Synthesis of β3‐Peptides and Mixed α/β3‐Peptides by Thioligation

Five β‐peptide thioesters ( 1 – 5 , containing 3, 4, 10 residues) were prepared by manual solid‐phase synthesis and purified by reverse‐phase preparative HPLC. A β‐undecapeptide ( 6 ) and an α‐undecapeptide ( 7 ) with N‐terminal β³‐HCys and Cys residues were prepared by manual and machine synthesis, respectively. Coupling of the thioesters with the cysteine derivatives in the presence of PhSH (Scheme and Fig. 1) in aqueous solution occurred smoothly and quantitatively. Pentadeca‐ and heneicosapeptides ( 8 – 10 ) were isolated, after preparative RP‐HPLC purification, in yields of up to 60%. Thus, the so‐called native chemical ligation works well with β‐peptides, producing larger β³‐ and α/β³‐mixed peptides. Compounds 1 – 10 were characterized by high‐resolution mass spectrometry (HR‐MS) and by CD spectroscopy, including temperature and concentration dependence. β‐Peptide 9 with 21 residues shows an intense negative Cotton effect near 210 nm but no zero‐crossing above 190 nm, (Figs. 2–4), which is characteristic of β‐peptidic 3₁₄‐helical structures. Comparison of the CD spectra of the mixed α/β‐pentadecapeptide ( 10 ) and a helical α‐peptide (Fig. 5) indicate the presence of an α‐peptidic 3.6₁₃ helix.     

Synthesis of β‐Hexa‐ and β‐Heptapeptides Containing Novel β2,3‐Amino Acids with Two Serine or Two Cysteine Side Chains – CD‐ and NMR‐Spectroscopic Evidence for 314‐Helical Secondary Structures in Water

Two representatives of a new type of β‐amino acids, carrying two functionalized side chains, one in the 2‐ and one in the 3‐position, have been prepared stereoselectively: a β‐Ser derivative with an additional CH₂OH group in the 2‐position (for β‐peptides with better water solubility; Scheme 2) and a β‐HCys derivative with an additional CH₂SBn group in the 2‐position (for disulfide formation and metal complexation with the derived β‐peptides; Scheme 3). Also, a simple method for the preparation of α‐methylidene‐β‐amino acids is presented (see Boc‐2‐methylidene‐β‐HLeu‐OH, 8 in Scheme 3). The two amino acids with two serine or two cysteine side chains are incorporated into a β‐hexa‐ and two β‐heptapeptides ( 18 and 23/24 , resp.), which carry up to four CH₂OH groups. Disulfide formation with the β‐peptides carrying two CH₂SH groups generates very stable 1,2‐dithiane rings in the centre of the β‐heptapeptides, and a cyclohexane analog was also prepared (cf. 27 in Scheme 6). The CD spectra in H₂O clearly indicate the presence of 3₁₄‐helical structures of those β‐peptides ( 18 , 23 , 24 , 27b ) having the `right' configurations at all stereogenic centers (Fig. 2). NMR Measurements (Tables 1 and 2, and Fig. 4) in aqueous solution of one of the new β‐peptides ( 24 ) are interpreted on the assumption that the predominant secondary structure is the 3₁₄‐helix, a conformation that has been found to be typical for β‐peptides in MeOH or pyridine solution, according to our previous NMR investigations.     

Preparation of Protected β2‐ and β3‐Homocysteine, β2‐ and β3‐Homohistidine,and β2‐Homoserine for Solid‐Phase Syntheses

