Turn-Mimic Hydantoin-Based Loops Constructed by a Sequential Multicomponent Reaction

A collection of peptidomimetics characterized by having an aspartic acid motif embedded in a rigid hydantoin heterocycle are synthesized through a sequential multicomponent domino process followed by standard regioselective deprotection/coupling reactions based on acid–base liquid/liquid purification protocols. 1H nuclear magnetic resonance experiments, molecular modeling, and X-ray analysis showed that the resulting hydantoin-based loops I (in particular) and II (to a lesser extent) can be considered novel β-turn inducer motifs being able to project two peptide-like strands in a U-shaped conformation driven by the formation of intermolecular hydrogen bonds.


■ INTRODUCTION
One of the drawbacks of the use of short peptides as drugs is their inability to retain organized secondary structures essential for the biological activity. 1 For this reason, different motifs have been developed to stabilize protein secondary structures, thus improving peptide leads.In particular, the quest for scaffolds capable of mimicking turn motifs has garnered significant attention, as turns play a crucial role in stabilizing conformations involved in many biological molecular recognition events. 2 With the turn motif being characterized by the dihedral angles of the amide backbone, the typical strategy to induce a turn conformation in peptidomimetics is to design scaffolds that favor the intramolecular formation of hydrogen bonds (H-bonds) involving the C�O belonging to the peptide bond (Figure 1, top left). 3Turn conformations have also been observed to be triggered by intramolecular H-bonds involving the C�O groups of aspartic acid (Asp) or asparagine (Asn) side chains (Figure 1, top right). 4However, Asx-turns are very often formed along a second secondary structure, mainly β-turn, providing a well-defined H-bonddriven intersected conformation referred to as Asx-β-turn. 5To date, the debate on which turn pre-exists, thus fostering the formation of the second, is still open. 6Nevertheless, recent in silico research has shown that Asx−Gly sequence in a small dipeptide model is able to induce type II′ β-turns independently. 7Since an effective approach to stabilize turn conformation is the synthesis of rigid peptide turn surrogates, we present herein the design, synthesis, and conformational analysis of novel hydantoin-based loops, where (1) the structure of Asn is embedded into a more rigid hydantoin five-membered ring, and (2) a flexible glycine or an even more flexible ethylenediamino residue is tethered to the Nα of Asn (or N1 of the hydantoin ring), respectively, in Hyd-loop I and Hyd-loop II (Figure 1, bottom).We reasoned that if a turn structure can exist in a flexible Asp−Gly sequence, even if triggered by a pre-existing β-turn, the designed hydantoin loops could possibly induce in itself a defined turn conformation with a 9-membered ring intramolecular Hbond.The synthesis of such peptidomimetics is achieved by exploiting a sequential multicomponent (MC) process followed by standard hydrolysis/coupling reactions using liquid−liquid acid/base extraction protocols for the purification of the intermediates. 8The strategy described herein is suitable for the combinatorial synthesis of wide libraries of such compounds, thus overcoming the drawbacks of tedious multistep, time-consuming synthetic pathways often exploited for the synthesis of turn motifs.
■ RESULTS AND DISCUSSION Synthesis.In medicinal chemistry, hydantoin heterocycle is considered to be a privileged scaffold from which it is possible to prepare compounds with a wide spectrum of biological activities. 9Moreover, thanks to its planar conformation with two carbonyl groups that could be involved in hydrogen bonding, it has been exploited as a building block for the synthesis of peptidomimetics with well-defined secondary structures. 10Due to the wide interest raised by this heterocycle during the time, in the past decade, we have developed a MC process for the synthesis of differently substituted hydantoin derivatives with biological interest. 11Taking into account all these features, we have designed Hyd-loop I and Hyd-loop II frameworks as possible inducers of turn structures, which would be accessible with the MC process developed by us.Compared to the structure of the hydantoin-based universal peptidomimetic previously reported 8,10c where the extended strands are attached to the N3 nitrogen of the hydantoin ring and to the C5 hydantoin carbon, the Hyd-loop I and Hydloop II scaffolds bear the extended strands in the vicinal N1 hydantoin nitrogen and C5 hydantoin carbon.The synthetic pathway foresees the in situ formation of carbodiimides 3 by Staudinger/aza-Wittig reaction between primary azides 1 and commercially available tertiary tert-butyl or adamantyl (Ad) isocyanates 2 (Table 1).After completion of the reaction, fumaric acid mono-p-nitrophenylester 4 was added to the reaction mixture providing the formation of hydantoin intermediates 5 through a domino process involving a regiospecific condensation/intramolecular aza-Michael/O−N acyl migration domino process.The regiocontrol of the reaction is due to the preferential nucleophilic attack of the primary amine moiety compared to the sterically hindered tertiary amine moiety during the intramolecular aza-Michael step. 12Finally, the addition in situ of amines, either alkyl, aryl, or glycine derivatives, yields hydantoin-based peptidomimetics 6 by nucleophilic displacement of p-nitrophenol at room temperature.
