Amino Acid‐Specific,Ribonucleotide‐Promoted Peptide Formation in the Absence of Enzymes

Nucleic acids and polypeptides are at the heart of life. It is interesting to ask whether the monomers of these biopolymers possess intrinsic reactivity that favors oligomerization in the absence of enzymes. We have recently observed that covalently linked peptido RNA chains form when mixtures of monomers react in salt‐rich condensation buffer. Here, we report the results of a screen of the 20 proteinogenic amino acids and four ribonucleotides. None of the amino acids prevent phosphodiester formation, so all of them are compatible with genetic encoding through RNA chain growth. A reactivity landscape was found, in which peptide formation strongly depends on the structure of the amino acid, but less on the nucleobase. For example, proline gives ribonucleotide‐bound peptides most readily, tyrosine favors pyrophosphate and phosphodiester formation, and histidine gives phosphorimidazolides as dominant products. When proline and aspartic acid were allowed to compete for incorporation, only proline was found at the N‐terminus of peptido chains. The reactivity described here links two fundamental classes of biomolecules through reactions that occur without enzymes, but with amino acid specificity.     

Ribonucleotides and RNA Promote Peptide Chain Growth

All known forms of life use RNA‐mediated polypeptide synthesis to produce the proteins encoded in their genes. Because the principal parts of the translational machinery consist of RNA, it is likely that peptide synthesis was achieved early in the prebiotic evolution of an RNA‐dominated molecular world. How RNA attracted amino acids and then induced peptide formation in the absence of enzymes has been unclear. Herein, we show that covalent capture of an amino acid as a phosphoramidate favors peptide formation. Peptide coupling is a robust process that occurs with different condensation agents. Kinetics show that covalent capture can accelerate chain growth over oligomerization of the free amino acid by at least one order of magnitude, so that there is no need for enzymatic catalysis for peptide synthesis to begin. Peptide chain growth was also observed on phosphate‐terminated RNA strands. Peptide coupling promoted by ribonucleotides or ribonucleotide residues may have been an important transitional form of peptide synthesis that set in when amino acids were first captured by RNA.     

How Small Heterocycles Make a Reaction Network of Amino Acids and Nucleotides Efficient in Water

Organisms use enzymes to ensure a flow of substrates through biosynthetic pathways. How the earliest form of life established biosynthetic networks and prevented hydrolysis of intermediates without enzymes is unclear. Organocatalysts may have played the role of enzymes. Quantitative analysis of reactions of adenosine 5’‐monophosphate and glycine that produce peptides, pyrophosphates, and RNA chains reveals that organocapture by heterocycles gives hydrolytically stabilized intermediates with balanced reactivity. We determined rate constants for 20 reactions in aqueous solutions containing a carbodiimide and measured product formation with cyanamide as a condensing agent. Organocapture favors reactions that are kinetically slow but productive, and networks, over single transformations. Heterocycles can increase the metabolic efficiency more than two‐fold, with up to 0.6 useful bonds per fuel molecule spent, boosting the efficiency of life‐like reaction systems in the absence of enzymes.     

Wet and Functional Adhesives from One‐Step Aqueous Self‐Assembly of Natural Amino Acids and Polyoxometalates

Sessile organisms have undergone long‐term evolution to develop the unique ability by positioning themselves on wet solid surface through secreting adhesive proteins. The present study reveals that natural amino acid monomers can also exhibit similar adhesion capacity. This kind of biomimetic adhesives were created by the one‐step aqueous assembly of basic amino acids with assistance of anionic polyoxometalates. The polyoxometalates not only serve as multivalent scaffold to initiate the supramolecular cross‐linking of amino acid molecules, but also function as a redox component, bestowing the wet adhesives with electrochromic features.     

Asymmetric Strecker Reaction Arising from the Molecular Orientation of an Achiral Imine at the Single‐Crystal Face: Enantioenriched l‐ and d‐Amino Acids

Strecker synthesis has long been considered one of the prebiotic reactions for the synthesis of α‐amino acids. However, the correlation between the origin of chirality and highly enantioenriched α‐amino acids through this method remains a puzzle. In the reaction, it may be conceivable that the handedness of amino acids has been determined at the formation stage of the chiral intermediate α‐aminonitrile, that is, the enantioselective addition of hydrogen cyanide to an imine. Herein, an enantiotopic crystal surface of an achiral imine acted as an origin of chirality for the enantioselective formation of α‐aminonitriles by the addition of HCN. In conjunction with the amplification of the enantiomeric excess and multiplication of enantioenriched aminonitrile, a large amount of near enantiopure α‐amino acids, with the l ‐ and d ‐handedness corresponding to the molecular orientation of the imine, is reported.     