The Ser, Cys, and His side chains play decisive roles in the syntheses, structures, and functions of proteins and enzymes. For our structural and biomedical investigations of β‐peptides consisting of amino acids with proteinogenic side chains, we needed to have reliable preparative access to the title compounds. The two β³‐homoamino acid derivatives were obtained by Arndt–Eistert methodology from Boc‐His(Ts)‐OH and Fmoc‐Cys(PMB)‐OH (Schemes 2–4), with the side‐chain functional groups' reactivities requiring special precautions. The β²‐homoamino acids were prepared with the help of the chiral oxazolidinone auxiliary DIOZ by diastereoselective aldol additions of suitable Ti‐enolates to formaldehyde (generated in situ from trioxane) and subsequent functional‐group manipulations. These include OH→O^tBu etherification (for β²hSer; Schemes 5 and 6), OH→STrt replacement (for β²hCys; Scheme 7), and CH₂OH→CH₂N₃→CH₂NH₂ transformations (for β²hHis; Schemes 9–11). Including protection/deprotection/re‐protection reactions, it takes up to ten steps to obtain the enantiomerically pure target compounds from commercial precursors. Unsuccessful approaches, pitfalls, and optimization procedures are also discussed. The final products and the intermediate compounds are fully characterized by retention times (t_R), melting points, optical rotations, HPLC on chiral columns, IR, ¹H‐ and ¹³C‐NMR spectroscopy, mass spectrometry, elemental analyses, and (in some cases) by X‐ray crystal‐structure analysis.     

β‐Peptidic Secondary Structures Fortified and Enforced by Zn2+ Complexation – On the Way to β‐Peptidic Zinc Fingers?

The correlation between β²‐, β³‐, and β^2,3‐amino acid‐residue configuration and stability of helix and hairpin‐turn secondary structures of peptides consisting of homologated proteinogenic amino acids is analyzed (Figs. 1–3). To test the power of Zn²⁺ ions in fortifying and/or enforcing secondary structures of β‐peptides, a β‐decapeptide, 1 , four β‐octapeptides, 2 – 5 , and a β‐hexadecapeptide, 10 , have been devised and synthesized. The design was such that the peptides would a) fold to a 14‐helix ( 1 and 3 ) or a hairpin turn ( 2 and 4 ), or form neither of these two secondary structures (i.e., 5 ), and b) carry the side chains of cysteine and histidine in positions, which will allow Zn²⁺ ions to use their extraordinary affinity for RS^? and the imidazole N‐atoms for stabilizing or destabilizing the intrinsic secondary structures of the peptides. The β‐hexadecapeptide 10 was designed to a) fold to a turn, to which a 14‐helical structure is attached through a β‐dipeptide spacer, and b) contain two cysteine and two histidine side chains for Zn complexation, in order to possibly mimic a Zn‐finger motif. While CD spectra (Figs. 6–8 and 17) and ESI mass spectra (Figs. 9 and 18) are compatible with the expected effects of Zn²⁺ ions in all cases, it was shown by detailed NMR analyses of three of the peptides, i.e., 2, 3, 5 , in the absence and presence of ZnCl₂, that i) β‐peptide 2 forms a hairpin turn in H₂O, even without Zn complexation to the terminal β³hHis and β³hCys side chains (Fig. 11), ii) β‐peptide 3 , which is present as a 14‐helix in MeOH, is forced to a hairpin‐turn structure by Zn complexation in H₂O (Fig. 12), and iii) β‐peptide 5 is poorly ordered in CD₃OH (Fig. 13) and in H₂O (Fig. 14), with far‐remote β³hCys and β³hHis residues, and has a distorted turn structure in the presence of Zn²⁺ ions in H₂O, with proximate terminal Cys and His side chains (Fig. 15).     

NMR‐Structural Investigations of a β3‐Dodecapeptide with Proteinogenic Side Chains in Methanol and in Aqueous Solutions

