP optimization of sizes and properties easily matches with the interest of study. Recent results indicate that streptavidin conjugated to gold nanoparticles in the size range 5e30 nm retains its biotin binding activity and in particular layer thickness values suggest that streptavidin monolayers are formed on 20 and 30 nm spheres, whereas a partially loss of tertiary structure has been observed for NPs with diameter < 15 nm [42]. DLS measurements for NPs indicated that the prepared gold NPs presented the well monodisperse characteristic (PdI0.15) and the average diameter was 18.2 nm (Fig. 8). The stability of the synthesized NPs was analyzed by Zeta potential measurements (Table 4). However, these values indicated negative surface properties of the gold nanoparticles and good stability in suspension as reported for similar systems [43]. NPs were successfully coated by avidin and streptavidin as suggested by several techniques. In the UVevisible spectra, a strong absorption by nude gold NPs at around 520 nm was observed, moreover the absorption peak changed after the particles were coated with streptavidin (lmax ¼ 524 nm) and avidin (lmax ¼ 527 nm) (Fig. 9). It has been reported that the peak intensity and the position of the surface plasmon resonance absorption are dependent on NP size [44,45]. Furthermore, no

% HABA binding

90

10 -10 -6.2

-5.2

IC50

-4.2

% HABA binding

90 70 50 30 10 -10 -6.2

-5.2

IC50

-4.2

log[Ligand] Fig. 6. Competitive assay for the titrations of avidineHABA (upper graph) and streptavidineHABA (lower graph) complexes with biotin (A) and carnosineebiotin conjugate (C). IC50 is graphically shown in the case of biotin as a ligand.

the reliability of our experimental procedure. Free Car added with Bio did not modify the recognition process between Bio and Av (data not shown). However, the covalent BioCar conjugate has slight lower affinity (IC50 ¼ 15.5 mM) than natural Bio. Such result means that the carnosine moiety covalently bound to Bio does not drastically modify the binding properties of the cofactor to Av. This statement is also valid to describe the different affinity of both tested compounds for SAv (Fig. 6). The IC50 value fitted for the Bio binding curve to SAv (20.0 mM) is lower than that of BioCar (30.0 mM). However, the difference between Bio and BioCar in terms of affinity for the protein binding sites is smaller for Av (15.5e12.3 ¼ 3.2) than that for SAv (30.2e20.0 ¼ 10.2). Moreover, the IC50 values of Bio and BioCar for Av are significantly lower than those shown for SAv. The Hill slope constant value (nH) indicates if the reaction between the protein and the ligand takes place in a positively (>1), negatively (