Greening the synthesis of peptide therapeutics: an industrial perspective.

Solid-phase peptide synthesis (SPPS) is generally the method of choice for the chemical synthesis of peptides, allowing routine synthesis of virtually any type of peptide sequence, including complex or cyclic peptide products. Importantly, SPPS can be automated and is scalable, which has led to its widespread adoption in the pharmaceutical industry, and a variety of marketed peptide-based drugs are now manufactured using this approach. However, SPPS-based synthetic strategies suffer from a negative environmental footprint mainly due to extensive solvent use. Moreover, most of the solvents used in peptide chemistry are classified as problematic by environmental agencies around the world and will soon need to be replaced, which in recent years has spurred a movement in academia and industry to make peptide synthesis greener. These efforts have been centred around solvent substitution, recycling and reduction, as well as exploring alternative synthetic methods. In this review, we focus on methods pertaining to solvent substitution and reduction with large-scale industrial production in mind, and further outline emerging technologies for peptide synthesis. Specifically, the technical requirements for large-scale manufacturing of peptide therapeutics are addressed.

Click-Chemistry-Mediated Synthesis of Selective Melanocortin Receptor 4 Agonists.

The melanocortin receptor 4 (MC4R) subtype of the melanocortin receptor family is a target for therapeutics to ameliorate metabolic dysfunction. Endogenous MC4R agonists possess a critical pharmacophore (HFRW), and cyclization of peptide agonists often enhances potency. Thus, 17 cyclized peptides were synthesized by solid phase click chemistry to develop novel, potent, selective MC4R agonists. Using cAMP measurements and a transcriptional reporter assay, we observed that several constrained agonists generated by a cycloaddition reaction displayed high selectivity (223- to 467-fold) toward MC4R over MC3R and MC5R receptor subtypes without compromising agonist potency. Significant variation was also observed between the EC50 values for the two assays, with robust levels of reporter expression measured at lower concentrations than those effecting appreciable increases in cAMP levels for the majority of the compounds tested. Collectively, we characterized significant elements that modulate the activity of the core pharmacophore for MC4R and provide a rationale for careful assay selection for agonist screening.

Novel diversity-oriented synthesis-derived respiratory syncytial virus inhibitors identified via a high throughput replicon-based screen.

Respiratory syncytial virus (RSV) infections affect millions of children and adults every year. Despite the significant disease burden, there are currently no safe and effective vaccines or therapeutics. We employed a replicon-based high throughput screen combined with live-virus triaging assays to identify three novel diversity-oriented synthesis-derived scaffolds with activity against RSV. One of these small molecules is shown to target the RSV polymerase (L protein) to inhibit viral replication and transcription; the mechanisms of action of the other small molecules are currently unknown. The compounds described herein may provide attractive inhibitors for lead optimization campaigns.

Triazole-containing N-acyl homoserine lactones targeting the quorum sensing system in Pseudomonas aeruginosa.

In an attempt to devise new antimicrobial treatments for biofilm infections, the bacterial cell-cell communication system termed quorum sensing has emerged as an attractive target. It has proven possible to intercept the communication system by synthetic non-native ligands and thereby lower the pathogenesis and antibiotic tolerance of a bacterial biofilm. To identify the structural elements important for antagonistic or agonistic activity against the Pseudomonas aeruginosa LasR protein, we report the synthesis and screening of new triazole-containing mimics of natural N-acyl homoserine lactones. A series of azide- and alkyne-containing homoserine lactone building blocks was used to prepare an expanded set of 123 homoserine lactone analogues through a combination of solution- and solid-phase synthesis methods. The resulting compounds were subjected to cell-based quorum sensing screening assays, thereby revealing several bioactive compounds, including 13 compounds with antagonistic activity and 9 compounds with agonistic activity.

Combining the Petasis 3-component reaction with multiple modes of cyclization: a build/couple/pair strategy for the synthesis of densely functionalized small molecules.

A build/couple/pair strategy for the synthesis of complex and densely functionalized small molecules is presented. The strategy relies on synthetically tractable building blocks (build), that is, diversely substituted hydrazides, α-hydroxy aldehydes, and boronic acids, which undergo Petasis 3-component reactions (couple) to afford densely functionalized anti-hydrazido alcohols. The resulting scaffolds can subsequently be converted via chemoselective cyclization reactions (pair), including intramolecular Diels-Alder or Ru-alkylidene catalyzed ring-closing metathesis, into sets of structurally diverse heterocycles in good yields in only 3-4 steps.

Synthesis of a natural product-like compound collection through oxidative cleavage and cyclization of linear peptides.

