Fully synthetic, tunable poly(α-amino acids) as the base of bioinks curable by visible light.

Bioinks play a crucial role in tissue engineering, influencing mechanical and chemical properties of the printed scaffold as well as the behavior of encapsulated cells. Recently, there has been a shift from animal origin materials to their synthetic alternatives. In this context, we present here bioinks based on fully synthetic and biodegradable poly(α,L-amino acids) (PolyAA) as an alternative to animal-based gelatin methacrylate (Gel-Ma) bioinks. Additionally, we first reported the possibility of the visible light photoinitiated incorporation of the bifunctional cell adhesive RGD peptide into the PolyAA hydrogel matrix. The obtained hydrogels are shown to be cytocompatible, and their mechanical properties closely resemble those of gelatin methacrylate-based scaffolds. Moreover, combining the unique properties of PolyAA-based bioinks, the photocrosslinking strategy, and the use of droplet-based printing allows the printing of constructs with high shape fidelity and structural integrity from low-viscosity bioinks without using any sacrificial components. Overall, presented PolyAA-based materials are a promising and versatile toolbox that extends the range of bioinks for droplet bioprinting.

Design, synthesis, and evaluation of novel arecoline-linked amino acid derivatives for insecticidal and antifungal activities.

A series of arecoline derivatives with amino acid moieties were designed and synthesised using an acylamide condensation strategy, taking arecoline as the foundational structure. The insecticidal efficacy of these compounds against Aphis craccivora and Tetranychus cinnabarinus was evaluated. Notably, derivatives 3h and 3i demonstrated superior insecticidal activity compared with arecoline. Additionally, 3h and 3i showed good fungicidal effectiveness against two types of plant fungi. Moreover, molecular docking analyses suggested that 3h and 3i could affect the nervous systems of A. craccivora and T. cinnabarinus by binding to neuronal nicotinic acetylcholine receptors. These findings suggest that compounds 3h and 3i represent promising leads for further development in insecticide and fungicide research.

Pyramidalization of the Carboxamide sp2-Center in Peptide Structures.

A CSD search in the Cambridge Crystallographic Database for the substructure N-CαH-C'(═O)-N gave 24,180 peptide structures for analysis of the pyramidalization of the sp2-hybridized carboxamide group C'(═O)NCα, which had not been investigated before. The dependence of the pyramidalization θ = O-N-C'-Cα on the rotation angle ψ = O═C'-Cα-N about bond C'-Cα resulted in a curve with three maxima, three minima, and six zero-crossings. Surprisingly, the ψ/θ analysis of the individual amino acid building blocks showed that all of them exhibited similar curves, irrespective of their different R substituents. This unusual behavior is explained by a 3-fold short-range potential set up by the three covalent bonds, emanating from Cα. The tie-up of the rotation angle ψ and the pyramidalization θ in a rigid coupling is remarkable. In the 24,180 peptide structures, subjected to X-ray crystallography, there is no dynamics. For peptides in solution, the rotation/pyramidalization curve ψ/θav determines the degree of pyramidalization θ, when the rotation angle ψ runs through a full 360° circle. Density functional theory (DFT) calculations of alaninamide supported the analysis.

Natural Amino Acid-Bearing Carbamate Prodrugs of Daidzein Increase Water Solubility and Improve Phase II Metabolic Stability for Enhanced Oral Bioavailability.

Daidzein (DAN) is an isoflavone, and it is often found in its natural form in soybean and food supplements. DAN has poor bioavailability owing to its extremely low water solubility and first-pass metabolism. Herein, we hypothesized that a bioactivatable natural amino acid-bearing carbamate prodrug strategy could increase the water solubility and metabolic stability of DAN. To test our hypothesis, nine amino acid prodrugs of DAN were designed and synthesized. Compared with DAN, the optimal prodrug (daidzein-4'-O-CO-N-isoleucine, D-4'-I) demonstrated enhanced water solubility and improved phase II metabolic stability and activation to DAN in plasma. In addition, unlike the passive transport of DAN, D-4'-I maintained high permeability via organic anion-transporting polypeptide 2B1 (OATP2B1)-mediated transport. Importantly, D-4'-I increased the oral bioavailability by 15.5-fold, reduced the gender difference, and extended the linear absorption capacity in the pharmacokinetics of DAN in rats. Furthermore, D-4'-I exhibited dose-dependent protection against liver injury. Thus, the natural amino acid-bearing carbamate prodrug strategy shows potential in increasing water solubility and improving phase II metabolic stability to enhance the oral bioavailability of DAN.

