An Orthogonal D2O‐Based Induction System that Provides Insights into d‐Amino Acid Pattern Formation by Radical S‐Adenosylmethionine Peptide Epimerases

Radical S‐adenosyl methionine peptide epimerases (RSPEs) are an enzyme family that accomplishes regiospecific and irreversible introduction of multiple d ‐configured residues into ribosomally encoded peptides. Collectively, RSPEs can generate diverse epimerization patterns in a wide range of substrates. Previously, the lack of rapid methods to localize epimerized residues has impeded efforts to investigate the function and applicative potential of RSPEs. An efficient mass spectrometry‐based assay is introduced that permits characterization of products generated in E. coli . Applying this to a range of non‐natural peptide‐epimerase combinations, it is shown that the d ‐amino acid pattern is largely but not exclusively dictated by the core peptide sequence, while the epimerization order is dependent on the enzyme‐leader pair. RSPEs were found to be highly promiscuous, which allowed for modular introduction of peptide segments with defined patterns.     

Identification of the Enzymes Mediating the Maturation of the Seryl‐tRNA Synthetase Inhibitor SB‐217452 during the Biosynthesis of Albomycins

Albomycin δ₂ is a sulfur‐containing sideromycin natural product that shows potent antibacterial activity against clinically important pathogens. The l ‐serine‐thioheptose dipeptide partial structure, known as SB‐217452, has been found to be the active seryl‐tRNA synthetase inhibitor component of albomycin δ₂. Herein, it is demonstrated that AbmF catalyzes condensation between the 6′‐amino‐4′‐thionucleoside with the d ‐ribo configuration and seryl‐adenylate supplied by the serine adenylation activity of AbmK. Formation of the dipeptide is followed by C3′‐epimerization to produce SB‐217452 with the d ‐xylo configuration, which is catalyzed by the radical S‐adenosyl‐l ‐methionine enzyme AbmJ. Gene deletion suggests that AbmC is involved in peptide assembly linking SB‐217452 with the siderophore moiety. This study establishes how the albomycin biosynthetic machinery generates its antimicrobial component SB‐217452.     

Butelase‐Mediated Macrocyclization of d‐Amino‐Acid‐Containing Peptides

Macrocyclic compounds have received increasing attention in recent years. With their large surface area, they hold promise for inhibiting protein–protein interactions, a chemical space that was thought to be undruggable. Although many chemical methods have been developed for peptide macrocyclization, enzymatic methods have emerged as a promising new economical approach. Thus far, most enzymes have been shown to act on l ‐peptides; their ability to cyclize d ‐amino‐acid‐containing peptides has rarely been documented. Herein we show that macrocycles consisting of d ‐amino acids, except for the Asn residue at the ligating site, were efficiently synthesized by butelase 1, an Asn/Asp‐specific ligase. Furthermore, by using a peptide‐library approach, we show that butelase 1 tolerates most of the d ‐amino acid residues at the P1′′ and P2′′ positions.     

N‐(l‐2‐Aminobutyryl)‐l‐alanine and 2‐(l‐alanylamino)‐l‐butyric acid 0.33‐hydrate

The crystal structure of N‐(l ‐2‐amino­butyryl)‐l ‐alanine, C₇H₁₄N₂O₃, is closely related to the structure of l ‐alanyl‐l ‐alanine, both being tetragonal, while the retro‐analogue 2‐(l ‐alanyl­amino)‐l ‐butyric acid 0.33‐hydrate, C₇H₁₄N₂O₃·­0.33H₂O, forms a new type of molecular columnar structure with three peptide mol­ecules in the asymmetric unit.     

Structure‐guided RP‐HPLC chromatography of diastereomeric α‐helical peptide analogs substituted with single amino acid stereoisomers

An α‐helical model peptide (Ac‐EAEKAAKE‐X‐EKAAKEAEK‐amide) was used as a template to examine the efficacy of conventional reversed‐phase high‐performance liquid chromatography (RP‐HPLC) in separating peptide analogs with single substitutions (at position X) of diasteromeric amino acids Ile, allo‐Ile, d ‐Ile and d ‐allo‐Ile. We compared differences in peptide retention behavior on a C₈ column and a C₁₈ column at different temperatures. We demonstrated how subtle differences in peptide secondary structure affected by the different substitutions of amino acids with identical overall hydrophobicity enabled effective resolution of these peptide analogs. We also demonstrated the ability of RP‐HPLC to separate Ile‐ and allo‐Ile‐substituted analogs of a 26‐residue α‐helical antimicrobial peptide (AMP), with the substitution site towards the C‐terminus of the α‐helix. These peptides show different values of antibacterial activity and hemolytic activity, and different selectivity against bacteria and human cells. Our results underline the ability of RP‐HPLC to resolve even difficult diasteromeric peptide mixtures as well as its value in monitoring very subtle hydrophobicity changes in de novo‐designed AMP. Copyright © 2013 John Wiley & Sons, Ltd.     

