'100 years of peptide synthesis': ligation methods for peptide and protein synthesis with applications to beta-peptide assemblies.

A brief survey of the history of peptide chemistry from Theodore Curtius to Emil Fischer to Bruce Merrifield is first presented. The discovery and development of peptide ligation, i.e. of actual chemical synthesis of proteins are described. In the main chapter, 'Synthesis of Proteins by Chemical Ligation' a detailed discussion of the principles, reactivities and mechanisms involved in the various coupling strategies now applied (ligation, chemical ligation, native chemical ligation) is given. These include coupling sites with cysteine and methionine (as well as the seleno analogs), histidine, glycine and pseudo-prolines, 'unrestricted' amino-acid residues (using the Staudinger reaction), as well as solid-phase segment coupling by thioligation of unprotected peptides. In another section, 'Synthesis of beta-peptides by Thioligation', couplings involving beta2- and beta3-peptides are described (with experimental details).

Differential effects of temperature on E. coli and synthetic polyhydroxybutyrate/polyphosphate channels.

Complexes of poly-(R)-3-hydroxybutyrate and inorganic polyphosphate (PHB/polyP), isolated from the plasma membranes of Escherichia coli or prepared synthetically (HB(128)/polyP(65)), form Ca(2+)-selective ion channels in planar lipid bilayers that exhibit indistinguishable gating and conductance characteristics at 22 degrees C. Here we examine the gating and conductance of E. coli and synthetic PHB/polyP complexes in planar lipid bilayers as a function of temperature from 15 to 45 degrees C. E. coli PHB/polyP channels remained effectively open throughout this range, with brief closures that became more rare at higher temperatures. Conversely, as temperatures were gradually increased, the open probability of HB(128)/polyP(65) channels progressively decreased. The effect was fully reversible. Channel conductance exhibited three distinct phases. Below 25 degrees C, as PHB approached its glass temperature (ca. 10 degrees C), the conductance of both E. coli and synthetic channels remained at about the same level (95-105 pS). Between 25 degrees C and ca. 40 degrees C, the conductance of E. coli and synthetic channels increased gradually with temperature coefficients (Q(10)) of 1.45 and 1.42, respectively. Above 40 degrees C, E. coli channel conductance increased sharply, whereas the conductance of HB(128)/polyP(65) channels leveled off. The discontinuities in the temperature curves for E. coli channels coincide with discontinuities in thermotropic fluorescence spectra and specific growth rates of E. coli cells. It is postulated that E. coli PHB/polyP complexes are associated with membrane components that inhibit their closure at elevated temperatures.

Computational, ReactIR-, and NMR-spectroscopic investigations on the chiral formyl anion equivalent N-(alpha-lithiomethylthiomethyl)-4-isopropyl-5,5-diphenyloxazolidin-2-one and related compounds.

The 4-isopropyl-3-methylthiomethyl-5,5-diphenyloxazolidin-2-one is readily lithiated in THF on the exocyclic CH2 group (1 --> 2) to give a synthetically useful chiral nucleophilic formylating reagent. We have now studied the lithiation reaction by ReactIR spectroscopy and the structure of the organolithium reagent by computational methods and by NMR-spectroscopic measurements. The lithiation is complete at -78 degrees C within 90 seconds, and it is accompanied by a decrease of the C=O wavenumber by 50 cm(-1). The NMR data (collected in [D8]THF) give no evidence for 13C,6Li coupling or for aggregation; from DPFGSE-ROE spectra the single diastereoisomer of the lithium compound 2 seen in the NMR spectra (NMR-spectroscopic measurements from -105 to -20 degrees C) is assigned like configuration; the 13C=O signal of the oxazolidinone undergoes a 7 ppm downfield shift upon lithiation (1 --> 2); line-shape analyses of the signals from the diastereotopic CH2 and CMe2 protons in lithiated 4,4-dimethyl-3-methylthiomethyloxazolidin-2-one (model compound 7) reveal a deltaH(double dagger) of 8.9 +/- 0.2 kcal mol(-1) for enantiomerization. The theoretical calculations provide an energy-minimum structure for the lithium compound 2 with coordination of the carbonyl oxygen to lithium, with an antiperiplanar arrangement of the C,Li and S,CH3 bonds, and with relative like configuration of the two stereocenters--in perfect agreement with the conclusions from the IR- and NMR-spectroscopic measurements!

