LongLarf

Nature Reviews Chemistry, Published online: 16 May 2023; doi:10.1038/s41570-023-00503-z

Nature Chemical Biology, Published online: 15 May 2023; doi:10.1038/s41589-023-01338-x

Difficult but worthy. Peptide macrobicycles are more difficult to obtain than their monocyclic counterparts, but their remarkable biological applications make their synthesis worthwhile. This report describes solution and solid-phase strategies that rely on multicomponent reactions to assemble two dissimilar types of macrobicyclic peptide scaffolds. NMR and circular dichroism studies show they may occur in stable tertiary folds.

Abstract

Macrocyclization of peptides is typically used to fix specific bioactive conformations and improve their pharmacological properties. Recently, macrobicyclic peptides have received special attention owing to their capacity to mimic protein structures or be key components of peptide-drug conjugates. Here, we describe the development of novel synthetic strategies for two distinctive types of peptide macrobicycles. A multicomponent macrocyclo-dimerization approach is introduced for the production of interconnected β-turns, allowing two macrocyclic rings to be formed and dimerized in one pot. Also, an on-resin double stapling strategy is described for the assembly of lactam-bridged macrobicycles with stable tertiary folds.

Publication date: 8 June 2023

Source: Chem, Volume 9, Issue 6

Author(s): Grace A. Lutovsky, Samuel N. Gockel, Mark W. Bundesmann, Scott W. Bagley, Tehshik P. Yoon

An unconventional approach to peptide cyclization involving the use of acyl ammonium species was developed. Rapid and epimerization/dimerization-free cyclization of synthetically challenging peptides was possible, including a difficult cyclization reaction involving N-methyl amide bond formation. The approach is characterized by ease of purification of the products, high productivity, and high reaction mass efficiencies.

Abstract

Although cyclic peptides have become increasingly important as drugs, the most conventional peptide cyclization method using moderately active coupling agents suffers from a lot of waste and high cost as well as long reaction times and burdensome purification. Herein, we report an unconventional approach to peptide cyclization that uses acylammonium species generated from inexpensive and less wasteful Me₂NBn and ClCO₂ i-Pr. Using this approach, we observed the desired rapid activation of the C-terminus of cyclization precursors by an acylammonium ion for rapid and epimerization/dimerization-free cyclization of synthetically challenging peptides, including a difficult cyclization involving N-methyl amide bond formation. The ease of purification, productivities, and reaction mass efficiencies of our approach were significantly superior to those in previous reports. We synthesized a previously reported versicotide D analogue, and our data indicated that its assigned stereostructure should be revised.

A straightforward two-step protocol was developed to convert linear or α-branched carboxylic acids into non-racemic N-Boc-protected α-monosubstituted (asymmetric catalysis) or α,α-disubstituted (enantioconvergent catalysis) α-amino acids.

Abstract

N-Boc-protected α-amino acids are synthesized in two steps from linear or branched carboxylic acid feedstocks. In the first step, the carboxylic acid is coupled with tert-butyl aminocarbonate (BocNHOH) to generate azanyl ester (acyloxycarbamate) RCO₂NHBoc. In the second step, this azanyl ester undergoes a stereocontrolled iron-catalyzed 1,3-nitrogen migration to generate the N-Boc-protected non-racemic α-amino acid. This straightforward protocol is applicable to the catalytic asymmetric synthesis of α-monosubstituted α-amino acids with aryl, alkenyl, and alkyl side chains. Furthermore, α,α-disubstituted α-amino acids are accessible in an enantioconvergent fashion from racemic carboxylic acids. The new method is also advantageous for the synthesis of α-deuterated α-amino acids. N-Boc-protected α-amino acids synthesized using this two-step protocol are ready-to-use building blocks.

Cyclize or diversify. The novel amino acid pCPF is compatible with in vitro translation and allows spontaneous macrocyclization with peptides containing cysteine or diversification through its reactivity with thiols.