The structural properties of an all‐β³‐dodecapeptide with the sequence H‐β‐HLys(N^ε‐CO(CH₂)₃‐S Acm)‐β‐HPhe‐β‐HTyr‐β‐HLeu‐β‐HLys‐β‐HSer‐β‐HLys‐β‐HPhe‐β‐HSer‐β‐HVal‐β‐HLys‐β‐HAla‐OH ( 1 ) have been studied by two‐dimensional homonuclear ¹H‐NMR and by CD spectroscopy. In MeOH solution, high‐resolution NMR spectroscopy showed that the β‐dodecapeptide forms an (M)‐3₁₄‐helix, and the CD spectrum corresponds to the pattern expected for an (M)‐3₁₄‐helical secondary structure. In aqueous solution, however, the peptide adopts a predominantly extended conformation without regular secondary‐structure elements, which is in agreement with the absence of the characteristic trough near 215 nm in the CD spectrum. The NMR and CD measurements with solutions of 1 in MeOH containing 3M urea further indicated that the peptide retains the regular secondary structural elements under these conditions, whereas, after addition of 40% (v/v) H₂O to the MeOH solution, the large ¹H‐chemical‐shift dispersion indicative of a defined spatial peptide fold was lost. The β³‐dodecapeptide is – so far – the longest β‐peptide shown to adopt a regular (M)‐3₁₄‐helix conformation in an organic solvent. The observation that the structure of this long β³‐peptide is not maintained in aqueous solution indicates that the (M)‐3₁₄‐fold is primarily stabilized by short‐range interactions.     

cyclo(β‐Asp‐β3‐hVal‐β3‐hLys) – Solid‐Phase Synthesis and Solution Structure of a Water Soluble β‐Tripeptide. Preliminary Communication

The H₂O‐soluble cyclic β³‐tripeptide cyclo(β‐Asp‐β³‐hVal‐β³‐hLys) ( 4 ) was obtained by on‐resin cyclization of the side‐chain‐anchored β‐peptide 3 (Scheme). In aqueous solution, 4 adopts a structure with uniformly oriented amide bonds and all side chains in lateral positions (Fig. 3).     

Stereoselective Preparation of 3‐Amino‐2‐fluoro Carboxylic Acid Derivatives,and Their Incorporation in Tetrahydropyrimidin‐4(1H)‐ones,and in Open‐Chain and Cyclic β‐Peptides

The preparation of (2S,3S)‐ and (2R,3S)‐2‐fluoro and of (3S)‐2,2‐difluoro‐3‐amino carboxylic acid derivatives, 1 – 3 , from alanine, valine, leucine, threonine, and β³h‐alanine (Schemes 1 and 2, Table) is described. The stereochemical course of (diethylamino)sulfur trifluoride (DAST) reactions with N,N‐dibenzyl‐2‐amino‐3‐hydroxy and 3‐amino‐2‐hydroxy carboxylic acid esters is discussed (Fig. 1). The fluoro‐β‐amino acid residues have been incorporated into pyrimidinones ( 11 – 13 ; Fig. 2) and into cyclic β‐tri‐ and β‐tetrapeptides 17 – 19 and 21 – 23 (Scheme 3) with rigid skeletons, so that reliable structural data (bond lengths, bond angles, and Karplus parameters) can be obtained. β‐Hexapeptides Boc[(2S)‐β³hXaa(αF)]₆OBn and Boc[β³hXaa(α,αF₂)]₆‐OBn, 24 – 26 , with the side chains of Ala, Val, and Leu, have been synthesized (Scheme 4), and their CD spectra (Fig. 3) are discussed. Most compounds and many intermediates are fully characterized by IR‐ and ¹H‐, ¹³C‐ and ¹⁹F‐NMR spectroscopy, by MS spectrometry, and by elemental analyses, [α]_D and melting‐point values.     

Preparation of β2‐Homotryptophan Derivatives for β‐Peptide Synthesis

In view of the prominent role of the 1H‐indol‐3‐yl side chain of tryptophan in peptides and proteins, it is important to have the appropriately protected homologs H‐β² HTrp OH and H‐β³ HTrp OH (Fig.) available for incorporation in β‐peptides. The β²‐HTrp building block is especially important, because β²‐amino acid residues cause β‐peptide chains to fold to the unusual 12/10 helix or to a hairpin turn. The preparation of Fmoc and Z β²‐HTrp(Boc) OH by Curtius degradation (Scheme 1) of a succinic acid derivative is described (Schemes 2–4). To this end, the (S)‐4‐isopropyl‐3‐[(N‐Boc‐indol‐3‐yl)propionyl]‐1,3‐oxazolidin‐2‐one enolate is alkylated with Br CH₂CO₂Bn (Scheme 3). Subsequent hydrogenolysis, Curtius degradation, and removal of the Evans auxiliary group gives the desired derivatives of (R)‐H β²‐HTrp OH (Scheme 4). Since the (R)‐form of the auxiliary is also available, access to (S)‐β²‐HTrp‐containing β‐peptides is provided as well.     

