N-alkylated Amino Acids Research Articles

Enhancement of N-Methyl Amino Acid Incorporation into Proteins and Peptides Using Modified Bacterial Ribosomes and Elongation Factor P.

N-Methylated amino acids are constituents of natural bioactive peptides and proteins. Nα-methylated amino acids appear abundantly in natural cyclic peptides, likely due to their constraint of peptide conformation and contribution to peptide stability. Peptides containing Nα-methylated amino acids have long been prepared by chemical synthesis. While such natural peptides are not produced ribosomally, recent ribosomal strategies have afforded Nα-methylated peptides. Presently, we define new strategies for the ribosomal incorporation of Nα-methylated amino acids into peptides and proteins. First, we identify modified ribosomes capable of facilitating the incorporation of six N-methylated amino acids into antibacterial scorpion peptide IsCT. Also synthesized analogously was a protein domain (RRM1) from hnRNP LL; improved yields were observed for nearly all tested N-methylated amino acids. Computational modeling of the ribosomal assembly illustrated how the distortion imposed by N-methylation could be compensated by altering the nucleotides in key 23S rRNA positions. Finally, it is known that incorporation of multiple prolines (an N-alkylated amino acid) ribosomally can be facilitated by bacterial elongation factor P. We report that supplementing endogenous EF-P during IsCT peptide and RRM1 protein synthesis gave improved yields for most of the N-methylated amino acids studied.

Asymmetric Synthesis of N-Alkyl Amino Acids through a Biocatalytic Dynamic Kinetic Resolution of PEGylated N-Alkyl Amino Esters.

The first examples of a practical procedure for a lipase-catalyzed dynamic kinetic resolution of PEGylated N-alkyl amino esters is reported. This method allows for the preparation of a broad range of aromatic and aliphatic enantiomerically enriched N-alkyl unnatural amino acids in up to 98% yield and 99% ee. We have found that PEGylated esters have a significant solubility advantage and improved reactivity over traditional hydrophobic lipase substrates, thereby allowing for efficient and scalable dynamic kinetic resolution (DKR) under aqueous conditions.

Creating a Defined Chirality in Amino Acids and Cyclic Dipeptides by Photochemical Deracemization.

2,5-Diketopiperazines are cyclic dipeptides displaying a wide range of applications. Their enantioselective preparation has now been found possible from the respective racemates by a photochemical deracemization (53 examples, 74% to quantitative yield, 71-99% ee). A chiral benzophenone catalyst in concert with irradiation at λ= 366 nm enables to establish the configuration at the stereogenic carbon atom C6 at will. If other stereogenic centers are present in the diketopiperazines they remain unaffected and a stereochemical editing is possible at a single position. Consecutive reactions, including the conversion into N-aryl or N-alkyl amino acids or the reduction to piperazines, occur without compromising the newly created stereogenic center. Transient absorption spectroscopy revealed that the benzophenone catalyst processes one enantiomer of the 2,5-diketopiperazines preferentially and enables a reversible hydrogen atom transfer that is responsible for the deracemization process. The remarkably long lifetime of the protonated ketyl radical implies a yet unprecedented mode of action.

Resolution, quantification, and reliable determination of enantiomeric excess of proteinogenic and non-proteinogenic amino acids by comprehensive two-dimensional gas chromatography.

This work proposes a comprehensive two-dimensional gas chromatography method for the resolution and quantification of 27 amino acids, including 17 enantiomeric pairs, as stable N-trifluoroacetyl-O-methyl ester derivatives. The derivatization approach in combination with enantioselective two-dimensional gas chromatography has proven to be highly responsive with a method detection limit of 1-7pg even for sterically hindered amino acids such as α,α-dialkylated, and N-alkylated amino acids. Accurate determination of the enantiomeric excess was achieved with errors in the range of ±0.5%-2.5% (1σ) at concentrations ≥10-6 M. A thorough study of the mass spectra of the amino acid derivatives allowed the fragmentation pathways to be distinguished, enabling chromatographic peaks to be unambiguously assigned. The proposed method is particularly suited for applications that require the precise determination of enantiomeric excesses such as those concerning the role of d-amino acid enantiomers in humans, animals, and the environment, as well as for analyses of extraterrestrial samples aimed at understanding the selection of amino acids in stereochemical l-configuration.

