β-Peptides: Synthesis by Arndt-Eistert homologation with concomitant peptide coupling. Structure determination by NMR and CD spectroscopy and by X-ray crystallography. Helical secondary structure of a β-hexapeptide in solution and its stability towards pepsin

The β-hexapeptide (H-β-HVal-β-HAla-β-HLeu)₂-OH ( 2 ) was prepared from the component L -β-amino acids by conventional peptide synthesis, including fragment coupling. A cyclo-β-tri- and a cyclo-β-hexapeptide were also prepared. The β-amino acids were obtained from α-amino acids by Arndt-Eistert homologation. All reactions leading to the β-peptides occur smoothly and in high yields. The β-peptides were characterized by their CD and NMR spectra (COSY, ROESY, TOCSY, and NOE-restricted modelling), and by an X-ray crystal-structure analysis. β-Sheet-type structures (in the solid state) and a compact, left-handed or (M) 3₁ helix of 5-Å pitch (in solution) were discovered. Comparison with the analogous secondary structures of α-peptides shows fundamental differences, the most surprising one at this point being the greater stability of β-peptide helices. There are structural relationships of β-peptides with oligomers of β-hydroxyalkanoic acids, and dissimilarities between the two classes of compounds are a demonstration of the power of H-bonding. The β-hexapeptide 2 is stable to cleavage by pepsin at pH 2 in H₂O for at least 60 h at 37°, while the corresponding α-peptide H-(Val-Ala-Leu)₂-OH is cleaved instantaneously under these conditions. The implication of the described results are discussed.     

Preparation and Structure of β-Peptides Consisting of Geminally Disubstituted β2,2- and β3,3-Amino Acids: A Turn Motif for β-Peptides

We report on the synthesis of new and previously described β-peptides ( 1 – 6 ), consisting of up to twelve β^2,2- or β^3,3-geminally disubstituted β-amino acids which do not fit into any of the secondary structural patterns of β-peptides, hitherto disclosed. The required 2,2- and 3,3-dimethyl derivatives of 3-aminopropanoic acid are readily obtained from 3-methylbut-2-enoic acid and ammonia (Scheme 1) and from Boc-protected methyl 3-aminopropanoate by enolate methylation (Scheme 2). Protected (Boc for solution-, Fmoc for solid-phase syntheses) 1-(aminomethyl)cycloalkanecarboxylic-acid derivatives (with cyclopropane, cyclobutane, cyclopentane, and cyclohexane rings) are obtained from 1-cyanocycloalkanecarboxylates and the corresponding dihaloalkanes (Scheme 3). Fully ¹³C- and ¹⁵N-labeled 3-amino-2,2-dimethylpropanoic-acid derivatives were prepared from the corresponding labeled precursors (see asterixed formula numbers and Scheme 4). Coupling of these amino acids was achieved by methods which we had previously employed for other β-peptide syntheses (intermediates 18 – 23 ). Crystal structures of Boc-protected geminally disubstituted amino acids ( 16a – d ) and of the corresponding tripeptide ( 23a ), as well as NMR and IR spectra of an isotopically labeled β-hexapeptide ( 2a* ) are presented (Figs. 1 – 4) and discussed. The tripeptide structure contains a ten-membered H-bonded ring which is proposed to be a turn-forming motif for β-peptides (Fig. 2).     

(S)-β3-Homolysine- and (S)-β3-Homoserine-Containing β-Peptides: CD Spectra in Aqueous Solution

For further structural studies and for physiological investigations of β-peptides, it is necessary to have H₂O-soluble derivatives. Thus, we have prepared β-hexa-, β-hepta-, and β-nonapeptides ( 1 – 6 ) with two, three, and seven side chains of lysine and serine. To detect possible π-π interactions, we also included the β-amino acid β²-HHop, resulting from homologation of so-called homophenylalanine (Hop) ( 5 and 6 ). The Fmoc-β²- and β³-amino-acid derivatives ( 11 – 14 and 19 ), and the corresponding β-peptides were prepared by methods previously described (solid-phase peptide coupling; HPLC-pure samples, Fig. 1). Circular-dichroism spectra (Fig. 2) indicate the presence of less pronounced secondary structures (especially of the lysine analogues with multiple positive charge) in H₂O as compared to MeOH. The β³-heptapeptide ( 3 ) with two serine side chains is well soluble in H₂O and exhibits the CD pattern typical of the 3₁-helical structure.     