Preparation of the β2‐Homoselenocysteine Derivatives Fmoc‐(S)‐β2hSec(PMB)‐OH and Boc‐(S)‐β2hSec(PMB)‐OH for Solution and Solid‐Phase Peptide Synthesis

Fmoc‐β²hSer(^tBu)‐OH was converted to Fmoc‐β²hSec(PMB)‐OH in five steps. To avoid elimination of HSeR, the selenyl group was introduced in the second last step (Fmoc‐β²hSer(Ts)‐OAll→Fmoc‐β²hSec(PMB)‐OAll). In a similar way, the N‐Boc‐protected compound was prepared. With the β²hSe‐derivatives, 21 β²‐amino‐acid building blocks with proteinogenic side chains are now available for peptide synthesis.     

Isotopically Labelled and Unlabelled β‐Peptides with Geminal Dimethyl Substitution in 2‐Position of Each Residue: Synthesis and NMR Investigation in Solution and in the Solid State

The preparation of (S)‐β^2,2,3‐amino acids with two Me groups in the α‐position and the side chains of Ala, Val, and Leu in the β‐position (double methylation of Boc‐β‐HAla‐OMe, Boc‐β‐Val‐OMe, and Boc‐β‐Leu‐OMe, Scheme 2) is described. These β‐amino acids and unlabelled as well as specifically ¹³C‐ and ¹⁵N‐labelled 2,2‐dimethyl‐3‐amino acid (β^2,2‐HAib) derivatives have been coupled in solution (Schemes 1, 3 and 4) to give protected (N‐Boc, C‐OMe), partially protected (N‐Boc/C‐OH, N‐H/C‐OMe), and unprotected β^2,2‐ and β^2,2,3‐hexapeptides, and β^2,2‐ and β^2,2,3‐heptapeptides 1 – 7 . NMR Analyses in solution (Tables 1 and 2, and Figs. 2–4) and in the solid state (2D‐MAS NMR measurements of the fully labelled Boc‐(β^2,2‐HAib)₆‐OMe ([¹³C₃₀, ¹⁵N₆]‐ 1e ; Fig. 5), and TEDOR/REDOR NMR investigations of mixtures (Fig. 6) of the unlabelled Ac‐(β^2,2‐HAib)₇‐OMe ( 4 ) and of a labelled derivative ([¹³C₄,¹⁵N₂]‐ 5 ; Figs. 7–11, and 19), a molecular‐modeling study (Figs. 13–15), and a search in the Cambridge Crystallographic Data Base (Fig. 16) allow the following conclusions: i) there is no evidence for folding (helix or turn) or for aggregation to sheets of the geminally dimethyl substituted peptide chains in solution; ii) there are distinct conformational preferences of the individual β^2,2‐ and β^2,2,3‐amino acid residues: close to eclipsing around the C(O) C(Me₂(CHR)) bond (τ_1,2), almost perfect staggering around the C(2) C(3) ethane bond (τ_2,3), and antiperiplanar arrangement of H(C3) and H(N) (τ_3,N; Fig. 12) in the solid state; iii) the β^2,2‐peptides may be part of a turn structure with a ten‐membered H‐bonded ring; iv) the main structure present in the solid state of F₃CCO(β^2,2‐HAib)₇‐OMe is a nonfolded chain (>30 Å between the termini and >20 Å between the N‐terminus and the CH₂ group of residue 5) with all CO bonds in a parallel alignment (±10°). With these structural parameters, a simple modelling was performed producing three (maybe four) possible chain geometries: one fully extended, two with parallel peptide planes (with zick‐zack and crankshaft‐type arrangement of the peptide bonds), and (possibly) a fourth with meander‐like winding ( D – G in Figs. 17 and 18).     

