Syntheses and CD‐Spectroscopic Investigations of Longer‐Chain β‐Peptides: Preparation by Solid‐Phase Couplings of Single Amino Acids,Dipeptides, and Tripeptides

The synthesis and CD‐spectroscopic analysis of eleven water‐soluble β‐peptides composed of all‐β³ or alternating β²‐ and β³‐amino acids is described. Different approaches for the efficient syntheses of longer‐chain β‐peptides (>9 residues) were investigated. They were synthesized on solid phase with Fmoc‐protected amino acids or Fmoc‐protected di‐ or tripeptide fragments (assembled using solution‐phase synthesis). The use of preformed fragments significantly increased the purity of the crude peptides and facilitated purification. Especially, the use of Fmoc‐protected β²/β³‐dipeptides for the synthesis of a ‘mixed' β²/β³‐nonapeptide proved to be remarkably effective, yielding the crude peptide in 95% purity and without detectable epimerization of the β²‐amino acid residues. This is a significant improvement over previously reported procedures for the solid‐phase synthesis of β‐peptides, and foreshadows that the field of β‐peptide research will now switch from synthesis to the design and study of complex functional ‘β‐proteins'.     

Preparation of (S,S)‐Fmoc‐β2hIle‐OH, (S)‐Fmoc‐β2hMet‐OH,and (S)‐Fmoc‐β2hTyr(tBu)‐OH for Solid‐Phase Syntheses of β2‐ and β2/β3‐Peptides

The preparation of three new N‐Fmoc‐protected (Fmoc=[(9H‐fluoren‐9‐yl)methoxy]carbonyl) β²‐homoamino acids with proteinogenic side chains (from Ile, Tyr, and Met) is described, the key step being a diastereoselective amidomethylation of the corresponding Ti‐enolates of 3‐acyl‐4‐isopropyl‐5,5‐diphenyloxazolidin‐2‐ones with CbzNHCH₂OMe/TiCl₄ (Cbz=(benzyloxy)carbonyl) in yields of 60–70% and with diastereoselectivities of >90%. Removal of the chiral auxiliary with LiOH or NaOH gives the N‐Cbz‐protected β‐amino acids, which were subjected to an N‐Cbz/N‐Fmoc (Fmoc=[(9H‐fluoren‐9‐yl)methoxy]carbonyl) protective‐group exchange. The method is suitable for large‐scale preparation of Fmoc‐β²hXaa‐OH for solid‐phase syntheses of β‐peptides. The Fmoc‐amino acids and all compounds leading to them have been fully characterized by melting points, optical rotations, IR, ¹H‐ and ¹³C‐NMR, and mass spectra, as well as by elemental analyses.     

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.     

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.     

β‐Hairpins Generated from Hybrid Peptide Sequences Containing both α‐ and β‐Amino Acids

The incorporation of the β‐amino acid residues into specific positions in the strands and β‐turn segments of peptide hairpins is being systematically explored. The presence of an additional torsion variable about the C(α) C(β) bond (θ) enhances the conformational repertoire in β‐residues. The conformational analysis of three designed peptide hairpins composed of α/β‐hybrid segments is described: Boc‐Leu‐Val‐Val‐^DPro‐β Phe ‐Leu‐Val‐Val‐OMe ( 1 ), Boc‐Leu‐Val‐β Val ‐^DPro‐Gly‐β Leu ‐Val‐Val‐OMe ( 2 ), and Boc‐Leu‐Val‐β Phe ‐Val‐^DPro‐Gly‐Leu‐β Phe ‐Val‐Val‐OMe ( 3 ). 500‐MHz ¹H‐NMR Analysis supports a preponderance of β‐hairpin conformation in solution for all three peptides, with critical cross‐strand NOEs providing evidence for the proposed structures. The crystal structure of peptide 2 reveals a β‐hairpin conformation with two β‐residues occupying facing, non‐H‐bonded positions in antiparallel β‐strands. Notably, βVal(3) adopts a gauche conformation about the C(α) C(β) bond (θ=+65°) without disturbing cross‐strand H‐bonding. The crystal structure of 2 , together with previously published crystal structures of peptides 3 and Boc‐β Phe ‐β Phe ‐^DPro‐Gly‐β Phe ‐β Phe ‐OMe, provide an opportunity to visualize the packing of peptide sheets with local ‘polar segments' formed as a consequence of reversal peptide‐bond orientation. The available structural evidence for hairpins suggests that β‐residues can be accommodated into nucleating turn segments and into both the H‐bonding and non‐H‐bonding positions on the strands.     