Massive efforts in molecular library synthesis have strived for the development of synthesis methodology which systematically delivers natural product-like compounds of high spatial complexity. Herein, we present a conceptually simple approach that builds on the power of solid-phase peptide synthesis to assemble precursor peptides (oligomers) designed to undergo oxidative cascade reactions. By harnessing the structural side-chain diversity and inherent stereochemical features offered by readily available amino acids (monomers), a proof-of-concept collection of 54 skeletally and stereochemically diverse compounds was generated, and selected compounds were elaborated into isoform-selective metalloprotease inhibitors.

Diversity-oriented synthesis yields a new drug lead for treatment of chagas disease.

A phenotypic high-throughput screen using ∼100,000 compounds prepared using Diversity-Oriented Synthesis yielded stereoisomeric compounds with nanomolar growth-inhibition activity against the parasite Trypanosoma cruzi, the etiological agent of Chagas disease. After evaluating stereochemical dependence on solubility, plasma protein binding and microsomal stability, the SSS analogue (5) was chosen for structure-activity relationship studies. The p-phenoxy benzyl group appended to the secondary amine could be replaced with halobenzyl groups without loss in potency. The exocyclic primary alcohol is not needed for activity but the isonicotinamide substructure is required for activity. Most importantly, these compounds are trypanocidal and hence are attractive as drug leads for both acute and chronic stages of Chagas disease. Analogue (5) was nominated as the molecular libraries probe ML341 and is available through the Molecular Libraries Probe Production Centers Network.

Solid-phase synthesis and biological evaluation of N-dipeptido L-homoserine lactones as quorum sensing activators.

Bacteria use small signaling molecules to communicate in a process termed "quorum sensing" (QS), which enables the coordination of survival strategies, such as production of virulence factors and biofilm formation. In Gram-negative bacteria, these signaling molecules are a series of N-acylated L-homoserine lactones. With the goal of identifying non-native compounds capable of modulating bacterial QS, a virtual library of N-dipeptido L-homoserine lactones was screened in silico with two different crystal structures of LasR. The 30 most promising hits were synthesized on HMBA-functionalized PEGA resin and released through an efficient acid-mediated cyclative release mechanism. Subsequent screening for modulation of QS in Pseudomonas aeruginosa and E. coli identified six moderately strong activators. A follow-up library designed from the preliminary derived structure-activity relationships was synthesized and evaluated for their ability to activate the QS system in this bacterium. This resulted in the identification of another six QS activators (two with low micromolar activity) thus illuminating structural features required for QS modulation.

Ruthenium hydride/Brønsted acid-catalyzed tandem isomerization/N-acyliminium cyclization sequence for the synthesis of tetrahydro-ß-carbolines.

This paper describes an efficient tandem sequence for the synthesis of 1,2,3,4-tetrahydro-ß-carbolines (THBCs) relying on a ruthenium hydride/Brønsted acid-catalyzed isomerization of allylic amides to N-acyliminium ion intermediates which are trapped by a tethered indole nucleophile. The methodology provides not only a convenient "aldehyde-free" alternative to the classical Pictet-Spengler reaction but also attractive possibilities for total synthesis, including rapid generation of molecular complexity and formation of quaternary stereogenic centers. TBHCs can also be accessed by harnessing the Suzuki cross-coupling reaction to the isomerization/N-acyliminium cyclization sequence. Finally, diastereo- and enantioselective versions of the title reaction have been examined using substrate control (with dr >15: 1) and asymmetric catalysis (ee up to 57%), respectively.

Synthesis and biological evaluation of triazole-containing N-acyl homoserine lactones as quorum sensing modulators.

Many bacterial species are capable of assessing their local population densities through a cell-cell signaling mechanism termed quorum sensing (QS). This intercellular communication process is mediated by small molecule or peptide ligands and their cognate protein receptors. Numerous pathogens use QS to initiate virulence once they achieve a threshold cell number on a host. Consequently, approaches to intercept QS have attracted considerable attention as potential anti-infective therapies. Our interest in the development of small molecule tools to modulate QS pathways motivated us to evaluate triazole-containing analogs of natural N-acyl L-homoserine lactone (AHL) signals as non-native QS agonists and antagonists in Gram-negative bacteria. We synthesized 72 triazole derivatives of five broad structure types in high yields and purities using efficient Cu(I)-catalyzed azide-alkyne couplings. These compounds were evaluated for their ability to activate or inhibit two QS receptors from two prevalent pathogens - LasR from Pseudomonas aeruginosa and AbaR from Acinetobacter baumannii- using bacterial reporter strains. Several triazole derivatives were identified that were capable of strongly modulating the activity of LasR and AbaR. These compounds represent a new and synthetically accessible class of AHL analogs, and could find utility as chemical tools to study QS and its role in bacterial virulence.