Preparation of dendritic and non-dendritic styryl-substituted Salens for cross-linking suspension copolymerization with styrene and multiple use of the corresponding Mn and Cr complexes in enantioselective epoxidations and hetero-Diels-Alder reactions.

Following work with TADDOLs and BINOLs, we have now prepared Salen derivatives (2, 3, 14, 15, 18, 19, 20, 21) carrying two to eight styryl groups for cross-linking copolymerization with styrene. The Salen cores are either derived from (R,R)-diphenyl ethylene diamine (3, 15, 19, 21) or from (R,R)-cyclohexane diamine (2, 14, 18, 20). The styryl groups are attached to the salicylic aldehyde moieties, using Suzuki (cf. 1) or Sonogashira cross-coupling (cf. 11), and/or phenolic etherification (cf. 5, 7) with dendritic styryl-substituted Fréchet-type benzylic branch bromides. Subsequent condensation with the diamines provides the chiral Salens. Corresponding Salens lacking the peripheral vinyl groups (cf. 12, 13, 16, 17) were also prepared for comparison of catalytic activities in homogeneous solution with those in polystyrene. Cross-linking radical suspension copolymerization of styrene and the styryl Salens, following a procedure by Itsuno and Fréchet, gave beads (ca. 400 microm diameter) which were loaded with Mn or Cr (ca. 0.2 mmol of complex per g of polymer), with more than 95% of the Salen incorporated being actually accessible for complexation (by elemental analysis). The polymer-bound Mn and Cr complexes were used as catalysts for epoxidations of six phenyl-substituted olefins (m-CPBA/NMO; products 22a-f), and for dihydropyranone formation from the Danishefsky diene and aldehydes (PhCHO, C5H11CHO, C6H11CHO, products 23a-c). There are several remarkable features of the novel immobilized Salens: i) The dendritic branches do not slow down the catalytic activity of the complexes in solution; ii) the reactions with Salen catalysts incorporated in polystyrene give products of essentially the same enantiopurity as those observed in homogeneous solution with the dendritically substituted or with the original Jacobsen - Katsuki complexes; iii) some Mn-loaded beads have been stored for a year, without loss of activity; iv) especially the biphenyl- and the acetylene-linked Salen polymers (p-2, -3, -20, -21, Figure 2, 3) give Mn complexes of excellent performance: after ten uses (without re-charging with Mn!) there is no loss of enantioselectivity or degree of conversion under the standard conditions.

Peptide folding induces high and selective affinity of a linear and small beta-peptide to the human somatostatin receptor 4.

beta-Peptides with side chains in the 2- and 3-positions on neighboring residues (of (S) configuration) are known to fold and form a turn (similar to an alpha-peptidic beta-turn). Thus, we have synthesized an appropriately substituted beta-tetrapeptide derivative to mimic the hormone somatostatin in its binding to the human receptors hsst(1-5), which is known to rest upon a turn containing the amino acid residues Thr, Lys, Trp, and Phe. The N-acetyl-peptide amide Ac-beta(3)-HThr-beta(2)-HLys-beta(3)-HTrp-beta(3)-HPhe-NH(2) (1) indeed shows all characteristics of the targeted turn-mimic: Lys CH(2) groups are in the shielding cone of the Trp indole ring (by NMR analysis, Figure 2) and there is high and specific nanomolar affinity for hsst(4) receptor (Table 1). In contrast, the isomer 2 bearing the Lys side chain in 3-, rather than in the 2-position, has a 1000-fold smaller affinity to hsst(4). The syntheses of the required Fmoc-protected beta-amino acids (8-11, 17) are described (Schemes 1-3). Coupling of the beta-amino acids was achieved by the manual solid-phase technique, on Rink resin.

The beta-peptide hairpin in solution: conformational study of a beta-hexapeptide in methanol by NMR spectroscopy and MD simulation.