The process worked efficiently starting from glycine azide tert-butylester (entry 4, Table 1) and diglycine azide methylamide (entries 1−3, Table 1) providing the formation of four peptidomimetics based on Hyd-loop I (compounds 6a−d, Table 1) and with N-Boc ethylenediamine mono azide (entries 5, 7, 9, Table 1) or N-Ac-ethylenediamine mono azide (entries 6, 8, Table 1) yielding other five peptidomimetics based on Hyd-loop II (compounds 6e−i, Table 1).It is worth noting that peptidomimetics 6d, g, i, having terminal functional groups orthogonally protected, are prone to further elongation.Indeed, we synthesized 6 new peptidomimetics 8− 10, 12, 14−15 bearing two artificial dipeptide strands through classical deprotection/condensation procedures using liquid− liquid acid/base extraction protocols for the purification of the intermediates in high yields (Scheme 1).More specifically, the benzyl ester of hydantoin 6d was selectively hydrolyzed by treatment with hydrogen in the presence of a catalytic amount of Pd/C, and the resulting carboxylic acid coupled with H− Gly−CONH−iBu providing intermediate 7. Thus, the tertbutyl ester was cleaved upon treatment with TFA in DCM, and the resulting carboxylic acid coupled with p-Cl-benzyl amine, 2,2-diphenyl-ethylamine, and p-I-phenylamine affording peptidomimetics 8−10, respectively, in good yields.Analogously, after selective hydrogenolysis of the benzyl ester, the upper strand of hydantoin 6g was elongated with H−Gly−CONH− PMB yielding intermediate 11, which was Boc deprotected with TFA and coupled with AcNH−Gly−OH producing peptidomimetic 12.We could also first elongate the lower arm of 6g by tert-butyloxycarbonyl (Boc)-deprotection of the amino function (TFA/DCM) and coupling with AcNH−Gly− OH yielding 13, followed by hydrogenolysis of the benzyl ester and coupling with p-Cl-benzyl amine leading to peptidomimetic 14.Lastly, the corresponding adamantyl derivative 6i, having a benzyl ester on the upper strand and an amino group protected as Boc in the lower strand, was first submitted to hydrogenolysis and coupled with iso-propyl amine leading to the formation of 15 and then Boc deprotected and coupled with 3-phenyl-propanoyl chloride or Ph−CH 2 −CH 2 − CONH−Gly−OH affording 16 and 17, respectively.
