β-Peptides incorporating polyhydroxylated cyclohexane β-amino acids: synthesis and conformational study

We describe the synthesis of trihydroxylated cyclohexane β-amino acids from (–)-shikimic acid, in their cis and trans configuration, and the incorporation of the trans isomer into a trans -2- aminocyclohexanecarboxylic acid peptide chain. Subsequently, the hydroxyl groups were partially or totally deprotected. The structural study of the new peptides by FTIR, CD, solution NMR and DFT calculations revealed that they all fold into a 14-helix secondary structure, similarly to the homooligomer of trans -2-aminocyclohexanecarboxylic acid. This means that the high degree of substitution of the cyclohexane ring of the new residue is compatible with the adoption of a stable helical secondary structure and opens opportunities for the design of more elaborate peptidic foldamers with oriented polar substituents at selected positions of the cycloalkane residues.


Introduction
6][17] This has led, for instance, to the design of bioactive β-peptides [18][19][20] , or self-assembling nanomaterials [21][22][23][24] and catalysts. 25,26e folding properties of carbocyclic β-peptidic foldamers have been widely studied.Pioneering work demonstrated that homooligomers of trans-2-aminocyclohexanecarboxylic acid (trans-ACHC) fold as a robust 14-helix in short peptide sequences 15,27 while homooligomers of trans-2aminocyclopentanecarboxylic acid (trans-ACPC) fold as a 12-helix 16,28,29 in the crystal and in solution.Other secondary structures can be accessed by modification of the backbone stereochemical configuration 30,31 or the side chains 32,33 of homooligomers.Another strategy to tune the peptide secondary structure has been the combination of two or more β-amino acids into heterooligomers. 6,34- 37 ost of these studies have been carried out with highly hydrophobic compounds because the number of foldamers containing cyclic β-amino acid monomers functionalized with polar substituents is still limited. 6e most frequent strategy to obtain polar or water soluble foldamers based on cyclic β-amino acids has been the synthesis of heterooligomers of apolar trans-ACPC or trans-ACHC residues with structurally simple polar acyclic β-amino acids holding a polar group in their side chain (e.g.9][40][41][42] Other examples have the polar group in an otherwise structurally simple cyclic amino acid, as the trans-4-aminopyrrolidine-3-carboxylic acid (trans-APC) 18,20,34 , trans-DCHC 43,44 , or an aza-ACPC analogue. 45lyhydroxylated cycloalkane β-amino acids are particularly interesting targets, because their rich functionality makes them useful scaffolds for the access of a variety of lipo-or hydrosoluble peptides, by protection or deprotection of their hydroxy substituents.In addition, they can bear pharmacophoric groups in well defined spatial orientations, a property that may facilitate their interaction with biological receptors, or with substrates if the goal is to use them as peptidic catalysts.[48] In connection with our interest in polyhydroxylated cycloalkane amino acids and their peptides, 49,50 we present here an efficient stereoselective synthesis of two new polyhydroxylated cyclohexane βamino acids with cis-and trans-configurations, starting from commercially available (-)-shikimic acid.The newly synthesized trans-β-amino acid was incorporated into a short hybrid peptide also containing trans-ACHC and its structure in solution has been studied.

Results and discussion
Synthesis of polyhydroxylated cyclohexane β-amino acids 5 and 8 (-)-Shikimic acid is an attractive starting material on account of its convenient structural properties: a preformed cyclohexane ring bearing an α,β-unsaturated carboxylic acid moiety and three hydroxy substituents with well-defined spatial orientations. 51,52.The synthetic plan involved a stereoselective Michael addition of dibenzylamines to α,β-unsaturated esters, in this case methyl shikimates. 53dition of the known shikimate ester 1 50 to a cooled (-78 ºC) solution of lithium amide (R)-2 in THF provided, after 2h reaction followed by work-up with saturated aq.ammonium chloride solution, a mixture of cis-β-amino acid 3 (major component, 61% yield) and trans-β-amino acid 6 (minor component, 5% yield).In addition, reaction of compound 3 with a 2 M solution of NaOMe in MeOH resulted in its isomerization to compound 6 (89% yield).Compounds 3 and 6 were easily identified from their analytical and spectroscopic data.The structure of compound 3 was unambiguously established by X-ray crystallography.