Solid‐Phase Synthesis of a β3‐Icosapeptide Containing the Homologs of the Twenty Common Proteinaceous Amino Acids. Preliminary Communication

An icosapeptide, 1 , containing the β³‐amino acid residues with the 20 proteinogenic side chains has been assembled by manual solid‐phase synthesis, according to the Fmoc strategy. The sequence was chosen in such a way that a possible 3₁₄‐helical conformation (secondary structure) would be stabilized by salt bridges and have an amphipathic character (Fig. 1,a), and the N‐terminal β³hCys would lend itself to thioligations and disulfide formation ( 2 and 3 , in Figs. 1 and 2). The products 1 – 3 were pure according to RP‐HPLC, NMR, and MS analysis (Fig. 1,b and c, Fig. 2,c and d, and Fig. 3). With due caution, the CD spectra in aqueous solution (pH 7) and in MeOH (Fig. 4), with normalized Cotton effects θ =?14000 to ?16000 [deg?cm²?dmol^?1] between 209 and 210 nm, might be taken as an evidence for the presence of 3₁₄‐helical conformations. An evaluation of the data from a 700‐MHz 2D‐NMR measurement of the disulfide 2 in CD₃OH is in progress.     

Mixed β2/β3‐Hexapeptides and β2/β3‐Nonapeptides Folding to (P)‐Helices with Alternating Twelve‐ and Ten‐Membered Hydrogen‐Bonded Rings

The structural properties of four mixed β‐peptides with alternating β²/β³‐ or β³/β²‐sequences have been analyzed by two‐dimensional homonuclear ¹H‐NMR‐ and CD spectroscopic measurements. All four β‐peptides fold into (P)‐helices with twelve‐ and ten‐membered H‐bonded rings (Figs. 3–6). CD Spectra (Fig. 2) of the mixed β³/β²‐hexapeptide 4a and β³/β²‐nonapeptide 5a , indicating that peptides of this type also adopt the 12/10‐helical conformation, were confirmed by NMR structural analysis. For the deprotected β³/β²‐nonapeptide 5d , NOEs not consistent with the 10/12 helix have been observed, showing that the stability of the helix decreases upon N‐terminal deprotection. From the NMR structures obtained, an idealized helical‐wheel representation was generated (Fig. 7), which will be used for the design of further 12/10 or 10/12 helices.     

Evidence that the β‐Peptide 14‐Helix is Stabilized by β3‐Residues with Side‐Chain Branching Adjacent to the β‐Carbon Atom

Oligomers of β‐substituted β‐amino acids (‘β³‐peptides') are known to adopt a helical secondary structure defined by 14‐membered ring hydrogen bonds ('14‐helix'). Here, we describe a deca‐β³‐peptide, 1 , that does not adopt the 14‐helical conformation and that may prefer an alternative secondary structure. β³‐Peptide 1 is composed exclusively of residues with side chains that are not branched adjacent to the β‐C‐atom (β³‐hLeu, β³‐hLys, and β³‐hTyr). In contrast, an analogous β‐peptide, 2 , containing β³‐hVal residues in place of the β³‐hLeu residues of 1 , adopts a 14‐helical conformation in MeOH, according to CD data. These results illustrate the importance of side‐chain branching in determining the conformational preferences of β³‐peptides.     

Synthesis and Characterization of β‐Diketiminate Aluminum Compounds and Their Use in the Ring‐Opening Polymerization of ε‐Caprolactone