Determining the Diastereoselectivity of the Formation of Dipeptidonucleotides by NMR Spectroscopy

Proteins are composed of l -amino acids, but nucleic acids and most oligosaccharides contain d -sugars as building blocks. It is interesting to ask whether this is a coincidence or a consequence of the functional interplay of these biomolecules. One reaction that provides an opportunity to study this interplay is the formation of phosphoramidate-linked peptido RNA from amino acids and ribonucleotides in aqueous condensation buffer. Here we report how the diastereoselectivity of the first peptide coupling of the peptido RNA pathway can be determined in situ by NMR spectroscopy. When a racemic mixture of an amino acid ester was allowed to react with an 5′-aminoacidyl nucleotide, diastereomeric ratios of up to 72 : 28 of the resulting dipeptido nucleotides were found by integration of ³¹P- or ¹H-NMR peaks. The highest diastereomeric excess was found for the homochiral coupling product d -Ser-d -Trp, phosphoramidate-linked to adenosine 5′-monophosphate with its d -ribose ring. When control reactions with an N-acetyl amino acid and valine methyl ester were run in organic solvent, the diastereoselectivity was found to be lower, with diastereomeric ratios≤62 : 38. The results from the exploratory study thus indicate that the ribonucleotide residue not only facilitates the coupling of lipophilic amino acids in aqueous medium but also the formation of a homochiral dipeptide. The methodology described here may be used to search for other stereoselective reactions that shed light on the origin of homochirality.     

Introduction of d‐Amino Acids in Minimalistic Peptide Substrates by an S‐Adenosyl‐l‐Methionine Radical Epimerase

Post‐translational modifying enzymes from the S‐adenosyl‐l ‐methionine (AdoMet) radical superfamily garner attention due to their ability to accomplish challenging biochemical reactions. Among them, a family of AdoMet radical epimerases catalyze irreversible l ‐ to d ‐amino acid transformations of diverse residues, including 18 sites in the complex sponge‐derived polytheonamide toxins. Herein, the in vitro activity of the model epimerase OspD is reported and its catalytic mechanism and substrate flexibility is investigated. The wild‐type enzyme was capable of leader‐independent epimerization of not only the stand‐alone core peptide, but also truncated and cyclic core variants. Introduction of d ‐amino acids can drastically alter the stability, structure, and activity of peptides; thus, epimerases offer opportunities in peptide bioengineering.     

Mixed Anhydride Intermediates in the Reaction of 5(4H)‐Oxazolones with Phosphate Esters and Nucleotides

5(4H)‐Oxazolones can be formed through the activation of acylated α‐amino acids or of peptide C termini. They constitute potentially activated intermediates in the abiotic chemistry of peptides that preceded the origin of life or early stages of biology and are capable of yielding mixed carboxylic‐phosphoric anhydrides upon reaction with phosphate esters and nucleotides. Here, we present the results of a study aimed at investigating the chemistry that can be built through this interaction. As a matter of fact, the formation of mixed anhydrides with mononucleotides and nucleic acid models is shown to take place at positions involving a mono‐substituted phosphate group at the 3’‐ or 5’‐terminus but not at the internal phosphodiester linkages. In addition to the formation of mixed anhydrides, the subsequent intramolecular acyl or phosphoryl transfers taking place at the 3’‐terminus are considered to be particularly relevant to the common prebiotic chemistry of α‐amino acids and nucleotides.     

Palladium‐Mediated Arylation of Lysine in Unprotected Peptides

A mild method for the arylation of lysine in an unprotected peptide is presented. In the presence of a preformed biarylphosphine‐supported palladium(II)–aryl complex and a weak base, lysine amino groups underwent C−N bond formation at room temperature. The process generally exhibited high selectivity for lysine over other amino acids containing nucleophilic side chains and was applicable to the conjugation of a variety of organic compounds, including complex drug molecules, with an array of peptides. Finally, this method was also successfully applied to the formation of cyclic peptides by macrocyclization.     

Control Mechanism for cis Double‐Bond Formation by Polyunsaturated Fatty‐Acid Synthases

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) are essential fatty acids for humans. Some microorganisms biosynthesize these PUFAs through PUFA synthases composed of four subunits with multiple catalytic domains. These PUFA synthases each create a specific PUFA without undesirable byproducts, even though the multiple catalytic domains in each large subunit are very similar. However, the detailed biosynthetic pathways and mechanisms for controlling final‐product profiles are still obscure. In this study, the FabA‐type dehydratase domain (DH_FabA) in the C‐subunit and the polyketide synthase‐type dehydratase domain (DH_PKS) in the B‐subunit of ARA synthase were revealed to be essential for ARA biosynthesis by in vivo gene exchange assays. Furthermore, in vitro analysis with truncated recombinant enzymes and C₄‐ to C₈‐acyl ACP substrates showed that ARA and EPA synthases utilized two types of DH domains, DH_PKS and DH_FabA, depending on the carbon‐chain length, to introduce either saturation or cis double bonds to growing acyl chains.     