Preparation of N-Fmoc-Protected β2- and β3-Amino Acids and their use as building blocks for the solid-phase synthesis of β-peptides

N-Fmoc-Protected (Fmoc = (9H-fluoren-9-ylmethoxy)carbonyl) β-amino acids are required for an efficient synthesis of β-oligopeptides on solid support. Enantiomerically pure Fmoc-β³-amino acids β³: side chain and NH₂ at C(3)(= C(β)) were prepared from Fmoc-protected (S)- and (R)-α-amino acids with aliphatic, aromatic, and functionalized side chains, using the standard or an optimized Arndt-Eistert reaction sequence. Fmoc-β²- Amino acids (β² side chain at C(2), NH₂ at C(3)(= C(β))) configuration bearing the side chain of Ala, Val, Leu, and Phe were synthesized via the Evans' chiral auxiliary methodology. The target β³-heptapeptides 5–8 , a β³- pentadecapeptide 9 and a β²-heptapeptide 10 were synthesized on a manual solid-phase synthesis apparatus using conventional solid-phase peptide synthesis procedures (Scheme 3). In the case of β³-peptides, two methods were used to anchor the first β-amino acid: esterification of the ortho-chlorotrityl chloride resin with the first Fmoc-β-amino acid 2 (Method I, Scheme 2) or acylation of the 4-(benzyloxy)benzyl alcohol resin (Wang resin) with the ketene intermediates from the Wolff rearrangement of amino-acid-derived diazo ketone 1 (Method II, Scheme 2). The former technique provided better results, as exemplified by the synthesis of the heptapeptides 5 and 6 (Table 2). The intermediate from the Wolff rearrangement of diazo ketones 1 was also used for sequential peptide-bond formation on solid support (synthesis of the tetrapeptides 11 and 12 ). The CD spectra of the β²- and β³-peptides 5 , 9 , and 10 show the typical pattern previously assigned to an (M) 3₁ helical secondary structure (Fig.). The most intense CD absorption was observed with the pentadecapeptide 9 (strong broad negative Cotton effect at ca. 213 nm); compared to the analogous heptapeptide 5 , this corresponds to a 2.5 fold increase in the molar ellipticity per residue!     

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).     

Oligomers of β2- and of β3-Homoproline: What are the Secondary Structures of β-Peptides Lacking H-Bonds?

To study the role of H-bonds in stabilizing β-peptidic secondary structures, we have synthesized β-oligopeptides (up to the octadecamer 12 ) consisting of β²- and β³-homoproline, i.e., β-peptides lacking amide protons. The enantiomer purity of the building block β²-homoproline (nipecotic acid, 4 ) was determined by HPLC analysis of the N-(2,4-dinitrophenyl) derivative 5 on a Chiralcel-OD column (cf. Fig. 2). The CD spectra of the all-(S)-β²- and all-(S)-β³-HPro-containing β-peptides display novel and intensive CD patterns which may be indicative of a secondary structure (cf. Fig. 3). It is noteworthy that a distinct CD pattern was observed with the β³-HPro derivatives containing as few as three residues ( 7a ). The crystal structure of a N-deprotected β³-HPro-tripeptide 7c is presented (cf. Figs. 4 and 5), and a model for the structure of β-peptides consisting of β³-HPro is discussed (cf. Figs. 6 and 7).     

