Synthesis of Intrinsically Blue-Colored bis-Nitronyl Nitroxide

Aug 31, 2017 - Institute Lavoisier de Versailles, UMR 8180, University of Versailles St-Quentin en Yvelines, 78035 Versailles, France. §. ICB, Padova...
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Synthesis of Intrinsically Blue-Colored bis-Nitronyl Nitroxide Peptidomimetic Templates and Their Conformational Preferences as Revealed by a Combined Spectroscopic Analysis Marta De Zotti,*,† Karen Wright,*,‡ Edouard d’Aboville,‡ Antonio Toffoletti,† Claudio Toniolo,†,§ Giovanna Longhi,∥ Giuseppe Mazzeo,∥ Sergio Abbate,∥ and Fernando Formaggio†,§ †

Department of Chemistry, University of Padova, 35131 Padova, Italy Institute Lavoisier de Versailles, UMR 8180, University of Versailles St-Quentin en Yvelines, 78035 Versailles, France § ICB, Padova Unit, CNR, Department of Chemistry, University of Padova, 35131 Padova, Italy ∥ Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy ‡

S Supporting Information *

ABSTRACT: The intrinsically blue-colored Ullman imidazolinyl nitronyl nitroxide (NN) mono-radicals have found various applications, in particular as spin probes and organic magnetic materials. Here, we present the solution-phase synthesis, extensive characterization, and conformational analysis of the first peptidomimetics with two pendant, chiral nitronyl nitroxide free radical units. Two (R)-Aic(NN) residues, where Aic(NN) is 2-amino-5-nitronylnitroxide-indan-2-carboxylic acid, have been inserted at positions i and i+3 of the pentapeptide Boc-(R)-Aic(NN)-(Ala)2-(R)-Aic(NN)-Ala-OMe and the hexapeptide Boc-[Ala-(R)-Aic(NN)-Ala]2-OMe as well. The two compounds were obtained in good yields and high purities. Thanks to a combination of several spectroscopic techniques (IR absorption, NMR, VCD, and EPR) we gained clear evidence that both compounds adopt a right-handed 310-helical conformation with both nitronyl nitroxide pendants positioned on the same side of the helix. This peptidomimetic/free radical system is a potentially excellent template for the preparation of a set of appropriate analogs with exciting applications in the area of host−guest organic chemistry, or to spectroscopically evaluate in-depth the intramolecular exchange interactions in this type of probe.



flexibility in the side chains of the amino acid probes is in general acceptable or may even be beneficial. It is principally for this reason that we concentrated our efforts on C αtetrasubstituted residues with quasi-rigid side-chain probes. A very promising example of such an approach was the utilization of EPR spectroscopy and the free-radical 2,2,6,6-tetramethylpiperidine-1-oxy-4-amino-4-carboxylic acid (TOAC) residue,8 with only a limited mobility in its 6-membered ring piperidino side chain. In particular, incorporation of two strongly helicogenic TOAC residues in a 310-helix with different i, i + n separations in the sequence allowed us and our collaborators to deeply investigate by EPR techniques nitroxide···nitroxide radical interactions in great detail under a variety of experimental conditions.9,10 Indeed, the nitroxide probe, as present in TOAC, has been demonstrated to be very useful in a variety of EPR investigations to characterize the 3D-structure of synthetic peptides and their precise immersion depth in model and biological membranes.8−10 Our view on the usefulness of this approach (conformationally restricted peptide systems and two free-radical probes) is supported by the myriads of

INTRODUCTION

Conformationally restricted molecular platforms (templates) offer the possibility of obtaining precise information on the distance between and relative orientations of two suitable probes. This opportunity greatly helps our correct understanding of chemical and physicochemical processes relying on 3D-structural dependence. Constructs based on peptidomimetics of short to moderate length provide a significant advantage over other types of organic backbones because they are relatively easily prepared by chemical synthesis.1,2 To achieve this goal, we recently focused our attention on short peptidomimetics characterized by 3D-structurally constrained Cα-tetrasubstituted (quaternary) α-amino acids,3 in particular on members of their subclass adopting welldeveloped 310-helical4,5 main chains, generated by a series of consecutive, regular, type-III β-bend conformations.6,7 [The backbone φ, ψ torsion angles for the peptide type-III β-bend and the related 310-helix are −57°, −30°, while those for the αhelix are −63°, −42°.5] In this 3-fold helix the side chain of any amino acid at position i overlaps almost perfectly that of residue i+3. This short distance maximizes the interaction between the two probes, and consequently enhances greatly the intensities of their spectroscopic signals In this connection, a modest © 2017 American Chemical Society

Received: June 16, 2017 Published: August 31, 2017 10033

DOI: 10.1021/acs.joc.7b01498 J. Org. Chem. 2017, 82, 10033−10042

Article

The Journal of Organic Chemistry Scheme 1. Syntheses of the bis-Aic(CN) Tetra-, Penta-, and Hexapeptidesa

a

Reagents and conditions: (a) Boc-Ala-OH (Boc, tert-butyloxycarbonyl), HATU, DIEA (diisopropylethylamine), THF (tetrahydrofuran), r.t; (b) NaOH, THF/MeOH(methanol)/H2O, r.t. (c) H-Ala-OMe (OMe, methoxy), HATU, DIEA, THF, r.t.; (d) TFA(trifluoroacetic acid)/CH2Cl2 9:1; 0 °C; (e) Boc-(R)-Aic(CN)−OH, HATU, DIEA, THF, r.t.