The structural and thermodynamic properties of a 6-residue beta-peptide that was designed to form a hairpin conformation have been studied by NMR spectroscopy and MD simulation in methanol solution. The predicted hairpin would be characterized by a 10-membered hydrogen-bonded turn involving residues 3 and 4, and two extended antiparallel strands. The interproton distances and backbone torsional dihedral angles derived from the NMR experiments at room temperature are in general terms compatible with the hairpin conformation. Two trajectories of system configurations from 100-ns molecular-dynamics simulations of the peptide in solution at 298 and 340 K have been analyzed. In both simulations reversible folding to the hairpin conformation is observed. Interestingly, there is a significant conformational overlap between the unfolded state of the peptide at each of the temperatures. As already observed in previous studies of peptide folding, the unfolded state is composed of a (relatively) small number of predominant conformers and in this case lacks any type of secondary-structure element. The trajectories provide an excellent ground for the interpretation of the NMR-derived data in terms of ensemble averages and distributions as opposed to single-conformation interpretations. From this perspective, a relative population of the hairpin conformation of 20% to 30% would suffice to explain the NMR-derived data. Surprisingly, however, the ensemble of structures from the simulation at 340 K reproduces more accurately the NMR-derived data than the ensemble from the simulation at 298 K, a question that needs further investigation.

Lithiated 4-isopropyl-3-(methylthiomethyl)-5,5-diphenyloxazolidin-2-one: a chiral formyl anion equivalent for enantioselective preparations of 1,2-diols, 2-amino alcohols, 2-hydroxy esters, and 4-hydroxy-2-alkenoates.

The heterocyclic compound specified in the title (and readily prepared from commercial precursors) has a sterically protected C==O group, so that direct lithiation by BuLi at the exocyclic CH(2) group is possible (3 --> Li-3). The lithiated N,S-acetal derivative (Li-3) adds diastereoselectively to aldehydes (Table 2), unsymmetrical ketones (Table 3), chalcone (1,4-addition, Scheme 2), and N-phosphinoyl- and N-sulfonylimines (Table 4). Protection of the newly formed OH groups (Scheme 3) and/or MeS/OH displacement by Hg(O(2)CCF(3))(2) in aqueous THF/acetonitrile converts the N,S-acetals into hemiaminals (--> 20) which, in turn, are readily cleaved to aldehydes, with recovery of the chiral auxiliary (1, Scheme 4). The aldehydes (especially those lacking alpha-carbonyl hydrogens) may be isolated, or they are trapped in situ by reduction to (selectively protected) diols or amino alcohols, by addition of Grignard or Li reagents, which provides diols with two stereogenic centers, by oxidation to give 2-hydroxy esters, or by olefination to provide 4-hydroxy-2-alkenoates (Scheme 5). The scope and limitations of the new, overall enantioselective transformation are determined, and the readily recovered chiral auxiliary used is compared with oxazolidinones of other substitution patterns (Scheme 7). The configuration of a number of products has been assigned by single-crystal X-ray diffraction (cf. Figure 5). These structures and similarities of NMR data led to configurational assignment of the other products (see formulas in the schemes and tables) by analogy. A simple mechanistic model for the stereochemical course of the addition of Li-3 to aldehydes and ketones is presented (Figure 6).

The outstanding biological stability of beta- and gamma-peptides toward proteolytic enzymes: an in vitro investigation with fifteen peptidases.

A series of 36 linear and cyclic beta- and gamma-peptides consisting of as few as two, and as many as 15 residues, was offered as substrates to 15 commercially available proteases of bacterial, fungal, and eukaryotic origin, including a beta-lactamase and amidases, as well as most vigorous, nonspecific proteases, such as the 20S proteasome from human erythrocytes. For comparison, an alpha-eicosapeptide and standard substrates of the proteolytic enzymes were included in the investigation. Under conditions of complete cleavage of the alpha-peptide within 15 min the beta- and gamma-peptides were stable for at least 48 h. Inhibition studies with seven beta- and gamma-peptides and alpha-chymotrypsin show that the residual enzyme activity toward succinyl-Ala-Ala-Pro-Phe-p-nitroanilide is unchanged within experimental error after incubation for 15 min with the peptide analogues. Thus, beta- and gamma-peptides with proteinogenic side chains, that is, consisting of the singly or doubly homologated natural alpha-amino acids (one or two CH(2) groups inserted in the backbone of each residue), are completely stable to common proteases, without inhibiting their normal activity (as demonstrated for alpha-chymotrypsin). This proteolytic stability of peptides built of homologated amino acids is a prerequisite for their potential use as drugs.