Broadly Applicable and Comprehensive Synthetic Method for N-Alkyl-Rich Drug-like Cyclic Peptides.

We report a versatile and durable method for synthesizing highly N-alkylated drug-like cyclic peptides. This is the first reported method for synthesizing such peptides in parallel with a high success rate and acceptable purity that does not require optimizations for a particular sequence. We set up each reaction condition by overcoming the following issues: (1) diketopiperazine (DKP) formation, (2) insufficient peptide bond formation due to the steric hindrance of the N-alkylated amino acid, and (3) instability of highly N-alkylated peptides under acidic conditions. Using this newly established method, we successfully synthesized thousands of cyclic peptides to explore the scope of this modality in drug discovery. We here demonstrate the syntheses of a hundred representative examples, including our first clinical N-alkyl-rich cyclic peptide (LUNA18) that inhibits an intracellular tough target (RAS), in 31% total yield and 97% purity on average after 23 or 24 reaction steps.

An Exploration of the Effect of the Kleier Model and Carrier-Mediated Theory to Design Phloem-Mobile Pesticides Based on Researching the N-Alkylated Derivatives of Phenazine-1-Carboxylic Acid-Glycine.

The Kleier model and Carrier-mediated theory are effective for molecularly designing pesticides with phloem mobility. However, the single Kleier model or Carrier-mediated theory cannot achieve a reliable explanation of the phloem mobility of all exogenous substances. A detailed investigation of the two models and the scope of their applications can provide a more accurate and highly efficient basis for the guidance of the design and development of phloem-mobile pesticides. In the present paper, a strategy using active ingredient-amino acid conjugates as mode compounds is developed based on Carrier-mediated theory. An N-alkylated amino acid is used to improve the pesticide’s physicochemical properties following the Kleier model, thus allowing the conjugates to fall on the predicted and more accessible transportation region of phloem. Moreover, the influence of this movement on phloem is inspected by the Kleier model and Carrier-mediated theory. To verify this strategy, a series of N-alkylated phenazine-1-carboxylic acid-glycine compounds (PCA-Gly) were designed and synthesized. The results related to the castor bean seeds (R. communis L.) indicated that all the target compounds (4a–4f) had phloem mobility. The capacity for phloem mobility shows that N-alkylated glycine containing small substituents can significantly improve PCA phloem mobility, such as 4c(i-C3H7-N) > 4a(CH3-N) ≈ 4b(C2H5-N) > 4d (t-C4H9-N) > PCA-Gly > 4e(C6H5-N) > 4f(CH2COOH-N), with an oil–water partition coefficient between 1.2~2.5. In particular, compounds 4a(CH3-N), 4b(C2H5-N), and 4c(i-C3H7-N) present better phloem mobility, with the average concentrations in phloem sap of 14.62 μΜ, 13.98 μΜ, and 17.63 μΜ in the first 5 h, which are 8 to 10 times higher than PCA-Gly (1.71 μΜ). The results reveal that the Kleier model and Carrier-mediated theory play a guiding role in the design of phloem-mobile pesticides. However, the single Kleier model or Carrier-mediated theory are not entirely accurate. Still, there is a synergism between Carrier-mediated theory and the Kleier model for promoting the phloem transport of exogenous compounds. Therefore, we suggest the introduction of endogenous plant compounds as a promoiety to improve the phloem mobility of pesticides through Carrier-mediated theory. It is necessary to consider the improvement of physicochemical properties according to the Kleier model, which can contribute to a scientific theory for developing phloem-mobile pesticides.

Metabolic Engineering of Pseudomonas putida for Fermentative Production of l-Theanine.