Conformational Analysis: NMR.To get insights on the possible conformations that Hyd-loop I and Hyd-loop II could trigger in solution, we sought to identify the presence of possible intramolecular H-bonds through proton nuclear magnetic resonance ( 1 H NMR) experiments.The first feature to take in consideration is the chemical shift of the amidic protons −CONH− in relatively nonpolar solvents such as CDCl 3 .Indeed, it is well documented that the amidic protons resonating at lower fields, typically around 8.0 ppm, are likely to be involved in intramolecular hydrogen bonding, whereas hydrogens resonating at higher fields are not. 13The chemical shifts of the amidic protons NH A −NH D of Hyd-loop I peptidomimetics and NH A , NH B′ , NH C , NH D′ belonging to

The Journal of Organic Chemistry
Hyd-loop II peptidomimetics for which it was possible to recover the 1 H NMR spectra in CDCl 3 were assigned through correlation spectroscopy (COSY) experiments (see the Supporting Information) and are reported in Table 2. 14  Interestingly, in short, peptidomimetics 6, having only one amidic proton in the upper strand, namely NH A , and one or two amidic protons in the lower arm (NH B/B′ and NH D/D′ ), only the Hyd-loop I seems to trigger the formation of an intramolecular H-bond involving NH B , which resonates at 8.02 and 7.78 ppm for compounds 6a,b, respectively (entries 1 and The Journal of Organic Chemistry 2, Table 2), whereas all the amidic protons of peptidomimetics of type 6 built on the Hyd-loop II resonate at ppm lower than 7.5 (entries 3−8, Table 2).These results strongly suggest that the Hyd-loop I likely induces the formation of a H-bonddriven β-turn conformation in solution, while the presence of a more flexible ethylenediamino residue in Hyd-loop II hampers the formation of strong intramolecular H-bonds, at least in short sequences.The capability of the Hyd-loop I to induce turn conformations in solution is maintained also in peptidomimetics having two artificial dipeptide arms, such as 8 (entry 9, Table 2).Indeed, amidic protons NH A and NH B are likely involved in intramolecular H-bond, whereas proton NH C , resonating at 6.53 ppm, is clearly free in solution.It is noteworthy that also proton NH D (7.66 ppm) seems involved to some extent in the formation of an intramolecular H-bond, corroborating the results obtained from X-ray analysis (see below).Very interestingly, also Hyd-loop II seems to be able to induce the formation of a defined intramolecular hydrogen bonding network when the lower arm is elongated by a glycinamide residue.Indeed, the presence of a further amide functional group in the lower arm seems to trigger the formation of two intramolecular H-bonds involving protons NH A and NH B′ , which resonate at ppm higher than 8.0 in peptidomimetics 12, 14, and 16 (entries 10, 12, 15, respectively, Table 2).On the contrary, when Hyd-loop II is elongated only in the upper arm, such as in peptidomimetics 15 and 16 (entries 13 and 14, respectively, Table 2), there is no evidence of the presence of any H-bonds being all the amidic protons resonating at ppm lower than 7.0, whereas weaker intramolecular H-bonds involving proton NH A and NH B′ seem to be present in peptidomimetic 13 having an extended lower arm (entry 11, Table 2).
Notably, compared to that of the precursors 6g and 13, the chemical shifts of amidic protons NH A and NH B′ in peptidomimetic 14 were shifted downfield pronouncedly by, Scheme 1. Synthesis of Elongated Hydantoin-Based 12,14,16,17 3).Next, we performed variable temperature (VT) 1 H NMR experiments on model peptidomimetics 6a, 8, based on Hydloop I and 14 based on Hyd-loop II (Table 3).We measured the variation of the chemical shifts of the amidic protons in 2.0   The Journal of Organic Chemistry mM CDCl 3 solution of 6a, 8, and 14, and in 2.0 mM dimethyl sulfoxide (DMSO)-d 6 solution of 6a and 8. Indeed, the variation of the chemical shifts of amidic protons within the rising of the temperature is a parameter often considered to get insights on the presence/strength of intramolecular H-bonds.More in detail, in low-polarity solvents, temperature coefficient significantly larger than 2.4 ppb/K can be assigned to protons engaged in intramolecular H-bonds, which are weakened upon increasing the temperature, whereas values lower than 2.4 ppb/ K are not really indicative because they can be assigned to protons either accessible or not accessible to the solvent.15 On the other hand, in polar DMSO-d 6 values of δΔ/δT < 5 ppb/K denote not accessible protons, thus already involved in Hbond.16 The temperature coefficient measured in CDCl 3 solutions were not very useful to clarify the presence of intramolecular H-bonds.Actually, the value of δΔ/δT obtained for NH B of peptidomimetic 6a based on Hyd-loop I, i.e., the proton that seemed to be involved in intramolecular hydrogen bond considering its chemical shift (Table 3), was equal to 11.4 ppb/K, higher than the reference of 2.4 ppb/K but not as high as those we observed in the previous study.10c Moreover, the values obtained for all the amidic protons in peptidomimetic 8 having an extended upper strand were even lower.However, the temperature coefficient measured for the same peptidomimetics in DMSO-d 6 solutions were smaller than 5.0 ppb/K, suggesting the possibility for these peptidomimetics to adopt different conformations where the amidic NH could be involved in hydrogen bonding.The results obtained for peptidomimetic 14 based on Hyd-loop II demonstrate a certain tendency of proton NH A , NH B′ , and NH D′ to be involved in H-bonding, having δΔ/δT values respectively of 18.1, 13.4, and 13.2 ppb/K.Given that we have recorded the spectra of Hyd-loop I peptidomimetics 6a and 8 in both 2.5 mM CDCl 3 and DMSOd 6 solutions, we calculated the difference in the chemical shifts of the amidic proton as a measure of their accessibility (Figure 4).17 In both cases, hydrogen H B showed the smallest difference, suggesting its engagement in intramolecular hydrogen bonding.