Synthesis,Crystal Structures,and Modelling of β-Oligopeptides Consisting of 1-(Aminomethyl)cyclopropanecarboxylic Acid: Ribbon-Type Arrangement of Eight-Membered H-Bonded Rings

Partially and fully protected, and unprotected β-oligopeptides ( 3 – 9 ) were prepared from 1-(aminomethyl)cyclopropanecarboxylic acid, which, in turn, is readily available from cyanoacetate and dibromoethane. N-Boc and C-OMe protection were applied for the fragment-coupling (1-hydroxy-1H-benzotriazole (HOBt)/1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC)) solution synthesis. X-Ray crystal structures of the dimer ( 3 ), trimer ( 5 ), and tetramer ( 6 ) are described, and compared with those of the Boc-protected building blocks ( 2 ) and of the corresponding trimer ( 10 ) consisting of 1-(aminomethyl)cyclohexanecarbonyl residues (cf. Figs. 1 and 2). While the cyclohexane derivative forms ten-membered hydrogen-bonded rings, the characteristic secondary-structural motif in the cyclopropane derivatives is an eight-membered ring with H-bonding between next neighbors (Fig. 1). All cyclopropanecarbonyl moieties in the reported structures have the – generally more stable – s-cis (`bisected') conformation of the C=O groups on the three-membered rings (not preferred with the cyclohexane analog, the exocyclic CO group of which may be in an s-trans, a perpendicular, an axial, or an equatorial position). The bisecting effect and the large exocyclic bond angle (120°) in the cyclopropane units are proposed to provide the `ordering' elements – on top of the staggering effect of the C(2)−C(3) ethane bond in all β-peptides – which lead to the observed substituent-induced turn formation. A high degree of intramolecular H-bonding is evident also from IR spectroscopy (Fig. 3), and concentration- and temperature-dependent NMR measurements (Fig. 4) of CHCl₃ and CD₂Cl₂ solutions, indicating that the boat-type arrangement of the eight-membered rings with their unusual H-bonding geometry (Fig. 1, f ) is also present in solution. A possible structure of a poly[1-(aminomethyl)cyclopropane-carboxylic acid] consisting of a flight of stairs formed by folded H-bonded eight-membered rings is modelled, using the oligomer X-ray data (Fig. 5). The type of secondary structure found in the solid state of the β^2,2-peptides reported here is unprecedented in the realm of α-peptides and proteins.     

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.     

β-Peptides as Inhibitors of Small-Intestinal Cholesterol and Fat Absorption

Selective lipid transport through the brush-border membrane in the small intestine of mammals is mediated by membrane-bound proteins, the so-called scavenger receptors of class B, type I or II (SR-BI or -BII). These, in turn, are inhibited by certain proteins and synthetic α-peptides that have an amphipathic helix as the binding motif (Fig. 1). In whole cells (test with human colonic carcinoma cells, CaCo-2), on the other hand, the inhibitors are subject to proteolysis. We have now tested six β-peptides (hexa-, hepta-, and nonamers 1 – 6 ), each carrying one to seven water-solubilizing side chains of either Ser or Lys, with a brush-border-membrane (BBM) vesicle model system (rate and IC₅₀ values in Figs. 2 and 3) and with a tightly packed monolayer of CaCo-2 cells (rate in Fig. 4), to find that the rate of transport of cholesterol can be reduced to what may be considered the passive diffusion (`background') level. There is a correlation between the ability of the β-peptides to form an amphipathic-type 3₁₄-helical secondary structure in MeOH and their inhibitory effect (Table 1 and Fig. 5). Although the inhibitory activity of the β-peptides is in only the mM range (Table 2), it is to be compared with no activity at all of previously tested α-peptides and proteins (built of L -amino acids) in CaCo-2 cells. Furthermore, these active β-peptides ( 1 , 5 , and 6 ) contain only seven or nine residues and must be considered simple, first-generation models capable of mimicking the biological activity of amphipathic α-peptide helices in living whole cells.     