N-alkylated amino acids are intermediates of natural biological pathways and can be found incorporated in peptides or have physiological roles in their free form. The N-ethylated amino acid l-theanine shows taste-enhancing properties and health benefits. It naturally occurs in green tea as major free amino acid. Isolation of l-theanine from Camilla sinensis shows low efficiency, and chemical synthesis results in a racemic mixture. Therefore, biochemical approaches for the production of l-theanine gain increasing interest. Here, we describe metabolic engineering of Pseudomonas putida KT2440 for the fermentative production of l-theanine from monoethylamine and carbon sources glucose, glycerol, or xylose using heterologous enzymes from Methylorubrum extorquens for l-theanine production and heterologous enzymes from Caulobacter crescentus for growth with xylose. l-Theanine (15.4 mM) accumulated in shake flasks with minimal medium containing monoethylamine and glucose, 15.2 mM with glycerol and 7 mM with xylose. Fed-batch bioreactor cultures yielded l-theanine titers of 10 g L-1 with glucose plus xylose, 17.2 g L-1 with glycerol, 4 g L-1 with xylose, and 21 g L-1 with xylose plus glycerol, respectively. To the best of our knowledge, this is the first l-theanine process using P. putida and the first compatible with the use of various alternative carbon sources.

Sustainable Production of N-methylphenylalanine by Reductive Methylamination of Phenylpyruvate Using Engineered Corynebacterium glutamicum

N-alkylated amino acids occur widely in nature and can also be found in bioactive secondary metabolites such as the glycopeptide antibiotic vancomycin and the immunosuppressant cyclosporine A. To meet the demand for N-alkylated amino acids, they are currently produced chemically; however, these approaches often lack enantiopurity, show low product yields and require toxic reagents. Fermentative routes to N-alkylated amino acids like N-methyl-l-alanine or N-methylantranilate, a precursor of acridone alkaloids, have been established using engineered Corynebacterium glutamicum, which has been used for the industrial production of amino acids for decades. Here, we describe metabolic engineering of C. glutamicum for de novo production of N-methylphenylalanine based on reductive methylamination of phenylpyruvate. Pseudomonas putida Δ-1-piperideine-2-carboxylate reductase DpkA containing the amino acid exchanges P262A and M141L showed comparable catalytic efficiencies with phenylpyruvate and pyruvate, whereas the wild-type enzyme preferred the latter substrate over the former. Deletion of the anthranilate synthase genes trpEG and of the genes encoding branched-chain amino acid aminotransferase IlvE and phenylalanine aminotransferase AroT in a strain engineered to overproduce anthranilate abolished biosynthesis of l-tryptophan and l-phenylalanine to accumulate phenylpyruvate. Upon heterologous expression of DpkAP262A,M141L, N-methylphenylalanine production resulted upon addition of monomethylamine to the medium. In glucose-based minimal medium, an N-methylphenylalanine titer of 0.73 ± 0.05 g L−1, a volumetric productivity of 0.01 g L−1 h−1 and a yield of 0.052 g g−1 glucose were reached. When xylose isomerase gene xylA from Xanthomonas campestris and the endogenous xylulokinase gene xylB were expressed in addition, xylose as sole carbon source supported production of N-methylphenylalanine to a titer of 0.6 ± 0.04 g L−1 with a volumetric productivity of 0.008 g L−1 h−1 and a yield of 0.05 g g−1 xylose. Thus, a fermentative route to sustainable production of N-methylphenylalanine by recombinant C. glutamicum has been established.

Dedicated to a peptide chemist of few words and immense impact: Louis A. Carpino

Dedicated to a peptide chemist of few words and immense impact: Louis A. Carpino

Fermentative N-Methylanthranilate Production by Engineered Corynebacterium glutamicum.

The N-functionalized amino acid N-methylanthranilate is an important precursor for bioactive compounds such as anticancer acridone alkaloids, the antinociceptive alkaloid O-isopropyl N-methylanthranilate, the flavor compound O-methyl-N-methylanthranilate, and as a building block for peptide-based drugs. Current chemical and biocatalytic synthetic routes to N-alkylated amino acids are often unprofitable and restricted to low yields or high costs through cofactor regeneration systems. Amino acid fermentation processes using the Gram-positive bacterium Corynebacterium glutamicum are operated industrially at the million tons per annum scale. Fermentative processes using C. glutamicum for N-alkylated amino acids based on an imine reductase have been developed, while N-alkylation of the aromatic amino acid anthranilate with S-adenosyl methionine as methyl-donor has not been described for this bacterium. After metabolic engineering for enhanced supply of anthranilate by channeling carbon flux into the shikimate pathway, preventing by-product formation and enhancing sugar uptake, heterologous expression of the gene anmt encoding anthranilate N-methyltransferase from Ruta graveolens resulted in production of N-methylanthranilate (NMA), which accumulated in the culture medium. Increased SAM regeneration by coexpression of the homologous adenosylhomocysteinase gene sahH improved N-methylanthranilate production. In a test bioreactor culture, the metabolically engineered C. glutamicum C1* strain produced NMA to a final titer of 0.5 g·L−1 with a volumetric productivity of 0.01 g·L−1·h−1 and a yield of 4.8 mg·g−1 glucose.

Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.

SummaryRNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5′-3′) mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3′-5′) is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation.

Katalin Barta

Katalin Barta

Conversion of "Customizable Units" into N-Alkyl Amino Acids and Generation of N-Alkyl Peptides.

An efficient conversion of hydroxyproline "customizable" units into new amino acids with a variety of N-alkyl substituents is described. The process is versatile and can afford valuable N-methyl amino acids and N, O-acetals. In addition, it allows the introduction of N-homoallylic substituents and N-chains with terminal ester, ketone, or cyano groups. These chains could be used for peptide extension or conjugation to other molecules (e.g., by olefin metathesis, peptide ligation, etc.). The transformation is carried out in just two (for R = CH2OAc) or three steps (scission of the pyrrolidine ring, manipulation of the α-chain, and the N-substituent) under mild, metal-free conditions, affording products with high optical purity.

Fermentative Production of N-Methylglutamate From Glycerol by Recombinant Pseudomonas putida.

N-methylated amino acids are present in diverse biological molecules in bacteria, archaea and eukaryotes. There is an increasing interest in this molecular class of alkylated amino acids by the pharmaceutical and chemical industries. N-alkylated amino acids have desired functions such as higher proteolytic stability, enhanced membrane permeability and longer peptide half-lives, which are important for the peptide-based drugs, the so-called peptidomimetics. Chemical synthesis of N-methylated amino acids often is limited by incomplete stereoselectivity, over-alkylation or the use of hazardous chemicals. Here, we describe metabolic engineering of Pseudomonas putida KT2440 for the fermentative production of N-methylglutamate from simple carbon sources and monomethylamine. P. putida KT2440, which is generally recognized as safe and grows with glucose and the alternative feedstock glycerol as sole carbon and energy source, was engineered for the production of N-methylglutamate using heterologous enzymes from Methylobacterium extorquens. About 3.9 g L−1 N-methylglutamate accumulated within 48 h in shake flask cultures with minimal medium containing monomethylamine and glycerol. A fed-batch cultivation process yielded a N-methylglutamate titer of 17.9 g L−1.

Biocatalytic Synthesis of Chiral N-Functionalized Amino Acids.

N-Functionalized amino acids are important building blocks for the preparation of diverse bioactive molecules, including peptides. The development of sustainable manufacturing routes to chiral N-alkylated amino acids remains a significant challenge in the pharmaceutical and fine-chemical industries. Herein we report the discovery of a structurally diverse panel of biocatalysts which catalyze the asymmetric synthesis of N-alkyl amino acids through the reductive coupling of ketones and amines. Reactions have been performed on a gram scale to yield optically pure N-alkyl-functionalized products in high yields.

Leveraging a "Catch-Release" Logic Gate Process for the Synthesis and Nonchromatographic Purification of Thioether- or Amine-Bridged Macrocyclic Peptides.

Macrocyclic peptides containing N-alkylated amino acids have emerged as a promising therapeutic modality, capable of modulating protein-protein interactions and an intracellular delivery of hydrophilic payloads. While multichannel automated solid-phase peptide synthesis (SPPS) is a practical approach for peptide synthesis, the requirement for slow and inefficient chromatographic purification of the product peptides is a significant limitation to exploring these novel compounds. Herein, we invent a "catch-release" strategy for the nonchromatographic purification of macrocyclic peptides. A traceless catch process is enabled by the invention of a dual-functionalized N-terminal acetate analogue, which serves as a handle for capture onto a purification resin and as a leaving group for macrocyclization. Displacement by a C-terminal nucleophilic side chain thus releases the desired macrocycle from the purification resin. By design, this catch/release process is a logic test for the presence of the key components required for cyclization, thus removing impurities which lack the required functionality, such as common classes of peptide impurities, including hydrolysis fragments and truncated sequences. The method was shown to be highly effective with three libraries of macrocyclic peptides, containing macrocycles of 5-20 amino acids, with either thioether- or amine-based macrocyclic linkages; in this latter class, the reported method represents an enabling technology. In all cases, the catch-release protocol afforded significant enrichment of the target peptides purity, in many cases completely obviating the need for chromatography. Importantly, we have adapted this process for automation on a standard multichannel peptide synthesizer, achieving an efficient and completely integrated synthesis and purification platform for the preparation of these important molecules.

