Volume 7 - 2019 | https://doi.org/10.3389/fchem.2019.00180 This article is part of the Research TopicFolded Synthetic Peptides for Biomedical Applications View all 12 articles The insertion of azobenzene moiety in complex molecular protein or peptide systems can lead to molecular switches to be used to determine kinetics of folding/unfolding properties of secondary structures absorption of light induces a reversible trans ↔ cis isomerization which in turns generates a strain or a structure relaxation in the chain that causes peptide folding/unfolding In particular azobenzene may permit reversible conformational control of hairpin formation In the present work a synthetic photochromic azobenzene amino acid derivative was incorporated as a turn element to modify the synthetic peptide [Pro7,Asn8,Thr10]CSF114 previously designed to fold as a type I β-turn structure in biomimetic HFA/water solution the P-N-H fragment at positions 7–9 was replaced by an azobenzene amino acid derivative (synthesized ad hoc) to investigate if the electronic properties of the novel peptidomimetic analog could induce variations in the isomerization process The absorption spectra of the azopeptidomimetic analog of the type I β-turn structure and of the azobenzene amino acid as control were measured as a function of the irradiation time exciting into the respective first ππ* and nπ* transition bands Isomerization of the azopeptidomimetic results strongly favored by exciting into the ππ* transition conformational changes induced by the cis↔ trans azopeptidomimetic switch were investigated by NMR in different solvents compounds containing a methyl spacer between the phenyl ring and the amino group as [3-(3-aminomethyl)-phenylazo]phenylacetic acid (AMPP) acid or (4-aminomethyl)phenylazobenzoic acid (AMPB) give more flexibility to the chemical structure of a Xaa–AMPB–Yaa fragment than the rigid chromophore β-Hairpins are very interesting structures as they are involved in many biological processes i.e., often constitute binding epitopes and are implied in protein–protein or protein–DNA interactions (Hillier et al., 1999; Gajiwala et al., 2000; Wong et al., 2000; Schumacher et al., 2001; Zavala-Ruiz et al., 2004) the development of highly stable β-hairpins based on introduction of molecules as azobenzene allowed to control the hairpin structure and initiate a folding or unfolding transition with high isomerization yield a modified sequence of [Pro7,Asn8,Thr10]CSF114 was selected as an optimized type I β-turn structure Aim of the present work is the design and synthesis of a photocontrolled probe based on AMPB azobenzene as a turn element in the central part of the amino acid sequence to investigate if the electronic properties of the new molecule could induce variations in the isomerization process of the azobenzene unit and to study the effect of the photoswitch on its conformation DIC (N,N′-Diisopropylcarbodiimide) and Oxyma were purchased from Iris Biotech GmbH (Marktredwitz The following amino acid side-chain-protecting groups were used: OtBu (Asp Peptide-synthesis grade N,N-dimethylformamide (DMF) was purchased from Scharlau (Barcelona Spain); acetonitrile (ACN) from Carlo Erba (Milan piperidine were purchased from Sigma-Aldrich (Milan or micro-cleavages performed with a microwave apparatus CEM Discover™ single-mode MW reactor (CEM Corporation Final cleavage was performed using a mixture of TFA/TIS/H2O (95:2.5:2.5 v:v:v) for 3 h at room temperature The crude azopeptide was pre-purified by Reverse Phase Liquid Chromatography (RP-HPLC) using a Li-Chroprep C-18 column on an Armen Instrument (Armen Instrument France) working at 20 ml/min with H2O (MilliQ) and CH3CN as solvent systems The second step of purification was performed by semipreparative RP-HPLC on a Waters instrument (Separation Module 2,695 detector diode array 2,996) using a Phenomenex (Torrance at 4 mL/min with solvent systems A (0.1% TFA in H2O) and B (0.1% TFA in CH3CN) The azopeptide 1 was characterized by RP-HPLC-ESI-MS, obtaining a final purity ≥ 98% (Figure S1) HPLC: tr = 3.17 min (cis isomer) and 3.78 min (trans isomer) gradient 35–55% of B in 5 min; Mr = calcd for C116 H170 N29 O25 S1: 2402,88 ESI-MS: m/z: 1202,47 [M+2H]2+; 802,02 [M+3H]3+ RP-HPLC system is an Alliance Chromatography (Waters USA) with a Bioshell A160 C18 (Sigma Aldrich Milano Italy; 1.7 μm 2.1 × 50 mm) column at 35°C at 0.6 mL/min coupled to a single quadrupole ESI-MS Micromass ZQ (Waters The solvent systems used were A (0.1% TFA in H2O) and B (0.1% TFA in CH3CN) Peptides were lyophilized using an Edward Modulyo lyophilizer (Richmond scientific Ldt. The experimental set up used for the irradiation procedure consists of a Xe “Ozone free” Orion lamp emitting 450 W in the spectral range 200–2,000 nm The light is focalized through a lens (f = 200 mm) onto the entrance slit of a monochromator is shaped by means of a pin-hole and collimated with a 50 mm lens on the sample cuvette (quartz A magnetic stirrer placed inside the cuvette ensures that the irradiating beam always interacts with fresh solution Incident beam cross section has been estimated 0.8 × 0.8 cm2 Incident power has been measured with a Coherent Field Max II power meter The power we used for the irradiation was around 500 μW for all the three excitation wavelengths used 313 Absorption spectra have been obtained with a Varian Cary 5 spectrophotometer Azobenzene was purchased by Sigma-Aldrich (purity 98%). [(4-aminomethylphenyl)diazenyl] phenylacetic acid was synthesized as described by Juodaityte and Sewald (2004) and azopeptide 1 was synthesized as described Solution concentrations were all around 10−4 M Unknown molar extinctions were determined for the new synthetic azopeptide 1 in the trans form (amino derivative εt,(326) = 9,000 cm−1M−1; aminopeptide εt,(330) = 5,090 cm−1M−1) The extinctions of the corresponding cis form was calculated considering still valid the ratio εcis/εtrans in azobenzene 3 NMR chemical shifts were calibrated with respect to the residual protiated solvent signal on 1D 1H or 2D 1H-13C HSQC experiments synthesized and studied the reversible cis ↔ trans photoisomerization of the [Pro7,Asn8,Thr10]CSF114 analog peptide 1 where from the original sequence the P-N-H tripeptide was replaced by the photoswitch (4-aminomethyl)phenylazobenzoic acid (AMPB) The photoisomerization of the synthetic azopeptide 1 was explored and its reversibility was compared with more simple systems the azobenzene amino acid 2 and the azobenzene 3 have been reported as molecules able to isomerize reversibly when exposed to light of appropriate wavelength The 21-mer peptide [Pro7,Asn8,Thr10]CSF114, derived from the family of structure-based designed β-turn peptides termed CSF114(Glc), is characterized by a type I β-turn motif (Carotenuto et al., 2006, 2008) In this sequence the β-turn structure was shown around the proline and asparagine residues in positions 7-8 The role of conformation in the recognition and binding of this synthetic antigenic probe to autoantibodies in the context of an immunoenzymatic assay (ELISA) was previously determined to be fundamental Because of the importance of the conformation and of the correct exposure of epitopes involved in autoantibody recognition, the light-induced conformational change of the synthetic peptide [Pro7,Asn8,Thr10]CSF114, after the introduction of the azobenzene moiety into the sequence is the object of the present study (Figure 1) Starting from the [Pro7,Asn8,Thr10]CSF114 sequence the P-N-H segment was targeted for replacement by an AMPB azobenzene amino acid (A) azopeptide 1 (X = T-P-R-V-E-R-;Y = T-V-F-L-A-P-Y-G-W-M-V-K); trans-(4-aminomethyl)phenylazobenzoic (AMPB) acid (X = CH2NH2,Y = CH2COOH) 2; trans-azobenzene (X,Y = H) 3 The photoswitch (4-aminomethyl)phenylazobenzoic acid (AMPB) was obtained by the condensation of a 4-nitrophenylacetic acid with 4-aminobenzylamine as described previously (Ulysse and Chmielewski, 1994; Juodaityte and Sewald, 2004; Aemissegger et al., 2005) The amino function of 4-aminobenzylamine was protected as Fmoc to obtain 4-[2-[4-[[[(9H-fluorenyl-9-methoxy)carbonyl]amino]methyl]phenyl]diazenyl]benzenacetic acid to be used in Fmoc solid-phase peptide synthesis Its incorporation into the peptide sequence proceeded into a straightforward manner applying the standard Fmoc-solid phase methodology The synthesis of the azobenzene-containing peptide 1 was carried out on a 0.1 mmol scale following the standard Fmoc/tBu solid phase peptide synthesis (SPPS) starting from Fmoc-Lys(Boc)-Wang resin After coupling the amino acids of the sequence protected for SPPS the peptide was cleaved from the resin and purified and characterized by RP-HPLC coupled to Mass Spectrometry The ability of AMPB to induce variations in the isomerization process was elucidated by UV-Vis and NMR In order to propose the synthetic azobenzene as a molecular switch, a deep characterization of its response to light excitation is mandatory to fully understand the effect of substituents on isomerization (Figure 2) Left side: UV-Vis absorption spectra of the trans-azobenzene 3 (solid line) and the equilibrium mixture trans/cis (dash-dotted line) Right side: UV-Vis