Identification of LasR ligands through a virtual screening approach.

With the widespread occurrence of bacterial resistance to antibiotics, the development of new strategies beyond conventional treatments is a pursuit taken by public health institutions worldwide. LasR, a transcription factor that controls quorum sensing in Pseudomonas aeruginosa, has emerged as an attractive therapeutic target for the next generation of antimicrobial agents. In the present study, a virtual screening workflow combining pharmacophore- and structure-based approaches was used to identify new LasR ligands. Five novel inducers and three inhibitors of LasR activity were validated experimentally by use of a cell-based assay. Interestingly, these compounds are molecularly distinct from the native signal molecule, N-3-oxododecanoyl-L-homoserine lactone (OHN), and may serve as lead structures for the design of new drugs. The binding modes of these compounds to the OHN binding site in LasR were predicted and used to identify the key interactions that contribute to the induction and inhibition of LasR activity.

Synthesis of tetrahydro-ß-carbolines via isomerization of N-allyltryptamines: a metal-catalyzed variation on the Pictet-Spengler theme.

An efficient and broadly applicable alternative to the classical Pictet-Spengler synthesis of tetrahydro-ß-carbolines is presented. The method relies on metal-catalyzed isomerization of allylic amines to form reactive iminium intermediates which can be trapped by a tethered indole nucleophile.

Build/couple/pair strategy combining the Petasis 3-component reaction with Ru-catalyzed ring-closing metathesis and isomerization.

A "build/couple/pair" pathway for the systematic synthesis of structurally diverse small molecules is presented. The Petasis 3-component reaction was used to synthesize anti-amino alcohols displaying pairwise reactive combinations of alkene moieties. Upon treatment with a ruthenium alkylidene-catalyst, these dienes selectively underwent ring-closing metathesis reactions to form 5- and 7-membered heterocycles and cyclic aminals via a tandem isomerization/N-alkyliminium cyclization sequence.

Petasis three-component coupling reactions of hydrazides for the synthesis of oxadiazolones and oxazolidinones.

An application of readily available hydrazides in the Petasis 3-component coupling reaction is presented. An investigation of the substrate scope was performed to establish a general, synthetically useful protocol for the formation of hydrazido alcohols, which were selectively converted to oxazolidinone and oxadiazolone ring systems through triphosgene-mediated cyclization reactions.

Solid-phase synthesis of smac peptidomimetics incorporating triazoloprolines and biarylalanines.

Apoptotic induction mechanisms are of crucial importance for the general homeostasis of multicellular organisms. In cancer the apoptotic pathways are downregulated, which, at least partly, is due to an abundance of inhibitors of apoptosis proteins (IAPs) that block the apoptotic cascade by deactivating proteolytic caspases. The Smac protein has an antagonistic effect on IAPs, thus providing structural clues for the synthesis of new pro-apoptotic compounds. Herein, we report a solid-phase approach for the synthesis of Smac-derived tetrapeptide libraries. On the basis of a common (N-Me)AVPF sequence, peptides incorporating triazoloprolines and biarylalanines were synthesized by means of Cu(I)-catalyzed azide-alkyne cycloaddition and Pd-catalyzed Suzuki cross-coupling reactions. Solid-phase procedures were optimized to high efficiency, thus accessing all products in excellent crude purities and yields (both typically above 90%). The peptides were subjected to biological evaluation in a live/dead cellular assay which revealed that structural decorations on the AVPF sequence indeed are highly important for cytotoxicity toward HeLa cells.

N-acyliminium intermediates in solid-phase synthesis.

N-Acyliminium ions are powerful intermediates in synthetic organic chemistry. Examples of their use are numerous in solution-phase synthesis, but there are unmerited few reports on these highly reactive electrophiles in solid-phase synthesis. The present review covers the literature to date and illustrates the methods used to generate N-acyliminium intermediates on solid support and their further elaboration to a range of pharmacologically interesting peptidomimetics, heterocycles, and other small molecules.

Ralph F. Hirschmann award address 2009: Merger of organic chemistry with peptide diversity.