Hydrogen‐bonding features in the 1:2 adduct of 4‐aminobenzoic acid and l‐proline

The title adduct, 4‐aminobenzoic acid–l ‐proline–water (1/2/1), C₇H₇NO₂·2C₅H₉NO₂·H₂O, contains two independent proline chains with a C(5) motif, each of the head‐to‐tail type and each held together by N—H...O hydrogen bonds, propagated parallel to the b and c axes of the unit cell. Thus, the proline residues aggregate parallel to the ac plane. 4‐Aminobenzoic acid (PABA) residues are arranged on both sides of the proline aggregate and are connected through water O atoms, which act as acceptors for PABA and as hydrogen‐bond donors to the amino acids. The characteristic features of PABA, viz. twisting of the carboxyl plane from the aromatic ring and the formation of a head‐to‐tail chain motif [C(8)] along the b axis, are observed. A distinct feature of the structure is that no proton transfer occurs between proline and PABA.     

A Diene‐Containing Noncanonical Amino Acid Enables Dual Functionality in Proteins: Rapid Diels–Alder Reaction with Maleimide or Proximity‐Based Dimerization

Here, we describe a diene‐containing noncanonical amino acid (ncAA) capable of undergoing fast and selective normal electron‐demand Diels–Alder (DA) reactions following its incorporation into antibodies. A cyclopentadiene derivative of lysine (CpHK) served as the reactive handle for DA transformations and the substrate for genetic incorporation. CpHK incorporated into antibodies with high efficiency and was available for maleimide conjugation or self‐reaction depending on position in the amino acid sequence. CpHK at position K274 reacted with the maleimide drug‐linker AZ1508 at a rate of ≈79 m ^?1 s^?1 to produce functional antibody–drug conjugates (ADCs) in a one‐step process. Incorporation of CpHK at position S239 resulted in dimerization, which covalently linked antibody heavy chains together. The diene ncAA described here is capable of producing therapeutic protein conjugates with clinically validated and widely available maleimide compounds, while also enabling proximity‐based stapling through a DA dimerization reaction.     

Organocatalysts Derived from Unnatural α‐Amino Acids: Scope and Applications

The organocatalytic properties of unnatural α‐amino acids are reviewed. Post‐translational derivatives of natural α‐amino acids include 4‐hydroxy‐l ‐proline and 4‐amino‐l ‐proline scaffolds, and also proline homologues. The activity of synthetic unnatural α‐amino acid‐based organocatalysts, such as β‐alkyl alanines, alanine‐based phosphines, and tert‐leucine derivatives, are reviewed herein. The organocatalytic properties of unnatural monocyclic, bicyclic, and tricyclic proline derivatives are also reviewed. Several families of these organocatalysts permit the efficient and stereoselective synthesis of complex natural products. Most of the reviewed organocatalysts accelerate the reported reactions through covalent interactions that raise the HOMO (enamine intermediates) or lower the LUMO (iminium intermediates).     

Simultaneous Chain‐Growth and Step‐Growth Polymerization—A New Route to Cyclic Polymers

Reinvestigation of numerous ring‐opening polymerizations by means of MALDI‐TOF mass spectrometry has evidenced that cyclic polymers were formed as the only reaction products or, at least, in large fractions. This finding is ascribed to the intermediate formation of difunctional chains having active end groups that can react with each other. Due to the low concentration of these difunctional chains cyclization is favored over chain extension according to the Ruggli–Ziegler dilution principle. A polymerization mechanism which usually favors the formation of cyclic polymers is the zwitterionic polymerization, but an exception from this rule is known. The following classes of monomers were discussed: α‐amino acid, N‐carboxyanhydrides (oxazolidine‐2,5‐diones), dithiolane‐2,4‐diones, 5,5‐dimethyl‐1,3,2‐dioxathiolan‐4‐one‐2‐oxide, salicylic acid O‐carboxyanhydride, L ‐lactide and D ,L ‐lactide, hexamethyl cyclotrisiloxane, and macrocyclic dithiocarbamates.