Radical S‐Adenosyl Methionine Epimerases: Regioselective Introduction of Diverse D‐Amino Acid Patterns into Peptide Natural Products

PoyD is a radical S‐adenosyl methionine epimerase that introduces multiple D ‐configured amino acids at alternating positions into the highly complex marine peptides polytheonamide A and B. This novel post‐translational modification contributes to the ability of the polytheonamides to form unimolecular minimalistic ion channels and its cytotoxic activity at picomolar levels. Using a genome mining approach we have identified additional PoyD homologues in various bacteria. Three enzymes were expressed in E. coli with their cognate as well as engineered peptide precursors and shown to introduce diverse D ‐amino acid patterns into all‐L peptides. The data reveal a family of architecturally and functionally distinct enzymes that exhibit high regioselectivity, substrate promiscuity, and irreversible action and thus provide attractive opportunities for peptide engineering.     

Mechanistic Insights into the Radical S‐adenosyl‐l‐methionine Enzyme NosL From a Substrate Analogue and the Shunt Products

The radical S‐adenosyl‐l ‐methionine (SAM) enzyme NosL catalyzes the transformation of l ‐tryptophan into 3‐methyl‐2‐indolic acid (MIA), which is a key intermediate in the biosynthesis of a clinically interesting antibiotic nosiheptide. NosL catalysis was investigated by using the substrate analogue 2‐methyl‐3‐(indol‐3‐yl)propanoic acid (MIPA), which can be converted into MIA by NosL. Biochemical assays with different MIPA isotopomers in D₂O and H₂O unambiguously indicated that the 5′‐deoxyadenosyl (dAdo)‐radical‐mediated hydrogen abstraction is from the amino group of l ‐tryptophan and not a protein residue. Surprisingly, the dAdo‐radical‐mediated hydrogen abstraction occurs at two different sites of MIPA, thereby partitioning the substrate into different reaction pathways. Together with identification of an α,β‐unsaturated ketone shunt product, our study provides valuable mechanistic insight into NosL catalysis and highlights the remarkable catalytic flexibility of radical SAM enzymes.     

Enantioseparation of aromatic amino acids using CEC monolith with novel chiral selector,N‐methacryloyl‐l‐histidine methyl ester

A new type of polymethacrylate‐based monolithic column with chiral stationary phase was prepared for the enantioseparation of aromatic amino acids, namely d ,l ‐phenylalanine, d ,l ‐tyrosine, and d ,l ‐tryptophan by CEC. The monolithic column was prepared by in situ polymerization of butyl methacrylate (BMA), N‐methacryloyl‐l ‐histidine methyl ester (MAH), and ethylene dimethacrylate (EDMA) in the presence of porogens. The porogen mixture included DMF and phosphate buffer. MAH was used as a chiral selector. FTIR spectrum of the polymethacrylate‐based monolith showed that MAH was incorporated into the polymeric structure via in situ polymerization. Some experimental parameters including pH, concentration of the mobile phase, and MAH concentration with regard to the chiral CEC separation were investigated. Single enantiomers and enantiomer mixtures of the amino acids were separately injected into the monolithic column. It was observed that l ‐enantiomers of aromatic amino acids migrated before d ‐enantiomers. The reversal enantiomer migration order for tryptophan was observed upon changing of pH. Using the chiral monolithic column (100 μm id and 375 μm od), the best chiral separation was performed in 35:65% ACN/phosphate buffer (pH 8.0, 10 mM) with an applied voltage of 12 kV in CEC. SEM images showed that the chiral monolithic column has a continuous polymeric skeleton and large through‐pore structure.     