Since the VT experiments did not clearly demonstrate the presence of strong intramolecular H-bonds, we performed DMSO titration experiments on peptidomimetics 6a and 14 by recording the chemical shifts of the amidic protons in CDCl 3 solutions (2.0 mM) upon addition of small aliquots (5 μL) of DMSO.In this experiment, the protons engaged in hydrogen bonding did not experience an evident chemical shift change after dilution with a coordinating solvent like DMSO, whereas sizable downfield shifts are recorded for protons free in solution, as a result of increasing H-bond with DMSO. 18As evidenced in Figure 5, in compound 6a, proton H B essentially did not shift upon dilution, whereas proton H D and to a larger extent proton H A showed a significant downfield shift.This result further corroborates the presence of a secondary structure triggered by an intramolecular H-bond involving H B for peptidomimetics built on Hyd-loop I. Concerning Hyd-loop II peptidomimetic 14, we did not record evident downfield shifted proton resonances for H A and H B′ , indicating their involvement in H-bonds, whereas for protons H C and H D′ , we observed a clear increase of chemical shifts due to their increased interaction with the titrating solvent.However, nuclear Overhauser enhancement spectroscopy (NOESY) experiment performed on compound 14 did not reveal clear long-range contacts supporting the possibility that the β-turn conformation promoted by Hyd-loop II could not be very robust.
Conformational Analysis: Computation.Computational tools were employed to gather more information on the conformational behavior of the proposed turn mimics.In accordance with the commonly accepted definition of β-turn conformation, the presence of both an interatomic distance dα  The Journal of Organic Chemistry < 7 Å and the absolute value of the dihedral angle C1−C2− C3−N4 |β| < 60°were strictly considered as a necessary condition for the presence of this specific turn.Different techniques were used at different levels as molecular mechanics (MM), ab initio density functional theory (DFT), and molecular dynamics (MD).First, a conformational search by a combined MM−Monte Carlo (MM−MC) approach was performed.Structures 6a−c (Hyd-loop I) and 6e−i (Hydloop II) were submitted to the MM−MC analysis using both MMFF94 and MMFFaq force fields; the latter was chosen to evaluate the presence of an aqueous environment on the formation of the turn.For each structure, the conformers within 10 kcal/mol from the minimum were considered.
In addition to the dα and β values, the formation of an intramolecular H-bond is also indicative of the presence of a βturn conformation; in a classical β-turn, this H-bond results in the formation of an 11-membered ring.In our case, two possible intramolecular H-bonds could be identified for the two Hyd-loop types.For Hyd-loop I, two 9-membered intramolecular hydrogen bond rings involving NH A or NH B could be formed, whereas for Hyd-loop II, a 9-membered (involving NH B′ ) and a 11-membered (involving H A ) hydrogen bond rings can be defined (Figure 6).Due to the important contribution of these H-bonds in stabilizing the turn conformation, their presence was also evaluated.
Results from this study are reported in Table 4 as percentage of conformers that meet the requirements.
All the structures showed a good propensity toward the adoption of a β-turn conformation, with about 30% of all conformers being within the desired values.The effect of water seemed to be insignificant, with only a small deviation from the values of the in vacuo calculations.As expected, a significant percentage of the conformers also have the considered intramolecular H-bonds, thus confirming the importance of this parameter.It must be noticed that for compounds 6a,b, an extra H-bond involving the H D hydrogen, resulting in a 12membered ring, is possible.Indeed, this H-bond could be found in 13,11, and 3% of conformers for 6a, 6b, and 6c respectively.Anyway, a closer inspection of the results showed some differences in the global minimum conformers between Hyd-loop I and Hyd-loop II.In Table 5, the relative energies of the first β-turn conformers are reported.As can be seen, the only compounds for which the global minimum structures are a β-turn both in vacuo and in water are Hyd-loop I 6a,b (in 6c, the presence of a terminal arylamide negatively affects the formation of the H-bond).In the case of Hyd-loop II, a less defined situation is present, with most of the β-turn conformations not being the global minimum.Even if in some cases the ΔE are not so high, this result points out the importance of the C 3 −C�O−N 4 carbonyl moiety in favoring the stabilization of the turn.