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.     

Zn2+‐Complexation by a β‐Peptidic Helix and Hairpin Containing β3hCys and β3hHis Building Blocks: Evidence from CD Measurements. Preliminary Communication

Two new β³‐homohistidine‐ and β³‐homocysteine‐containing β‐peptides have been prepared by solid‐phase synthesis. A β‐octapeptide ( 2 ) contains seven β³‐amino acids and one β²‐amino acid. The β²/β³ segment has been placed in the middle of this peptide, which contains β³‐amino acids of alternating configuration, to induce the formation of a hairpin secondary structure. A β‐decapeptide ( 3 ) has been designed to fold to a 3₁₄‐helical secondary structure with neighboring His side chains in 6‐ and 9‐positions. Circular‐dichroism (CD) measurements show the capability of both peptides to bind Zn²⁺ ions in aqueous solution. In the case of the β‐octapeptide, binding of Zn²⁺ causes a dramatic change of the CD spectrum, indicating a change or a stabilization of its secondary structure. Zn²⁺ Ions clearly stabilize the 3₁₄‐helix of the β‐decapeptide, in neutral and basic solution. For the construction of the two new β‐peptides, we needed to have a supply of the β‐amino acid derivatives Fmoc‐β³hCys(Trt)‐OH and Fmoc‐β³hHis(Trt)‐OH, the preparation of which is described herein.     

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.     

β‐Peptide Conjugates: Syntheses and CD and NMR Investigations of β/α‐Chimeric Peptides,of a DPA‐β‐Decapeptide,and of a PEGylated β‐Heptapeptide

β³‐Peptides consisting of six, seven, and ten homologated proteinogenic amino acid residues have been attached to an α‐heptapeptide (all d‐ amino acid residues; 4 ), to a hexaethylene glycol chain (PEGylation; 5c ), and to dipicolinic acid (DPA derivative 6 ), respectively. The conjugation of the β‐peptides with the second component was carried out through the N‐termini in all three cases. According to NMR analysis (CD₃OH solutions), the (M)‐3₁₄‐helical structure of the β‐peptidic segments was unscathed in all three chimeric compounds (Figs. 2, 4, and 5). The α‐peptidic section of the α/β‐peptide was unstructured, and so was the oligoethylene glycol chain in the PEGylated compound. Thus, neither does the appendage influence the β‐peptidic secondary structure, nor does the latter cause any order in the attached oligomers to be observed by this method of analysis. A similar conclusion may be drawn from CD spectra (Figs. 1, 3, and 5). These results bode well for the development of delivery systems involving β‐peptides.     

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.     

A Defined α‐Helix in the Bifunctional O‐Glycosylated Natriuretic Peptide TcNPa from the Venom of Tropidechis carinatus

Natriuretic peptides (NP) play important roles in human cardiac physiology through their guanylyl cyclase receptors NPR‐A and NPR‐B. Described herein is a bifunctional O‐glycosylated natriuretic peptide, TcNPa, from Tropidechis carinatus venom and it unusually targets both NPR‐A and NPR‐B. Characterization using specific glycosidases and ETD‐MS identified the glycan as galactosyl‐β(1‐3)‐N‐acetylgalactosamine (Gal‐GalNAc) and was α‐linked to the C‐terminal threonine residue. TcNPa contains the characteristic NP 17‐membered disulfide ring with conserved phenylalanine and arginine residues. Both glycosylated and nonglycosylated forms were synthesized by Fmoc solid‐phase peptide synthesis and NMR analysis identified an α‐helix within the disulfide ring containing the putative pharmacophore for NPR‐A. Surprisingly, both forms activated NPR‐A and NPR‐B and were relatively resistant towards proteolytic degradation in plasma. This work will underpin the future development of bifunctional NP peptide mimetics.     