Probing the Helical Secondary Structure of Short-Chain β-Peptides

Structural prerequisites for the stability of the 3₁ helix of β-peptides can be defined from inspection of models (Figs. 1 and 2): lateral non-H-substituents in 2- and 3-position on the 3-amino-acid residues of the helix are allowed, axial ones are forbidden. To be able to test this prediction, we synthesized a series of heptapeptide derivatives Boc-(β-HVal-β-HAla-β-HLeu-Xaa-β-HVal-β-HAla-β-HLeu)-OMe 13–22 (Xaa = α- or β-amino-acid residue) and a β-depsipeptide 25 with a central (S)-3-hydroxybutanoic-acid residue (Xaa = –OCH(Me)CH₂C(O)–) (Schemes 1 3). Detailed NMR analysis (DQF-COSY, HSQC, HMBC, ROESY, and TOCSY experiments) in methanol solution of the β-hexapeptide H(-β-HVal-β-HAla-β-HLeu)₂-OH ( 1 ) and of the β-heptapeptide H-β-HVal-β-HAla-β-HLeu-(S,S)-β-HAla(αMe)-β-HVal-β-HAla- β-HLeu-OH ( 22 ), with a central (2S,3S)-3-amino-2-methylbutanoic-acid residue, confirm the helical structure of such β-peptides (previously discovered in pyridine solution) (Fig.3 and Tables 1–5). The CD spectra of helical β-peptides, the residues of which were prepared by (retentive) Arndt-Eistert homologation of the (S)- or L -α-amino acids, show a trough at 215 nm. Thus, this characteristic pattern of the CD spectra was taken as an indicator for the presence of a helix in methanol solutions of compounds 13–22 and 25 (including partially and fully deprotected forms) (Figs.4–6). The results fully confirm predicted structural effects: incorporation of a single ‘wrong’ residue ((R)-β-HAla, β-HAib, (R,S)-β-HAla(α Me), or N-Me-β-HAla) in the central position of the β-heptapeptide derivatives A (see 17, 18, 20 , or 21 , resp.) causes the CD minimum to disappear. Also, the β-heptadepsipetide 25 (missing H-bond) and the β-heptapeptide analogs with a single α-amino-acid moiety in the middle ( 13 and 14 ) are not helical, according to this analysis. An interesting case is the heptapeptide 15 with the central achiral, unsubstituted 3-aminopropanoic-acid moiety: helical conformation appears to depend upon the presence or absence of terminal protection and upon the solvent (MeOH vs. MeOH/H₂O).     

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 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.     

How To Stabilize or Break β-Peptidic Helices by Disulfide Bridges: Synthesis and CD Investigation of β-Peptides with Cysteine and Homocysteine Side Chains

all-L -β³-Penta-, hexa-, and heptapeptides with the proteinogenic side chains of valine, leucine, serine, cysteine, and methionine have been prepared by previously described procedures ( 12 , 13 , 14 , 15 ; Schemes 2 – 5). Thioether cleavage with Na/NH₃ in β-HMet residues has also provided a β³-hexapeptide with homocysteine (CH₂CH₂S) side chains ( 13e ). The HS−(CH₂)_n groups were positioned on the β-peptidic backbone in such a way that, upon disulfide-bridge formation, the corresponding β-peptide was expected to maintain either a 3₁-helical secondary structure ( 1 , 2 ) (Fig. 1) or to be forced to adopt another conformation ( 3 , 4 ). The 13-, 17-, 19-, and 21-membered-ring macrocyclic disulfide derivatives and their open-chain precursors, as well as all synthetic intermediates, were purified (crystallization, flash or preparative HPL chromatography; Fig. 5) and fully characterized (m.p., [α]_D, CD, IR, NMR, FAB or ESI mass spectroscopy, and elemental analysis, whenever possible; Fig. 2 and Exper. Part). The structures in MeOH and H₂O of the new β-peptides were studied by CD spectroscopy (Figs. 3 and 4), where the characteristic 215-nm-trough/200-nm-peak pattern was used as an indicator for the presence or absence of (M)-3₁-helical conformations. A CH₂−S₂−CH₂ and, somewhat less so, a (CH₂)₂−S₂−(CH₂)₂ bracket between residues i and i+3 ( 1 vs. 12d , and 2 vs. 13e in Fig. 3) give rise to CD spectra which are compatible with the presence of 3₁-helical structures, while CH₂−S₂−CH₂ brackets between residues i and i+2 ( 3 vs. 14c ) or i and i+4 ( 4 vs. 15c in Fig. 4) do not.     

