Peptide cyclisation promoted by supramolecular complex formation.

Phenol ester activated dipeptides that are reluctant to ring-close have been cyclised with the aid of sterically shielding metallo-porphyrins avoiding unwanted intermolecular reactions. The binding of ZnTPP to the dipyridine-functionalised activating phenolic ester was studied by NMR titrations and modelling. Staudinger-mediated cyclisations in the presence of ZnTPP increased the yield of the cyclic dipeptide from 16% to 40%.

[2]Catenane Synthesis via Covalent Templating.

After earlier unsuccessful attempts, this work reports the application of covalent templating for the synthesis of mechanically interlocked molecules (MiMs) bearing no supramolecular recognition sites. Two linear strands were covalently connected in a perpendicular fashion by a central ketal linkage. After subsequent attachment of the first strand to a template via temporary benzylic linkages, the second was linked to the template in a backfolding macrocyclization. The resulting pseudo[1]rotaxane structure was successfully converted to a [2]catenane via a second macrocyclization and cleavage of the ketal and temporary linkages.

Synthetic Evidence of the Amadori-Type Alkylation of Biogenic Amines by the Neurotoxic Metabolite Dopegal.

The neurotransmitter metabolite 3,4-dihydroxy-phenylglycolaldehyde (dopegal) damages neurons and the myocardium by protein cross-linking, resulting in conglomerations and cell death. We investigated this process on a synthetic scale, leading to the discovery of an Amadori-type rearrangement of dopegal in the reaction with several amino acids and neuropeptides. This alkylation also occurs with neurotransmitters, suggesting an influence of dopegal on neurochemical processes. The rearrangement occurs readily under physiological conditions.

A Covalent and Modular Synthesis of Homo- and Hetero[n]rotaxanes.

Incorporation of 2,5-dihydroxyterephthalate as a covalent scaffold in the axis of a 30-membered all-carbon macrocycle provides access to a modular series of rotaxanes. Installment of tethered alkynes or azides onto the terephthalic phenolic hydroxyl functionalities, which are situated at opposite sides of the macrocycle, gives versatile prerotaxane building blocks. The corresponding [2]rotaxanes are obtained by introduction of bulky stoppering ("capping") units at the tethers and subsequent cleavage of the covalent ring/thread ester linkages. Extension of this strategy via coupling of two prerotaxanes bearing complementary linker functionalities (i.e., azide and alkyne) and follow-up attachment of stopper groups provide efficient access to [n]rotaxanes. The applicability and modular nature of this novel approach were demonstrated by the synthesis of a series of [2]-, [3]-, and [4]rotaxanes. Furthermore, it is shown that the prerotaxanes allow late-stage functionalization of the ring fragment introducing further structural diversity.

Efficient Enzymatic Cyclization of Disulfide-Rich Peptides by Using Peptide Ligases.

Disulfide-rich macrocyclic peptides-cyclotides, for example-represent a promising class of molecules with potential therapeutic use. Despite their potential their efficient synthesis at large scale still represents a major challenge. Here we report new chemoenzymatic strategies using peptide ligase variants-inter alia, omniligase-1-for the efficient and scalable one-pot cyclization and folding of the native cyclotides MCoTI-II, kalata B1 and variants thereof, as well as of the θ-defensin RTD-1. The synthesis of the kB1 variant T20K was successfully demonstrated at multi-gram scale. The existence of several ligation sites for each macrocycle makes this approach highly flexible and facilitates both the larger-scale manufacture and the engineering of bioactive, grafted cyclotide variants, therefore clearly offering a valuable and powerful extension of the existing toolbox of enzymes for peptide head-to-tail cyclization.

Stereoselective C-terminal peptide elongation from Chan-Lam-Evans reaction generated isopropenyl esters.

C-Terminal dipeptide isopropenyl esters were synthesised by a Cu(ii)-mediated Chan-Lam-Evans enol esterification of peptide carboxylic acids and isoprenyl boroxine. These shelf stable peptide esters could be coupled stereoselectively with a variety of amino acid and dipeptide nucleophiles in high yield and purity in the presence of pyrazole/DBU as the catalyst.

A One-Pot "Triple-C" Multicyclization Methodology for the Synthesis of Highly Constrained Isomerically Pure Tetracyclic Peptides.