Preparation of the β3‐Homoselenocysteine Derivatives Fmoc‐β3hSec(PMB)‐OH and Boc‐β3hSec(PMB)‐OH for Solution and Solid‐Phase‐Peptide Synthesis and Selenoligation

The title compounds, 4 and 7 , have been prepared from the corresponding α‐amino acid derivative selenocystine ( 1 ) by the following sequence of steps: cleavage of the Se? Se bond with NaBH₄, p‐methoxybenzyl (PMB) protection of the SeH group, Fmoc or Boc protection at the N‐atom and Arndt–Eistert homologation (Schemes 1 and 2). A β³‐heptapeptide 8 with an N‐terminal β³‐hSec(PMB) residue was synthesized on Rink amide AM resin and deprotected (‘in air’) to give the corresponding diselenide 9 , which, in turn, was coupled with a β³‐tetrapeptide thiol ester 10 by a seleno‐ligation. The product β³‐undecapeptide was identified as its diselenide and its mixed selenosulfide with thiophenol (Scheme 3). The differences between α‐ and β‐Sec derivatives are discussed.     

Facile Synthesis of Internal and C‐Terminal Peptide α‐Ketoamides with Fmoc‐Solid Phase Peptide Synthesis

We report a general and operationally simple method for the solid phase synthesis of α‐ketoamide peptides using standard Fmoc solid phase peptide synthesis. The method delivers deprotected peptide α‐ketoamides directly upon resin cleavage without any additional steps, and tolerates all side chain functional groups. A small collection of C‐terminal and internal α‐ketoamide peptides – including two reported protease inhibitors (HCV and SUB1) – were prepared in good yields. In addition, we demonstrate that our method serves as versatile platform for the convenient preparation of cyclic α‐ketoamide peptides, photocagged peptide α‐ketoamides, and fluorescently labeled peptides.     

Quaternary β2,2‐Amino Acids: Catalytic Asymmetric Synthesis and Incorporation into Peptides by Fmoc‐Based Solid‐Phase Peptide Synthesis

β‐Amino acid incorporation has emerged as a promising approach to enhance the stability of parent peptides and to improve their biological activity. Owing to the lack of reliable access to β^2,2‐amino acids in a setting suitable for peptide synthesis, most contemporary research efforts focus on the use of β³‐ and certain β^2,3‐amino acids. Herein, we report the catalytic asymmetric synthesis of β^2,2‐amino acids and their incorporation into peptides by Fmoc‐based solid‐phase peptide synthesis (Fmoc‐SPPS). A quaternary carbon center was constructed by the palladium‐catalyzed decarboxylative allylation of 4‐substituted isoxazolidin‐5‐ones. The N?O bond in the products not only acts as a traceless protecting group for β‐amino acids but also undergoes amide formation with α‐ketoacids derived from Fmoc‐protected α‐amino acids, thus providing expeditious access to α‐β^2,2‐dipeptides ready for Fmoc‐SPPS.     

Synthesis,and Helix or Hairpin‐Turn Secondary Structures of ‘Mixed’ α/β‐Peptides Consisting of Residues with Proteinogenic Side Chains and of 2‐Amino‐2‐methylpropanoic Acid (Aib)

Twelve peptides, 1 – 12 , have been synthesized, which consist of alternating sequences of α‐ and β‐amino acid residues carrying either proteinogenic side chains or geminal dimethyl groups (Aib). Two peptides, 13 and 14 , containing 2‐methyl‐3‐aminobutanoic acid residues or a ‘random mix’ of α‐, β²‐, and β³‐amino acid moieties were also prepared. The new compounds were fully characterized by CD (Figs. 1 and 2), and ¹H‐ and ¹³C‐NMR spectroscopy, and high‐resolution mass spectrometry (HR‐MS). In two cases, 3 and 14 , we discovered novel types of turn structures with nine‐ and ten‐membered H‐bonded rings forming the actual turns. In two other cases, 8 and 11 , we found 14/15‐helices, which had been previously disclosed in mixed α/β‐peptides containing unusual β‐amino acids with non‐proteinogenic side chains. The helices are formed by peptides containing the amino acid moiety Aib in every other position, and their backbones are primarily not held together by H‐bonds, but by the intrinsic conformations of the containing amino acid building blocks. The structures offer new possibilities of mimicking peptide–protein and protein–protein interactions (PPI).     