both (TOAC and Aic(NN)-containing) families of compounds showed a slightly higher puckering mobility for the TOAC 6membered ring with respect to that of the external Aic(NN) 5membered ring. To expand the extremely scarce repertoire of known nitronyl nitroxide α-amino acid building blocks, we have recently reported the solution-phase synthesis and the UV−vis absorption, electronic CD (ECD), IR absorption, NMR, EPR, magnetic susceptibility, and X-ray diffraction properties of the first peptidomimetic, a tripeptide, based on a single Cαtetrasubstituted α-amino acid with a nitronyl nitroxide monoradical moiety incorporated into one of its side chains.35 The chiral, optically resolved residue used is 2-amino-5-nitronylnitroxide-indan-2-carboxylic acid, abbreviated as Aic(NN). Not surprisingly, Aic(NN) exhibits a remarkably effective peptide folding propensity being a member (like TOAC) of the subclass of Cα,α-cyclized, Cα-tetrasubstituted α-amino acids.3 The sequence of the tripeptide was Boc-Ala-(R)-Aic(NN)-AlaOMe (where Boc is tert-butyloxycarbonyl and OMe is methoxy). In this article, we describe the synthesis and complete characterization of the first two peptidomimetics each characterized by two Aic(NN) residues and long enough to potentially fold into fully developed 310-helical structures.3−5 As a first step in the study of these bis-nitronyl nitroxide peptidomimetic templates, a large array of spectroscopic techniques was exploited.

publications that exploit it, particularly in biochemistry (proteins) and physical chemistry (model compounds for indepth EPR studies). In addition, a currently extensively used EPR methodology (called either PELDOR or DEER) has been developed theoretically and experimentally precisely for this purpose. Specifically, this methodology allows one to determine rigorously (particularly with quasi-rigid free radical-based, sidechain probes) intramolecular distances and relative orientations between different parts (or segments) of the same molecule. It is quite clear that for the beneficial extension of this research project (efficient helix-forming peptidomimetics with quasi-rigid, appropriately free-radical functionalized, side chains as templates) a larger arsenal of conformationally restricted building blocks is required.

Another quite interesting, related class of organic, stable mono-radicals is that of the usually intrinsically blue-colored imidazolinyl nitronyl nitroxides, introduced by Ullman and coworkers almost 50 years ago11−15 [for the only other class known of intrinsically blue-colored peptides, based on the Trp side-chain analog β-(1-azulenyl)-L-alanine, see ref 16]. Only a limited number of peptides have been (mono)labeled with this type of radical, either at the N-terminal amino function or covalently linked to a few α-amino acid side chains with the purpose of investigating their biological activities and radical scavenging properties.17−20 Moreover, published organic compounds containing nitronyl nitroxide bi-radicals are relatively rare15,21−33 (specifically, those based on a peptide template have not been reported to date). The properties of such systems have been studied by use of UV−vis absorption, EPR, NMR, magnetic susceptibility, and electrochemistry, and in a few cases their ability to function as EPR-active sensors for metal cations has been noted.22,32 Nitroxide and nitronyl nitroxide free-radical probes, rather than being considered an alternative, are complementary. For instance, nitronyl nitroxides absorb deeply in the Vis region and exhibit intriguing magnetic susceptibility/electrochemical properties/EPR features (e.g., number of spectral lines) which either are missing or simply quite different from those of nitroxides. Particularly convenient for PELDOR experiments are the nitronyl nitroxide spin relaxation times (both T1 and T2) that are longer than those of nitroxides.34 Also, our X-ray diffraction structures of



RESULTS AND DISCUSSION

Peptide Synthesis. The synthesis in solution of the bisAic(NN)-containing penta- and hexapeptides was performed from their respective synthetic precursors, each with two Aic(CN) units. While it is feasible to exploit the Aic(NN) residue directly in peptide synthesis, the acid- and lightsensitive nature of the nitronyl nitroxide radicals makes the use of its Aic(CN) precursor more straightforward for this purpose.35 The coupling reagent O-(7-azabenzo-1,2,3-triazol1-yl)-1,1,3,3-tetramethyluronium (HATU) hexafluorophosphate36 was used to synthesize the Aic(CN)-based peptides in a stepwise manner. Despite the severe steric hindrance of the Aic(CN) residue, the coupling reactions resulted in moderate to good yields (61% for the tetra-, 66% for the penta-, and 54% for the hexapeptides). Syntheses and characterizations of the diand tripeptides were already reported.35 Details of the synthetic strategy employed to obtain the tetra-, penta-, and hexapeptides are outlined in Scheme 1. Reduction of the two cyano groups in the penta- and hexapeptides to bis-aldehydes was carried out by Raney/Ni in the presence of sodium hypophosphite.37 Condensation of the 10034

DOI: 10.1021/acs.joc.7b01498 J. Org. Chem. 2017, 82, 10033−10042

Article

The Journal of Organic Chemistry Scheme 2. Synthesis of the bis-Aic(NN) Hexapeptidea

Reagents and conditions: (a) Raney/Ni, NaH2PO2, pyridine/acetic acid/H2O, 40 °C; (b) 2,3-diamino-2,3-dimethylbutane, CHCl3, 60 °C; (c) (i) mCPBA, CH2Cl2/NaHCO3 aq.; (ii) NaIO4 aq., 0 °C.

a

Figure 1. (A) FT-IR absorption spectra of the Boc-Ala-(R)-Aic(NN)-Ala-OMe tripeptide (- · -) (taken from ref 35), the bis-(R)-Aic(NN) pentapeptide (---), and the bis-(R)-Aic(NN) hexapeptide (−) in CDCl3 solution in the amide N−H stretching region. The vertical scale is deliberately not reported to give particular emphasis to the ratios between the areas under the two peaks of each curve. (B) FT-IR absorption spectrum of the bis-(R)-Aic(NN) hexapeptide in the N−O stretching region. Peptide concentration: 1 mM.

