Search

Cyanophycin is a natural biopolymer produced by a wide range of bacteria, consisting of a chain of poly-l-Asp residues with l-Arg residues attached to the β-carboxylate sidechains by isopeptide bonds. Cyanophycin is synthesized from ATP, aspartic acid and arginine by a homooligomeric enzyme called cyanophycin synthetase (CphA1). CphA1 has domains that are homologous to glutathione synthetases and muramyl ligases, but no other structural information has been available. Here, we present cryo-electron microscopy and X-ray crystallography structures of cyanophycin synthetases from three different bacteria, including cocomplex structures of CphA1 with ATP and cyanophycin polymer analogs at 2.6 Å resolution. These structures reveal two distinct tetrameric architectures, show the configuration of active sites and polymer-binding regions, indicate dynamic conformational changes and afford insight into catalytic mechanism. Accompanying biochemical interrogation of substrate binding sites, catalytic centers and oligomerization interfaces combine with the structures to provide a holistic understanding of cyanophycin biosynthesis.

ABSTRACTCellular drug delivery can improve efficacy and render intracellular pathogens susceptible to compounds that cannot permeate cells. The transport of physiologically active compounds across membranes into target cells can be facilitated by cell-penetrating peptides (CPPs), such as oligoarginines. Here, we investigated whether intracellular delivery of the drug fosmidomycin can be improved by combination with the CPP octaarginine. Fosmidomycin is an antibiotic that inhibits the second reaction in the nonmevalonate pathway of isoprenoid biosynthesis, an essential pathway for many obligate intracellular pathogens, including mycobacteria and apicomplexan parasites. We observed a strict correlation between octaarginine host cell permeability and its ability to improve the efficacy of fosmidomycin. Plasmodium bergheiliver-stage parasites were only partially susceptible to an octaarginine-fosmidomycin complex. Similarly, Toxoplasma gondiiwas only susceptible during the brief extracellular stages. In marked contrast, a salt complex of octaarginine and fosmidomycin greatly enhanced efficacy against blood-stage Plasmodium falciparum. This complex and a covalently linked conjugate of octaarginine and fosmidomycin also reverted resistance of Mycobacteriato fosmidomycin. These findings provide chemical genetic evidence for vital roles of the nonmevalonate pathway of isoprenoid biosynthesis in a number of medically relevant pathogens. Our results warrant further investigation of octaarginine as a delivery vehicle and alternative fosmidomycin formulations for malaria and tuberculosis drug development.

Connecting experimental observables with the underlying conformational ensemble is a long‐standing problem in the structure determination of biomolecules. The simulations described in this article attempt to resolve a seeming discrepancy between the conformational features derived from measured NOE intensities, 3J‐coupling constants, and circular dichroism (CD) spectra for two β‐peptides differing in a linker between two side‐chains. Although both peptides are very similar in terms of the r−6averaged distances between atom pairs involved in the observed NOEs, the molecular dynamics trajectories suggest why the CD spectra show a greater 314‐helical propensity for the linked, cyclic peptide than for the linear one, whereas slightly more NMR NOE peaks are observed and assigned for the latter. The nine 100 ns unrestrained simulations show better agreement with the observed experimental data than the single conformations derived from the published NMR structures by additional energy minimization with the GROMOS force field. They show why the seemingly contradictory quantities obtained by NMR and CD spectroscopy can arise from a single conformational ensemble.

Methods for replacing the carbonyl oxygen by two new substituents (CO→CR1R2) are discussed in this Minireview, whereby R may be H, NR2, alkyl, allyl, benzyl, vinyl, alkynyl, aryl, heteroaryl, or acyl groups. The most frequently used starting materials for geminal disubstitution with the formation of two CC bonds (R1,R2≠H, NR2) are amides and thioamides, which react with organometallic nucleophiles RM (M=Li, MgX, CeX2, TiX3, ZrX3) to give tertiary sec‐ and tert‐alkylamines. Quaternary centers can be built directly from ketones by treatment with Me3Al, MeTiCl3, or Me2TiCl2(R1R2CO→R1R2CMe2). The scope and limitations of the various methods and mechanistic models are briefly discussed. The remarkable variety and diversity of structures thus accessible are demonstrated by numerous examples.

