Synthesis Of Noncanonical Amino Acids Research Articles

Stereoselective Synthesis of Noncanonical Amino Acids Enabled by Photoredox-Pyridoxal Radical Biocatalysis

Stereoselective Synthesis of Noncanonical Amino Acids Enabled by Photoredox-Pyridoxal Radical Biocatalysis

Expanding the β-substitution reactions of serine synthase through mutagenesis of aromatic active site residues

The Gram-negative bacterium, Fusobacterium nucleatum, possesses a fold II type pyridoxal 5′-phosphate-dependent enzyme that catalyzes the reversible β-replacement of l-cysteine and l-serine, generating H2S and H2O, respectively. This enzyme, termed serine synthase (FN1055), contains an active site Asp232 that serves as a general base in the activation of a water molecule for nucleophilic attack of the ⍺-aminoacrylate intermediate. A network of hydrophobic residues surrounding Asp232 are key to catalysis as they increase the basicity of the side chain. However, these residues severely restrict the range of nucleophilic substrates that can react with the ⍺-aminoacrylate, making the enzyme an ineffective biocatalyst for noncanonical amino acid biosynthesis. Herein, we systematically substituted four aromatic active residues (Trp99, Phe125, Phe148 and Phe234) to an alanine to determine their catalytic importance in serine/cysteine synthase reactions and if their substitution could broaden the scope of nucleophiles that could react with the ⍺-aminoacrylate intermediate. All four single site mutants W99A, F125A, F148A, and F234A could form the ⍺-aminoacrylate intermediate upon reaction with either l-cysteine or l-serine; however, the rate constant associated with the elimination of the β-hydroxyl group from l-serine was 150 to 200-fold lower in the F125A and F148A variants. Substitution of Phe125 and Phe148, situated ∼3–4 Å from the general base, also abolished the serine synthase reaction due to their inability to activate a water molecule for nucleophilic attack of the ⍺-aminoacrylate. Overall, the mutational studies indicate that the clustering of aromatic residues disproportionately benefits the serine synthase reaction as they increase the binding affinity for l-cysteine, decrease the binding of the product, l-serine, and promote the activation of a water molecule. Notably, the aminoacrylate species present in F125A and F148A was able to react with thiophenol, signifying that serine synthase has biocatalytic potential in the synthesis of noncanonical amino acids.

Diversifying Amino Acids and Peptides via Deaminative Reductive Cross-Couplings Leveraging High-Throughput Experimentation.

A deaminative reductive coupling of amino acid pyridinium salts with aryl bromides has been developed to enable efficient synthesis of noncanonical amino acids and diversification of peptides. This method transforms natural, commercially available lysine, ornithine, diaminobutanoic acid, and diaminopropanoic acid to aryl alanines and homologated derivatives with varying chain lengths. Attractive features include ability to transverse scales, tolerance of pharma-relevant (hetero)aryls and biorthogonal functional groups, and the applicability beyond monomeric amino acids to short and macrocyclic peptide substrates. The success of this work relied on high-throughput experimentation to identify complementary reaction conditions that proved critical for achieving the coupling of a broad scope of aryl bromides with a range of amino acid and peptide substrates including macrocyclic peptides.

Gram-Scale Asymmetric Synthesis of Fluorinated Amino Acids Using a Chiral Nickel(II) Complex.

Fluorinated amino acids play an important role in the field of peptide and protein engineering. Although numerous syntheses have been published in recent decades, strategies that allow routine access to fluorinated amino acids on a gram-scale have been poorly described. Furthermore, the described pathways that gain fluorinated amino acids are based on different synthetic strategies, making a uniform approach that uses similar starting materials highly beneficial. Chiral Ni(II) complexes were introduced as powerful tools in the synthesis of noncanonical amino acids. In this work, we present a strategy for the synthesis of a diverse range of fluorinated amino acids based on the corresponding Ni(II) complex from which the products can be obtained in enantiopure form (99% ee) on a gram-scale. In addition, we describe an optimized procedure for the synthesis of alkyl iodide building blocks that are required for the alkylation reactions with the corresponding Ni(II) complex. Finally, we characterized the synthesized fluorinated amino acids with regard to their hydrophobicity and α-helix propensity.

Glucose-Limited Fed-Batch Cultivation Strategy to Mimic Large-Scale Effects in Escherichia coli Linked to Accumulation of Non-Canonical Branched-Chain Amino Acids by Combination of Pyruvate Pulses and Dissolved Oxygen Limitation

Insufficient mixing in large-scale bioreactors provokes gradient zones of substrate, dissolved oxygen (DO), pH, and other parameters. E. coli responds to a high glucose, low oxygen feeding zone with the accumulation of mixed acid fermentation products, especially formate, but also with the synthesis of non-canonical amino acids, such as norvaline, norleucine and β-methylnorleucine. These amino acids can be mis-incorporated into recombinant products, which causes a problem for pharmaceutical production whose solution is not trivial. While these effects can also be observed in scale down bioreactor systems, these are challenging to operate. Especially the high-throughput screening of clone libraries is not easy, as fed-batch cultivations would need to be controlled via repeated glucose pulses with simultaneous oxygen limitation, as has been demonstrated in well controlled robotic systems. Here we show that not only glucose pulses in combination with oxygen limitation can provoke the synthesis of these non-canonical branched-chain amino acids (ncBCAA), but also that pyruvate pulses produce the same effect. Therefore, we combined the enzyme-based glucose delivery method Enbase® in a PALL24 mini-bioreactor system and combined repeated pyruvate pulses with simultaneous reduction of the aeration rate. These cultivation conditions produced an increase in the non-canonical branched chain amino acids norvaline and norleucine in both the intracellular soluble protein and inclusion body fractions with mini-proinsulin as an example product, and this effect was verified in a 15 L stirred tank bioreactor (STR). To our opinion this cultivation strategy is easy to apply for the screening of strain libraries under standard laboratory conditions if no complex robotic and well controlled parallel cultivation devices are available.