Four aluminum alkyl compounds, [CH{(CH₃)CN‐2,4,6‐MeC₆H₂}₂AlMe₂] ( 1 ), [CH{(CH₃)CN‐2,4,6‐MeC₆H₂}₂AlEt₂] ( 2 ), [CH{(CH₃)CN‐2‐iPrC₆H₄}₂AlMe₂] ( 3 ), and [CH{(CH₃)CN‐2‐iPrC₆H₄}₂AlEt₂] ( 4 ), bearing β‐diketiminate ligands [CH{(Me)CN‐2,4,6‐MeC₆H₂}]₂ (L¹H) and [CH{(Me)CN‐2‐ⁱPrC₆H₄}]₂ (L²H) were obtained from the reactions of trimethylaluminum, triethylaluminum with the corresponding β‐diketiminate, respectively. All compounds were characterized by ¹H NMR and ¹³C NMR spectroscopy, single‐crystal X‐ray structural analysis, and elemental analysis. Compounds 1 – 4 were found to catalyze the ring‐opening polymerization (ROP) of ε‐caprolactone (ε‐CL) with good activity.     

Synthesis,CD Spectra,and Enzymatic Stability of β2‐Oligoazapeptides Prepared from (S)‐2‐Hydrazino Carboxylic Acids Carrying the Side Chains of Val,Ala, and Leu

β‐Peptides offer the unique possibility to incorporate additional heteroatoms into the peptidic backbone (Figs. 1 and 2). We report here the synthesis and spectroscopic investigations of β²‐peptide analogs consisting of (S)‐3‐aza‐β‐amino acids carrying the side chains of Val, Ala, and Leu. The hydrazino carboxylic acids were prepared by a known method: Boc amidation of the corresponding N‐benzyl‐L ‐α‐amino acids with an oxaziridine (Scheme 1). Couplings and fragment coupling of the 3‐benzylaza‐β²‐amino acids and a corresponding tripeptide (N‐Boc/C‐OMe strategy) with common peptide‐coupling reagents in solution led to β²‐di, β²‐tri‐, and β²‐hexaazapeptide derivatives, which could be N‐debenzylated ( 4 – 9 ; Schemes 2–4). The new compounds were identified by optical rotation, and IR, ¹H‐ and ¹³C‐NMR, and CD spectroscopy (Figs. 4 and 5) and high‐resolution mass spectrometry, and, in one case, by X‐ray crystallography (Fig. 3). In spite of extensive measurements under various conditions (temperatures, solvents), it was not possible to determine the secondary structure of the β²‐azapeptides by NMR spectroscopy (overlapping and broad signals, fast exchange between the two types of NH protons!). The CD spectra of the N‐Boc and C‐OMe terminally protected hexapeptide analog 9 in MeOH and in H₂O (at different pH) might arise from a (P)‐3₁₄‐helical structure. The N‐Boc‐β²‐tri and N‐Boc‐β²‐hexaazapeptide esters, 7 and 9 , were shown to be stable for 48 h against the following peptidases: pronase, proteinase K, chymotrypsin, trypsin, carboxypeptidase A, and 20S proteasome.     

New Open‐Chain and Cyclic Tetrapeptides,Consisting of α‐, β2‐, and β3‐Amino‐Acid Residues,as Somatostatin Mimics – A Survey

Cyclo‐β‐tetrapeptides are known to adopt a conformation with an intramolecular transannular hydrogen bond in solution. Analysis of this structure reveals that incorporation of a β²‐amino‐acid residue should lead to mimics of ‘α‐peptidic β‐turns’ (cf. A, B, C ). It is also known that short‐chain mixed β/α‐peptides with appropriate side chains can be used to mimic interactions between α‐peptidic hairpin turns and G protein‐coupled receptors. Based on these facts, we have now prepared a number of cyclic and open‐chain tetrapeptides, 7 – 20 , consisting of α‐, β²‐, and β³‐amino‐acid residues, which bear the side chains of Trp and Lys, and possess backbone configurations such that they should be capable of mimicking somatostatin in its affinity for the human SRIF receptors (hsst_1–5). All peptides were prepared by solid‐phase coupling by the Fmoc strategy. For the cyclic peptides, the three‐dimensional orthogonal methodology (Scheme 3) was employed with best success. The new compounds were characterized by high‐resolution mass spectrometry, NMR and CD spectroscopy, and, in five cases, by a full NMR‐solution‐structure determination (in MeOH or H₂O; Fig. 4). The affinities of the new compounds for the receptors hsst_1–5 were determined by competition with [¹²⁵I]LTT‐SRIF₂₈ or [¹²⁵I] [Tyr¹⁰]‐CST₁₄. In Table 1, the data are listed, together with corresponding values of all β‐ and γ‐peptidic somatostatin/Sandostatin^® mimics measured previously by our groups. Submicromolar affinities have been achieved for most of the human SRIF receptors hsst_1–5. Especially high, specific binding affinities for receptor hsst₄ (which is highly expressed in lung and brain tissue, although still of unknown function!) was observed with some of the β‐peptidic mimics. In view of the fact that numerous peptide‐activated G protein‐coupled receptors (GPCRs) recognize ligands with turn structure (Table 2), the results reported herein are relevant far beyond the realm of somatostatin: many other peptide GPCRs should be ‘reached’ with β‐ and γ‐peptidic mimics as well, and these compounds are proteolytically and metabolically stable, and do not need to be cell‐penetrating for this purpose (Fig. 5).     