Methoden zum Ersatz des Carbonylsauerstoffs durch zwei neue Substituenten (CO→CR1R2) werden beschrieben. Die Substituenten R können H, NR2, Alkyl‐, Allyl‐, Benzyl‐, Vinyl‐, Alkinyl‐, Aryl‐, Heteroaryl‐ oder Acyl‐Gruppen sein. Häufigste Ausgangsverbindungen für die geminale Disubstitution unter Bildung zweier neuer C,C‐Bindungen (R1,R2≠H, NR2) sind Amide und Thioamide, die mit metallorganischen Nucleophilen R‐M (M=Li, MgX, CeX2, TiX3, ZrX3) in tertiäre sec‐ und tert‐Alkylamine überführt werden. Mit Me3Al, MeTiCl3oder Me2TiCl2und Ketonen können andererseits direkt quartäre Zentren aufgebaut werden (R1R2CO→R1R2CMe2). Vor‐ und Nachteile der verschiedenen Methoden und Reaktionsmechanismen werden diskutiert. Die erstaunliche Vielfalt der so zugänglichen Strukturen wird an zahlreichen Beispielen demonstriert.

The β,αdipeptide Lcarnosine occurs in high concentrations in longlived innervated mammalian tissues and is widely sold as a food additive. On a large scale Lcarnosine is produced by chemical synthesis procedures. We have established two aqueous enzymatic reaction systems for the preparation of Lcarnosine using the dissolved bacterial βaminopeptidases DmpA from Ochrobactrum anthropiand BapA from Sphingosinicella xenopeptidilyticaas catalysts and investigated the kinetics of the enzymecatalyzed peptide couplings. DmpA catalyzed the formation of Lcarnosine from Cterminally activated βalanine derivatives acyl donor and Lhistidine acyl acceptor in an aqueous reaction mixture at pH10 with high catalytic rates Vmax19.2μmolmin−1per mg of protein, kcat12.9s−1, whereas Vmaxin the BapAcatalyzed coupling reaction remained below 1.4μmolmin−1per mg of protein kcat0.87s−1. Although the equilibrium of this reaction lies on the side of the hydrolysis products, the reaction is under kinetic control and Lcarnosine temporarily accumulated to concentrations that correspond to yields of more than 50% with respect to the employed acyl donor. However, competing nucleophiles caused unwanted hydrolysis and coupling reactions that led to decreased product yield and to formation of various peptidic byproducts. The substitution of Lhistidine for Lhistidine methyl ester as acyl acceptor shifted the pKaof the amino functionality from 9.25 to 6.97, which caused a drastic reduction in the amount of coupling byproducts in an aqueous reaction system at pH8.

The interaction of β-peptides with the DNA duplexes of dA20dT20 and a GCN4-binding CRE sequence was examined. To gauge the factors that govern these interactions, two β-pentadecapeptides, 1 and 2, a β-dodecapeptide, 3, three β-decapeptides, 4–6, three β-heptapeptides, 7–9, and β-octaarginine 10 were designed and synthesized. The β-peptides were conceived to adopt a β-peptide 314 helix, in which the side chains at position i and i + 3 are aligned vertically along one side of the helix. The side chains of Lys, Asn, and Arg were positioned such that potential H-bonding sites were created for a helical conformation to interact with the base pairs of DNA. CD Analysis showed that β-peptides 1, 2, and 10 interacted with dA20dT20. In addition, β-peptides 1 and 2 showed significant interaction with a DNA-duplex 20mer containing the ATF/CREB recognition sequence for the regulatory protein GCN4. It is impossible, at this stage of the investigation, to make a safe proposal about the actual nature of the interaction of the structures(s) of the complexes, the formation of which is suggested by the CD spectra reported herein.

No Abstract

No Abstract

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. 4;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).

β-Peptides and, to a certain extent, also mixed α,β-peptides, are resistant to degradation by a variety of proteolytic enzymes that rapidly degrade natural α-peptides. This is one of many characteristics that make β-peptides an attractive class of compounds for drug-discovery studies. On the other hand, modern organometallic reactions such as the Suzuki–Miyaura cross-coupling have become standard tools in industry laboratories to derivatize side chains of α-peptidic compounds to build up libraries of unnatural peptides. Combining both features, we prepared (4-bromo)-β3-homophenylalanine derivatives 3–5 and 12 as precursors for Suzuki–Miyaura couplings. From these bromo compounds, we synthesized biaryl-substituted β-homoamino acids 6, and analogs 13 and 15 of the anti-AIDS drug Saquinavir.