To better evaluate this effect, selected compounds 6a and 6f,g were investigated by DFT (Table 6).The energy minima of both the first β-turn and open (not β-turn) conformations, as obtained from MM−MC, were submitted to a full energy optimization (with the calculation of vibrational energies to add for zero point energy correction) with DFT at the B3LYP 6-31+G(d,p) level.Energies were calculated in vacuo and with the contribution of water solvation.For compound 6a, the βturn is favored both in water and in vacuo, and the first open conformation is found at a substantially higher energy (5.33 and 6.22 kcal/mol for in vacuo and in water geometries ,respectively); these ΔE values virtually ensure a total preference for the turn conformation at room temperature.In this conformation, H D is involved in the H-bond; this is     The Journal of Organic Chemistry agreement in with what was observed by solid-state X-ray analysis (vide infra).For compounds 6f,g, the situation is less defined.The ΔE are quite small (0.60 to 1.46 kcal/mol), thus supporting the concurrent presence of both conformations at room temperature.For 6g, the β-turn is preferred in water, whereas in vacuo, the open conformation is more stable.In this case, it can be noticed that for the open conformation, an acceptable dα = 5.95 Å value is found, but the high β angle value and lack of the H-bond gave this compound an open conformation.In conclusion, we can suggest that both the Hyd-loop I and Hyd-loop II are able to stabilize a β-turn conformation, but the presence of a carbonyl within the turn in compounds 6a,b seems to have a great positive effect in stabilizing the turn.
The role of a β-turn is to generate an inversion of the protein chain direction of about 180°and to stabilize the two parallel chains.For this reason, it was also important to investigate with similar tools the effect of the chain elongation on Hyd-loop I and Hyd-loop II.Two representative compounds were selected for the two different loops, namely compounds 8 and 14.Briefly, as predicted, both structures could generate the turn in the 41−53% of cases with little differences between Hyd-loop I and Hyd-loop II and between in vacuo an in-water environments.Similarly, from DFT calculations, both compounds showed a great preference for the β-turn conformation (see the Supporting Information for details).
To further assess the stability of the turn conformation, we decided to further investigate the Hyd-loop I and Hyd-loop II by MD (Figure 7).Compounds 6a,f, 8, and 14 were selected as reference for the two Hyd-loop types both in the minimal and the peptidomimetic forms.The most stable β-turn conformations, as obtained from MM−MC, were therefore submitted to 200 ns of MD simulation in water.The value of dα and β and the presence of the intramolecular H-bond were analyzed through the simulation.In Figure 7, the plots of dα and |β| vs time are reported.As predicted, there is a strong correlation between these two parameters: a change in the dα value beyond the limit value of 7 Å is associated with an increase of the |β| over the 60°limit.Remarkably, from this analysis, a great difference between Hyd-loop I and Hyd-loop II was revealed.Compounds 6a and 8 (Hyd-loop I) showed a greater tendency toward the β-turn conformation, which was rather stable during the dynamic; moreover, a sharp switch to the less favored open conformation is found.For compounds 6f and 14, the β turn is very unstable, and once this conformation is lost, its recovery is not easy.These findings are in good agreement with the results from MM−MC and DFT, according to which Hyd-loop II is less disposed to stabilize the β-turn conformation, especially in a water environment.
Finally, the comparison to classical β-turn types (I, I′, II, and II′) has been made by superimposition of the amide backbones with compound 6a.The best pairing was found with a type I turn with an acceptable root-mean-square deviation (rmsd) = 0.74 Å (type II rmsd = 1.04 Å; type I′ rmsd = 1.25 Å; type II′ rmsd = 1.09Å) In conclusion, from computational investigation, it can be assumed that both the Hyd-loop I and Hyd-loop II scaffolds are able to achieve the desired β-turn conformation, but this tendency is more significant for Hyd-loop I, especially when water is considered as the solvent.