Scheme 1
Catalytic hydrogenation of compound 3, using Pd(OH)2, provided amino acid ester 4, which upon reaction with 6 M HCl resulted in the formation of the new trihydroxylated cis-2aminocyclohexanecarboxylic acid 5 (76% yield over the two steps).Similarly, compound 6 provided the new trihydroxylated trans-2-aminocyclohexanecarboxylic acid 8 (77% yield over the three steps), via compound 7.

Compound 3 ORTEP diagram
The favoured Michael addition of the chiral amide (R)-2 to the si-face of the α,β-unsaturated carboxylic acid ester moiety of 1 can be justified as a case of double chiral induction by the reagent and the substrate, leading to the stereospecific formation of the intermediate enolate shown in Scheme 2 by the complexation of the litium ion with the carbonyl oxygen and the amino atoms.Then, protonation of this enolate can occur according either to path a (more favoured) or path b (less favoured), the result being the formation of a mixture of compound 3a (kinetic isomer, major component) and compound 6 (thermodynamic isomer, minor component) (Scheme 2). 54,55 chose to introduce the nitrogen substituent with amide (R)-2 because it highly favours the formation of the cis-β-amino ester 3 (kinetic product), which can then be quantitatively transformed into the trans-β-amino ester 6 (thermodynamic product).Although dibenzylamine could also be tried, our previous experience with similar compounds indicated that it would likely lead to a mixture of both isomers in uncertain proportions. 50

Synthesis of pentamers 14, 15 and 16, containing trans-polyhydroxylated cyclohexane β-amino acid 8
Oligomers of trans-ACHC are known to adopt the 14-helix secondary structure. 15,16,27We synthesized pentamer 14 that has the functionalized β-amino acid 8 in the central position, flanked by trans-ACHC residues.Sequential deprotection of the hydroxyls of 14 led to pentamers 15 and 16, which allowed us to study the influence of their degree of polarity and steric hindrance on their propensity to adopt the 14-helix secondary structure.

Scheme 3
Pentamer 14 was transformed into the monohydroxylated pentamer 15 in 76 % yield by treatment with TBAF.Finally, pentamer 15 was transformed into the trihydroxylated pentamer 16 when submitted to a hydrolysis reaction catalyzed by acetic acid.All three pentamers 14-16 were purified and then subjected to structural studies in solution.

Infrared spectroscopy
We measured the ATR-FTIR spectra of dimer 9, trimer 11 and the three pentamers 14-16 in the solid state to assess the formation of hydrogen bonds.Significant differences were observed for the vibration of the Amide A band, with clearly lower wavenumber values for pentamers 14 (3281 cm -1 ) and 16 (3269 cm -1 ) than for dimer 9 (3297 cm -1 ) and trimer 11 (3327 cm -1 ) as shown in Figure 1A.
The vibration Amide II band shifts to higher wave numbers from dimer 9 (1533 cm -1 ) and trimer 11 (1528 cm -1 ) to pentamers 14 (1536 cm -1 ), 15 (1547 cm -1 ) and 16 (1547 cm -1 ) (Figure 1B).Differences in the Amide I band of the five compounds were negligible.The signals of Amide A and Amide II are compatible with the increase of hydrogen bonding as the polyamide chain gets longer.Such behaviour is the expected for a higher level of secondary structure formation as oligomer length increases.Upon formation of H-bonds, vibration frequencies of Amide A shift to lower wavenumbers, while vibrations shift of Amide II shift to higher wavenumber.
However, this information is not sufficient to determine what fold (e.g.what helix type) is adopted by the peptide. 56,57