Planting a chemical flag on antigens.

Next-generation live vaccines are created by autonomous production of nitrated antigens.

Preparation of amino acid chiral ionic liquid and visual chiral recognition of glutamine and phenylalanine enantiomers.

In this paper, the amino acid chiral ionic liquid (AACIL) was prepared with L-phenylalanine and imidazole. It was characterized by CD, FT-IR, 1H NMR, and 13C NMR spectrum. The chiral recognition sensor was constructed with AACIL and Cu(II), which exhibited different chiral visual responses (solubility or color difference) to the enantiomers of glutamine (Gln) and phenylalanine (Phe). The effects of solvent, pH, time, temperature, metal ions, and other amino acids on visual chiral recognition were optimized. The minimum concentrations of Gln and Phe for visual chiral recognition were 0.20 mg/ml and 0.28 mg/ml, respectively. The mechanism of chiral recognition was investigated by FT-IR, TEM, SEM, TG, XPS, and CD. The location of the host-guest inclusion or molecular placement has been conformationally searched based on Gaussian 09 software.

Preparation, assay, and application of 4-fluorothreonine transaldolase from Streptomyces sp. MA37 for ß-hydroxyl amino acid derivatives.

ß-Hydroxy-α-amino acids (ßHAAs) are an essential class of building blocks of therapeutically important compounds and complex natural products. They contain two chiral centers at Cα and Cß positions, resulting in four possible diastereoisomers. Many innovative asymmetric syntheses have been developed to access structurally diverse ßHAAs. The main challenge, however, is the control of the relative and absolute stereochemistry of the asymmetric carbons in a sustainable way. In this respect, there has been considerable attention focused on the chemoenzymatic synthesis of ßHAAs via a one-step process. Nature has evolved different enzymatic routes to produce these valuable ßHAAs. Among these naturally occurring transformations, L-threonine transaldolases present potential biocatalysts to generate ßHAAs in situ. 4-Fluorothreonine transaldolase from Streptomyces sp. MA37 (FTaseMA) catalyzes the cross-over transaldolation reaction between L-Thr and fluoroacetaldehyde to give 4-fluorothreonine and acetaldehyde (Ad). It has been demonstrated that FTaseMA displays considerable substrate plasticity toward structurally diverse aldehyde acceptors, leading to the production of various ßHAAs. In this chapter, we describe methods for the preparation of FTaseMA, and the chemoenzymatic synthesis of ßHAAs from various aldehydes and L-Thr using FTaseMA.

Synthesis of fluorinated amino acids by low-specificity, promiscuous aldolases coupled to in situ fluorodonor generation.

Fluorine (F) is an important element in the synthesis of molecules broadly used in medicine, agriculture, and materials. F addition to organic structures represents a unique strategy for tuning molecular properties, yet this atom is rarely found in Nature and approaches to produce fluorometabolites (such as fluorinated amino acids, key building blocks for synthesis) are relatively scarce. This chapter discusses the use of L-threonine aldolase enzymes (LTAs), a class of enzymes that catalyze reversible aldol addition to the α-carbon of glycine. The C-C bond formation ability of LTAs, together with their known substrate promiscuity, make them ideal for in vitro F biocatalysis. Here, we describe protocols to harness the activity of the low-specificity LTAs isolated from Escherichia coli and Pseudomonas putida on 2-fluoroacetaldehyde to efficiently synthesize 4-fluoro-L-threonine in vitro. This chapter also provides a comprehensive account of experimental protocols to implement these activities in vivo. These methods are illustrative and can be adapted to produce other fluorometabolites of interest.

Rapid room-temperature phosphorescence chiral recognition of natural amino acids.

Chiral recognition of amino acids is very important in both chemical and life sciences. Although chiral recognition with luminescence has many advantages such as being inexpensive, it is usually slow and lacks generality as the recognition module relies on structural complementarity. Here, we show that one single molecular-solid sensor, L-phenylalanine derived benzamide, can manifest the structural difference between the natural, left-handed amino acid and its right-handed counterpart via the difference of room-temperature phosphorescence (RTP) irrespective of the specific chemical structure. To realize rapid and reliable sensing, the doped samples are obtained as nanocrystals from evaporation of the tetrahydrofuran solutions, which allows for efficient triplet-triplet energy transfer to the chiral analytes generated in situ from chiral amino acids. The results show that L-analytes induce strong RTP, whereas the unnatural D-analytes produce barely any afterglow. The method expands the scope of luminescence chiral sensing by lessening the requirement for specific molecular structures.