NMR‐Solution Structures of Fluoro‐Substituted β‐Peptides: A 314‐Helix and a Hairpin Turn. The First Case of a 90° OC?C?F Dihedral Angle in an α‐Fluoro‐Amide Group

To further study the preference of the antiperiplanar (ap) conformation in α‐fluoro‐amide groups, two β‐peptides, 1 and 2 , containing a (2‐F)‐β³hAla and a (2‐F)‐β²hPhe residue, have been synthesized. Their NMR‐solution structures in CD₃OH were determined and compared with those of non‐F‐substituted analogs, 3 and 4a . While we have found in a previous investigation (Helv. Chim. Acta 2005 , 88, 266) that a stereospecifically introduced F‐substituent in the central position of a β‐heptapeptide is capable of ‘breaking’ the 3₁₄‐helical structure by enforcing the F? C? C?O ap‐conformation, we could now demonstrate that the same procedure leads to a structure with the unfavorable ca. 90° F? C? C?O dihedral angle, enforced by the 3₁₄‐helical folding in a β‐tridecapeptide (cf. 1 ; Fig. 4). This is interpreted as a consequence of cooperative folding in the longer β‐peptide. A F‐substituent placed in the turn section of a β‐peptidic hairpin turn was shown to be in an ap‐arrangement with respect to the neighboring C?O bond (cf. 2 ; Fig. 7). Analysis of the non‐F‐substituted β‐tetrapeptides (with helix‐preventing configurations of the two central β²/β³‐amino acid residues) provides unusually tight hairpin structural clusters (cf. 3 and 4a ; Figs. 8 and 9). The skeleton of the β‐tetrapeptide H‐(R)β³hVal‐(R)β²hVal‐(R)β³hAla‐(S)β³hPhe‐OH ( 4a ) is proposed as a novel, very simple backbone structure for mimicking α‐peptidic hairpin turns.     

Efficient Access to Enantiopure γ4‐Amino Acids with Proteinogenic Side‐Chains and Structural Investigation of γ4‐Asn and γ4‐Ser in Hybrid Peptide Helices