CD spectroscopy
Circular dichroism (CD) spectroscopy can be used to establish the secondary structure of polyamide chains, since each type of secondary structure gives rise to a characteristic CD spectrum in the far ultraviolet region (190-240 nm). 58In fact, it has been used routinely to establish the secondary structure of peptide α-amino acids and even establish the proportion of each of these secondary structures in natural proteins. 59For the case of polyamides constituted by other than α-amino acids, there is no simple way to correlate far-ultraviolet CD spectra with their specific conformations, 60 but comparison of CD data from new and existing polyamides can provide useful information if structures are similar.
The CD spectra of dimer 9, trimer 11 and pentamer 14, at 1 mM concentration in methanol (Figure 2a), present an ellipticity maximum in the far ultraviolet region around 220 nm, that arises from the backbone amide groups.The intensity of this maximum increases with β-peptide length.We then compared the CD spectra of the three differently protected pentamers 14-16, at 1 mM concentration in methanol solution (Figure 2a).The three pentamers present an absorption maximum in the far ultraviolet region of about 220 nm.The slight shift of θmax (221 nm for 14, 221 nm for 15 and 217 nm for 16) can be justified assuming that the conformation of the helices are slightly different when the substituted cyclohexane ring is strained by the acetonide group (14 and 15) to when this acetonide is not present (16).Indeed, the value of θmax of 16 is identical to the reported value for the unsubstituted trans-ACHC oligomers (217 nm).Slight shifts of the CD maxima have also been attributed to different populations of the 14-helix averaging with other conformers existing in solution. 27,41ditionally, we performed CD experiments with pentamer 14 by changing the concentration and temperature (Figure 2b).Increasing the concentration resulted in higher intensity but no shift of the θmax, consistent with the molecule not aggregating in this concentration range.More interestingly, negligible changes occurred upon increasing temperature from 0 ºC to 25 ºC, demonstrating the overall stability of the structure.

NMR spectroscopy
We analyzed the structure of pentamers 14-16 by NMR spectroscopy in CDCl3, DMSO-d6, and methanol-d3 solutions.Dispersion of chemical shifts in DMSO-d6 solution was sufficient to analyze the NMR spectra of compounds 14-16. 61Amide HN resonances were well dispersed in the 1D proton spectra, suggesting a high population of a single well-defined conformation of each compound.Residue-specific assignments of the backbone HN, Hα, and Hβ protons were made based on a combination of COSY, TOCSY, and ROESY spectra.The other side-chain resonances were typically not assigned due to extensive overlap (see tables of assignments in SI).The backbone fold of the peptides was determined from NOE contacts characteristic of the 14-helix secondary structure, like the strong long-range Hα(i)/Hβ(i+3) and sequential HN(i)/Hα(i-1) NOEs, that correspond to H-H distances of ≈ 2.6 Å in a 14-helix. 15,37,40,61Further support to the 14-helix fold comes from the HN(i)/Hβ(i+2) and the HN(i)/Hβ(i+3) NOE peaks of medium intensity, that correspond to distances in the range of 3.0-3.5Å (see the SI for tables of NOEs of the three peptides).Some NOEs from residue 3 to the terminal t-Bu and OBn groups are also compatible with the 14-helix conformation Å (see the SI for tables of NOEs of the three peptides).Restrained molecular dynamics calculated with XPLOR-NIH 62,63 using the NOE and scalar coupling data led to structures with the 14-helical fold for peptides 14-16.These models were further optimized using density functional theory (DFT) calculations that employed the hybrid density functional M05-2X 64 with the 6-31+G(d) basis set.DFT calculations were performed using Gaussian 09. 65 analyze the alignment of the atoms intervening in hydrogen bonding, we considered the distances and angles listed in Table 1.In principle, as it is stated in the IUPAC definition for hydrogen bonds, the closer the X-H•••Y angles angle is to 180º, the stronger is the hydrogen bond and the shorter is the H•••Y distance. 66The structure of 16 resembled very closely the geometry of the crystallographic structure of the trans-ACHC homohexamer, 15    We studied peptide 16 in CD3-OH solution at 500 and 750 MHz between 273 and 298 K.The amide HN peaks in the 1D 1 H spectrum were consistent with the existence of a major conformer and at least one minor conformations.Assignment of backbone resonances was not possible due to the overlap of the Hα(i) peaks even with spectra recorded at 750 MHz.Furthermore, the HN(i)/Hβ(i), HN(i)/Hβ(i+2) and HN(i)/Hβ(i+3) peaks were very weak in the ROESY and NOESY spectra recorded in methanol-d3, even when the sample was cooled to 273 K.This can be explained if the structure is not too rigid, such that the amide HN protons can exchange and that the major conformer is in equilibrium with other conformations or aggregates in the methanol solution.
In summary, the FTIR, CD, NMR and DFT calculations data support that pentamers 14-16 adopt the expected 14-helical fold, similarly to homooligomers of trans-ACHC, regardless the functionalization of residue 3. The strain of the cyclohexane ring due to the fused five membered acetonide ring (in pentamers 14-15) or the bulky hydrophobic TBS group protecting the Oε(3) atom (in pentamer 14) do not impede the adoption of a backbone conformation compatible with the 14-helix (i.e. that placing the carbonyl and the amino groups in equatorial orientation), at least in these short peptides.Although CD spectra can not be measured in DMSO, but only in methanol, the solution NMR analysis of peptide 16 in DMSO-d6 and methanol-d3 revealed that it adopts essentially the same fold in both solvents.