absorption spectra of the trans isomers of azobenzene 3 (solid line) aminoazobenzene 2 (dash-dotted line) and azopeptide 1 (short dash line) The trans-azobenzene 3 spectrum showed in Figure 2 (left) is characterized by a strong absorption centered around 317 nm and a medium one at 230 nm both assigned as ππ* electronic transitions a weak band is observed due to the nπ* state Appearance of the cis form is revealed by the intensity decrease of the main band and a simultaneous growing of a medium intensity absorption at 238 nm The absorption spectra of the trans isomers of amino-azobenzene 2 and azopeptide 1 are shown in Figure 2 (right) While the correspondence of the electronic transitions is maintained a small red shift of the bands is observed in the amino-derivative probably due to a moderate conjugation and/or electron-donor effect induced by the substituents on the aromatic rings on the blue side of the aromatic ππ* transition whose maximum is red-shifted at 330 nm the characteristic absorption of tryptophan is also observed at 290 nm UV-Vis absorption spectra at several irradiation time for (A) azobenzene 3 exciting at the wavelength of the absorption maxima (313 nm for azobenzene and aminoazobenzene indicating the presence of the trans-cis equilibrium in solution Azobenzene absorption spectrum shows up to four isosbestic points On the opposite the azopeptide 1 shows only one isosbestic point at longer wavelength due to the overlap in the blue side of the spectrum where the trans→cis and cis→trans isomerization rates equalize and no further variation is observed in the absorption intensity In Figure 4, the absorption maxima are plotted as a function of the irradiation time at those wavelengths. From this graph, quantitative information can be gained about the isomerization kinetics and the relative photochemical yields, following known kinetic procedure (Zimmermann et al., 1958; Bortolus and Monti, 1979) Absorbance decrease of the ππ* transition as a function of the irradiation time (azobenzene The time evolution is due to the conversion between the trans and cis forms In the inset the photoconversion quantum yields are reported along with the respective decay times obtained by fitting the absorbance data (solid lines in the graph) where [cis] = C0·Y, being C0 the initial molar concentration of the trans isomer and Y the cis molar fraction appearing in time. Φt is the photochemical yield of the trans→cis reaction, while Φc of the cis→trans one. k is the constant relative to the thermic isomerization. Since this last mechanism is much slower than the photochemistry, it can be neglected in our experiment (Zimmermann et al., 1958) Iλ is the power density absorbed by the trans/cis isomer the kinetic equation assumes the following aspect: where εt is the extinction coefficient of the trans form, F = A/(1–10−A), being A the absorbance plotted in Figure 4. This last equation may be integrated giving a function whose time dependence is linear. The slope m of this line is related to the photochemical yield of the trans isomer (Zimmermann et al., 1958): By using this kinetic analysis, the photochemical yield Φt shown in the inset of Figure 4 is obtained In agreement with previous data (Bortolus and Monti, 1979; Siampiringue et al., 1987; Satzger et al., 2004) azobenzene photochemical yield in ethanol was found to be 0.15 the ring substitution has the effect to increase the yield to 0.22 The major change is observed in the case of azopeptide 1 where the isomerization results strongly favored giving a yield of 0.70 Further investigations on thermal cis→trans reconversion kinetics confirm the stability of the final compound, accordingly to the NMR results. In fact, keeping irradiated solutions of the azopeptide 1 48 h in the dark at 50°C, only half trans form is thermally recovered, while for azobenzene and α-helix short peptide chain derivatives the recovery is complete, on this time scale, also at room temperature (Kumita et al., 2000) we also decided to investigate the use of ACN/water 1:1 mixture to analyze the effect of organic cosolvent on azopeptide folding Structure of the cis-azopeptide 1 with observed NOEs indicated by arrows A comparison of backbone Hα and HN chemical shift differences between cis and trans forms is shown in Figure 6 Large chemical shift variation is observed for the azobenzene group and neighboring residues Arg6 and Thr9 more distant residues in the two peptide arms are also affected Similar trends are observed under both solvent conditions The chemical shift changes can be ascribed to magnetic susceptibility