Abstract

Macrocyclization has proven to be a beneficial strategy to improve upon some of the disadvantages of peptides as therapeutics. Nevertheless, many peptide cyclization strategies are not compatible with in vitro display technologies like mRNA display. Here we describe the novel amino acid p-chloropropynyl phenylalanine (pCPF). pCPF is a substrate for a mutant phenylalanyl-tRNA synthetase and its introduction into peptides via in vitro translation leads to spontaneous peptide macrocyclization in the presence of peptides containing cysteine. Macrocyclization occurs efficiently with a wide variety of ring sizes. Moreover, pCPF can be reacted with thiols after charging onto tRNA, enabling the testing of diverse ncAAs in translation. The versatility of pCPF should facilitate downstream studies of translation and enable the creation of novel macrocyclic peptide libraries.

Computational enzyme design is a cornerstone of rational protein engineering, and a plethora of methods exists. But how do these tools deal with enzymes containing cofactors, nucleic, or non-canonical amino acids? Here, we review important computational tools and well-known and less-documented caveats and remedies in engineering enzymes with non-proteogenic parts.

Abstract

Enzyme engineering aims to improve or install a new function in biocatalysts for applications ranging from chemical synthesis to biomedicine. For decades, computational techniques have been developed to predict the effect of protein changes and design new enzymes. However, these techniques may have been optimized to deal with proteins composed of the standard amino acid alphabet, while the function of many enzymes relies on non-proteogenic parts like cofactors, nucleic acids, and post-translational modifications. Enzyme systems containing such molecules might be handled or modeled improperly by computational tools, and thus be unsuitable, or require additional tweaking, parameterization, or preparation. In this review, we give an overview of common and recent tools and workflows available to computational enzyme engineers. We highlight the various pitfalls that come with including non-proteogenic compounds in computations and outline potential ways to address common issues. Finally, we showcase successful examples from the literature that computationally engineered such enzymes.

In the biosynthetic pathway of the first natural brexane-type compound, chlororaphen, farnesyl pyrophosphate (FPP) is methylated and cyclized by a non-canonical FPP methyltransferase (FPP-MT), furnishing γ-presodorifen pyrophosphate (γ-PSPP). Methylation by the γ-PSPP-MT yields α-prechlororaphen pyrophosphate (α-PCPP), which is converted into the bishomosesquiterpene chlororaphen by the chlororaphen synthase (ChloS).

Abstract

A non-canonical biosynthetic pathway furnishing the first natural brexane-type bishomosesquiterpene (chlororaphen, C₁₇H₂₈) was elucidated in the γ-proteobacterium Pseudomonas chlororaphis O6. A combination of genome mining, pathway cloning, in vitro enzyme assays, and NMR spectroscopy revealed a three-step pathway initiated by C10 methylation of farnesyl pyrophosphate (FPP, C₁₅) along with cyclization and ring contraction to furnish monocyclic γ-presodorifen pyrophosphate (γ-PSPP, C₁₆). Subsequent C-methylation of γ-PSPP by a second C-methyltransferase furnishes the monocyclic α-prechlororaphen pyrophosphate (α-PCPP, C₁₇), serving as the substrate for the terpene synthase. The same biosynthetic pathway was characterized in the β-proteobacterium Variovorax boronicumulans PHE5-4, demonstrating that non-canonical homosesquiterpene biosynthesis is more widespread in the bacterial domain than previously anticipated.

Hyperpolarized ¹³C NMR spectroscopy by dissolution dynamic nuclear polarization provides a rich and resolved ¹³C metabolic profile on urine samples at natural abundance while retaining precious quantitative information using a standard addition workflow.

Abstract

Hyperpolarized nuclear magnetic resonance (NMR) offers an ensemble of methods that remarkably address the sensitivity issues of conventional NMR. Dissolution Dynamic Nuclear Polarization (d-DNP) provides a unique and general way to detect ¹³C NMR signals with a sensitivity enhanced by several orders of magnitude. The expanding application scope of d-DNP now encompasses the analysis of complex mixtures at natural ¹³C abundance. However, the application of d-DNP in this area has been limited to metabolite extracts. Here, we report the first d-DNP-enhanced ¹³C NMR analysis of a biofluid -urine- at natural abundance, offering unprecedented resolution and sensitivity for this challenging type of sample. We also show that accurate quantitative information on multiple targeted metabolites can be retrieved through a standard addition procedure.

Discovery of the biosynthetic gene cluster for the α-diazoester natural product azaserine is reported. Isotope feeding and biochemical experiments implicate generation of a hydrazonoacetic acid intermediate that is oxidized and transferred to l-serine. This pathway represents a distinct biosynthetic strategy for diazo formation.