Poly(styrene‐co‐N‐methacryloyl‐l‐phenylalanine methyl ester)‐functionalized magnetic nanoparticles as sorbents for the analysis of sodium benzoate in beverages

In this study, poly(styrene‐co‐N‐methacryloyl‐l ‐phenylalanine methyl ester)‐functionalized magnetic nanoparticles were constructed and used as magnetic solid‐phase extraction sorbents for analysis of food preservatives in beverages. To prepare the poly(amino acid)‐based sorbents, N‐methacryloyl‐l ‐phenylalanine methyl ester, and styrene served as the functional monomers and modified onto the magnetic nanoparticles via free radical polymerization. Interestingly, compared with propylparaben and potassium sorbate, the proposed poly(amino acid)‐based sorbents showed a good selectivity to sodium benzoate. The adsorption capacity of the sorbents to sodium benzoate was 6.08 ± 0.31 mg/g. Moreover, the fast adsorption equilibrium could be reached within 5 min. Further, the resultant poly(amino acid)‐based sorbents were applied in the analysis of sodium benzoate in real beverage samples. The results proved that the proposed magnetic solid‐phase extraction sorbents have a great potential for the analysis of preservatives in food samples.     

Rose Bengal‐Sensitized Photooxidation of the Dipeptides l‐Tryptophyl‐l‐Phenylalanine,l‐Tryptophyl‐l‐Tyrosine and l‐Tryptophyl‐l‐Tryptophan: Kinetics,Mechanism and Photoproducts¶

The Rose Bengal‐sensitized photooxidations of the dipeptides l ‐tryptophyl‐l ‐phenylalanine (Trp‐Phe), l ‐tryptophyl‐l ‐tyrosine (Trp‐Tyr) and l ‐tryptophyl‐l ‐tryptophan (Trp‐Trp) have been studied in pH 7 water solution using static photolysis and time‐resolved methods. Kinetic results indicate that the tryptophan (Trp) moiety interacts with singlet molecular oxygen (O₂(¹Δ_g)) both through chemical reaction and through physical quenching, and that the photooxidations can be compared with those of equimolecular mixtures of the corresponding free amino acids, with minimum, if any, influence of the peptide bond on the chemical reaction. This is not a common behavior in other di‐ and polypeptides of photooxidizable amino acids. The ratio between chemical (k_r) and overall (k_t) rate constants for the interaction O₂(¹Δ_g)‐dipeptide indicates that Trp‐Phe and Trp‐Trp are good candidates to suffer photodynamic action, with k_rlk_t values of 0.72 and 0.60, respectively (0.65 for free Trp). In the case of Trp‐Tyr, a lower k_rlk_t value (0.18) has been found, likely as a result of the high component of physical deactivation of O₂(¹Δ_g) by the tyrosine moiety. The analysis of the photooxidation products shows that the main target for O₂(¹Δ_g) attack is the Trp group and suggests a much lower accumulation of kynurenine‐type products, as compared with free Trp. This is possibly because of the occurrence of another accepted alternative pathway of oxidation that gives rise to 3a‐oxidized hydrogenated pyrrolo[2,3‐b]indoles.     

Design of peptides with α,β‐de­hydro residues: pseudo‐tripeptide N‐benzyl­oxy­carbonyl–ΔLeu–l‐Ala–l‐Leu–OCH3

The title peptide N‐benzyl­oxy­carbonyl–ΔLeu–l ‐Ala–l ‐Leu–OCH₃ [methyl N‐(benzyl­oxy­carbonyl)‐α,β‐de­hydro­leucyl‐l ‐alanyl‐l ‐leucinate], C₂₄H₃₅N₃O₆, was synthesized in the solution phase. The peptide adopts a type II′β‐turn conformation which is stabilized by an intramolecular 4 1 N—H?O hydrogen bond. The crystal packing is stabilized by two intermolecular N—H?O hydrogen bonds.     

A Combined Effect of Thermal and Enzymatic Racemization for d‐Aspartic Acid to l‐Aspartic Acid by High‐Performance Liquid Chromatography Assay

The racemization of d ‐aspartic acid to l ‐aspartic acid has been successfully performed with a coupled enzyme system at 90 °C and a pH of about 4.0 by the assay of high‐performance liquid chromatography. This coupled enzymatic racemization is a successive two‐step reaction first induced by d ‐amino acid oxidase and a subsequent coupled reaction by an aminotransferase clonezyme with the help of coenzyme pyridoxal 5′‐phosphate and cosubstrate l ‐glutamate. Due to the very high temperature, part of the l ‐aspartic acid is produced by the thermal effect. In fact the thermal racemization for aspartic acid can proceed from either d ‐ or l ‐aspartic acid via an intermediate fumaric acid and leads to the formation of d ,l ‐malic acid. The formation of α‐oxalacetic acid formed irreversibly from d ‐aspartic acid with d ‐amino acid oxidase can induce a side reaction to l ‐alanine. The thermal effect may also be responsible for the production of d ‐, and l ‐alanine.     