Helical polymers of isocyanopeptides derived from β‐amino acids have been synthesized and their architectures have been studied in detail. Similar to their α‐amino acid analogues, the helical conformation in these macromolecules is stabilized by internal hydrogen‐bonding arrays along the polymeric backbone. Unexpectedly, the flexibility of the β‐peptide side arms results in a rearrangement of the initial macromolecular architecture, leading to a more stable helical structure possessing a better defined hydrogen‐bonding pattern, as was concluded from IR and temperature‐dependent circular dichroism studies. Based on these results we propose a dynamic helical model for the β‐amino acid derived polyisocyanopeptides; this model is in contrast to the kinetically stable helical macromolecules that are formed upon polymerization of α‐amino acid based isocyanopeptides.

The correlation between β2-, β3-, and β2,3-amino acid-residue configuration and stability of helix and hairpin-turn secondary structures of peptides consisting of homologated proteinogenic amino acids is analyzed (Figs. 4;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. 4;6–8 and 17) and ESI mass spectra (Figs. 4;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. 4;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. 4;12), and iii) β-peptide 5 is poorly ordered in CD3OH (Fig. 4;13) and in H2O (Fig. 4;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. 4;15).

The principal secondary structural motifs adopted by peptides assembled from β‐amino acid units are discussed: the 14‐, 12‐, 10‐, 12/10‐, and 8‐helices, as well as the hairpin turn, extended structures, stacks, and sheets. Features that promote a particular folding propensity are outlined and illustrated by structures determined in solution (NMR) and in the solid‐state (x‐ray). The N–Cβ–Cα–CO dihedral angles from molecular dynamics simulations, which are indicative of a particular secondary structure, are presented. A brief description of a helix and a turn of γ‐peptides is also given. © 2005 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 84: 23–37, 2006This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

The influence of charged side chains on the folding–unfolding equilibrium of β‐peptides was investigated by means of molecular dynamics simulations. Four different peptides containing only negatively charged side chains, positively charged side chains, both types of charged side chains (with the ability to form stabilizing salt bridges) or no charged side chains were studied under various conditions (different simulation temperatures, starting structures and solvent environment). The NMR solution structure in methanol of one of the peptides (A) has already been published; the synthesis and NMR analysis of another peptide (B) is described here. The other peptides (Cand D) studied herein have hitherto not been synthesized. All four peptides A–Dare expected to adopt a left‐handed 314‐helix in solution as well as in the simulations. The resulting ensembles of structures were analyzed in terms of conformational space sampled by the peptides, folding behavior, structural properties such as hydrogen bonding, side chain–side chain and side chain–backbone interactions and in terms of the level of agreement with the NMR data available for two of the peptides. It was found that the presence of charged side chains significantly slows down the folding process in methanol solution due to the stabilization of intermediate conformers with side chain–backbone interactions. In water, where the solvent competes with the solute–solute polar interactions, the folding process to the 314‐helix is faster in the simulations.

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.

The β-heptapeptides H-βhVal-βhAla-βhLeu-βhAla(Xn)-βhVal-βhAla-βhLeu-OH 3–7 with central 3-amino-2-fluoro-, 3-amino-2,2-difluoro-, or 3-amino-2-hydroxybutanoic acid residues (βhAla(Xn)) of like and unlike configuration were subjected to a detailed NMR analysis in MeOH solution. For the geminal difluoro and for the F- and OH-substituted derivatives of u-configuration (see 5, 4, and 7, resp.), 14-helices were found, i.e., with axial disposition of the hetero atoms on the helix. The two compounds containing the central l-configured β-amino acid moieties (see 3 and 6) are not helical over the full lengths of the chains; they have ‘quasi-helical’ termini and a central turn consisting of a ten-membered H-bonded ring (Fig. 4;2, d and e). Quantum-mechanical calculations with l- and u-AcNH-CHMe-CHF-CONH2 confirm the observed preference for a conformation with antiperiplanar arrangement of the F&bond;C and the C&dbond;O bond. The calculated energy difference between the observed ‘non-helical’ geometry of this moiety and a hypothetical helical one is 6.4 4;kcal/mol (Fig. 4;3).

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 4;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→OtBu etherification (for β2hSer; Schemes 4;5 and 6), OH→STrt replacement (for β2hCys; Scheme 4;7), and CH2OH→CH2N3→CH2NH2 transformations (for β2hHis; Schemes 4;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 (tR), 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.