anisotropy effects as the two aromatic groups get closer in space and/or to conformational effects Δδ chemical shift differences between the cis and the trans forms of azopeptide 1 calculated for Hα (A) and HN protons (B) The analysis of backbone and sequential NOEs reveals the presence of complex equilibria between extended and turn/helical folded conformations HN-Hα sequential NOEs are stronger than intraresidual ones indicating that extended backbone conformations are largely populated the observation of sequential HN-HN NOEs (V4/E5 G16/W17 in particular) can be ascribed to turn or helical conformations These folded conformations are further supported by weak NOEs of azobenzene aromatic protons with methyl protons of Val4 and Leu12 no NOEs could be detected between the two peptide arms in both forms This result is in agreement with the observation that the trans→cis isomerization does not stabilize a β-hairpin structure In this work photoisomerization of the azopeptide 1 was explored and its reversibility was compared with more simple systems such as azobenzene amino acid 2 and azobenzene 3 To this aim azopeptide 1 was modified introducing the photoswitch (4-aminomethyl)phenylazobenzoic acid (AMPB) to replace in [Pro7,Asn8,Thr10]CSF114 the P-N-H tripeptide on the tip of the β-hairpin and azopeptide 1 were measured as a function of irradiation time exciting into the ππ* band The major differences are observed in the case of 1 where the isomerization results favored by exciting into the ππ* transition and the corresponding cis isomer results strongly stabilized Detailed NMR structural studies of azopeptide 1 confirmed that the AMPB chromophore insertion into the sequence allowed reversible control of peptide conformation in solution but the trans→cis isomerization does not stabilize a β-hairpin structure incorporation of different photocontrolled switches will require further investigations to verify their possible role in controlling β-hairpin conformations No datasets were generated or analyzed for this study performed and interpreted the data of the UV/Vis experiments performed and interpreted the data of the NMR experiments LS provided technical support to the experiments This work was supported by the Fondazione Ente Cassa di Risparmio Firenze (grant n The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2019.00180/full#supplementary-material Synthesis and application of an azobenzene amino acid as a light-switchable turn element in polypeptides CrossRef Full Text | Google Scholar Optimizing the photocontrol of bZIP coiled coils with azobenzene crosslinkers: role of the crosslinking site Photoisomerization in different classes of azobenzene Photo-control of peptide conformation on a timescale of seconds with a conformationally constrained Photomodulation of the conformation of cyclic peptides with azobenzene moieties in the peptide backbone (1994) NMR solution structure of the isolated N-terminal fragment of protein-G B1 domain Evidence of trifluoroethanol induced native-like beta-hairpin formation Cis-trans photoisomerization of azobenzene CrossRef Full Text | Google Scholar Shape-shifting azo dye polymers: towards sunlight-driven molecular devices Designed glycopeptides with different beta-turn types as synthetic probes for the detection of autoantibodies as biomarkers of multiple sclerosis Conformation-activity relationship of designed glycopeptides as synthetic probes for the detection of autoantibodies Theoretical study of the isomerization mechanism of azobenzene and disubstituted azobenzene derivatives Light-triggered aggregation and disassembly of amyloid-like structures Near-infrared photoswitching of azobenzenes under physiological conditions Red-shifting azobenzene photoswitches for in vivo use (2006) A photocontrolled b-hairpin peptide Structure of the winged-helix protein hRFX1 reveals a new mode of DNA binding Goulet-Hanssens (2013) Photo-control of biological systems with azobenzene polymers CrossRef Full Text | Google Scholar Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex Synthesis of photoswitchable amino acids based on azobenzene chromophores: building blocks with potential for photoresponsive biomaterials Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides Biological activity of photoisomerizable diarylethenes Photo-control of helix content in a short peptide NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy The glycopeptide CSF114(Glc) detects serum antibodies in multiple sclerosis An N-glucosylated peptide detecting disease-specific autoantibodies Photoswitchable metal coordinating