FT-IR Absorption and VCD. Figure 1A shows the FT-IR absorption spectra in the backbone amide A (N−H stretching) region of the Boc-Ala-(R)-Aic(NN)-Ala-OMe tripeptide35 and the bis-(R)-Aic(NN) penta- and hexapeptides in CDCl3, a solvent of low polarity. The ratio between the areas under the absorptions near 3330 cm−1 (H-bonded NH groups) and 3425 cm−1 (free, solvated NH groups)39 is slightly lower in the pentapeptide. This trend is confirmed by the observation of the even lower ratio for the Boc-Ala-(R)-Aic(NN)-Ala-OMe tripeptide under the same experimental conditions. None of these spectra changes significantly upon dilution (to 0.1 mM concentration, Figure S7, S.I.). Taken together, these results strongly support the view that all Aic(NN)-containing peptides investigated tend to fold in CDCl3 and that the conformation adopted (either a single turn or a short helix) is stabilized by CO···H−N intramolecular H-bond(s). This conformational propensity can be safely attributed to the Cα-tetrasubstituted Aic unit specifically characterized by its five-membered (cyclopentene) ring structure.3 Moreover, Figure 1B corrobo-

bis-aldehyde groups with 2,3-diamino-2,3-dimethylbutane afforded the corresponding bis-tetramethyl-imidazolidine intermediates (not isolated) which were subsequently oxidized by means of 3-chloroperbenzoic acid (mCPBA)/NaIO438 to produce the desired, final bis-nitronyl nitroxide peptides. The synthesis of the bis-Aic(NN) hexapeptide is shown in Scheme 2. Preparation of the bis-Aic(NN) pentapeptide was performed following the same procedure. The three-step methodology to convert the bis-Aic(CN) peptides to the corresponding bisAic(NN) peptides is characterized by overall yields of 22−29%. Conformational Studies. We performed an in-depth conformational investigation in solution on the bis-(R)Aic(NN) and bis-(R)Aic(CN) penta- and hexapeptides by means of combined FT-IR absorption, vibrational CD (VCD), near-UV/ vis absorption, ECD, NMR, and EPR spectroscopic techniques. Unfortunately, our numerous attempts to grow single crystals suitable for X-ray diffraction analysis from the final compounds failed. 10035

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against interfering transitions, in particular those originating from aromatic chromophores,49,50 as in the ECD spectra of the compounds studied here. Figure 2B (bottom) shows the experimental VCD spectrum of the bis-(R)-Aic(CN) pentapeptide in CDCl3 (amide I and II spectral regions). Two strong bands are seen at 1679 and 1513 cm−1. The former band is well correlated with the amide I absorption maximum (1675 cm−1). The latter is broader and remarkably lower in energy than the corresponding one in the IR absorption spectrum (1533 cm−1). All these parameters were characteristically found by Keiderling and co-workers51,52 for the amides I and II transitions of 310helical peptides. Moreover, the remarkably more intense and negative amide II VCD band points to a right-handed screw sense for the observed helix. To better understand the experimental VCD data and to confirm the aforementioned stereochemical conclusions for all peptides investigated, we performed computations on the representative bis-(R)-Aic(CN) pentapeptide in CHCl3 solution by the DFT methodology [iefpcm B3LYP(6-31G*) level] starting from ideal geometries with backbone torsion angles characteristic of either an α- or a 310-helix5 each of them with either right or left handedness (Table S1 and Figure S1, S.I.). Energy considerations allowed us to conclude that (i) for the peptide backbone, the right-handed 310-helix is the most stable 3D-structure (e.g., an initial α-helix converges to a 310-helix after optimization) and (ii) for the (R)-Aic(CN) side chains, the conformer with the two aromatic moieties residing on the same face of the helix is the most populated. The VCD weighted average spectra of the optimized conformers were calculated (Figure 2B; for details on the VCD spectra of the various conformers see Figure S2, S.I.). The results corroborate our view that in this chiral spectroscopy the intense amide II band is quite useful to discriminate between the right- versus the left-handed helical structure. We assigned the two negative peaks calculated at 1531 and 1514 cm−1 (scaled wavenumbers) to typical amide II vibrational modes delocalized over the four -Ala-Ala-(R)-Aic(CN)-Ala residues. The overall amide I signal is more complex, making the situation less clear. In Figure S3, S.I., we reported also the VCD spectrum of the bis-(R)-Aic(NN) hexapeptide which exhibits a very similar shape, thus suggesting that the two side-chain moieties (R)Aic(CN) and (R)-Aic(NN) do not influence differently the overall backbone conformation. For the biradical hexapeptide, the DFT-calculated, most stable 310-helix structure (conformer c), illustrated in Figures 7A and S4, S.I., exhibits an intramolecular NN····NN distance of 10.6 Å (Table S2, S.I.). Near-UV/vis Absorption and ECD. Two intense absorption bands (at approximately 280 and 370 nm) occur in the nearUV region (250−400 nm) and one (at about 600 nm), much broader and weak, is seen in the Vis (400−800 nm) region of the spectra of the bis-(R)-Aic(NN) penta- and hexapeptides in methanol (MeOH) solution (Figure S6, S.I., and Figure 3, respectively). The general shape of this spectrum is very close to that of the mono-Aic(NN) tripeptide.35 The absorption near 600 nm (n → π* transition of the nitronyl nitroxide chromophore) generates the blue color typical of all compounds of this class.15,40−44 The 370 and 280 nm bands are assigned to different π → π* transitions.41,44 In Figure 4 we report the ECD spectrum of the bis-(R)Aic(NN) hexapeptide above 380 nm in MeOH solution and compare it with that already published35 for the mono-(R)Aic(NN) tripeptide. It is quite clear that the two multiple-peak spectra are almost mirror images. Interestingly, in our previous

rates our notion that the unique N−O stretching signature of the nitroxyl nitroxide group falls near 1360 cm−1.15,40−44 In Figure 2A (bottom) the experimental IR absorption spectrum of the bis-(R)-Aic(CN) pentapeptide in CDCl3 is

Figure 2. Experimental (black) and calculated (for the 310-helical peptide, gray) (A) FT-IR absorption spectra and (B) VCD spectra of the bis-(R)-Aic(CN) pentapeptide, in CDCl3 in the 1900−1100 cm−1 region (peptide concentration: 8 mM). For a better comparison with experiments, in the simulations the calculated wavenumbers were multiplied by 0.97 on the horizontal axis.