The influence of valine side chains on the folding/unfolding equilibrium and, in particular, on the 314-helical propensity of β3-peptides were investigated by means of molecular-dynamics (MD) simulation. To that end, the valine side chains in two different β3-peptides were substituted by leucine side chains. The resulting four peptides, of which three have never been synthesized, were simulated for 150 to 200 4;ns at 298 and 340 4;K, starting from a fully extended conformation. The simulation trajectories obtained were compared with respect to structural preferences and folding behavior. All four peptides showed a similar folding behavior and were found to predominantly adopt 314-helical conformations, irrespective of the presence of valine side chains. No other well-defined conformation was observed at significant population in any of the simulations. Our results imply that β3-peptides show a structural preference for 314-helices independent of the branching nature of the side chains, in contrast to what has been previously proposed on the basis of circular-dichroism (CD) measurements.

Oligomers of (R)-3-hydroxybutanoate (OHB) have previously been shown to transport cations through lipid bilayers. The ion-transport activity has been attributed to the formation of hydrophobic aggregates or pores, which have been identified by fluorescence-microscopy measurements of membrane-incorporated fluorescence-labeled OHBs. To obtain more information about these aggregates, we describe here the synthesis of the specifically F-labeled HB oligomers II–IV for structural investigation by means of solid-state 19F-NMR spectroscopic techniques.

(S)-β2-Homoamino acids with the side chains of Asp, Glu, Asn, and Gln have been prepared and suitably protected (N-Fmoc, CO2tBu, CONHTrt) for solid-phase peptide syntheses. The key steps of the syntheses are: N-acylation of 5,5-diphenyl-4-isopropyl-1,3-oxazolidin-2-one (DIOZ) with succinic and glutaric anhydrides (Scheme 4;2), alkylation of the corresponding Li-enolates with benzyl iodoacetate and Curtius degradation (Scheme 4;4), and removal of the chiral auxiliary (Scheme 4;5). In addition, numerous functional-group manipulations (CO2H⇌;CO2tBu, CO2Bn⇌;CO2H, CbzNH→FmocNH, CO2H→CO2NH2→CONHTrt; Schemes 4;2, 4, 5, and 6) were necessary, in order to arrive at the four target structures. The configurational assignments were confirmed by X-ray crystal-structure determinations (Scheme 4;2 and Fig. 4;3). The enantiomeric purities of a β2hAsn and of a β2hGln derivative were determined by HPLC on a Chiralcel column to be 99.7 : 0.3 and >99 : 1, respectively (Fig. 4;4). Notably, it took up to twelve steps to prepare a suitably protected trifunctional product with a single stereogenic center (overall yield of 10% from DIOZ and succinic anhydride)!

Two different strategies have been employed for the synthesis of Fmoc‐protected β3‐homoarginine; the Arndt–Eistert homologation of α‐arginine and the guanidinylation of β3‐homoornithine. Solid‐phase β‐peptide synthesis was used for the preparation of β‐heptapeptide 1, which was designed to form a helix stabilized by electrostatic interactions through positively (β3hArg) and negatively charged (β3hGlu) amino acid residues. CD measurements and corresponding NMR investigations in MeOH and aqueous solutions do indeed show that the β‐peptidic 314‐helix can be stabilized by salt‐bridge formation.

Two different strategies have been employed for the synthesis of Fmoc-protected β³-homoarginine; the Arndt–Eistert homologation of α-arginine and the guanidinylation of β³-homoornithine. Solid-phase β-peptide synthesis was used for the preparation of β-heptapeptide 1, which was designed to form a helix stabilized by electrostatic interactions through positively (β³hArg) and negatively charged (β³hGlu) amino acid residues. CD measurements and corresponding NMR investigations in MeOH and aqueous solutions do indeed show that the β-peptidic 314-helix can be stabilized by salt-bridge formation.

Methylthiomethylation and subsequent sulfoxidation and pyrolysis of the N-Boc-protected ?3-amino acid methyl esters derived from Ala, Val, and Leu provided the ?-methylene esters; these were then used to synthesize the ?-peptides specified in the title (step-by-step and fragment coupling in solution). The novel ?-peptides were fully characterized, but neither solid-state nor solution structures were found.