Directed Evolution of a Cyclized Peptoid-Peptide Chimera against a Cell-Free Expressed Protein and Proteomic Profiling of the Interacting Proteins to Create a Protein-Protein Interaction Inhibitor.

N-alkyl amino acids are useful building blocks for the in vitro display evolution of ribosomally synthesized peptides because they can increase the proteolytic stability and cell permeability of these peptides. However, the translation initiation substrate specificity of nonproteinogenic N-alkyl amino acids has not been investigated. In this study, we screened various N-alkyl amino acids and nonamino carboxylic acids for translation initiation with an Escherichia coli reconstituted cell-free translation system (PURE system) and identified those that efficiently initiated translation. Using seven of these efficiently initiating acids, we next performed in vitro display evolution of cyclized peptidomimetics against an arbitrarily chosen model human protein (β-catenin) cell-free expressed from its cloned cDNA (HUPEX) and identified a novel β-catenin-binding cyclized peptoid-peptide chimera. Furthermore, by a proteomic approach using direct nanoflow liquid chromatography-tandem mass spectrometry (DNLC-MS/MS), we successfully identified which protein-β-catenin interaction is inhibited by the chimera. The combination of in vitro display evolution of cyclized N-alkyl peptidomimetics and in vitro expression of human proteins would be a powerful approach for the high-speed discovery of diverse human protein-targeted cyclized N-alkyl peptidomimetics.

Domino Process Achieves Site-Selective Peptide Modification with High Optical Purity. Applications to Chain Diversification and Peptide Ligation.

The development of peptide libraries by site-selective modification of a few parent peptides would save valuable time and materials in discovery processes but still is a difficult synthetic challenge. Herein, we introduce natural hydroxyproline as a convertible unit for the production of a variety of optically pure amino acids, including expensive N-alkyl amino acids, homoserine lactones, and Agl lactams, and to achieve the mild, efficient, and site-selective modification of peptides. A domino process is used to cleave the customizable Hyp unit under mild, metal-free conditions. Both terminal and internal positions can be modified, and similar customizable units can be differentiated. The resulting products possess two reactive chains which can be manipulated independently. The versatility and scope of this process is highlighted by its application to the ligation of two peptide chains, and the generation of peptides with several chains and peptides with conformational restrictions.

ChemInform Abstract: Model Foldamers: Applications and Structures of Stable Macrocyclic Peptides Identified Using in vitro Selection

AbstractReview: 46 refs.

Peptide Formation by N-Methyl Amino Acids in Translation Is Hastened by Higher pH and tRNAPro

Applications of N-methyl amino acids (NMAAs) in drug discovery are limited by their low efficiencies of ribosomal incorporation, and little is known mechanistically about the steps leading to incorporation. Here, we demonstrate that a synthetic tRNA body based on a natural N-alkyl amino acid carrier, tRNA(Pro), increases translation incorporation rates of all three studied NMAAs compared with tRNA(Phe)- and tRNA(Ala)-based bodies. We also investigate the pH dependence of the incorporation rates and find that the rates increase dramatically in the range of pH 7 to 8.5 with the titration of a single proton. Results support a rate-limiting peptidyl transfer step dependent on deprotonation of the N-nucleophile of the NMAA. Competition experiments demonstrate that several futile cycles of delivery and rejection of A-site NMAA-tRNA are required per peptide bond formed and that increasing magnesium ion concentration increases incorporation yield. Data clarify the mechanism of ribosomal NMAA incorporation and provide three generalizable ways to improve incorporation of NMAAs in translation.