reported in the mid- IR region (between 1900 and 1100 cm−1), where the amide I (carbonyl stretching mode) and amide II bands are typically seen.45 We observed the two absorption maxima at 1675 and 1533 cm−1, respectively. Strictly analogous results were recorded for a bis-(R)-Aic(NN) peptide (Figure S3, S.I.). Although it is well-known that it is difficult to distinguish the 310- from the α-helical structure using this technique, the occurrence of the amide I band moderately over 1670 cm−1 points to a prevailing 310-helix since the published range for the absorption maximum of this structure (1666− 1663 cm−1) is closer to the experimental value than that of the α-helix (1663−1658 cm−1).45 The IR absorption amide II band is less informative for this conformational discrimination. VCD is a chirospectroscopic technique of great potential for conformational and configurational analyses of polypeptide molecules.46−48 Its major advantage over the much more extensively exploited ECD is the intrinsic resolution capability of the IR absorption region which permits viable discrimination 10036

DOI: 10.1021/acs.joc.7b01498 J. Org. Chem. 2017, 82, 10033−10042

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The Journal of Organic Chemistry

Furthermore, the variety of contributions from the Aic(NN) aromatic and nitronyl nitroxide41,53,54 chromophores to the ECD in the far-UV (below 250 nm) region makes this usually extremely valuable spectroscopy much less straightforward for polypeptide conformational and screw-sense helix assignments due to severe overlapping of the numerous electronic transitions involved in this traditionally informative region. NMR. We performed a thorough 2D-NMR conformational analysis on both the synthetic, diamagnetic precursors bis-(R)Aic(CN) penta- and hexapeptides in CD3OH solution at 2 mM concentration. All NHi → NHi+1 cross-peaks, diagnostic of the onset of a helical structure,55 are clearly visible in the NOESY spectra (for the pentapeptide, see Figure 5A). Moreover, a 2D-

Figure 3. Near-UV/vis absorption spectra of the bis-(R)-Aic(NN) hexapeptide in MeOH solution. Peptide concentration: 1 mM. The specific electronic transitions of the nitronyl nitroxide chromophore for the three absorption bands are indicated.

Figure 4. Comparison between the CD spectra in the Vis region for the Boc-Ala-(R)-Aic(NN)-Ala-OMe tripeptide (···) (taken from ref 35) and the bis-(R)-Aic(NN) hexapeptide (−) in MeOH solution. Peptide concentration: 1 mM.

Figure 5. (A) Amide NH region and (B) fingerprint region of the NOESY spectrum of the bis-(R)-Aic(CN) pentapeptide (2 mM in CD3OH, 600 MHz). The αHi → NHi+2 cross-peaks are highlighted in red.

paper on the (R)-Aic(NN) tripeptide,35 we demonstrated by Xray diffraction that the φ, ψ backbone torsion angles of this Cαtetrasubstituted residue are characteristic of a left-handed helical structure. On the basis of the “enantiomeric” ECD spectra observed for the tri- versus the hexapeptide, where the chirality of all Aic(NN) residues is the same, we conclude that the (R)-Aic(NN) backbone torsion angles in the longer oligomer would be reminiscent of the right-handed helix, in agreement with the outcome of the above-described VCD experiments.

NMR study in the fingerprint region provided unambiguous evidence for the occurrence of αHi → NHi+2 cross-peaks. Two of them are shown in the pentapeptide spectrum reported in Figure 5B, indicative of a 310-helical structure.55 No spectral changes were observed upon dilution of the peptide solution to 0.5 mM (see S.I.), thus confirming the intramolecular nature of the correlations. In addition, the conformationally informative, experimental αCHi → NHi+2 cross peaks 3HA-5HN and 2HA4HN (Figure 5) observed in the NOESY spectrum of the 10037

DOI: 10.1021/acs.joc.7b01498 J. Org. Chem. 2017, 82, 10033−10042

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Figure 6. (A) Experimental cw EPR spectrum of the bis-(R)-Aic(NN) hexapeptide in toluene at room temperature (full line). Peptide concentration: 0.2 mM. The simulated spectrum is shown as a dashed line. (B) Experimental cw EPR of the same peptide in frozen toluene at 140 K (full line). Peptide concentration: 0.2 mM. The simulated spectrum is shown as a dashed line.

Figure 7. (A) DFT-calculated 310-helix structure with the lowest Gibbs free energy for our bi-radical hexapeptide in CHCl3 solution. (B) Suggested model for our 310-helical bi-radical hexapeptide estimated from its cw EPR data.

Aic(NN) residue, containing the mono-radical nitronyl nitroxide moiety, has unpaired electron hyperfine interactions with the two equivalent 14N nuclear spins (I = 1). In this case, in liquid solution, because of the isotropic couplings, we expected a quintet of hyperfine lines with a relative intensity ratio 1:2:3:2:1. However, our recorded spectrum is characterized by nine hyperfine lines which arise from large intramolecular electron−electron exchange interactions Jexch occurring in our bi-radical system. A Jexch value larger than the other magnetic interactions makes the unpaired electron spin interact equally with all of the four 14N nuclear spins in the molecule, thus affording a nine-line EPR spectrum. The Figure also shows the spectrum calculated with the routines of the EasySpin software running in the MATLAB environment. The simulation, based on the best fit values of the αN constant (αN = 0.746 mT) and with an exchange constant J much larger than αN, reproduces very well the experimental curve. In this Figure we show the simulation calculated with g = 2.0068 and J = 10 mT. Also, these data agree closely with those published for similar bisimidazolinyl nitronyl nitroxides in solvents of low polarity.15,21−33 Moreover, we acquired the spectrum of the same bi-radical hexapeptide in frozen (140 K) toluene solution. Under these conditions, the spectral width is determined mainly by the electron dipolar interaction characterized by the parameter D. Using the point dipole approximation, one can write the relation D = (3/2)g2β2/R3, where R is the distance between the two unpaired electrons located on the Aic(NN) residues. From a comparison between the experimental and simulated spectra (Figure 6B), we obtained D = 1.9 mT. From this result, we