Structural bioinformatics studies of glutamate transporters and their AlphaFold2 predicted water-soluble QTY variants and uncovering the natural mutations of L->Q, I->T, F->Y and Q->L, T->I and Y->F.

Glutamate transporters play key roles in nervous physiology by modulating excitatory neurotransmitter levels, when malfunctioning, involving in a wide range of neurological and physiological disorders. However, integral transmembrane proteins including the glutamate transporters remain notoriously difficult to study, due to their localization within the cell membrane. Here we present the structural bioinformatics studies of glutamate transporters and their water-soluble variants generated through QTY-code, a protein design strategy based on systematic amino acid substitutions. These include 2 structures determined by X-ray crystallography, cryo-EM, and 6 predicted by AlphaFold2, and their predicted water-soluble QTY variants. In the native structures of glutamate transporters, transmembrane helices contain hydrophobic amino acids such as leucine (L), isoleucine (I), and phenylalanine (F). To design water-soluble variants, these hydrophobic amino acids are systematically replaced by hydrophilic amino acids, namely glutamine (Q), threonine (T) and tyrosine (Y). The QTY variants exhibited water-solubility, with four having identical isoelectric focusing points (pI) and the other four having very similar pI. We present the superposed structures of the native glutamate transporters and their water-soluble QTY variants. The superposed structures displayed remarkable similarity with RMSD 0.528Å-2.456Å, despite significant protein transmembrane sequence differences (41.1%->53.8%). Additionally, we examined the differences of hydrophobicity patches between the native glutamate transporters and their QTY variants. Upon closer inspection, we discovered multiple natural variations of L->Q, I->T, F->Y and Q->L, T->I, Y->F in these transporters. Some of these natural variations were benign and the remaining were reported in specific neurological disorders. We further investigated the characteristics of hydrophobic to hydrophilic substitutions in glutamate transporters, utilizing variant analysis and evolutionary profiling. Our structural bioinformatics studies not only provided insight into the differences between the hydrophobic helices and hydrophilic helices in the glutamate transporters, but they are also expected to stimulate further study of other water-soluble transmembrane proteins.

Expansive discovery of chemically diverse structured macrocyclic oligoamides.

Small macrocycles with four or fewer amino acids are among the most potent natural products known, but there is currently no way to systematically generate such compounds. We describe a computational method for identifying ordered macrocycles composed of alpha, beta, gamma, and 17 other amino acid backbone chemistries, which we used to predict 14.9 million closed cycles composed of >42,000 monomer combinations. We chemically synthesized 18 macrocycles predicted to adopt single low-energy states and determined their x-ray or nuclear magnetic resonance structures; 15 of these were very close to the design models. We illustrate the therapeutic potential of these macrocycle designs by developing selective inhibitors of three protein targets of current interest. By opening up a vast space of readily synthesizable drug-like macrocycles, our results should considerably enhance structure-based drug design.

Practical Approaches to Genetic Code Expansion with Aminoacyl-tRNA Synthetase/tRNA Pairs.

Genetic code expansion (GCE) can enable the site-selective incorporation of non-canonical amino acids (ncAAs) into proteins. GCE has advanced tremendously in the last decade and can be used to create biorthogonal handles, monitor and control proteins inside cells, study post-translational modifications, and engineer new protein functions. Since establishing our laboratory, our research has focused on applications of GCE in protein and enzyme engineering using aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs. This topic has been reviewed extensively, leaving little doubt that GCE is a powerful tool for engineering proteins and enzymes. Therefore, for this young faculty issue, we wanted to provide a more technical look into the methods we use and the challenges we think about in our laboratory. Since starting the laboratory, we have successfully engineered over a dozen novel aaRS/tRNA pairs tailored for various GCE applications. However, we acknowledge that the field can pose challenges even for experts. Thus, herein, we provide a review of methodologies in ncAA incorporation with some practical commentary and a focus on challenges, emerging solutions, and exciting developments.

Genome-Wide Screen for Enhanced Noncanonical Amino Acid Incorporation in Yeast.