Conformational Analysis: X-ray.Single-Crystal X-ray Structure of 6a.A colorless single crystal of 6a suitable for single-crystal X-ray diffraction (SC-XRD) was obtained by evaporation from AcOEt/MeOH at room temperature.During the crystal handling, no crystal degradation was noticed.The diffraction data was recorded at a synchrotron facility (ALBA, No intramolecular H-bond is observed in the solid state, confirming the minor tendency of Hyd-loop II to adopt Hbond-triggered β-turn conformation. Chloroform molecules are stabilized in the crystal lattice via weak electrostatic interactions O2•••H1S−C1S: 3.086(1) Å.The packing of 6f is shown in Figure 11, where the CHCl 3 solvent molecules form layers separating the layers formed of two 6f molecules.The 6f crystals including chloroform are rather stable at room temperature conditions since we did not observe any crystal decay after the chloroform solution of 6f was evaporated completely in the crystallization process.

■ CONCLUSIONS
In conclusion, we synthesized a collection of racemic peptidomimetics based on two different loops, namely Hydloop I and Hy-loop II, having the structure of Asn embedded into a rigid hydantoin five-membered ring, a flexible glycine or an even more flexible ethylenediamine residue tethered to the Nα of Asn (or N1 of the hydantoin ring), respectively, and two strands of different lengths.The synthesis is straightforward, exploiting a sequential MC domino process followed by deprotection/coupling strategy based on acid/base liquid− liquid purification procedures, thus not requiring further chromatographic purification steps of the intermediates, which is suitable for the possible synthesis of large libraries of such compounds.The tendency to trigger β-turn conformation in vacuo, through MC−MM and Ab initio computations, in solution, through 1 H NMR experiments, and in solid-state, through X-ray analysis, have been performed. 1H NMR experiments showed that Hyd-loop I can trigger a βturn conformation in solution due to the presence of a quite strong intramolecular hydrogen bond involving amidic NH B , regardless of the length of the upper and lower strands.This result is corroborated by molecular modeling that showed that this framework can adopt favored intramolecular H-bonddriven conformations mimicking the β-turn.Quite surprisingly, in solid-state, the β-turn conformation resists even if triggered by a different intramolecular H-bond involving the amidic NH D .Also, more flexible Hyd-loop II can induce the formation of β-turn conformation even if to a lesser extent.Indeed, from molecular modeling, 1 H NMR analysis, and Xray, it seems that the β-turn conformation is favored only when the two peptide-like strands are elongated, in which case, amidic hydrogens NH A and NH B′ could be involved in the formation of intramolecular H-bond favoring the arrangement of the two strands in parallel chains.All these features demonstrate that Hyd-loop I and Hyd-loop II, designed in this work to embed Asp motif in a rigid hydantoin scaffold, could be considered as novel frameworks to promote a more (Hyd-loop I) or less (Hyd-loop II) robust folded β-turn conformation.The synthesis of nonracemic, enantiomerically pure Hyd-loop I-and Hyd-loop II-based peptidomimetics and their biological application are ongoing in our laboratories.

■ MATERIALS AND METHODS
Materials.Commercially available reagent-grade solvents were employed without purification.Compound 4 was synthesized as described in ref 8. Azides 1 and isocyanates 2 are commercially available.Thin-layer chromatography (TLC) was run on silica gel 60 F254 Merck.Visualization of the developed chromatogram was achieved with UV light and ceric ammonium molybdate (CAM) or ninhydrin stains.Flash chromatography (FC) was performed with silica gel 60 (60−200 μm, Merck). 1 H-, and 13 C NMR spectra were run at 400 MHz.Chemical shifts are expressed in ppm (δ), using tetramethylsilane (TMS) as the internal standard for 1 H and 13 C nuclei (δH and δC = 0.00).Structural assignments were made with additional information from gradient COSY (gCOSY) experiments.Electrospray ionization (ESI) mass spectroscopy was performed by a Bruker Esquire 3000+ instrument equipped with an MS detector composed by an ESI ionization source and a single quadrupole mass selective detector or by an Agilent Technologies 1200 Series HPLC system equipped with a diode array detector (DAD) and a 6120 MS detector composed by an ESI ionization source and a single quadrupole mass selective detector.
Chem.Chem. Phys., 8, 3172−3191 (2006)] by means of the "conformer distribution" function, using the MC search method.The Merck molecular force field (MMFF) in vacuo and MMFaq force field were used for the energy minimization of the found structures.The structures were then clustered according to the default setting of the software (which prunes out higher-energy conformers, keeping a diverse set of the low-energy conformers using the rms-torsion definition of nearness).Full geometry optimizations were performed with DFT at the B3LYP 6-31+G (d,p) level in vacuo.All energies were corrected by adding the ZPE as obtained by frequency calculation at the same level.MD simulations were realized with the Yasara program using the AMBER14 force field.The structures were analyzed through a 200 ns simulation in water at pH 7.4 and in physiological ion concentration (0.9% NaCl).