Aic(CN) pentapeptide have been integrated and properly converted into interproton distances (for details, see the Experimental Section). The obtained values (3.40 ± 0.34 Å and 3.46 ± 0.35 Å, respectively) are definitely closer to the corresponding distances calculated for the 310-helix model (3.79 and 3.83 Å) than to those calculated for the α-helix model (4.40 and 4.41 Å). The two models were constructed using the canonical φ, ψ peptide backbone torsion angles, as reported in ref 5. These folded structures in such short oligopeptides are reasonably induced by the presence of the two (R)-Aic(CN) residues in each of them, thus clearly stressing the helix promoter character of these residues. This result is not surprising since the basic unit of any Aic-type Cα-tetrasubstituted α-amino acid is 1-aminocyclohexane-1-carboxylic acid, well-known for the very high tendency to induce type-III βbends and 310-helical structures.3 It is also our view that this 3D-structural conclusion can be safely extrapolated to their two closely related (R)-Aic(NN) penta- and hexapeptides, where the only chemical change has been introduced at the side-chain level. Continuous Wave (cw) EPR. The experimental cw EPR spectrum of the bis-(R)-Aic(NN) hexapeptide in toluene solution at room temperature is reported in Figure 6A. An appreciable self-association of our helical peptide molecules was excluded in view of the observation that the hyperfine structure of the EPR lines is well resolved and the line width of each hyperfine component is small (ca. 0.15 mT). Moreover, a 10fold decrease of the peptide concentration from 0.20 mM to 0.025 mM does not modify the overall hyperfine pattern, but just reduces the signal intensities (Figure S8, S.I.). An (R)10038

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could estimate a value of 11.4 ± 0.4 Å for the intramolecular distance R between the two nitronyl nitroxide moieties (Figure 7), calculated under the assumptions of the electron spin density concentrated midway along each N−O bond. This finding is in agreement with the peptide adopting a 310-helix with the two (R)-Aic(NN) side chains bent in opposite directions. Such a conformer is indeed the most probable one according to the Boltzmann distribution of Gibbs free energies evaluated by DFT computations (presented in the S.I.).56,57

Article

EXPERIMENTAL SECTION

Peptide Synthesis and Characterization. Boc-Ala-Ala-(R)Aic(CN)-Ala-OMe. The tripeptide Boc-Ala-(R)-Aic(CN)-Ala-OMe35 (195 mg, 0.43 mmol) was dissolved in CH2Cl2 (4 mL). A solution of HCl in diethyl ether (Et2O) (12 mL, 1.7 M, 20,4 mmol) was added and the mixture was stirred at r.t. for 18 h. The mixture was concentrated under reduced pressure. The resulting residue was taken up in THF (20 mL) and the reaction mixture was cooled on an ice bath. Boc-Ala-OH (97 mg, 0.52 mmol) was added, then DIEA (0.22 mL, 1.29 mmol) and HATU (196 mg, 0.52 mmol). The resulting mixture was stirred at r.t. for 5 h. The mixture was concentrated under reduced pressure and the residue was dissolved in CH2Cl2. The solution was washed with 0.5 M aqueous HCl, then with a saturated aqueous NaCl solution, and finally with a saturated aqueous NaHCO3 solution. The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography using CH2Cl2/MeOH (95:5) as eluant to give the desired product (139 mg, 61%) as a solid. Rf = 0.32 (CH2Cl2/ MeOH 95:5); mp 190 °C; [α]D25 = +8 (c 0.51, CH2Cl2); 1H NMR (300 MHz, CDCl3, 25 °C): δ = 7.40−7.45 (m, 4H; 2 ArH, 2 NH); 7.27 (d, 1H, J = 8.1 Hz; ArH); 6.63 (d, 1H, J = 5.9 Hz; NH); 5.10 (d, 1H, J = 4.2 Hz; NH); 4.45−4.54 (m, 1H; CH); 4.13−4.22 (m, 1H; CH); 3.92−4.01 (m, 1H; CH); 3.67−3.83 (m, 5H; OCH3, CH2); 3.38−3.49 (m, 2H; CH2); 1.33−1.45 (m, 18H; tBu, CH3); 13C NMR (75 MHz, CDCl3, 25 °C): δ = 173.3, 173.2, 172.1, 156.1 (CO), 146.1, 142.1, 130.9, 128.2, 125.4 (ArC), 119.2 (CN), 110.4 (ArC), 81.1 (C(CH3)3), 67.1 (Cα), 52.2 (OCH3), 51.3, 50.2, 48.6 (CH), 43.6, 42.6 (CH2), 28.2 (C(CH3)3), 17.5, 17.2, 16.9 (CH3). HRMS (ESI): m/z calcd. for C26H36N5O7+: 530.2615 [M+H]+; found 530.2614. Boc-(R)-Aic(CN)-Ala-Ala-(R)-Aic(CN)-Ala-OMe. The tetrapeptide Boc-Ala-Ala-(R)-Aic(CN)-Ala-OMe (139 mg, 0.26 mmol) was dissolved in CH2Cl2 (6 mL). The solution was cooled on an ice bath and TFA (1.5 mL) was added. The mixture was stirred at 0 °C for 6 h and concentrated under reduced pressure. Toluene was added to the residue, and the resulting mixture concentrated again. Boc-(R)Aic(CN)−OH (85 mg, 0.32 mmol) and THF (6 mL) were added to the residue, and the mixture was cooled on an ice bath. DIEA (0.17 mL, 0. 96 mmol) and HATU (121 mg, 0.32 mmol) were added and the mixture was stirred at r.t. for 18 h and concentrated under reduced pressure. The residue was dissolved in CH2Cl2. The solution was washed with 0.5 M aqueous HCl, then with saturated aqueous NaCl solution, and finally with saturated aqueous NaHCO3 solution. The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography using CH2Cl2/MeOH (92.5:7.5) as eluant to give the title product (143 mg, 66%) as a solid. Rf = 0.46 (CH2Cl2/MeOH 92.5:7.5); mp 120−122 °C; [α]D25 = +89 (c 0.50, CH2Cl2); 1H NMR (300 MHz, CDCl3, 25 °C): δ = 7.73 (d, 1H, J = 6.4 Hz; NH); 7.48− 7.58 (m, 3H; ArH, 2 NH); 7.26−7.42 (m, 5H; ArH, NH); 7.18 (d, 1H, J = 7.8 Hz; ArH); 6.02 (bs, 1H; NH); 4.44−4.54 (m, 1H; CH); 4.19−4.28 (m, 1H; CH); 4.05−4.15 (m, 1H; CH); 3.68−3.87 (m, 5H; OCH3, CH2); 3.40−3.62 (m, 4H; CH2); 3.01−3.21 (m, 2H; CH2); 1.26−1.47 (m, 18H; tBu, CH3); 13C NMR (75 MHz, CDCl3, 25 °C): δ = 173.9, 173.3, 172.9, 172.8, 172.7, 156.2 (CO), 146.4, 143.7, 142.4, 142.0, 131.3, 130.7, 128.5, 128.1, 125.6, 125.1 (ArC), 119.3, 118.8 (CN), 111.5, 110.3 (ArC), 82.1 (C(CH3)3), 67.0, 66.9 (Cα), 52.2 (OCH3), 51.7, 50.5, 48.6 (CH), 44.1, 43.9, 42.5, 42.0 (CH2), 28.2 (C(CH3)3), 17.5, 17.1, 16.5 (CH3). HRMS (ESI): m/z calcd. for C37H44N7O8+: 714.3250 [M+H]+; found 714.3251. Boc-Ala-(R)-Aic(CN)-Ala-Ala-(R)-Aic(CN)-Ala-OMe. The pentapeptide Boc-(R)-Aic(CN)-Ala-Ala-(R)-Aic(CN)-Ala-OMe (120 mg, 0.16 mmol) was dissolved in CH2Cl2 (4 mL). The solution was cooled on an ice bath and TFA (1 mL) was added. The mixture was stirred at 0 °C for 8 h and concentrated under reduced pressure. Toluene was added to the residue, and the resulting mixture concentrated again. Boc-Ala-OH (36 mg, 0.19 mmol) and THF (4 mL) were added to the residue, and the mixture was cooled on an ice bath. DIEA (0.03 mL, 0.19 mmol) and HATU (73 mg, 0.19 mmol) were added and the mixture was stirred at r.t. for 3 days, then concentrated under reduced