The Cyclo‐β‐Tetrapeptide (β‐HPhe‐β‐HThr‐β‐HLys‐β‐HTrp): Synthesis,NMR Structure in Methanol Solution,and Affinity for Human Somatostatin Receptors

The known solid‐state structure (Fig. 1, top) of cyclo(β‐HAla)₄ was used to model the structure of the title compound 1 as a prospective somatostatin mimic (Fig. 1, bottom). The synthesis started with the N‐protected natural amino acids Boc‐Phe‐OH, Boc‐Trp‐OH, Boc‐Lys(2‐Cl‐Z)‐OH, and Boc‐Thr(OBn)‐OH, which were homologated to the corresponding β‐amino‐acid derivatives (Scheme 1) and coupled to the β‐tetrapeptide Boc‐β‐HTrp‐β‐HPhe‐β‐HThr(OBn)‐β‐HLys(2‐Cl‐Z)‐OMe ( 16 ); the (N‐Me)‐β‐HThr‐(N‐Me)‐β‐HPhe analog 17 was also prepared. C‐ and N‐terminal deprotection and cyclization through the pentafluorophenyl ester gave the insoluble β‐tetrapeptide with protected Thr and Lys side chains ( 18 ). Solubilization and debenzylation could only be effected in LiCl‐containing THF (ca. 10% yield; with ca. 55% recovery). HPLC Purification provided a sample of the title compound 1 , the structure of which, as determined by NMR‐spectroscopy (Fig. 2, left) was drastically different from the `theoretical' model (Fig. 1). There is a transannular H‐bond dividing the macrocyclic 16‐membered ring, thus forming a ten‐ and a twelve‐membered H‐bonded ring, the former mimicking, or actually being superimposable on, an α‐peptidic so‐called β‐turn. Still, the four side chains occupy equatorial positions on the ring, as planned, albeit with somewhat different geometry as compared to the `original'. The cyclo‐β‐tetrapeptide has micromolar affinities to the human somatostatin receptors (hsst 1 – 5). Thus, we have demonstrated for the first time that it is possible to mimic a natural peptide hormone with a small β‐peptide. Furthermore, we have discovered a simple way to construct the ubiquitous β‐turn motif with β‐peptides (which are known to be stable to mammalian peptidases).     

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).     

A Designed Three‐Stranded β‐Sheet in an α/β Hybrid Peptide

The incorporation of β‐amino acid residues into the antiparallel β‐strand segments of a multi‐stranded β‐sheet peptide is demonstrated for a 19‐residue peptide, Boc‐LV^βFV^DPGL^βFVVL^DPGLVL^βFVV‐OMe (BBH19). Two centrally positioned ^DPro–Gly segments facilitate formation of a stable three‐stranded β‐sheet, in which β‐phenylalanine (^βPhe) residues occur at facing positions 3, 8 and 17. Structure determination in methanol solution is accomplished by using NMR‐derived restraints obtained from NOEs, temperature dependence of amide NH chemical shifts, rates of H/D exchange of amide protons and vicinal coupling constants. The data are consistent with a conformationally well‐defined three‐stranded β‐sheet structure in solution. Cross‐strand interactions between ^βPhe3/^βPhe17 and ^βPhe3/Val15 residues define orientations of these side‐chains. The observation of close contact distances between the side‐chains on the N‐ and C‐terminal strands of the three‐stranded β‐sheet provides strong support for the designed structure. Evidence is presented for multiple side‐chain conformations from an analysis of NOE data. An unusual observation of the disappearance of the Gly NH resonances upon prolonged storage in methanol is rationalised on the basis of a slow aggregation step, resulting in stacking of three‐stranded β‐sheet structures, which in turn influences the conformational interconversion between type I′ and type II′ β‐turns at the two ^DPro–Gly segments. Experimental evidence for these processes is presented. The decapeptide fragment Boc‐LV^βFV^DPGL^βFVV‐OMe (BBH10), which has been previously characterized as a type I′ β‐turn nucleated hairpin, is shown to favour a type II′ β‐turn conformation in solution, supporting the occurrence of conformational interconversion at the turn segments in these hairpin and sheet structures.     