Conclusions
In conclusion, we present here a stereocontrolled synthesis of highly functionalized cyclohexane βamino acids from (-)-shikimic acid that has allowed the synthesis, on the gram scale, of the first trihydroxylated cyclohexane β-amino acids described having cis-or trans-relative configurations in their free form (5 and 8) or orthogonally protected for their incorporation into peptides (4 and 7, respectively).The availability of highly functionalized β-amino acids is useful for obtaining cycloalkane β-peptides with polar side chains, including the previously unreported polyhydroxylated cycloalkane b-amino acids, of potential interest in chemical biology and material sciences.As an example, herein we present the synthesis of a pentameric β-peptide containing the orthogonally protected trans β-amino acid 7 at an internal position, flanked by apolar trans-ACHC residues.We also demonstrate the chemical stability of this class of peptides (by sequentially deprotecting the hydroxyl groups of pentamer 14 to give pentamers 15 and 16), and their conformational stability, as the structural studies revealed that they adopt a 14-helix fold despite the bulky substituents of the cyclohexane ring.Work to construct more elaborate peptides containing combinations of this new polar residue with apolar ones is in progress to obtain functionalized and potentially amphiphilic peptides.

General information
All non-aqueous reactions were carried out under a positive atmosphere of argon in flamedried glassware unless otherwise stated.Air-and moisture-sensitive liquid reagents were added by dry syringe or cannula.Anhydrous tetrahydrofuran (THF) was freshly distilled from sodium/benzophenone under argon and all other solvents and reagents were used as obtained from commercial sources without further purification unless stated.Flash chromatography was performed using 60 Merck 230-400 mesh (flash, 0.04-0.063)silica.Thin layer chromatography (tlc) was carried out on aluminium backed sheets coated with 60 GF254 silica.Plates were developed using a spray of 0.2% w/v cerium(IV) sulfate and 5% ammonium molybdate in 2 M sulfuric acid, or in 5% w/v ninhydrin in methanol.Only the characteristic peaks are quoted (in units of cm -1 ), st, m, and br designate strong, medium, and broad, respectively.All the spectra were measured in KBr unless stated.Optical rotations were measured on a Jasco DIP-370 polarimeter with a path length of 0.5 dm and Na (589 nm) lamp.Concentrations are given in g/100 mL.

Tripeptide 12
LiOH•H2O (50 mg, 1.180 mmol) was added to a solution of dipeptide 11 (336 mg, 0.472 mmol) in THF/MeOH/H2O (1:1:1) (8 mL) at 0 °C.The mixture was stirred over 24 h at room temperature.The solvent was removed under reduced pressure, the mixture was diluted with a 10 % citric acid solution (5 mL) and extracted with Et2O (3 x 10 mL).The combined organic layers were dried over Na2SO4 anhydrous, filtered and concentrated to afford Tripeptide 12 (301 mg, 92%) as a white solid that was used in the next reaction without further purification.
Pentapeptide 14 EDCI•HCl (54 mg, 0.282 mmol) was added over a solution of dipeptide 12 (89 mg, 0.128 mmol) in dry DMF (0.91 mL) and the mixture was stirred over 30 min.Then, Tripeptide 13 (46 mg, 0.128 mmol) in dry DMF (0.91 mL) and DMAP (47 mg, 0.384 mmol), were added to the mixture and the reaction was stirred 21 h more under an argon atmosphere.Then, the solvent was removed under vacuum, the residue was redissolved in DCM (5 mL) and washed with 5 mL portions of HCl (1M), NaCl (sat), NaHCO3 (sat.) and NaCl (sat).The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum.