CONCLUSIONS Some bis-imidazolinyl nitronyl nitroxide compounds have previously been prepared and their chemical, spectroscopic, and magnetic properties investigated.15,21−33 However, there are no published reports so far on two such nitronyl nitroxides covalently linked to the same peptidomimetic scaffold. In this article, we reported the syntheses by solution methods and full characterizations of two blue-colored peptidomimetics, each bearing two pendant, chiral, imidazolinyl nitronyl nitroxide free radical units. The semi-rigid (R)-Aic(NN) residue herein employed is a full member of the family of well-established helix-inducer, Cα,α-cyclized, Cα-tetrasubstituted α-amino acids.3 We have inserted two such mono-radical amino acids of the same Cα-configuration at positions i and i+3 in the sequence. Being separated by three intervening residues, we expected them to be positioned on the same face of a peptide 310-helix after one complete helical turn.4,5 By a combination of spectroscopic techniques, namely FT-IR absorption, VCD, ECD, NMR, and EPR, we indeed demonstrated the overwhelming occurrence of a stable 310-helix structure for our compounds, even as short as penta-/hexapeptides. The occurrence of significant peptide self-association was not documented in any of the solvents utilized for our spectroscopic analyses. We were not surprised by these findings because peptides rich in Cα-tetrasubstituted α-amino acids and shorter than heptapeptides are known to largely prefer 310-helix over α-helix and the monomeric state over the self-aggregated state.3 Moreover, VCD and EPR have unambiguously shown, respectively, that the screw sense adopted by their helices is right-handed and that in each peptide the two side-chain monoradical systems undergo a significant intramolecular interaction. Finally, the DFT calculated 310-helix structure with the lowest Gibbs free energy for the bi-radical hexapeptide is the one with the longest intramolecular distance between the two nitronyl nitroxide moieties. Taking together the results of our initial effort presented here, we believe that our conformationally restricted, bi-radical, 310-helical peptidomimetic templates could pave the way for the construction of a variety of related systems with predictable distances and orientations between the two nitronyl nitroxides (as a function of their relative i, i + n positions in the main chain) to be developed in the future. It is foreseen that additional work in this area will allow one to easily modulate the intramolecular exchange coupling between this type of radical and, as a result, to obtain a deeper insight into their spectroscopic, electronic, and magnetic properties. In addition, by taking advantage of the cavity formed by the 310-helical peptidomimetic backbone and the two pendant (R)-Aic(NN) side chains, which can act as a host for metal cations22,32 as appropriate guests, the construct described in this work is envisaged to potentially function as an EPR-active sensor. 10039

DOI: 10.1021/acs.joc.7b01498 J. Org. Chem. 2017, 82, 10033−10042

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The Journal of Organic Chemistry