     

Photocaged Quinone Methide Crosslinkers for Light‐Controlled Chemical Crosslinking of Protein–Protein and Protein–DNA Complexes

Small‐molecule crosslinkers are invaluable for probing biomolecular interactions and for crosslinking mass spectrometry. Existing chemical crosslinkers target only a small selection of amino acids, while conventional photo‐crosslinkers target almost all residues non‐specifically, complicating data analysis. Herein, we report photocaged quinone methide (PQM)‐based crosslinkers that target nine nucleophilic residues through Michael addition, including Gln, Arg, and Asn, which are inaccessible to existing chemical crosslinkers. PQM crosslinkers were used in vitro, in Escherichia coli, and in mammalian cells to crosslink dimeric proteins and endogenous membrane receptors. The heterobifunctional crosslinker NHQM could crosslink proteins to DNA, for which few crosslinkers exist. The photoactivatable reactivity of these crosslinkers and their ability to target multiple amino acids will enhance the use of chemical crosslinking for studies of protein–protein and protein–DNA networks and for structural biology.     

Pushing the Lewis Acidity Boundaries of Boron Compounds With Non‐Planar Triarylboranes Derived from Triptycenes

Bending the planar trigonal boron center of triphenylborane by connecting its aryl rings with carbon or phosphorus linkers gave access to a series of 9‐boratriptycene derivatives with unprecedented structures and reactivities. NMR spectroscopy and X‐ray diffraction of the Lewis adducts of these non‐planar boron Lewis acids with weak Lewis base revealed particularly strong covalent bond formation. The first Lewis adduct of a trivalent boron compounds with the Tf₂N^? anion illustrates the unrivaled Lewis acidity of these species. Increasing the pyramidalization of the boron center and using a cationic phosphonium linker resulted in an exceptional enhancement of Lewis acidity. Introduction of a phosphorus and a boron atom at each edge of a triptycene framework, allowed access to new bifunctional Lewis acid‐base 9‐phospha‐10‐boratriptycenes featuring promising reactivity for the activation of carbon‐halogen bonds.     

Geometry‐Retentive C‐Alkenylation of Lithiated α‐Aminonitriles: Quaternary α‐Alkenyl Amino Acids and Hydantoins

α‐Amino nitriles tethered to alkenes through a urea linkage undergo intramolecular C‐alkenylation on treatment with base by attack of the lithionitrile derivatives on the N′‐alkenyl group. A geometry‐retentive alkene shift affords stereospecifically the E or Z isomer of the 5‐alkenyl‐4‐iminohydantoin products from the corresponding starting E ‐ or Z ‐N ′‐alkenyl urea, each of which may be formed from the same N ‐allyl precursor by stereodivergent alkene isomerization. The reaction, formally a nucleophilic substitution at an sp² carbon atom, allows the direct regioselective incorporation of mono‐, di‐, tri‐, and tetrasubstituted olefins at the α‐carbon of amino acid derivatives. The initially formed 5‐alkenyl iminohydantoins may be hydrolyzed and oxidatively deprotected to yield hydantoins and unsaturated α‐quaternary amino acids.     

Highly Efficient Synthesis and Assay of Protein‐Imprinted Nanogels by Using Magnetic Templates

We report an approach integrating the synthesis of protein‐imprinted nanogels (“plastic antibodies”) with a highly sensitive assay employing templates attached to magnetic carriers. The enzymes trypsin and pepsin were immobilized on amino‐functionalized solgel‐coated magnetic nanoparticles (magNPs). Lightly crosslinked fluorescently doped polyacrylamide nanogels were subsequently produced by high‐dilution polymerization of monomers in the presence of the magNPs. The nanogels were characterised by a novel competitive fluorescence assay employing identical protein‐conjugated nanoparticles as ligands to reversibly immobilize the corresponding nanogels. Both nanogels exhibited K_d<10 pM for their respective target protein and low cross‐reactivity with five reference proteins. This agrees with affinities reported for solid‐phase‐synthesized nanogels prepared using low‐surface‐area glass‐bead supports. This approach simplifies the development and production of plastic antibodies and offers direct access to a practical bioassay.     

Liquid chromatographic resolution of proline and pipecolic acid derivatives on chiral stationary phases based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid

Two liquid chromatographic chiral stationary phases based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid were applied to the resolution of the amide derivatives of cyclic α‐amino acids including proline and pipecolic acid. Among the five amide derivatives of proline, aniline amide was resolved best on the first chiral stationary phase, which contains two N–H tethering amide groups, with the separation factor of 1.31 and the resolution of 2.60, and on the second chiral stationary phase, which contains two N–CH₃ tethering amide groups, with the separation factor of 1.57 and the resolution of 5.50. Among the five amide derivatives of pipecolic acid, 2‐naphthyl amide was resolved best on the first chiral stationary phase with the separation factor of 1.30 and the resolution of 1.75, but 1‐naphthylmethyl amide was resolved best on the second chiral stationary phase with the separation factor of 1.30 and the resolution of 2.26. In general, the second chiral stationary phase was found to be better than the first chiral stationary phase in the resolution of the amide derivatives of cyclic α‐amino acids. In this study, the second chiral stationary phase was first demonstrated to be useful for the resolution of secondary amino compounds.