A broadly applicable one-pot methodology for the facile transformation of linear peptides into tetracyclic peptides through a chemoenzymatic peptide synthesis/chemical ligation of peptides onto scaffolds/copper(I)-catalyzed reaction (CEPS/CLIPS/CuAAC; "triple-C") locking methodology is reported. Linear peptides with varying lengths (≥14 amino acids), comprising two cysteines and two azidohomoalanines (Aha), were efficiently cyclized head-to-tail by using the peptiligase variant omniligase-1 (CEPS). Subsequent ligation-cyclization with tetravalent (T41/2 ) scaffolds containing two bromomethyl groups (CLIPS) and two alkyne functionalities (CuAAC) yielded isomerically pure tetracyclic peptides. Sixteen different functional tetracycles, derived from bicyclic inhibitors against urokinase plasminogen activator (uPA) and coagulation factor XIIa (FXIIa), were successfully synthesized and their bioactivities evaluated. Two of these (FF-T41/2 ) exhibited increased inhibitory activity against FXIIa, compared with a bicyclic control peptide. The corresponding hetero-bifunctional variants (UF/FU-T41/2 ), with a single copy of each inhibitory sequence, exhibited micromolar activities against both uPA and FXIIa; thus illustrating the potential of the "bifunctional tetracyclic peptide" inhibitor concept.

Template-Directed Synthesis of an Inverted Spiro Architecture.

The regular bicyclic spiro motif is highly abundant given its synthetic accessibility while the diastereomer-virtually obtained through inversion at the central atom-is almost unknown. We have developed methodology to access the elusive inverted spiro architecture by employing a covalent template-directed approach. Comparison with the regular spiro bicycle analog unequivocally established the diastereomeric relationship, providing insight into the fascinating stereochemical and structural properties.

Total Synthesis of the Ortho-Hydroxylated Protoberberines ( S)-Govaniadine, ( S)-Caseamine, and ( S)-Clarkeanidine via a Solvent-Directed Pictet-Spengler Reaction.

The common para regioselectivity in Pictet-Spengler reactions with dopamine derivatives is redirected to the ortho position by a simple change of solvents. In combination with a chiral auxiliary on nitrogen, this ortho-selective Pictet-Spengler produced the 1-benzyltetrahydroisoquinoline alkaloids ( S)-crassifoline and ( S)-norcrassifoline and the bioactive 1,2-dioxygenated tetrahydroprotoberberine alkaloids ( S)-govaniadine, ( S)-caseamine, and ( S)-clarkeanidine with high enantiopurity. Ortho/para ratios up to 89:19 and diastereomeric ratios up to 85:15 were obtained during formation of the B-ring. The general applicability of this solvent-directed regioselectivity was demonstrated with a second Pictet-Spengler reaction as required for C-ring formation of caseamine (o/p = 14:86 in trifluoroethanol) and clarkeanidine (o/p = 86:14 in toluene).

Design of a substrate-tailored peptiligase variant for the efficient synthesis of thymosin-α1.

The synthesis of thymosin-α1, an acetylated 28 amino acid long therapeutic peptide, via conventional chemical methods is exceptionally challenging. The enzymatic coupling of unprotected peptide segments in water offers great potential for a more efficient synthesis of peptides that are difficult to synthesize. Based on the design of a highly engineered peptide ligase, we developed a fully convergent chemo-enzymatic peptide synthesis (CEPS) process for the production of thymosin-α1via a 14-mer + 14-mer segment condensation strategy. Using structure-inspired enzyme engineering, the thiol-subtilisin variant peptiligase was tailored to recognize the respective 14-mer thymosin-α1 segments in order to create a clearly improved biocatalyst, termed thymoligase. Thymoligase catalyzes peptide bond formation between both segments with a very high efficiency (>94% yield) and is expected to be well applicable to many other ligations in which residues with similar characteristics (e.g. Arg and Glu) are present in the respective positions P1 and P1'. The crystal structure of thymoligase was determined and shown to be in good agreement with the model used for the engineering studies. The combination of the solid phase peptide synthesis (SPPS) of the 14-mer segments and their thymoligase-catalyzed ligation on a gram scale resulted in a significantly increased, two-fold higher overall yield (55%) of thymosin-α1 compared to those typical of existing industrial processes.

General and Facile Route to Isomerically Pure Tricyclic Peptides Based on Templated Tandem CLIPS/CuAAC Cyclizations.