Hybrid peptides composed of α‐ and β‐amino acids have recently emerged as new class of peptide foldamers. Comparatively, γ‐ and hybrid γ‐peptides composed of γ⁴‐amino acids are less studied than their β‐counterparts. However, recent investigations reveal that γ⁴‐amino acids have a higher propensity to fold into ordered helical structures. As amino acid side‐chain functional groups play a crucial role in the biological context, the objective of this study was to investigate efficient synthesis of γ⁴‐residues with functional proteinogenic side‐chains and their structural analysis in hybrid‐peptide sequences. Here, the efficient and enantiopure synthesis of various N‐ and C‐terminal free‐γ⁴‐residues, starting from the benzyl esters (COOBzl) of N‐Cbz‐protected (E)‐α,β‐unsaturated γ‐amino acids through multiple hydrogenolysis and double‐bond reduction in a single‐pot catalytic hydrogenation is reported. The crystal conformations of eight unprotected γ⁴‐amino acids (γ⁴‐Val, γ⁴‐Leu, γ⁴‐Ile, γ⁴‐Thr(OtBu), γ⁴‐Tyr, γ⁴‐Asp(OtBu), γ⁴‐Glu(OtBu), and γ‐Aib) reveals that these amino acids adopted a helix favoring gauche conformations along the central C^γ? C^β bond. To study the behavior of γ⁴‐residues with functional side chains in peptide sequences, two short hybrid γ‐peptides P1 (Ac‐Aib‐γ⁴‐Asn‐Aib‐γ⁴‐Leu‐Aib‐γ⁴‐Leu‐CONH₂) and P2 (Ac‐Aib‐γ⁴‐Ser‐Aib‐γ⁴‐Val‐Aib‐γ⁴‐Val‐CONH₂) were designed, synthesized on solid phase, and their 12‐helical conformation in single crystals were studied. Remarkably, the γ⁴‐Asn residue in P1 facilitates the tetrameric helical aggregations through interhelical H bonding between the side‐chain amide groups. Furthermore, the hydroxyl side‐chain of γ⁴‐Ser in P2 is involved in the interhelical H bonding with the backbone amide group. In addition, the analysis of 87 γ⁴‐residues in peptide single‐crystals reveal that the γ⁴‐residues in 12‐helices are more ordered as compared with the 10/12‐ and 12/14‐helices.     

Conformational Study of Heteropentapeptides Containing an α‐Ethylated α,α‐Disubstituted Amino Acid: (S)‐Butylethylglycine (=2‐Amino‐2‐ethylhexanoic Acid) within a Sequence of Dimethylglycine (=2‐Aminoisobutyric Acid) Residues

Heteropentapeptides containing the α‐ethylated α,α‐disubstituted amino acid (S)‐butylethylglycine and four dimethylglycine residues, i.e., CF₃CO‐[(S)‐Beg]‐(Aib)₄‐OEt ( 4 ) and CF₃CO‐(Aib)₂‐[(S)‐Beg]‐(Aib)₂‐OEt ( 7 ), were synthesized by conventional solution methods. In the solid state, the preferred conformation of 4 was shown to be both a right‐handed (P) and a left‐handed (M) 3₁₀‐helical structure, and that of 7 was a right‐handed (P) 3₁₀‐helical structure. IR, CD, and ¹H‐NMR spectra revealed that the dominant conformation of both 4 and 7 in solution was the 3₁₀‐helical structure. These conformations were also supported by molecular‐mechanics calculations.     

NMR‐Solution Structures in Methanol of an α‐Heptapeptide,of a β3/β2‐Nonapeptide,and of an all‐β3‐Icosapeptide Carrying the 20 Proteinogenic Side Chains

The NMR‐solution structure of an α‐heptapeptide with a central Aib residue was investigated in order to verify that, in contrast to β‐peptides, short α‐peptides do not form a helical structures in MeOH. Although the central Aib residue was found to induce a bend in the experimentally determined structure, no secondary structure typical for longer α‐peptides or proteins was found. A β²/β³‐nonapeptide with polar, positively charged side chains was subjected to NMR analysis in MeOH and H₂O. Whereas, in MeOH, it folds into a 10/12‐helix very similar to the structure determined for a corresponding β²/β³‐nonapeptide with only aliphatic side chains, no dominant conformation could be determined in H₂O. Finally, the NMR analysis of a β³‐icosapeptide containing the side chains of all 20 proteinogenic amino acids in MeOH is described. It revealed that this 20mer folds into a 3₁₄‐helix over its whole length forming six full turns, the longest 3₁₄‐helix found so far. Together, our findings confirm that, in contrast to α‐peptides, β‐peptides not only form helices with just six residues, but also form helices that are longer than helical sections usually observed in proteins or natural peptides. The higher helix‐forming propensity of long β‐peptides is attributed to the conformation‐stabilizing effect of the staggered ethane sections in β‐peptides which outweighs the detrimental effect of the increasing macrodipole.