X-ray Analysis.6a was recorded using high-resolution synchrotron X-ray radiation at the Alba-Cells synchrotron (Barcelona) center.The wavelength used in the experiments was λ = 0.82656 Å.Data were indexed, integrated, and scaled using the xia2 program 19 with the DIALS pipeline for small molecule 20 at the B13-XALOC Macromolceular Crystallography beamline.The structure was determined using direct methods (SHELXTL 97) and refined (based on F2 using all independent data) by full-matrix least-squares methods (SHELX 2014). 21All non-hydrogen atoms were located from different Fourier maps and refined with anisotropic displacement parameters.Hydrogen atoms were added in riding positions.
The single-crystal X-ray data for 6f have been recorded at the Elettra Synchrotron facility (Italy).Single crystals of 6f dipped in NVH oil (Jena Bioscience, Jena, Germany), mounted at room temperature on Kapton loops (MiTeGen, Ithaca, USA) and flashfrozen in liquid nitrogen.Two data sets were recorded, and only the best data quality sample was used for refinement and structural analysis.
6f has been characterized at the XRD1 beamline 22 of the Elettra synchrotron, Trieste (Italy), through X-ray diffraction and the rotating crystal method.During data collection, the sample has been kept at 100 K via a gas nitrogen stream (Oxford Cryostream 700�Oxford Cryosystems Ltd., Oxford, United Kingdom).The diffraction images were acquired using a monochromatic wavelength of 0.700 Å and a Pilatus 2 M area detector (DECTRIS Ltd., Baden-Daettwil, Switzerland) for a total rotation of 180°.
The raw diffraction data were indexed and integrated using XDS. 23 The structure was solved by the dual space algorithm implemented in SHELXT. 24Fourier analysis and refinement were performed by the full-matrix least-squares methods implemented in SHELXL, 21 based on F2.
General Procedure for the MC Synthesis of Intermediates 6.To a stirred solution of azide 1 (2.5 mmol, 1.0 equiv) and isocyanate 2 (2.5 mmol, 1.0 equiv) in CH 3 CN (0.1 M), solid Ph 3 P (2.7 mmol, 1.1 equiv) was added, and the resulting solution was stirred overnight.2,4,6 Trimethylpyridine (TMP, 2.7 mmol, 1.1 equiv) followed by acid 3 (2.7 mmol, 1.1 equiv) were added at room temperature.Once intermediate 4 is formed (3−4 h, TLC monitoring), the amine component (2.7 mmol, 1.1 equiv) is added, and the mixture stirred at room temperature overnight.The solution was diluted with AcOEt and washed with a 1 M aqueous HCl solution, brine, a saturated aqueous solution of NaHCO 3 , and brine once again.The combined organic layers were dried on Na 2 SO 4 , filtered, and the organic solvent evaporated.The crude was purified by FC affording compounds 7 in 67−81% yields.