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).     

Temperature-Dependent NMR and CD Spectra of β-Peptides: On the Thermal Stability of β-Peptide Helices – Is the Folding Process of β-Peptides Non-cooperative?

Temperature-dependent NMR and CD spectra of methanol solutions of a β-hexapeptide and of a β-heptapeptide at temperatures between 298 and 393 K are reported. They establish the fact that the 3₁₄-helical secondary structures of the two β-peptides, 1 and 2 , do not `melt' in the temperature range investigated. This is in sharp contrast to the behavior of the helices of α-peptides and proteins which undergo cooperative unfolding (`denaturing') upon heating. A non-cooperative mechanism is proposed, with a stepwise, rather than an `un-zipping' opening of H-bonded rings (cf. Fig. 6). The experimental results are regarded as evidence that, of the three effects which have been identified as contributing to the stability of β-peptide helices, i.e., H-bonding, hydrophobic interactions, and ethane staggering, the latter one is predominant.     

Cyclo-β-peptides: Structure and tubular stacking of cyclic tetramers of 3-aminobutanoic acid as determined from powder diffraction data

The solid-state structures of three stereoisomer, 1–3 , of the cyclic tetramer of 3-aminobutanoic acid are presented. These cyclo-β-peptides were found to be highly insoluble materials, and it proved to be impossible to grow crystals of sufficient quality for X-ray single-crystal analysis. The samples of 1–3 were, however, suitable candidates for structure determination from powder diffraction data (Fig. 1), and the application of this method is described. All three isomers have been found to adopt tubular structures (Figs. 2–4) in a fashion similar to those already observed for certain cyclo-α-peptides. The stacks of 16-membered rings are held together by four nonlinear C?O…?H? N H-bonds between pairs of molecules (Fig.5).     

Linear,Peptidase‐Resistant β2/β3‐Di‐ and α/β3‐Tetrapeptide Derivatives with Nanomolar Affinities to a Human Somatostatin Receptor,Preliminary Communication

N‐Acyl‐β²/β³‐dipeptide‐amide somatostatin analogs, 5 – 8 , with β²‐HTrp‐β³‐HLys ('natural' sequence) and β²‐HLys‐β³‐HTrp (retro‐sequence) have been synthesized (in solution). Depending on their relative configurations and on the nature of the terminal N‐acyl and terminal C‐amino group, the linear β‐dipeptide derivatives have affinities for the human receptor hsst 4, ranging from 250 to >10000 nanomolar (Fig. 3). Also, N‐Ac‐tetrapeptide amides 9 and 10 , which contain one α‐ and three β‐amino acid residues (N‐β‐α‐β‐β‐C), have been prepared (solid‐phase synthesis), with the natural (Phe, Trp, Lys, Thr) and the retro‐sequence (Thr, Lys, Trp, Phe) of side chains and with two different configurations, each, of the two central amino acid residues. The novel ‘mixed', linear α/β‐peptides have affinities for the hsst 4 receptor ranging from 23 to >10000 nanomolar (Fig. 4), and, like ‘pure' β‐peptides, they are completely stable to a series of proteolytic enzymes. Thus, the peptidic turn of the cyclic tetradecapeptide somatostatin (Fig. 1) can be mimicked by simple linear di‐ and tetrapeptides. The tendency of β‐dipeptides for forming hydrogen‐bonded rings is confirmed by calculations at the B3LYP/6‐31G(d,p) level (Fig. 2). The reported results open new avenues for the design of low‐molecular‐weight peptidic drugs.     

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).     

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.     

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.