Solid‐Phase Synthesis of a β‐Dodecapeptide with Seven Functionalized Side Chains and CD‐Spectroscopic Evidence for a Dramatic Structural Switch When Going from Water to Methanol Solution

An all‐β³‐dodecapeptide with a protected N‐terminal thiol‐anchoring group and with seven side chains has been synthesized in multi‐mg amounts by the manual solid‐phase technique, applying Fmoc methodology and the Wang resin. The sequence is β‐HLys‐β‐HPhe‐β‐HTyr‐β‐HLeu‐β‐HLys‐β‐HSer‐β‐HLys‐β‐HPhe‐β‐HSer‐β‐HVal‐β‐HLys‐β‐HAla‐OH (from N‐ to C‐terminus; see 1 ). The functional groups in the side chains of the building blocks were Boc (β‐HLys) or t‐Bu ether (β‐HSer, β‐HTyr) protected to allow for simultaneous deprotection and detachment from the resin with trifluoroacetic acid. All coupling steps were achieved with HBTU (=O‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyl uronium hexafluorophosphate)/HOBt (=1‐hydroxy‐1H‐benzotriazole) in DMF. For Fmoc (=(9H‐fluoren‐9‐yl)methoxycarbonyl) deprotection, a protocol was developed to surmount the previously reported problems arising in solid‐phase synthesis of β‐peptides when the chain length exceeds seven or eight amino‐acid moieties: for up to seven amino acids, a 20% solution of piperidine in DMF was used for removal of Fmoc; for the subsequent five amino acids, DBU and piperidine were employed for complete deprotection. The crude product was purified by preparative reversed‐phase HPLC, and the yield of pure β‐dodecapeptide derivative ( 1 ) was 23%. As the compound is well‐soluble in H₂O, it was characterized by ¹H‐NMR (in MeOH and H₂O), ¹³C‐NMR (in MeOH), and CD spectroscopy (in MeOH and in H₂O at pH values ranging from 3.5 to 11), and its molecular weight and composition were confirmed by high‐resolution mass spectrometry (Figs. 1 – 4). In MeOH solution, the β‐dodecapeptide exhibits the expected CD pattern typical of an (M)‐3₁₄‐helical secondary structure. In H₂O, however, the characteristic trough near 215 nm is missing in the CD spectrum, only a strong positive Cotton effect at 202 nm was observed, indicating the presence of β‐peptidic secondary structures, containing ten‐membered H‐bonded rings, such as the 12/10 helix (Fig. 4, right) or the hairpin. Only a detailed NMR solution‐structure analysis will provide the clues necessary for understanding the effects leading to the observed dramatic structural change of the highly functionalized β‐dodecapeptide described.     

Theoretical Study of β‐Peptide Models: Intrinsic Preferences of Helical Structures

β‐Peptides form various secondary structures, such as 14‐helix, 12‐helix, 10/12‐helix, 10‐helix, 2₈‐ribbon, C6‐ribbon, and pleated‐sheet. Thus, it is useful to understand the intrinsic backbone conformational preferences of these basic structures. By using a simple repeating‐unit method, we have calculated the preferences of C6‐ribbon, β‐strand, 10/12‐helix, 14‐helix, 12‐helix, 10‐helix, and 2₈‐ribbon of a series of poly‐β‐alanine models, Ac‐(β‐Ala)_n‐NH₂, with n=1–9. Interactions among single amino acids result in cooperative residue energies. This is not found for the formations of β‐strands, 2₈‐ribbons, and C6‐ribbons, which possess constant residue energies. In contrast, the 12‐helix, 10‐helix, and 14‐helix are characterized by increasing residue energies as the peptide elongates. Therefore, there is a considerable positive cooperative impetus in the gas phase for their formation. The residue energy of the 10/12‐helix increases significantly for n=2 and 3, and then displays a zigzag pattern. Meanwhile, there is a good correlation between calculated residue energies and residue dipole moments, indicating the importance of long‐range electrostatic interactions to the cooperative residue energy. Efforts have been made to separate the electrostatic and torsional interactions between residues. Thereby, the 12‐, 10‐, and 10/12‐helices all benefit from electrostatic interactions, while the 14‐helix has the most intrinsic preference in terms of torsional interaction. The effect of MeOH on the secondary structures has also been evaluated by SCIPCM solvent model calculations.