Size‐Controlled Formation of Noble‐Metal Nanoparticles in Aqueous Solution with a Thiol‐Free Tripeptide

A combinatorial screening revealed the peptide H‐His‐d ‐Leu‐d ‐Asp‐NH₂ ( 1 ) as an additive for the generation of monodisperse, water‐soluble palladium nanoparticles with average diameters of 3 nm and stabilities of over 9 months. The tripeptide proved to be also applicable for the size‐controlled formation of other noble‐metal nanoparticles (Pt and Au). Studies with close analogues of peptide 1 revealed a specific role of each of the three amino acids for the formation and stabilization of the nanoparticles. These data combined with microscopic and spectroscopic analyses provided insight into the structure of the self‐assembled peptidic monolayer around the metal core. The results open interesting prospects for the development of functionalized metal nanoparticles.     

Synthesis and radical ring‐opening polymerization of vinylcyclopropanes derived from amino acids with hydrophobic moieties

Six 1,1‐disubstituted vinylcyclopropanes (VCP) were synthesized from glycine and amino acids bearing hydrophobic moieties, l ‐alanine, l ‐valine, l ‐leucine, l ‐isoleucine, and l ‐phenylalanine. These VCP derivatives efficiently underwent radical ring‐opening polymerization to afford the corresponding polymers bearing trans‐vinylene moiety in the main chains and the amino acid‐derived chiral moieties in the side chains. The polymers were film‐formable, and in the films of polymers bearing the glycine‐ and alanine‐derived side chains, presence of hydrogen bonding was confirmed by IR analysis. Thermogravimetric analysis of the polymers revealed that the temperatures of 5% weight loss were higher than 300 °C. Differential scanning calorimetry clarified that the polymers were amorphous ones showing glass transition temperatures in a range of 48–80 °C. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 3996–4002     

HPLC determination of acidic d‐amino acids and their N‐methyl derivatives in biological tissues

d ‐Aspartate (d ‐Asp) and N‐methyl‐d ‐aspartate (NMDA) occur in the neuroendocrine systems of vertebrates and invertebrates, where they play a role in hormone release and synthesis, neurotransmission, and memory and learning. N‐methyl‐d ‐glutamate (NMDG) has also been detected in marine bivalves. Several methods have been used to detect these amino acids, but they require pretreatment of tissue samples with o‐phthaldialdehyde (OPA) to remove primary amino acids that interfere with the detection of NMDA and NMDG. We report here a one‐step derivatization procedure with the chiral reagent N‐α‐(5‐fluoro‐2,4‐dinitrophenyl)‐(d or l )‐valine amide, FDNP‐Val‐NH₂, a close analog of Marfey's reagent but with better resolution and higher molar absorptivity. The diastereomers formed were separated by HPLC on an ODS‐Hypersil column eluted with TFA/water–TFA/MeCN. UV absorption at 340 nm permitted detection levels as low as 5–10 pmol. d ‐Asp, NMDA and NMDG peaks were not obscured by other primary or secondary amino acids; hence pretreatment of tissues with OPA was not required. This method is highly reliable and fast (less than 40 min HPLC run). Using this method, we detected d ‐Asp, NMDA and NMDG in several biological tissues (octopus brain, optical lobe and bucchal mass; foot and mantle of the mollusk Scapharca broughtonii), confirming the results of other researchers. Copyright © 2009 John Wiley & Sons, Ltd.     

A Simple and Efficient Procedure for Synthesis of Optically Active 1,2,4‐Triazolo‐[3,4‐b]‐1,3,4‐Thiadiazole Derivatives Containing l‐Amino Acid Moieties

Some new and optically active 1,2,4‐triazolo thiadiazoles bearing N‐phthaloyl‐l ‐amino acids were synthesized by reaction of 4‐amino‐5‐(3‐ or 4‐)pyridyl‐3‐mercapto‐(4H)‐1,2,4‐triazoles with N‐phthaloyl‐l ‐amino acids in the presence of phosphorus oxychloride. All the newly synthesized compounds were confirmed by IR, ¹H NMR, ¹³C NMR and elemental analysis.     