tweezers operated by light-harvesting dendrimers Kinetics of thermal cis-trans isomerization in a phototropic azobenzene-based single-component liquid crystal in its nematic and isotropic phases Designed glucopeptides mimetics of myelin protein epitopes as synthetic probes for the detection of autoantibodies A convenient microwave-assisted synthesis of N-glycosyl amino acids The use of post-translationally modified peptides for detection of biomarkers of immune-mediated diseases Use of azobenzene amino acids as photo-responsive conformational switches to regulate antibody-antigen interaction Photoisomerization dynamics and pathways of trans- and cis-azobenzene in solution from broadband femtosecond spectroscopies and calculations Time-resolved infrared studies of the unfolding of a light triggered β-hairpin peptide Mono- and bicyclic peptides with (4-amino)phenylazobenzoic acid as backbone constituent doi: 10.1002/1097-0282(200012)54:7<489::AID-BIP20>3.0.2-F Azobenzene as photoresponsive conformational switch in cyclic peptides Azobenzene as conformational switch in model peptides Synthesis of diastereomerically pure Lys(Nε-lipoyl) building blocks and their use in Fmoc/tBu solid phase synthesis of lipoyl-containing peptides for diagnosis of primary biliary cirrhosis Conventional and microwave-assisted SPPS approach: a comparative synthesis of PTHrP(1-34)NH(2) (2002) Mechanism by which 2,2,2-trifluoroethanol/water mixtures stabilize secondary-structure formation in peptides: a molecular dynamics study Excited-State dynamics of trans- and cis-azobenzene after UV excitation in the ππ* band CrossRef Full Text | Google Scholar Mechanism and dynamics of azobenzene photoisomerization a pleiotropic regulator of virulence genes in S The Cis-Trans photoisomerization of azobenzene: an experimental reexamination CrossRef Full Text | Google Scholar Spörlein Ultrafast spectroscopy reveals subnanosecond peptide conformational dynamics and validates molecular dynamics simulation The synthesis of a light-switchable amino acid for inclusion into conformationally mobile peptides bioorg CrossRef Full Text | Google Scholar Photoregulation of cyclic peptide conformation CrossRef Full Text | Google Scholar Structural basis of the recognition of the dishevelled DEP domain in the Wnt signaling pathway Coordination-driven macrocyclization for locking of photo- and thermal cis → trans isomerization of azobenzene A hairpin turn in a class II MHC-bound peptide orients residues outside the binding groove for T cell recognition (1958) The photochemical isomerization of azobenzene CrossRef Full Text | Google Scholar Pietraperzia G and Papini AM (2019) A Photochromic Azobenzene Peptidomimetic of a β-Turn Model Peptide Structure as a Conformational Switch Received: 10 January 2019; Accepted: 07 March 2019; Published: 29 March 2019 Copyright © 2019 Nuti, Gellini, Larregola, Squillantini, Chelli, Salvi, Lequin, Pietraperzia and Papini. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Anna Maria Papini, YW5uYW1hcmlhLnBhcGluaUB1bmlmaS5pdA== †These authors have contributed equally to this work Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish 2011 in the Life Care Center of Leominster daughter of the late Peter and Concetta (Dionise) Salamone courageously immigrated to the United States with their children Napoli centered her life around her family She and her husband enjoyed spending time with each other Peter Napoli and his wife Denise of Lexington Joseph Napoli and his wife Doreen of Marshfield and Philip Napoli and his wife Francesca of Johnston Rosa Travers and her husband Michael of Leominster; eleven grandchildren and Salvatore Perla; fifteen great grandchildren; one brother Burial will follow in Saint Bernard’s Cemetery 2013 at 1:02 am ET.css-79elbk{position:relative;}Vincent J he attended English High School and Northeastern University He previously worked as a clothing manufacturer for Girltown and was the owner of Cameron Sportswear He was a life member and former treasurer of Madonna Della Cava Society in the North End and a former member of the Pietraperzia Son’s of Italy Cammarata and her husband Mark Byrne of Canton and Helenann Civian and her husband Jamey of Canton Also he is survived by nine grandchildren and three great grandchildren Visiting hours at the Dockray & Thomas Funeral Home John the Evangelist Church Canton Wednesday morning at 10:30 Donations may be made in his memory to the Canton Veteran’s Service Department 801 Washington St Get more local news delivered straight to your inbox. 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