Synthesis of Non-canonical Amino Acids and Peptide Containing Them for Establishment of the Template for Drug Discovery.

Non-canonical amino acid derivatives are an attractive scaffold for novel drug candidates. Among the methods used to prepare this motif, the asymmetric Mannich-type reaction of α-imino carboxylic acid derivatives is a preeminent strategy because a wide variety of non-canonical amino acids can be accessed by changing only the nucleophile. Preparing the common substrate is difficult, however, which makes this method problematic. We developed a convenient method for synthesizing common substrates using MnO2-mediated oxidation of stable precursors. Peptides bearing non-canonical amino acids are another attractive synthetic target. We propose a new approach for synthesizing non-canonical amino acid-containing peptides by directly applying various organic reactions to peptidic substrates. Using hydrophobic anchor-supported peptides, we directly applied ring-closing metathesis and asymmetric Friedel-Crafts reactions to peptidic substrates. We also developed a novel recyclable organocatalyst according to the nature of the hydrophobic anchor tagged compound.

Recent Applications of Carbon-Nitrogen Lyases in Asymmetric Synthesis of Noncanonical Amino Acids and Heterocyclic Compounds.

Carbon‐nitrogen (C−N) lyases are enzymes that normally catalyze the cleavage of C−N bonds. Reversing this reaction towards carbon‐nitrogen bond formation can be a powerful approach to prepare valuable compounds that could find applications in everyday life. This review focuses on recent (last five years) applications of native and engineered C−N lyases, either as stand‐alone biocatalysts or as part of multienzymatic and chemoenzymatic cascades, in enantioselective synthesis of noncanonical amino acids and dinitrogen‐fused heterocycles, which are useful tools for neurobiological research and important synthetic precursors to pharmaceuticals and food additives.

Tackling Achilles' Heel in Synthetic Biology: Pairing Intracellular Synthesis of Noncanonical Amino Acids with Genetic-Code Expansion to Foster Biotechnological Applications.

For the last two decades, synthetic biologists have been able to unlock and expand the genetic code, generating proteins with unique properties through the incorporation of noncanonical amino acids (ncAAs). These evolved biomaterials have shown great potential for applications in industrial biocatalysis, therapeutics, bioremediation, bioconjugation, and other areas. Our ability to continue developing such technologies depends on having relatively easy access to ncAAs. However, the synthesis of enantiomerically pure ncAAs in practical quantitates for large-scale processes remains a challenge. Biocatalytic ncAA production has emerged as an excellent alternative to traditional organic synthesis in terms of cost, enantioselectivity, and sustainability. Moreover, biocatalytic synthesis offers the opportunity of coupling the intracellular generation of ncAAs with genetic-code expansion to overcome the limitations of an external supply of amino acid. In this minireview, we examine some of the most relevant achievements of this approach and its implications for improving technological applications derived from synthetic biology.

Nitroalkanes as Versatile Nucleophiles for Enzymatic Synthesis of Noncanonical Amino Acids.

C-C bond-forming reactions often require nucleophilic carbon species rarely compatible with aqueous reaction media, thus restricting their appearance in biocatalysis. Here we report the use of nitroalkanes as a structurally versatile class of nucleophilic substrates for C-C bond formation catalyzed by variants of the β-subunit of tryptophan synthase (TrpB). The enzymes accept a wide range of nitroalkanes to form noncanonical amino acids, here the nitro group can serve as a handle for further modification. Using nitroalkane nucleophiles greatly expands the scope of compounds made by TrpB variants and establishes nitroalkanes as a valuable substrate class for biocatalytic C-C bond formation.

Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble.

Allosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the synthesis of noncanonical amino acids (ncAA). Previously, we evolved the β-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA. However, the precise mechanism by which mutation activated TrpB to afford a stand-alone catalyst remained enigmatic. Here, we show that directed evolution caused a gradual change in the rate-limiting step of the catalytic cycle. Concomitantly, the steady-state distribution of the intermediates shifts to favor covalently bound Trp adducts, which have increased thermodynamic stability. The biochemical properties of these evolved, stand-alone TrpBs converge on those induced in the native system by allosteric activation. High-resolution crystal structures of the wild-type enzyme, an intermediate in the lineage, and the final variant, encompassing five distinct chemical states, show that activating mutations have only minor structural effects on their immediate environment. Instead, mutation stabilizes the large-scale motion of a subdomain to favor an otherwise transiently populated closed conformational state. This increase in stability enabled the first structural description of Trp covalently bound in a catalytically active TrpB, confirming key features of catalysis. These data combine to show that sophisticated models of allostery are not a prerequisite to recapitulating its complex effects via directed evolution, opening the way to engineering stand-alone versions of diverse allosteric enzymes.