Determination of l‐tryptophan and l‐kynurenine derivatized with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole by LC‐MS/MS on a triazole‐bonded column and their quantification in human serum

The concentrations of l ‐tryptophan (Trp) and the metabolite l ‐kynurenine (KYN) can be used to evaluate the in‐vivo activity of indoleamine 2,3‐dioxygenase (IDO) and tryptophan 2,3‐dioxygenase (TDO). As such, a novel method involving derivatization of l ‐Trp and l ‐KYN with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS) and separation by high‐performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection on a triazole‐bonded column (Cosmosil HILIC®) was developed to determine their concentrations. The optimized mobile phase, CH₃CN/10 mm ammonium formate in H₂O (pH 5.0) (90:10, v/v) eluted isocratically, resulted in satisfactory separation and MS/MS detection of the analytes. The detection limits of l ‐Trp and l ‐KYN were approximately 50 and 4.0 pm , respectively. The column temperature affected the retention behaviour of the Trp and KYN derivatives, with increased column temperatures leading to increased capacity factors; positive enthalpy changes were revealed by van't Hoff plot analyses. Using the proposed LC‐MS/MS method, l ‐Trp and l ‐KYN were successfully determined in 10 μL human serum using 1‐methyl‐l ‐Trp as an internal standard. The precision and recovery of l ‐Trp were in the ranges 2.85–9.29 and 95.8–113%, respectively, while those of l ‐KYN were 2.51–16.0 and 80.8–98.2%, respectively. The proposed LC‐MS/MS method will be useful for evaluating the in vivo activity of IDO or TDO. Copyright © 2016 John Wiley & Sons, Ltd.     

A pipecolic acid (Pip)‐containing dipeptide,Boc‐d‐Ala‐l‐Pip‐NHiPr

The title dipeptide, 1‐(tert‐butoxy­carbonyl‐d ‐alanyl)‐N‐iso­propyl‐l ‐pipecol­amide or Boc‐d ‐Ala‐l ‐Pip‐NHⁱPr (H‐Pip‐OH is pipecolic acid or piperidine‐2‐carboxylic acid), C₁₇H₃₁N₃­O₄, with a d –l heterochiral sequence, adopts a type II′β‐­turn conformation, with all‐trans amide functions, where the C‐terminal amide NH group interacts with the Boc carbonyl O atom to form a classical i+3 i intramolecular hydrogen bond. The C^α substituent takes an axial position [H^α (Pip) equatorial] and the trans pipecolamide function is nearly planar.     

A Biocatalytic Cascade for Versatile One‐Pot Modification of mRNA Starting from Methionine Analogues

Methyltransferases have proven useful to install functional groups site‐specifically in different classes of biomolecules when analogues of their cosubstrate S‐adenosyl‐l ‐methionine (AdoMet) are available. Methyltransferases have been used to address different classes of RNA molecules selectively and site‐specifically, which is indispensable for biophysical and mechanistic studies as well as labeling in the complex cellular environment. However, the AdoMet analogues are not cell‐permeable, thus preventing implementation of this strategy in cells. We present a two‐step enzymatic cascade for site‐specific mRNA modification starting from stable methionine analogues. Our approach combines the enzymatic synthesis of AdoMet with modification of the 5′ cap by a specific RNA methyltransferase in one pot. We demonstrate that a substrate panel including alkene, alkyne, and azido functionalities can be used and further derivatized in different types of click reactions.     

Rethinking Cysteine Protective Groups: S‐Alkylsulfonyl‐l‐Cysteines for Chemoselective Disulfide Formation

The ability to reversibly cross‐link proteins and peptides grants the amino acid cysteine its unique role in nature as well as in peptide chemistry. We report a novel class of S‐alkylsulfonyl‐l ‐cysteines and N‐carboxy anhydrides (NCA) thereof for peptide synthesis. The S‐alkylsulfonyl group is stable against amines and thus enables its use under Fmoc chemistry conditions and the controlled polymerization of the corresponding NCAs yielding well‐defined homo‐ as well as block co‐polymers. Yet, thiols react immediately with the S‐alkylsulfonyl group forming asymmetric disulfides. Therefore, we introduce the first reactive cysteine derivative for efficient and chemoselective disulfide formation in synthetic polypeptides, thus bypassing additional protective group cleavage steps.