A huge unleashed potential lies hidden in the large and diverse pool of encoded and particularly nonencoded chiral alpha-, beta-, and gamma-amino acids available today. Although these have been extensively exploited in peptide science, the community of organic chemistry has only used this source of diversity in a quite focused and targeted manner. The properties and behavior of peptides as functional molecules in biology are well documented and based on the ability of peptides to adapt a range of discrete conformers at a minimal entropic penalty and therefore ideally fitting their endogenous targets. The development of new organic reactions and chemistries that in a general and quantitative way transform peptides into new functional molecules, preferably on solid support, is a source of completely new classes of molecules with important and advantageous functional properties. The peptide diversity and the ability to perform chemistry on solid support add tremendously to the combinatorial scope of such reactions in pharmaceutical and materials screening scenario. In recent years, the need for "click" reactions to shape complex molecular architecture has been realized mainly with a basis in the world of peptides and DNA, and in polymer chemistry where connection of highly functionalized biologically active substances or property bearing fragments are assembled as molecular LEGO using quantitative and orthogonal click chemistries. In this article, three such new reactions originating in the Carlsberg Laboratory over the last decade taking advantage of organic transformations in the peptide framework is presented. Initially, the click reaction between azide and terminal alkynes catalyzed by Cu(1) (CuAAC-reaction) is described. This CuAAC "click" reaction was observed first at Carlsberg Laboratory in reactions of azido acid chlorides with alkynes on solid support. Second, the Electrophilic Aromatic Substitution Cyclization-Intramolecular Click-Cascade (EASCy-ICC) reaction will be presented. This quantitative stereo-selective cascade reaction provides a highly diverse set of interesting novel scaffolds from peptides. Finally, we describe the preparation of solid phase peptide phosphine- and carbene-based green catalysts (organozymes), which upon complex formation with transition metal perform with high turnovers under aqueous conditions. These catalysts thrive from the peptide folding and diversity, while phosphines and carbenes in the backbone provide for bidental complex formation with transition metals in a format providing an excellent entry into combinatorial catalyst chemistry.

Divergent pathway for the solid-phase conversion of aromatic acetylenes to carboxylic acids, alpha-ketocarboxylic acids, and methyl ketones.

An efficient divergent pathway for the selective and quantitative solid-phase conversion of aromatic acetylenes to the corresponding carboxylic acids, alpha-keto-carboxylic acids, and methyl ketones is presented. A range of aromatic trimethylsilyl-protected acetylene building blocks was synthesized in excellent yields using a Sonogashira cross-coupling protocol and used in solid-phase synthesis on PEGA resin. Dependent on the selection of conditions, the same solid-supported alkyne could be quantitatively converted to an aromatic carboxylic acid, an aromatic alpha-ketocarboxylic acid, or an aromatic methyl ketone. The conversion to carboxylic acid involved an OsO4/NaIO4/HMTA-mediated oxidative cleavage of the silyl-deprotected alkyne to provide the aromatic carboxylate in excellent yield. The alpha-ketocarboxylic acids were obtained by direct treatment of the trimethylsilyl-protected alkyne with OsO4/NMO/HMTA, while the ketones were obtained by simple acid-mediated hydration of the alkyne using aqueous TFA. In general, all products were obtained in excellent purities, typically above 90%. In addition, it was shown that the alkyne-containing building blocks could easily be incorporated into resin-bound peptides and after chemoselective conversion of the alkyne the new functional groups could be used for further derivatization into peptidomimetic compounds.

Solid-phase synthesis of aryl-substituted thienoindolizines: sequential Pictet-Spengler, bromination and Suzuki cross-coupling reactions of thiophenes.

The solid-phase synthesis of a range of novel heterocyclic scaffolds based on the thiophene ring system, including thienoindolizines and aryl-substituted thiophenes, is presented. Specifically, a sequential methodology for the decoration of thienoindolizine scaffolds has been developed. This method involves a highly efficient and diastereoselective intramolecular Pictet-Spengler reaction, a quantitative and regioselective bromination of the thiophene ring, and a final Suzuki cross-coupling with an arylboronic acid. Crude products were generally obtained in high purities (>90%). In addition, an investigation on the acidic and electronic effects governing the rate of the Pictet-Spengler reactions was performed. Finally, a range of substituted thiophenes was attached to solid supports and subjected to the regioselective bromination and Suzuki cross-coupling reactions, thus providing substituted thiophenes with high purities of crude products.

Scaffold diversity through intramolecular cascade reactions of solid-supported cyclic N-acyliminium intermediates.

The solid-phase synthesis of pharmacologically interesting heterocycles is presented. The formation of a series of (5,5)-, (5,6)-, (6,5)-, and (6,6)-fused bicyclic ring systems was systematically studied by implementation of a common strategy involving N-acyliminium intermediates. These are highly reactive and transformed further in intramolecular cascade reactions with strong as well as weak C, N, S, and O-nucleophiles. The methodology was successfully applied to the conversion of peptidomimetics into constrained small molecule core structures, such as the hexahydropyrrolo[2,1- b][1,3]oxazines, generally with full control of diastereoselectivity (>20:1) and in purities above 90%.