General Procedure for the Synthesis of Hyd-Loop II Peptidomimetic 12, 17.To a solution of compound 6g (or 6i) (0.50 mmol, 1.0 equiv) in a 1:1 mixture of AcOEt/MeOH (0.1 M), a catalytic amount of Pd/C (0.1 mmol, 0.2 equiv) was added.The mixture was stirred under a H 2 atmosphere until completion of the reaction (TLC monitoring).The mixture was filtered over a Celite pad, the solvent evaporated, and the crude submitted to the next step without further purification.The acid obtained as described above was dissolved in DMF (0.1 M solution), the temperature cooled to 0 °C, and HBTU (0.52 mmol, 1.05 equiv) was added.After 10 min, H− Gly−CONH−PMB (0.52 mmol, 1.05 equiv) followed by DIPEA (0.52 mmol, 1.05 equiv) were added, and the solution was stirred at room temperature overnight.The solution was diluted with a 1 M aqueous HCl solution and AcOEt and was extracted three times with AcOEt.The collected organic layers were washed with brine (once), a saturated aqueous solution of NaHCO 3 (twice), and brine (twice).The combined organic layers were dried on Na 2 SO 4 , filtered, and the organic solvent evaporated affording intermediate 11 (or 15), which was used in the following step without any further purification.Compound 11 (or 15) was dissolved in DCM (0.1 M solution), and TFA (10% in volume) was added.The solution was stirred until completion of the reaction (ca. 1 h, TLC monitoring).The solvents were evaporated and coevaporated twice with cyclo-hexane.The crude was dissolved in DMF (0.1 M solution), and HBTU (0.52 mmol, 1.05 equiv) followed by a solution of the amine (0.52 mmol, 1.05 equiv) dissolved in a minimum amount of DMF and DIPEA (0.52 mmol, 1.05 equiv) were added, and the resulting solution was stirred at room temperature overnight.The solution was diluted with a 1 M aqueous HCl solution and AcOEt and was extracted three times with AcOEt.The collected organic layers were washed with brine (once), a saturated aqueous solution of NaHCO 3 (twice), and brine (twice).The combined organic layers were dried on Na 2 SO 4 , filtered, and the organic solvent evaporated.The crude was purified by FC affording peptidomimetic 12 (or 17).
General Procedure for the Synthesis of Hyd-Loop II Peptidomimetic 14. Compound 6g (0.50 mmol, 1.0 equiv) was dissolved in DCM (0.1 M solution), and TFA (10% in volume) was added.The solution was stirred until completion of the reaction (ca. 1 h, TLC monitoring).The solvents were evaporated and coevaporated twice with cyclo-hexane.The crude was dissolved in DMF (0.1 M solution), and HBTU (0.52 mmol, 1.05 equiv) followed by a solution of the amine (0.52 mmol, 1.05 equiv) dissolved in a minimum amount of DMF and DIPEA (0.52 mmol, 1.05 equiv) were added, and the resulting solution was stirred at room temperature overnight.The solution was diluted with a 1 M aqueous HCl solution and AcOEt and was extracted three times with AcOEt.The collected organic layers were washed with brine (once), a saturated aqueous solution of NaHCO 3 (twice), and brine (twice).The combined organic layers were dried on Na 2 SO 4 , filtered, and the organic solvent evaporated affording intermediate 13, which was used in the following step without any further purification.Compound 13 was dissolved in a 1:1 mixture of AcOEt/MeOH (0.1 M), and a catalytic amount of Pd/C (0.1 mmol, 0.2 equiv) was added.The mixture was stirred under a H 2 atmosphere until completion of the reaction (TLC monitoring).The mixture was filtered over a Celite pad, the solvent evaporated, and the crude submitted to the next step without further purification.The acid obtained as described above was dissolved in DMF (0.1 M solution), the temperature cooled to 0 °C, and HBTU (0.52 mmol, 1.05 equiv) was added.After 10 min, a solution of the amine (0.52 mmol, 1.05 equiv) dissolved in a minimum amount of DMF followed by DIPEA (0.52 mmol, 1.05 equiv) were added, and the solution was stirred at room temperature overnight.The solution was diluted with a 1 M aqueous HCl solution and AcOEt and was extracted three times with AcOEt.The collected organic layers were washed with brine (once), a saturated aqueous solution of NaHCO 3 (twice), and brine (twice).The combined organic layers were dried on Na 2 SO 4 , filtered, and the organic solvent evaporated.The crude was purified by FC affording 14.

Figure 6 .
Figure 6.Geometrical parameters for the β-turn conformation for model compounds.

Figure 9 .
Figure 9. Crystal packing in 6a viewed along the b-axis.

Figure 10 .
Figure 10.Intermolecular H-bonds (black dashed lines) among two 6f molecules.Chloroform molecules have been removed for the sake of clarity.Distances in Å.

Figure 11 .
Figure 11.Crystal packing of 6f viewed along the a-axis including solvent CHCl 3 .

Table 2 .
Chemical Shifts of Amidic Protons CONH in Peptidomimetics Built on Hyd-Loops I and II a entry compound δ NH A δ NH B δ NH B′ δ NH C δ NH D δ NH D′

Table 4 .
Results from MM−MC Conformational Analysis: (a) In Vacuo and (b) in Water Results as Percentage of Conformers Meeting the Requirements

Table 6 .
Results from DFT Calculations a a The values of dα (Å) and β (deg) are also reported.b Not determined.