taken up in CH2Cl2 and filtered through a plug of silica gel, eluting with CH2Cl2/MeOH/TEA (90:10:1). The filtrate was evaporated to give the intermediate bis-imidazolidine. The residue was dissolved in CH2Cl2 (2 mL) and a saturated aqueous NaHCO3 solution (1 mL) was added. The mixture was cooled on an ice bath. A solution of mCPBA (70%, 49 mg, 0.2 mmol) in CH2Cl2 (0.5 mL) was added dropwise. The reaction mixture was stirred for 1 h at 0 °C. A solution of NaIO4 (26 mg, 0.12 mmol) in water (0.7 mL) was then added dropwise. The reaction mixture was stirred for 40 min at 0 °C, then diluted with CH2Cl2. The phases were separated. The aqueous phase was extracted once with CH2Cl2. The combined organic phases were washed with a saturated aqueous NaCl solution, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography using MeOH/EtOAc (3:97) as eluant to give the title product (23 mg, 29% over three steps) as a blue-colored solid. Rf = 0.36 (MeOH/CH2Cl2 7.5:92.5); mp 156−158 °C; [α]25 436 = −60 (c 0.08, CH2Cl2); HRMS (ESI): m/z calcd. for C52H72N10O13+H+: 1045.5359 [M+H+]; found: 1045.5344. High-resolution mass spectra (HRMS) were obtained on a Waters Micromass Q-Tof Micro instrument. NMR and ESI-MS spectra for all peptides are collected in the S.I. The presence of paramagnetic radicals causes a remarkable broadening in some of the NMR signals for Aic(NN)-containing peptides. This is the reason why we cannot provide the NMR characterization data for those compounds. Spectroscopic Measurements. UV−vis Absorption. UV−vis absorption spectra were recorded in 0.1 cm quartz cells on a Shimadzu UV-2501PC UV−Vis spectrophotometer. Electronic Circular Dichroism. The ECD spectra were measured on a Jasco (Hachioji City, Japan) model J-715 spectropolarimeter equipped with a Haake (Thermo Fisher Scientific, Waltham, MA) thermostat. Baselines were corrected by subtracting the solvent contribution. Fused quartz cells of 10- and 1 mm path length (Hellma, Mühlheim, Germany) were used. The values are expressed in terms of [θ]T, the total molar ellipticity (deg × cm2 × dmol−1). Spectrograde MeOH 99.9% (Acros Organic, Geel, Belgium) was used as solvent. Vibrational Circular Dichroism. The VCD spectra were recorded on a Jasco FVS 6000 spectrometer in CDCl3 solutions at concentrations of 7−9 mM in a BaF2 cells with a path length of 0.2 mm. The resolution was 4 cm−1, 6000 accumulations were taken, and the solvent spectrum was subtracted. DFT calculations were performed at the B3LYP/6-31G* level, within the iefpcm model and with the Gaussian 09 package.58 Harmonic frequencies, dipole, and rotational strengths were calculated by following the magnetic field perturbation method by Stephens.59,60 Lorentzian band shapes with a half width of 8 cm−1 were assumed. Infrared Absorption. The FT-IR absorption spectra were recorded at 293 K using a PerkinElmer model 1720X FT-IR spectrophotometer, nitrogen flushed, equipped with a sample-shuttle device, at 2 cm−1 nominal resolution, averaging 100 scans. Solvent (baseline) spectra were obtained under the same conditions. For spectral elaboration, the software SPECTRACALC, provided by Galactic (Salem, MA), was employed. Cells with path lengths of 1.0 and 10 mm (with CaF2 windows) were used. Spectrograde deuterated chloroform (99.8%,d2) was purchased from Merck (Darmstadt, Germany). Nuclear Magnetic Resonance. All NMR experiments were acquired on a Bruker Avance DMX-600 instrument using the TOPSPIN 1.3 software package, and recorded at 298 K. Suppression of the solvent signal was obtained applying a WATERGATE gradient program. CLEAN-TOCSY spectrum (spin lock pulse = 70 ms) was acquired by collecting 280 experiments of 48 scans each. The assignment of the Ala residues was accomplished following the Wüthrich procedure.55 The sequential assignment was performed by means of the NOESY spectrum, acquired by collecting 400 experiments, each one consisting of 96 scans (mixing time = 150 ms). The conformationally informative αCHi → NHi+2 cross-peaks of the NOESY spectrum of the Aic(CN) pentapeptide were integrated using the SPARKY 3.111 software package. The interproton distance (di) was obtained from the corresponding cross-peak volume Vi using the equation: di = [(d0·V0)/Vi]1/6, where d0 is the calibration distance

pressure. The residue was dissolved in CH2Cl2. The solution was washed with 0.5 M aqueous HCl, then with saturated aqueous NaCl solution, and finally with saturated aqueous NaHCO3 solution. The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography using CH2Cl2/MeOH (92.5:7.5) as eluant to give the desired product (68 mg, 54%) as a solid. Rf = 0.37 (CH2Cl2/MeOH 92.5:7.5); mp 112−114 °C; [α]D25 = +38 (c 0.50, CH2Cl2); 1H NMR (300 MHz, CDCl3, 25 °C): δ = 7.95 (d, 1H, J = 5.2 Hz; NH); 7.43−7.51 (m, 5H; ArH, 2 NH); 7.31−7.39 (m, 4H; ArH, NH); 7.18 (d, 1H, J = 7.8 Hz; ArH); 5.60 (bs, 1H; NH); 4.43−4.53 (m, 1H; CH); 4.27− 4.37 (m, 1H; CH); 4.08−4.17 (m, 1H; CH); 3.40−3.89 (m, 11H; OCH3, CH, CH2); 3.10−3.23 (m, 2H; CH2); 1.38−1.52 (m, 18H; tBu, CH3); 1.26 (d, 3H, J = 7.2 Hz; CH3); 13C NMR (75 MHz, CDCl3, 25 °C): δ = 175.4, 173.6, 173.3, 172.9, 172.9, 172.8, 156.9 (CO), 146.9, 143.6, 142.4, 141.8, 131.2, 130.7, 128.3, 127.9, 125.4, 125.2 (ArC), 119.3, 118.8 (CN), 111.2, 110.1 (ArC), 81.6 (C(CH3)3), 66.6, 66.5 (Cα), 52.2 (OCH3), 52.7, 51.8, 49.7, 48.4 (CH), 44.7, 43.5, 42.8, 42.0 (CH2), 28.2 (C(CH3)3), 17.5, 16.6, 16.5, 16.3 (CH3). HRMS (ESI): m/z calcd. for C40H49N8O9+: 785.3623 [M+H]+; found 785.3626. Boc-(R)-Aic(NN)-Ala-Ala-(R)-Aic(NN)-Ala-OMe. The pentapeptide Boc-(R)-Aic(CN)-Ala-Ala-(R)-Aic(CN)-Ala-OMe (125 mg, 0.17 mmol) was dissolved in pyridine (2 mL). AcOH (1 mL) and sodium hypophosphite hydrate (160 mg) were added. Then, a suspension of Raney/Ni 2800 (appx. 150 mg) in water (1 mL) was added. The reaction mixture was vigorously stirred at 45 °C for 1 h and allowed to cool. The supernatant was decanted and the remaining Ni washed twice with 95% EtOH. The combined organic phases were diluted with Et2O and washed with a saturated aqueous NaCl solution. The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting dialdehyde (89 mg, 0.12 mmol) was dissolved in CHCl3 (5 mL) and 2,3-diamino-2,3-dimethylbutane (57 mg, 0.49 mmol) was added. The reaction mixture was stirred at reflux for 24 h, allowed to cool, and dried over Na2SO4. The mixture was filtered and concentrated under reduced pressure. The residue was taken up in CH2Cl2 and filtered through a plug of silica gel, eluting with CH2Cl2/ MeOH/triethylamine (TEA) (90:10:1). The filtrate was evaporated to give the intermediate bis-imidazolidine. The residue was dissolved in CH2Cl2 (4 mL) and a saturated aqueous NaHCO3 solution (2 mL) was added. The mixture was cooled on an ice bath. A solution of m-CPBA (70%, 100 mg, 0.4 mmol) in CH2Cl2 (1 mL) was added dropwise. The reaction mixture was stirred for 1 h at 0 °C. A solution of NaIO4 (51 mg, 0.24 mmol) in water (1.4 mL) was then added dropwise. The reaction mixture was stirred for 45 min at 0 °C, then diluted with CH2Cl2. The phases were separated. The aqueous phase was extracted once with CH2Cl2. The combined organic phases were washed with a saturated aqueous NaCl solution, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography using MeOH/ethyl acetate (EtOAc) (5:95) as eluant to give the title product (36 mg, 22% over three steps) as a blue-colored solid. Rf = 0.33 (MeOH/CH2Cl2 7.5:92.5); mp 163−165 °C; [α]25 436 = +242 (c 0.08, CH2Cl2); HRMS (ESI): m/z calcd. for C49H67N9O12+H+: 974.4987 [M+H+]; found: 974.4980. Boc-Ala-(R)-Aic(NN)-Ala-Ala-(R)-Aic(NN)-Ala-OMe. The hexapeptide Boc-Ala-(R)-Aic(CN)-Ala-Ala-(R)-Aic(CN)-Ala-OMe (60 mg, 0.076 mmol) was dissolved in pyridine (1 mL). AcOH (0.5 mL) and sodium hypophosphite hydrate (70 mg) were added. Then, a suspension of Raney/Ni 2800 (appx. 80 mg) in water (0.5 mL) was added. The reaction mixture was vigorously stirred at 45 °C for 1 h and allowed to cool. The supernatant was decanted and the remaining Ni washed twice with 95% EtOH. The combined organic phases were diluted with Et2O and washed with a saturated aqueous NaCl solution. The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting dialdehyde (43 mg, 0.05 mmol) was dissolved in CHCl3 (4 mL) and 2,3-diamino-2,3-dimethylbutane (25 mg, 0.22 mmol) was added. The reaction mixture was stirred at reflux for 72 h, allowed to cool, and dried over Na2SO4. The mixture was filtered and concentrated under reduced pressure. The residue was 10040