We report a one-pot ligation/cyclization technology for the rapid and clean conversion of linear peptides into tricyclic peptides that is based on using tetravalent scaffolds containing two benzyl bromide and two alkyne moieties. These react via CLIPS/CuAAC reactions with cysteines and azides in the peptide. Flexibility in the scaffolds is key to the formation of isomerically pure products as the flexible scaffolds T41 and T42 mostly promote the formation of single isomeric tricycles while the rigid scaffolds T43 and T44 do not yield clean products. There seems to be no limitation to the number and types of amino acids present as 18 canonical amino acids were successfully implemented. We also observed that azides at the peptide termini and cysteine residues in the center gave better results than compounds with the functional groups placed the other way round.

In-Culture Cross-Linking of Bacterial Cells Reveals Large-Scale Dynamic Protein-Protein Interactions at the Peptide Level.

Identification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram-positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique interprotein cross-linked peptides with <1% false discovery rate by mass spectrometry and genome-wide database searching. Nearly 60% of the interprotein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the δ and ß' subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how δ might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth. Data are available via ProteomeXchange with identifier PXD006287.

Enzyme-catalyzed peptide cyclization.

The recent advancement of peptide macrocycles as promising therapeutics creates a need for novel methodologies for their efficient synthesis and (large scale) production. Within this context, due to the favorable properties of biocatalysts, enzyme-mediated methodologies have gained great interest. Enzymes such as sortase A, butelase 1, peptiligase and omniligase-1 represent extremely powerful and valuable enzymatic tools for peptide ligation, since they can be applied to generate complex cyclic peptides with exquisite biological activity. Therefore, the use of enzymatic strategies will effectively supplement the scope of existing chemical methodologies and will accelerate the development of future cyclic peptide therapeutics. The advantages and disadvantages of the different enzymatic methodologies will be discussed in this review.

Synthetic and Organocatalytic Studies of Quinidine Analogues with Ring-Size Modifications in the Quinuclidine Moiety.

Six highly enantiopure analogues of [2.2.2] were synthesized with five- or seven-membered rings in the (original) quinuclidine skeleton. Five of these compounds were prepared through epoxide opening by a secondary cyclic amine, providing the nor- and homoquinuclidine moieties through five- and six-membered ring formation. This method failed in the case of seven-membered ring formation, so for that particular ring size a different synthetic route starting from 3-quinuclidone was applied. The six novel analogues were examined as organocatalysts in four asymmetric conjugate addition reactions and the results compared with those of known cinchona alkaloid catalysts. This study shows that modification of the quinuclidine ring can have a substantial influence on catalyst activity and enantioselectivity. To acquire more insight into the characteristics of the new catalysts, the pKaH values were determined by means of fluorescence spectroscopy. Furthermore, relative reaction rates of conjugate thiol additions reactions catalyzed by these quinidine analogues were measured through polarimetry.

Formal Synthesis of Solanoeclepin A: Enantioselective Allene Diboration and Intramolecular [2+2] Photocycloaddition for the Construction of the Tricyclic Core.

An enantioselective synthesis of an intermediate in the Tanino total synthesis of solanoeclepin A has been developed. The key step was an intramolecular [2+2] photocycloaddition, which led to the tricyclo[5.2.1.0(1, 6)] decane core in six steps. The first photosubstrate, prepared through an indium-mediated Barbier-type reaction, gave an excellent [2+2] cycloaddition, but it could not be obtained in sufficient enantiopurity. The second photosubstrate, prepared through an asymmetric allene diborylation in high enantiomeric excess, gave the [2+2] cycloaddition product in high yield on irradiation at 365 nm on 20 g scale in a flow system. Other important steps were the replacement of a boronate group at the quaternary carbon by a vinyl group and diastereoselective cyclopropanation of an allylic alcohol.

Reconstructing the discontinuous and conformational ß1/ß3-loop binding site on hFSH/hCG by using highly constrained multicyclic peptides.

Making peptide-based molecules that mimic functional interaction sites on proteins remains a challenge in biomedical sciences. Here, we present a robust technology for the covalent assembly of highly constrained and discontinuous binding site mimics, the potential of which is exemplified for structurally complex binding sites on the "Cys-knot" proteins hFSH and hCG. Peptidic structures were assembled by Ar(CH2 Br)2-promoted peptide cyclizations, combined with oxime ligation and disulfide formation. The technology allows unprotected side chain groups and is applicable to peptides of different lengths and nature. A tetracyclic FSH mimic was constructed, showing >600-fold improved binding compared to linear or monocyclic controls. Binding of a tricyclic hCG mimic to anti-hCG mAb 8G5 was identical to hCG itself (IC50 =260 vs. 470 pM), whereas this mimic displayed an IC50 value of 149 nM for mAb 3468, an hCG-neutralizing antibody with undetectable binding to either linear or monocyclic controls.