Abstract

Azaserine is a bacterial metabolite containing a biologically unusual and synthetically enabling α-diazoester functional group. Herein, we report the discovery of the azaserine (aza) biosynthetic gene cluster from Glycomyces harbinensis. Discovery of related gene clusters reveals previously unappreciated azaserine producers, and heterologous expression of the aza gene cluster confirms its role in azaserine assembly. Notably, this gene cluster encodes homologues of hydrazonoacetic acid (HYAA)-producing enzymes, implicating HYAA in α-diazoester biosynthesis. Isotope feeding and biochemical experiments support this hypothesis. These discoveries indicate that a 2-electron oxidation of a hydrazonoacetyl intermediate is required for α-diazoester formation, constituting a distinct logic for diazo biosynthesis. Uncovering this biological route for α-diazoester synthesis now enables the production of a highly versatile carbene precursor in cells, facilitating approaches for engineering complete carbene-mediated biosynthetic transformations in vivo.

A chirality transfer strategy was established to access enantioenriched boron C,N-chelates stereogenic only at the B-atom. First, a library of chiral boron O,N-complexes was synthesized in diastereoselective fashion. The generated stereoinformation at boron was then transferred via the ate-complex into the boron C,N-chelates.Twitter affiliations: @LaschatGroup, @YStoeckl

Abstract

Molecules stereogenic only at tetrahedral boron atoms show great promise for applications, for example as chiroptical materials, but are scarcely investigated due to their synthetic challenge. Hence, this study reports a two-step synthesis of enantioenriched boron C,N-chelates. First, the diastereoselective complexation of alkyl/aryl borinates with chiral aminoalcohols furnished boron stereogenic heterocycles in up to 86 % yield and d.r. >98 : 2. Treatment of these O,N-complexes with chelate nucleophiles was surmised to transfer the stereoinformation via the ate-complex into the C,N-products. This chirality transfer succeeded by substitution of the O,N-chelates with lithiated phenyl pyridine to give boron stereogenic C,N-chelates in up to 84 % yield and e.r. up to 97 : 3. The chiral aminoalcohol ligands could be recovered after isolation of the C,N-chelates. The chirality transfer tolerated alkyl, alkynyl and (hetero-)aryl moieties at boron and could be further extended by post-modification: transformations such as catalytic hydrogenations or sequential deprotonation/electrophilic trapping were feasible while maintaining the stereochemical integrity of the C,N-chelates. Structural aspects of the boron chelates were studied by variable temperature NMR and X-ray diffraction.

The strategy of merging visible light photocatalysis and organocatalysis has been utilized and applied in a wide range of reactions. This review summarized the latest advances in the field of cooperative catalysis by the combination of organocatalysis and photocatalysis in recent organic synthesis.

Abstract

In recent years, the strategy of merging visible light photocatalysis and organocatalysis has been utilized and applied in a wide range of reactions. Taking advantage of synergistic visible light photocatalysis with organocatalysis, remarkable progress has recently been achieved in modern chemical synthesis. In these dual catalytic systems, photocatalysts or photosensitizers absorb visible light to induce their photo-excited states which can activate unreactive substrates via electron or energy transfer mechanisms, and organocatalysts are usually employed to control the chemical reactivities of the other substrates. This review mainly focuses on the recent development of cooperative catalysis by the combination of organocatalysis and photocatalysis in recent organic synthesis.

Abstract

Diazomethane is a powerful reagent for numerous chemical reactions such as esterifications and the homologation of carboxylic acids. Unfortunately, the synthetic utility of diazomethane is severely limited by its toxicity and highly explosive nature. Diazald® is typically used for ex situ synthesis, however it requires cumbersome and hazardous transfer of diazomethane from a caustic aqueous phase to the reaction medium. Herein, we present a low temperature and base-free in situ synthesis of diazomethane via a “click and release” reaction between an enamine and sulfonyl azide. Its utility is exemplified by the synthesis of diverse methyl esters in yields of up to 93%. Moreover, diazoketone synthesis from in situ generated diazomethane and acid chlorides was demonstrated for the first time. Finally, trideuteromethylation was achieved using acetone-d ₆ as the deuterium source. We anticipate that this method will enable the safer use of diazomethane in organic synthesis and drug discovery programs.