Although they are less abundant than their α-analogues, β-amino acids occur in nature both in free form and bound to peptides. Oligomers composed exclusively of β-amino acids (so-called β-peptides) might be the most thoroughly investigated peptidomimetics. Beside the facts that they are stable to metabolism, exhibit slow microbial degradation, and are inherently stable to proteases and peptidases, they fold into well-ordered secondary structures consisting of helices, turns, and sheets. In this respect, the most intriguing effects have been observed when β²-amino acids are present in the β-peptide backbone. This review gives an overview of the occurrence and importance of β²-amino acids in nature, placing emphasis on the metabolic pathways of β-aminoisobutyric acid (β-Aib) and the appearance of β²-amino acids as secondary metabolites or as components of more complex natural products, such as peptides, depsipeptides, lactones, and alkaloids. In addition, a compilation of the syntheses of both achiral and chiral β²-amino acids is presented. While there are numerous routes to achiral β²-amino acids, their EPC synthesis is currently the subject of many investigations. These include the diastereoselective alkylation and Mannich-type reactions of cyclic- or acyclic β-homoglycine derivatives containing chiral auxiliaries, the Curtius degradation, the employment of transition-metal catalyzed reactions such as enantioselective hydrogenations, reductions, C–H insertions, and Michael-type additions, and the resolution of rac. β²-amino acids, as well as several miscellaneous methods. In the last part of the review, the importance of β²-amino acids in the formation of β-peptide secondary structures is discussed. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 2004

Folding properties of β-peptides were investigated by means of NMR experiments and MD simulations of β-dipeptides, which serve as small test systems to study the influence of stereocenters and side chains on hydrogen-bond and consequently on secondary-structure formation. Two stereoisomers, SR and SS, of a Val-Phe dipeptide, and of the corresponding Ala-Ala dipeptide, and a Gly-Gly dipeptide were simulated in methanol for 40 ns. In agreement with experiment, the isomers of the Val-Phe dipeptide adopt quite different conformers at 298 K, the differences being reduced at 340 K. Interestingly, the SR isomer shows enhanced hydrogen bonding at the higher temperature. The adopted conformations are primarily determined by the R or S side chain substitution, and less by the type of side chain. Back-calculation of ¹H ROESY spectra and ³J coupling constants from the MD simulations and comparison with the experimental data for the Val-Phe dipeptides shows good agreement between simulation and experiment, and reveals possible problems and pitfalls, when deriving structural properties of a small and extremely flexible molecule from NMR data only. Inclusion of all aspects of internal dynamics is essential to the correct prediction of the NMR spectra of these small molecules. Cross comparison of calculated with experimental spectra for both isomers shows that only a few out of many ROESY peaks reflect the sizeable conformational differences between the isomers at 298 K.

Folding properties of β‐peptides were investigated by means of NMR experiments and MD simulations of β‐dipeptides, which serve as small test systems to study the influence of stereocenters and side chains on hydrogen‐bond and consequently on secondary‐structure formation. Two stereoisomers, SRand SS, of a Val‐Phe dipeptide, and of the corresponding Ala‐Ala dipeptide, and a Gly‐Gly dipeptide were simulated in methanol for 40 ns. In agreement with experiment, the isomers of the Val‐Phe dipeptide adopt quite different conformers at 298 K, the differences being reduced at 340 K. Interestingly, the SRisomer shows enhanced hydrogen bonding at the higher temperature. The adopted conformations are primarily determined by the Ror Sside chain substitution, and less by the type of side chain. Back‐calculation of 1H ROESY spectra and 3Jcoupling constants from the MD simulations and comparison with the experimental data for the Val‐Phe dipeptides shows good agreement between simulation and experiment, and reveals possible problems and pitfalls, when deriving structural properties of a small and extremely flexible molecule from NMR data only. Inclusion of all aspects of internal dynamics is essential to the correct prediction of the NMR spectra of these small molecules. Cross comparison of calculated with experimental spectra for both isomers shows that only a few out of many ROESY peaks reflect the sizeable conformational differences between the isomers at 298 K.

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.

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.

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.

Inspired by naturally occurring DNA-binding proteins and their artificial α-peptidic mimics reported to date, a research project was initiated aiming at creating a new class of β-peptides capable of binding to and ultimately regulating the functions of DNA. As an initial foray, a β³-pentadecapeptide 1, which bears H-bonding Asn side chains and positively charged Lys side chains, was designed and synthesized on the solid support. DNA-Complexation studies by means of circular dichroism and DNA-melting-temperature measurements revealed the first preliminary indications that support the existence of ordered interactions between β-peptides and DNA.

