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.     

Synthesis and CD Spectra of Fluoro‐ and Hydroxy‐Substituted β‐Peptides

β‐Amino acids 1 – 3 with OH and F substituents in the α‐position have been prepared (Scheme) from the natural (S)‐α‐amino acids alanine, valine, and leucine, and incorporated into β‐hexa‐ and β‐heptapeptides 4 – 12 . The peptide syntheses were performed according to a conventional solution strategy (Boc/Bn protection) with fragment coupling. The new β‐peptides with (series a ) and without (series b ) terminal protection were isolated in HPLC‐pure form and characterized by NMR spectroscopy and MALDI mass spectrometry. The chemical properties as well as the patterns of the CD spectra (Figs. 3–5) depend upon constitution (OH, F, F₂ substitution) and configuration (l or u) of the amino acid residues, upon the total number of OH and F substituents in the peptide chain, and upon the solvent used (H₂O, MeOH, CF₃CH₂OH, (CF₃)₂CHOH). No reliable clues regarding the structures can be obtained from these CD spectra. Only a full NMR analysis will be able to answer the questions: a) with which known secondary structures (Figs. 1 and 2) of β‐peptides are the OH and F derivatives compatible? b) Are new secondary structures enforced by the polar and/or H‐bonding backbone substituents? Furthermore, the β‐peptides described here will enable us to study changes in chemical, enzymatic, and metabolic stability, and in physiological properties caused by the heteroatoms.     

Conformational Preferences of a β‐Octapeptide as Function of Solvent and Force‐Field Parameters

The ability to design properly folded β‐peptides with specific biological activities requires detailed insight into the relationship between the amino acid sequence and the secondary and/or tertiary structure of the peptide. One of the most frequently used spectroscopic techniques for resolving the structure of a biomolecule is NMR spectroscopy. Because only signal intensities and frequencies are recorded in the experiment, a conformational interpretation of the recorded data is not straightforward, especially for flexible molecules. The occurrence of conformational and/or time averaging, and the limited amount and accuracy of experimental data hamper the precise conformational determination of a biomolecule. In addition, the relation between experimental observables with the underlying conformational ensemble is often only approximately known, thereby aggravating the difficulty of structure determination of biomolecules. The problematic aspects of structure refinement based on NMR nuclear Overhauser effect (NOE) intensities and ³J‐coupling data are illustrated by simulating a β‐octapeptide in explicit MeOH and H₂O as solvents using three different force fields. NMR Data indicated that this peptide would fold into a 3₁₄‐helix in MeOH and into a hairpin in H₂O. Our analysis focused on the conformational space visited by the peptide, on structural properties of the peptide, and on agreement of the MD trajectories with available NMR data. We conclude that 1) although the 3₁₄‐helical structure is present when the peptide is solvated in MeOH, it is not the only relevant conformation, and that 2) the NMR data set available for the peptide, when solvated in H₂O, does not provide sufficient information to derive a single secondary structure, but rather a multitude of folds that fulfill the NOE data set.     

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.     

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‐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).     

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).     

Synthesis and High‐Resolution NMR Structure of a β3‐Octapeptide with and without a Tether Introduced by Olefin Metathesis

Bridging between (i)‐ and (i+3)‐positions in a β³‐peptide with a tether of appropriate length is expected to prevent the corresponding 3₁₄‐helix from unfolding (Fig. 1). The β³‐peptide H‐β³hVal‐β³hLys‐β³hSer(All)‐β³hPhe‐β³hGlu‐β³hSer(All)‐β³hTyr‐β³hIle‐OH ( 1 ; with allylated βhSer residues in 3‐ and 6‐position), and three tethered β‐peptides 2 – 4 (related to 1 through ring‐closing metathesis) have been synthesized (solid‐phase coupling, Fmoc strategy, on chlorotrityl resin; Scheme). A comparative CD analysis of the tethered β‐peptide 4 and its non‐tethered analogue 1 suggests that helical propensity is significantly enhanced (threefold CD intensity) by a (CH₂)₄ linker between the β³hSer side chains (Fig. 2). This conclusion is based on the premise that the intensity of the negative Cotton effect near 215 nm in the CD spectra of β³‐peptides represents a measure of ‘helical content’. An NMR analysis in CD₃OH of the two β³‐octapeptide derivatives without (i.e., 1 ) and with tether (i.e., 4 ; Tables 1–6, and Figs. 4 and 5) provided structures of a degree of precision (by including the complete set of side chain–side chain and side chain–backbone NOEs) which is unrivaled in β‐peptide NMR‐solution‐structure determination. Comparison of the two structures (Fig. 5) reveals small differences in side‐chain arrangements (separate bundles of the ten lowest‐energy structures of 1 and 4 , Fig. 5, A and B ) with little deviation between the two backbones (superposition of all structures of 1 and 4 , Fig. 5, C ). Thus, the incorporation of a CH₂? O? (CH₂)₄? O? CH₂ linker between the backbone of the β³‐amino acids in 3‐ and 6‐position (as in 4 ) does accurately constrain the peptide into a 3₁₄‐helix. The NMR analysis, however, does not suggest an increase in the population of a 3₁₄‐helical backbone conformation by this linkage. Possible reasons for the discrepancy between the conclusion from the CD spectra and from the NMR analysis are discussed.     

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.     

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.     

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.     