Immobilization of BINOL by cross-linking copolymerization of styryl derivatives with styrene, and applications in enantioselective Ti and Al Lewis acid mediated additions of Et2Zn and Me3SiCN to aldehydes and of diphenyl nitrone to enol ethers

The chiral ligand 1,1'-bi-2-naphthol (BINOL) has been succesfully immobilized on polystyrene. Several dendritic and non-dendritic BINOL derivatives (3, and 13-17), bearing at least two polymerizable styryl groups, were prepared and fully characterized. Suspension copolymerization of the MOM- or TIPS-protected cross-linking BINOL ligands (MOM = methyloxymethyl, TIPS = triisopropylsilyl) with styrene, cleavage of the protecting-groups, and loading with a Lewis-acid afforded catalytically active polystyrene-supported BINOLates. The polymer-bound BINOLs p-3, and p-13-p-16 were tested in the Ti-BINOLate-mediated addition of Et2Zn to PhCHO. The enantioselectivities (up to 93%) and conversions obtained with the polymer-bound catalysts were in most cases identical (within experimental error) to those obtained with the unsubstituted 1,1'-bi-2-naphthol and with the non-polymerized BINOL cross-linkers under homogeneous conditions. Special focus was put on the reusability of the supported catalyst: the polymer-beads were used in up to 20 consecutive catalytic runs, with the best polymers showing no or only minor loss of selectivity. BINOL-polymers p-17, obtained by copolymerization of a 3,3'-distyryl-substituted BINOL 17a with styrene, were used in the BINOL. AlMe-mediated cycloaddition of diphenyl nitrone with alkyl vinyl ethers. In all cases the exo/endo selectivity (> or =92:8) and the enantioselectivities with which the exo-cycloadducts were formed (> or =95%) correspond to those observed in the homogeneous reactions. A dendritically cross-linked BINOL-polymer was also employed in the Ti-BINOLate-mediated cyanosilylation of pivalaldehyde. The enantiopurity of the cyanohydrine obtained in the first run was as high as in the homogeneous reaction (72%); surprisingly the catalytic performance of the supported catalyst increased steadily during the first catalytic cycles to reach 83%. Thus, cross-linking BINOLs can be succesfully incorporated into a polystyrene matrix (without racemization!) to give polymer-bound BINOL ligands that give excellent performance over many catalytic cycles with catalytic activities comparable with those of soluble analogues.

Synthetic substrates and inhibitors of beta-poly(L-malate)-hydrolase (polymalatase).

Polymalatase from Physarum polycephalum calalysed the hydrolysis of beta-poly[L-malate] and of the synthetic compounds beta-di(L-malate), beta-tetra(L-malate), beta-tetra(L-malate) beta-propylester, and L-malate beta-methylester. Cyclic beta-tri(L-malate), cyclic beta-tetra(L-malate), and D-malate beta-methylester were not cleaved, but were competitive inhibitors. The O-terminal acetate of beta-tetra(L-malate) was neither a substrate nor an inhibitor. L-Malate was liberated; the Km, Ki and Vmax values were measured. The appearance of comparable amounts of beta-tri(L-malate), and beta-di(L-malate) during the cleavage of beta-tetra(L-malate) indicated a distributive mechanism for small substrates. The accumulation of a series of oligomers, peaking with the 11-mer and 12-mer in the absence of higher intermediates, indicated that the depolymerization of beta-poly(L-malate) was processive. The results indicate that beta-poly(L-malate) is anchored at its OH-terminus by the highly specific binding of the penultimate malyl residue. The malyl moieties beyond 12 residues downstream from the OH-terminus extend into a diffuse second, electrostatic binding site. The catalytic site joins the first binding site, accounting for the cleavage of the polymer into malate residues. It is proposed that the enzyme does not dissociate from beta-poly(L-malate) during hydrolysis, when both sites are filled with the polymer. When only the first binding site is filled, the reaction partitions at each oligomer between hydrolysis and dissociation.

A valine-derived lithiated 3-methylthiomethyl-1,3-oxazolidin-2-one for enantioselective nucleophilic hydroxymethylation, formylation, and alkoxycarbonylation of aldehydes

The 3-methylthiomethyl-4-isopropyl-5,5-diphenyl-1,3-oxazolidin-2-one (I, prepared in three steps from Boc-valine ester) is lithiated and added to aldehydes, with protecting in situ trapping of the primary adducts, to give the N,S-acetal derivatives II of 2-hydroxy aldehydes in high yields and diastereoselectivities. Cleavage (with ready recovery of the oxazolidinone auxiliary) is possible, to afford, for instance, enantiopure 1,2-diols, selectively protected (OBn, OMOM, OTBS) in the 2-position.