Infrared experimental
Fourier-transform infrared spectroscopy (FTIR) spectra were acquired using a MIDAC Prospect FT-IR PerkinElmer Spectrum Two spectrometer.Samples were deposited on an ATR diamond accessory as a dry solid.

Circular Dichroism experimental
Circular dichroism (CD) spectra were acquired with an JASCO DIP-370 optical polarimeter using a 1 mm path length quartz cell (Hellma Analytics, Germany).Wavelength scans were collected between 260 and 195 nm with a 1nm bandwidth, 1 nm data interval and 100 nm/min scanning speed.
Samples were prepared by dissolving dry peptide in HPLC-grade methanol (Fisher Scientific) to give 1 mM solutions.Samples of 700, 500, and 100 μM concentration were prepared by serial dilution of the 1 mM stock.Three scans of each peptide solution were taken and averaged.Solvent blanks were subtracted from the raw spectra and smoothened over 17 data points prior to normalization to molar ellipticity.CD signal was converted into molar ellipticity ([Θ], deg cm 2 dmol -1 ) using the equation: [Θ] = 100•Ψ / (l•c) Where Ψ is the raw ellipticity in degrees, l is the path length in decimeters, and c the is molar concentration.
NMR spectroscopy and structure calculation of peptides 14-16.NMR spectra were recorded on Bruker Avance III 500 and NEO 750 spectrometers operating at 500 MHz and 750 MHz, respectively.The resonance of tetramethylsilane (TMS) was used as chemical shift reference in the 1 H NMR experiments (δTMS = 0.00 ppm).Samples for NMR experiments were prepared by dissolving peptides in 550 μL of deuterated solvent to a final concentration of 1-10 mM.Amide proton temperature coefficients were studied by recording 1D 1 H spectra at temperatures between 288 and 323 K in 10 K steps.Values are reported in ppb/K.Two-dimensional (2D) 1 H homonuclear spectra (COSY, TOCSY, ROESY, and NOESY) were recorded using standard pulse sequences.Each 2D spectrum was collected as a data matrix consisting of 2048 (t2) × 256 (t1) complex points and a sweep width of 5000 Hz.TOCSY spectra were recorded using the MLEV pulse sequence with a mixing time of 70 ms unless otherwise stated.ROESY experiments were acquired with mixing times of 120 and 200 ms.Spectra were processed using the programs TopSpin and MestreNova.Peptide resonance assignments were obtained using standard strategies based on two-dimensional NMR experiments.The NOEs were classified into three groups of strong, medium, and weak with upper bounds of 2.5, 3.5 and 5.0 Å, respectively.Structural models were calculated by restrained molecular dynamics with XPLOR-NIH 62,63 using the NOE and scalar coupling data.These models were further optimized using DFT calculations that employed the hybrid density functional M05-2X 64 with the 6-31+G(d) basis set.DFT calculations were performed using Gaussian 09. 65
the shorter O•••N and O•••H distances and larger C=O•••H angles.In contrast, the geometry of the hydrogen bonds of peptides 14 and 15 in the DFT optimized models was a bit distorted, while keeping the 14-helix fold, due to the steric constraints imposed by the substituents on positions γ, δ and ε of residue 3.This agrees well with the CD data recorded in methanol, as the maximum ellipticity of peptides 14 and 15 deviates a few nm from the values of the trans-ACHC homohexamer and peptide 16.
HCl aqueous solution (8 mL) is refluxed for 15 h.The solvent was concentrated in vacuum, and the residue was redissolved in H2O (1 mL).Activated Dowex 50WX4-50 was added, and the mixture was stirred for 1 h.Then, the Dowex was washed with water and MeOH.The compound was released of the resin with a 10 % aqueous solution of NH3.The combined aqueous layers were concentrated at vacuum to give 8 (0.031 g, 0.16 mmol, 84%) as pale-yellow oil.[α]D 23 : -26.9 (c 2.0, H2O).The mixture was stirred over hydrogen atmosphere for 16 h, then was filtered over celite and washed with MeOH.The solvent was removed under vacuum to afford dipeptide 10 (135 mg) as a white solid that was used in the next reaction without further purification.