Structure and Conformation of β‐Oligopeptide Derivatives with Simple Proteinogenic Side Chains: Circular Dichroism and Molecular Dynamics Investigations

A careful CD analysis (Figs. 1 – 3 and 5; MeOH or H₂O solutions) of β‐oligopeptides ( 1 – 6 , B , C ) containing four to seven β‐amino acids reveals that seemingly small structural changes cause a switch from the CD pattern (maxima of opposite sign near 215 and 200 nm) associated with a 3₁₄‐helical structure to the CD pattern (single Cotton effect at ca. 205 nm) considered characteristic of a so‐called 12/10‐helical structure, but also exhibited by a β‐peptide adopting a hair‐pin conformation with a ten‐membered H‐bonded ring as the turn motif. Comparison of these CD spectra with those of the trans‐2‐aminocyclohexanecarboxamide oligomers, which give rise to the long‐wavelength Cotton effect only, suggests that the H‐bonded 14‐, 12‐, and 10‐membered ring conformations of the β‐peptides, and not just the entire helix structures, might actually generate the Cotton effects. This interpretation would be compatible with our previous NMR structure determinations of β‐peptides and with previously reported temperature dependences of CD and NMR spectra of β‐peptides. To further substantiate this suggestion, we have performed a statistical analysis of the β‐peptidic conformations generated by molecular‐dynamics calculations (GROMOS96) for a β‐hexapeptide ( C ; the 12/10 helix) and a β‐heptapeptide ( 6 ; the 3₁₄ helix) in MeOH (Figs. 6 – 9). Up to 400,000 conformations at 0.5‐ps intervals were analyzed from up to 200‐ns simulations (at 298 to 360 K). The analysis reveals the co‐existence of the various H‐bonded rings. Remarkably, the central section of the β‐peptide 6 (containing a β^2,3‐amino‐acid residue of like‐configuration!) adopts a ten‐membered‐ring conformation for ca. 5% of the simulation time, while the central section of the β‐peptide C adopts a 14‐membered‐ring conformation for ca. 3% of the time, according to this computational analysis. Further experimental and theoretical work will be necessary to find out to which extent the components (H‐bonded rings) and the entire helical secondary structures of β‐peptides contribute to the observed Cotton effects.     

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.     

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.     

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.     

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.     

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).     

The Miraculous CD Spectra (and Secondary Structures?) of β‐Peptides as They Grow Longer,Preliminary Communication

The recently improved conditions for solid‐phase synthesis of β³‐peptides by the Fmoc strategy were used to synthesize a β‐tetracosapeptide ( 4 , Scheme) composed of eight different β‐amino acid residues; 11 of the 24 residues carry functionalized proteinogenic side chains (namely those of Glu, Lys, Ser, and Tyr). The highly H₂O‐soluble β‐tetracosapeptide was identified by ¹H‐NMR spectroscopy (in MeOH), analytical HPL chromatography, and ESI‐mass spectrometry (Fig. 1). The expected 3₁₄‐helical secondary structure of the new β‐peptide was designed to have one hydrophobic and two hydrophilic faces, and to be compared with other β‐peptides ( 1 – 3 ), two of which are also of amphipathic character in this secondary structure (Fig. 2). In the absence of NMR‐structural proof, the CD spectra of the four β‐peptides were compared (Figs. 3 and 4). The β‐tetracosapeptide exhibits an unprecedented CD pattern (in MeOH and in H₂O solution) that may arise from a new type of secondary structure or from an unordered conformation.     

Conformation of Peptides Containing a Chiral α‐Ethylated α,α‐Disubstituted α‐Amino Acid: (S)‐α‐Ethylleucine (=(2S)‐2‐Amino‐2‐ethyl‐4‐methylpentanoic Acid) within Sequences of Dimethylglycine and Diethylglycine Residues

An optically active (S)‐α‐ethylleucine ((S)‐αEtLeu) as a chiral α‐ethylated α,α‐disubstituted α‐amino acid was synthesized by means of a chiral acetal auxiliary of (R,R)‐cyclohexane‐1,2‐diol. The chiral α‐ethylated α,α‐disubstituted amino acid (S)‐αEtLeu was introduced into the peptides constructed from 2‐aminoisobutyric acid (=dimethylglycine, Aib), and also into the peptide prepared from diethylglycine (Deg). The X‐ray crystallographic analysis revealed that both right‐handed (P) and left‐handed (M) 3₁₀‐helical structures exist in the solid state of CF₃CO‐(Aib)₂‐[(S)‐αEtLeu]‐(Aib)₂‐OEt ( 14 ) and CF₃CO‐[(S)‐αEtLeu]‐(Deg)₄‐OEt ( 18 ), respectively. The IR, CD, and ¹H‐NMR spectra indicated that the dominant conformation of pentapeptides 14 and CF₃CO‐[(S)‐αEtLeu]‐(Aib)₄‐OEt ( 16 ) in solution is a 3₁₀‐helical structure, and that of 18 in solution is a planar C₅ conformation. The conformation of peptides was also studied by molecular‐mechanics calculations.     

Validation of the GROMOS 54A7 Force Field Regarding Mixed α/β‐Peptide Molecules

A molecular‐dynamics (MD) simulation study of two heptapeptides containing α‐ and β‐amino acid residues is presented. According to NMR experiments, the two peptides differ in dominant fold when solvated in MeOH: peptide 3 adopts predominantly β‐hairpin‐like conformations, while peptide 8 adopts a 14/15‐helical fold. The MD simulations largely reproduce the experimental data. Application of NOE atom? atom distance restraining improves the agreement with experimental data, but reduces the conformational sampling. Peptide 3 shows a variety of conformations, while still agreeing with the NOE and ³J‐coupling data, whereas the conformational ensemble of peptide 8 is dominated by one helical conformation. The results confirm the suitability of the GROMOS 54A7 force field for simulation or structure refinement of mixed α/β‐peptides in MeOH.