X-Ray, Molecular Diffusion, and NOESY NMR Studies of Chiral, Tetranuclear Cu(I) Catalysts Based on Monodentate Thiol Analogues of TADDOL.

The new, stable, chiral thiol-TADDOL Cu(I) catalysts 1 (X=OH, OMe, NMe(2)) reveal an unexpected monodentate complexation mode, both in solution and in the solid state. For the first time, the aggregation state of organocopper complexes has been determined by NMR diffusion measurements. NOESY NMR data on model isocyanide Cu complexes reveal a different conformation of the TADDOL moiety as a function of the second potential donor group. TADDOL=alpha,alpha,alpha',alpha'-tetraaryl-2,2-dimethyl-1,3-dioxolane-4,5-dimethanol.

Immobilization of TADDOL with a High Degree of Loading on Porous Silica Gel and First Applications in Enantioselective Catalysis.

The versatile TADDOL moiety has been immobilized for the first time on an inorganic support (highly porous silica gel). This gives access to Ti - Lewis acids (see picture), which turn out to be very efficient in two standard enantioselective reactions. X=OiPr, OTos.

Detection, synthesis, structure, and function of oligo(3-hydroxyalkanoates): contributions by synthetic organic chemists.

Two types of the biological macromolecules poly(R-3-hydroxyalkanoates) have been identified: the high-molecular-weight microbial storage material (sPHA) and a short-chain variety, consisting of butyrate and valerate residues, complexed with other biomacromolecules such as calcium polyphosphate or proteins (cPHB/PHV). While sPHA has attracted, and still enjoys, a lot of attention from numerous scientists around the world, research on cPHB and the structurally and functionally related polymalate (PMA) is still in its infancy. In this article, we present a review on the chemical synthesis, structure, function and interactions of monodisperse cPHAs, the oligo(3-hydroxyalkanoates), with emphasis on the butyrates (OHB); we report hitherto unpublished results on the enzymatic degradation of cPHB and PMA, on a new analytical method for HB/HV detection in biological samples, and on OHB-mediated Ca2+ transport through phospholipid bilayers of artificial vesicles; finally, we discuss possible mechanisms of ion transport through cell membranes, as caused by cPHB. The speculative--and provocative--question is asked whether the structurally simple PHAs may have evolved as storage materials and amphiphilic macromolecules before poly-peptides, -saccharides, and -nucleic acids, in the history of life, or under prebiotic conditions.

Nonapeptide analogues containing (R)-3-hydroxybutanoate and beta-homoalanine oligomers: synthesis and binding affinity to a class I major histocompatibility complex protein.

Crystal structures of antigenic peptides bound to class I MHC proteins suggest that chemical modifications of the central part of the bound peptide should not alter binding affinity to the MHC restriction protein but could perturb the T-cell response to the parent epitope. In our effort in designing nonpeptidic high-affinity ligands for class I MHC proteins, oligomers of (R)-3-hydroxybutanoate and(or) beta-homoalanine have been substituted for the central part of a HLA-B27-restricted T-cell epitope of viral origin. The affinity of six modified peptides to the B2705 allele was determined by an in vitro stabilization assay. Four out of the six designed analogues presented an affinity similar to that of the parent peptide. Two compounds, sharing the same stereochemistry (R,R,S,S) at the four stereogenic centers of the nonpeptidic spacer, bound to B2705 with a 5-6-fold decreased affinity. Although the chiral spacers do not strongly interact with the protein active site, there are configurations which are not accepted by the MHC binding groove, probably because of improper orientation of some lateral substituents in the bound state and different conformational behavior in the free state. However we demonstrate that beta-amino acids can be incorporated in the sequence of viral T-cell epitopes without impairing MHC binding. The presented structure-activity relationships open the door to the rational design of peptide-based vaccines and of nonnatural T-cell receptor antagonists aimed at blocking peptide-specific T-cell responses in MHC-associated autoimmune diseases.

Structure-based design of nonnatural ligands for the HLA-B27 protein.