Organocatalytic enantioselective Pictet-Spengler approach to biologically relevant 1-benzyl-1,2,3,4-tetrahydroisoquinoline alkaloids.

A general procedure for the synthesis of 1-benzyl-1,2,3,4-tetrahydroisoquinolines was developed, based on organocatalytic, regio- and enantioselective Pictet-Spengler reactions (86-92% ee) of N-(o-nitrophenylsulfenyl)-2-arylethylamines with arylacetaldehydes. The presence of the o-nitrophenylsulfenyl group, together with the MOM-protection in the catechol part of the tetrahydroisoquinoline ring system, appeared to be a productive combination. To demonstrate the versatility of this approach, 10 biologically and pharmaceutically relevant alkaloids were prepared using (R)-TRIP as the chiral catalyst: (R)-norcoclaurine, (R)-coclaurine, (R)-norreticuline, (R)-reticuline, (R)-trimemetoquinol, (R)-armepavine, (R)-norprotosinomenine, (R)-protosinomenine, (R)-laudanosine, and (R)-5-methoxylaudanosine.

Cinchona Alkaloid Catalyzed Sulfa-Michael Addition Reactions Leading to Enantiopure ß-Functionalized Cysteines.

Sulfa-Michael additions to α,ß-unsaturated N-acylated oxazolidin-2-ones and related α,ß-unsaturated α-amino acid derivatives have been enantioselectively catalyzed by Cinchona alkaloids functionalized with a hydrogen bond donating group at the C6' position. The series of Cinchona alkaloids includes known C6' (thio)urea and sulfonamide derivatives and several novel species with a benzimidazole, squaramide or a benzamide group at the C6' position. The sulfonamides were especially suited as bifunctional organocatalysts as they gave the products in very good diastereoselectivity and high enantioselectivity. In particular, the C6' sulfonamides catalyzed the reaction with the α,ß-unsaturated α-amino acid derivatives to afford the products in a diastereomeric ratio as good as 93:7, with the major isomer being formed in an ee of up to 99%. The products of the organocatalytic sulfa-Michael addition to α,ß-unsaturated α-amino acid derivatives were subsequently converted in high yields to enantiopure ß-functionalized cysteines suitable for native chemical ligation.

Enantioselective synthesis of tunable chiral Clickphine P,N-ligands and their application in Ir-catalyzed asymmetric hydrogenation.

A small library of highly tunable chiral Clickphine P,N-ligands has been prepared in an enantioselective fashion by Cu(I)-catalyzed asymmetric propargylic amination using a single chiral complex and a subsequent in situ cycloaddition click reaction. The scope of the propargylic amination to yield optically active triazolyl amines is described. The amines are transformed in a one-pot procedure to the corresponding Ir-Clickphine complexes, which serve as catalysts for the asymmetric hydrogenation of di-, tri-, and tetrasubstituted unfunctionalized alkenes. Enantioselectivities of up to 90% ee were obtained in these hydrogenations, which are among the best reported in the case of the tetrasubstituted substrate 2-(4'-methoxyphenyl)-3-methylbut-2-ene (9) (87% ee). This is a demonstration of the effective use of the chiral pool, as from one chiral catalyst a library of chiral Ir complexes has been synthesized that can hydrogenate various alkenes with high selectivity.

Organocatalytic enantioselective Pictet-Spengler reactions for the syntheses of 1-substituted 1,2,3,4-tetrahydroisoquinolines.

A series of 1-substituted 1,2,3,4-tetrahydroisoquinolines was prepared from N-(o-nitrophenylsulfenyl)phenylethylamines through BINOL-phosphoric acid [(R)-TRIP]-catalyzed asymmetric Pictet-Spengler reactions. The sulfenamide moiety is crucial for the rate and enantioselectivity of the iminium ion cyclization and the products are readily recrystallized to high enantiomeric purity. Using this methodology we synthesized the natural products (R)-crispine A, (R)-calycotomine and (R)-colchietine, the synthetic drug (R)-almorexant and the (S)-enantiomer of a biologically active (R)-AMPA-antagonist.