DOI: 10.1021/acs.joc.7b01498 J. Org. Chem. 2017, 82, 10033−10042

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The Journal of Organic Chemistry and V0 the corresponding volume. We calibrated the distances using non overlapping NHi → NHi+1 sequential correlations, set to a distance of 2.80 Å. A standard deviation of ±10% was estimated for all of the obtained distances. Continuous-Wave (cw) Electron Paramagnetic Resonance. About 300 μL of a 0.2 mM toluene solution of the peptide was transferred in an EPR quartz tube (i.d. 3 mm) which later was connected to a vacuum line. The tube was then sealed under vacuum in order to eliminate gaseous oxygen from the solution. EPR spectra were recorded with a Bruker ER 200-D X-band spectrometer (ν = 9÷10 GHz), equipped with a nitrogen flow cryostat to set the sample temperature (temp. controller: Bruker BVT 2000). Spectra were recorded at either T = 293 or 140 K with a field modulation amplitude of 0.04 mT and a microwave power of about 1 mW. The simulated spectra were obtained by means of the MATLAB program, according to the procedure described by Luckhurst.61 As for the simulation parameters, since the exchange constant J was much larger than the hyperfine interaction, it can possess only a limiting value of 52 G (for the estimated error, see text). The hyperfine coupling with 4 14N nuclei in two groups formed by two nuclei each was 7.46 G. (or 0.746 mT); the line width was 2.65 ± 0.05 G; the line shape was Lorentzian.



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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.7b01498. Tables and Figures supporting VCD simulations, experimental and calculated VCD spectra for the bis(R)-Aic(NN) hexapeptide and bis-(R)-Aic(CN) pentapeptide, and compound characterizations (1H NMR, 13C NMR, and mass spectra) (PDF)



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]; *E-mail: [email protected] ORCID

Marta De Zotti: 0000-0002-3302-6499 Giovanna Longhi: 0000-0002-0011-5946 Giuseppe Mazzeo: 0000-0002-3819-6438 Sergio Abbate: 0000-0001-9359-1214 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS Marta De Zotti is grateful to MIUR (Rome, Italy) for financial support (Futuro in Ricerca 2013, grant no. RBFR13RQXM). Giovanna Longhi gratefully acknowledges CINECA-Bologna (Italy) for the use of computer and software facilities and Regione Lombardia for financial support (LISA grant "LI08p_ChiPhyto" no. HPL13POZE1).



REFERENCES

(1) Spatola, A. F. In Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins; Weinstein, B., Ed.; Dekker: New York, NY, 1983; Vol. 7, pp 267−357. (2) Jamieson, A. G.; Boutard, N.; Sabatino, D.; Lubell, W. D. Chem. Biol. Drug Des. 2013, 81, 148−165. (3) Toniolo, C.; Crisma, M.; Formaggio, F.; Peggion, C. Biopolymers 2001, 60, 396−419. (4) Karle, I. L.; Balaram, P. Biochemistry 1990, 29, 6747−6756. (5) Tonlolo, C.; Benedetti, E. Trends Biochem. Sci. 1991, 16, 350− 353. (6) Venkatachalam, C. M. Biopolymers 1968, 6, 1425−1436. 10041

DOI: 10.1021/acs.joc.7b01498 J. Org. Chem. 2017, 82, 10033−10042

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DOI: 10.1021/acs.joc.7b01498 J. Org. Chem. 2017, 82, 10033−10042