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.

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.

Multigram amounts of suitably protected β²-amino acids with 17 of the 20 proteinogenic side chains are prepared by diastereoselective reactions of Li, B, or Ti enolates of the corresponding 3-acyl-4-isopropyl-5,5-diphenyloxazolidin-2-ones (acyl-DIOZ; 1) with appropriate electrophiles (amidomethylation, hydroxyalkylation, (benzyloxycarbonyl)methylation) in yields of 55–90% and with diastereoselectivities of 80 to >97% (Scheme). The primary products 2–8 thus obtained are converted to protected β²-amino acids by standard procedures (Table 1). Many of the DIOZ derivatives are highly crystalline compounds (31 X-ray crystal structures in Table 2). The chiral auxiliary DIOZ, readily prepared in either enantiomeric form, is recovered with high yield.

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'.

No Abstract

In vitro studies: In CaCo‐2 cell monolayers the β‐nonapeptide H(β‐HAla‐β‐HLys‐β‐HPhe)3‐OH·4HCl (1), 14C‐labeled on both C atoms of the CH2–CO moiety of the central β‐HPhe residue, showed a low intrinsic permeability (<1%) and is subject to a prominent efflux system. The β‐peptide (1) binds to human and rat plasma protein in vitroindependent of the concentration of 1and of the species (30–36% bound fraction at 50, 500, and 5000 ng/ml), and has only low affinity for the corresponding blood cells (less than 5% of compound 1in blood cells).

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)&bond;C(Me2(CHR)) bond (τ1,2), almost perfect staggering around the C(2)&bond;C(3) ethane bond (τ2,3), and antiperiplanar arrangement of H(C3) and H(N) (τ3,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&dbond;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).

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.

The preparation of three new chiral derivatives of DMPU (N,N'-dimethylpropyleneurea) is described (Schemes 2–4); one type of derivative carries 1-phenylethyl or 1-cyclohexylethyl groups at the N-atoms of the tetrahydropyrimidin-2(1H)-one ring (2 and 4), another type of derivative is substituted at C(4) and C(6) of the heterocyclic ring (7). The potential of these chiral Lewis bases as promoters in the regio- and/or enantioselective addition of 2-(1,3-dithianyl)lithium to cyclohex-2-en-1-one was explored; they are all unable to effect enantioselective addition; the derivatives with branched substituents at the N-atoms do not shift the addition mode from 1,2 to 1,4, while the 3,4,5,6-tetrahydro-1,3,4,6-tetramethylpyrimidin-2(1H)-one does (Scheme 5). The results provide useful information regarding the nature of the nucleophilic organolithium reagent: obviously, the steric hindrance to Li complexation on the C&dbond;O O-atom of the tetrahydropyrimidin-2(1H)-one by branched substituents at N-atoms (cf. X-ray crystal structure of 2 in the Fig.) prevents solvent-separated-ion-pair (SSIP) formation; this was confirmed by PM3 and B3LYP/3-21-G(d)//PM3 calculations (Scheme 6).

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.

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&bond;HTrp&bond;OH and H-β³&bond;HTrp&bond;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&bond; and Z&bond;β²-HTrp(Boc)&bond;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&bond;CH2CO2Bn (Scheme 3). Subsequent hydrogenolysis, Curtius degradation, and removal of the Evans auxiliary group gives the desired derivatives of (R)-H&bond;β²-HTrp&bond;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.

The crystal structures of 32 derivatives of 4-isopropyl-5,5-diphenyl-1,3-oxazolidin-2-one (A and 1–31) are presented (Fig. 2 and Tables 1–3). In all but four structures, the Me2CH group is in a disposition that mimick a Me3C group (Figs. 3–5). The five-membered ring shows conformations from an envelope form with the Ph2C group out of the plane containing the other four atoms to the twist form with the twofold axis through the C&dbond;O group (Fig. 6, and Table 2). In the entire series, the Me2CH and the neighboring trans Ph group are approximately antiperiplanar (average torsion angle 155°). The structural features are used to interpret the previously observed reactivity behavior of the diphenyl-oxazolidinone derivatives. The practical advantages of the title compound over classical Evans auxiliaries are outlined (Figs. 1 and 7, and Scheme 2): high crystallinity of all derivatives, steric protection of the C&dbond;O group in the ring, excellent stereoselectivities in reactions of its derivatives, and safe preparation and easy recovery of the auxiliary.