X-ray studies as well as structure-activity relationships indicate that the central part of class I MHC-binding nonapeptides represents the main interaction site for a T cell receptor. In order to rationally manipulate T cell epitopes, several nonpeptidic spacer have been designed from the X-ray structure of a MHC-peptide complex and substituted for the T cell receptor-binding part of several antigenic peptides. The binding of the modified epitopes to the HLA-B*2705 protein was studied by an in vitro stabilisation assay and the thermal stability of all complexes examined by circular dichroism spectroscopy. Depending on their chemical nature and length, the introduced spacers may be classified into two categories. Monofunctional spacers (11-amino undecanoate, (R)-3-hydroxybutyrate trimer) simply link two anchoring peptide positions (P3 and P9) but loosely contact the MHC binding groove, and thus decrease more or less the affinity of the altered epitopes to HLA-B*2705. Bifunctional spacers ((R)-3-hydroxybutyrate and beta-homoalanine combinations) not only bridges the two distant anchoring amino acids but also strongly interact with the binding cleft and lead to an increase in binding to the MHC protein. The presented modified ligands constitute interesting tools for perturbing the T cell response to the parent antigenic peptide.

Substituting nonpeptidic spacers for the T cell receptor-binding part of class I major histocompatibility complex-binding peptides.

X-ray diffraction studies as well as structure-activity relationships indicate that the central part of class I major histocompatibility complex (MHC)-binding nonapeptides represents the main interaction site for a T cell receptor. In order to rationally manipulate T cell epitopes, three nonpeptidic spacers have been designed from the x-ray structure of a MHC-peptide complex and substituted for the T cell receptor-binding part of several antigenic peptides. The binding of the modified epitopes to the human leukocyte antigen-B*2705 protein was studied by an in vitro stabilization assay, and the thermal stability of all complexes was examined by circular dichroism spectroscopy. Depending on their chemical nature and length, the introduced spacers may be classified into two categories. Monofunctional spacers (11-amino undecanoate, (R)-3-hydroxybutyrate trimer) simply link two anchoring peptide positions (P3 and P9) but loosely contact the MHC binding groove and thus decrease more or less the affinity of the altered epitopes to human leukocyte antigen-B*2705. A bifunctional spacer ((R)-3-hydroxybutyrate tetramer) not only bridges the two distant anchoring amino acids but also strongly interacts with the binding cleft and leads to a 5-fold increase in binding to the MHC protein. To our knowledge, this is the first report of a nonpeptidic modification of T-cell receptor binding residues that significantly enhances the binding of altered peptide ligands to their host MHC protein. The presented modified ligands constitute interesting tools for perturbing the T cell response to the parent antigenic peptide.

Reversible peptide folding in solution by molecular dynamics simulation.

Long-standing questions on how peptides fold are addressed by the simulation at different temperatures of the reversible folding of a peptide in solution in atomic detail. Molecular dynamics simulations correctly predict the structure that is thermodynamically stable at 298 K, irrespective of the initial peptide conformation. The rate of folding and the free energy of folding at different temperatures are estimated. Although the conformational space potentially accessible to the peptide is extremely large, very few conformers (10(1) to 10(2)) are significantly populated at 20 K above the melting temperature. This implies that the search problem in peptide (or even protein) folding is surmountable using dynamics simulations.

Proof for a nonproteinaceous calcium-selective channel in Escherichia coli by total synthesis from (R)-3-hydroxybutanoic acid and inorganic polyphosphate.

Traditionally, the structure and properties of natural products have been determined by total synthesis and comparison with authentic samples. We have now applied this procedure to the first nonproteinaceous ion channel, isolated from bacterial plasma membranes, and consisting of a complex of poly(3-hydroxybutyrate) and calcium polyphosphate. To this end, we have now synthesized the 128-mer of hydroxybutanoic acid and prepared a complex with inorganic calcium polyphosphate (average 65-mer), which was incorporated into a planar lipid bilayer of synthetic phospholipids. We herewith present data that demonstrate unambiguously that the completely synthetic complex forms channels that are indistinguishable in their voltage-dependent conductance, in their selectivity for divalent cations, and in their blocking behavior (by La3+) from channels isolated from Escherichia coli. The implications of our finding for prebiotic chemistry, biochemistry, and biology are discussed.