Expanding the genetic code beyond the 20 canonical amino acids enables access to a wide range of chemical functionality that is inaccessible within conventionally biosynthesized proteins. The vast majority of efforts to expand the genetic code have focused on the orthogonal translation systems required to achieve the genetically encoded addition of noncanonical amino acids (ncAAs) into proteins. There remain tremendous opportunities for identifying genetic and genomic factors that enhance ncAA incorporation. Here we describe genome-wide screening strategies to identify factors that enable more efficient addition of ncAAs to biosynthesized proteins. These unbiased screens can reveal previously unknown genes or mutations that can enhance ncAA incorporation and deepen our understanding of the translation apparatus.

Role of Protonation States in the Stability of Molecular Dynamics Simulations of High-Resolution Membrane Protein Structures.

Classical molecular dynamics (MD) simulations provide unmatched spatial and time resolution of protein structure and function. However, the accuracy of MD simulations often depends on the quality of force field parameters and the time scale of sampling. Another limitation of conventional MD simulations is that the protonation states of titratable amino acid residues remain fixed during simulations, even though protonation state changes coupled to conformational dynamics are central to protein function. Due to the uncertainty in selecting protonation states, classical MD simulations are sometimes performed with all amino acids modeled in their standard charged states at pH 7. Here, we performed and analyzed classical MD simulations on high-resolution cryo-EM structures of two large membrane proteins that transfer protons by catalyzing protonation/deprotonation reactions. In simulations performed with titratable amino acids modeled in their standard protonation (charged) states, the structure diverges far from its starting conformation. In comparison, MD simulations performed with predetermined protonation states of amino acid residues reproduce the structural conformation, protein hydration, and protein-water and protein-protein interactions of the structure much better. The results support the notion that it is crucial to perform basic protonation state calculations, especially on structures where protonation changes play an important functional role, prior to the launch of any conventional MD simulations. Furthermore, the combined approach of fast protonation state prediction and MD simulations can provide valuable information about the charge states of amino acids in the cryo-EM sample. Even though accurate prediction of protonation states in proteinaceous environments currently remains a challenge, we introduce an approach of combining pKa prediction with cryo-EM density map analysis that helps in improving not only the protonation state predictions but also the atomic modeling of density data.

Nontargeted Identification of D-Amino Acid-Containing Peptides Through Enzymatic Screening, Chiral Amino Acid Analysis, and LC-MS.

D-amino acid-containing peptides (DAACPs) in animals are a class of bioactive molecules formed via the posttranslational modification of peptides consisting of all-L-amino acid residues. Amino acid residue isomerization greatly impacts the function of the resulting DAACP. However, because isomerization does not change the peptide's mass, this modification is difficult to detect by most mass spectrometry-based peptidomic approaches. Here we describe a method for the identification of DAACPs that can be used to systematically survey peptides extracted from a tissue sample in a nontargeted manner.

Tuning Supramolecular Chirality in Iodinated Amphiphilic Peptides Through Tripeptide Linker Editing.

Protease-cleavable supramolecular oligopeptide nanofilaments are promising materials for targeted therapeutics and diagnostics. In these systems, single amino acid substitutions can have profound effects on the supramolecular structure and consequent proteolytic degradation, which are critical parameters for their intended applications. Herein, we describe changes to the self-assembly and proteolytic cleavage of iodine containing sequences for future translation into matrix metalloprotease (MMP-9)-activated supramolecular radio-imaging probes. We use a systematic single amino acid exchange in the tripeptide linker region of these peptide amphiphiles to provide insights into the role of each residue in the supramolecular assemblies. These modifications resulted in dramatic changes in the nature of the assembled structures formed, including an unexpected chiral inversion. By using circular dichroism, atomic force microscopy, Fourier transform infrared spectroscopy, and molecular dynamics simulations, we found that the GD loop, a common motif in ß-turn elements, induced a reversal of the chiral orientation of the assembled nanofibers. In addition to the impact on peptide packing and chirality, MMP-9-catalyzed hydrolysis was evaluated for the four peptides, with the ß-sheet content found to be a stronger determinant of enzymatic hydrolysis than supramolecular chirality. These observations provide fundamental insights into the sequence design in protease cleavable amphiphilic peptides with the potential for radio-labeling and selective biomedical applications.

Selective Hydrolysis of Ovalbumin by Zr-Based Lacunary Polyoxotungstate in Surfactant Solutions.

This study aims to design an artificial metalloprotease based on a Zr-containing polyoxometalate Na8[Zr(W5O18)2] [Zr(W5)2] for the hydrolysis of ovalbumin (OVA) in the presence of different surfactants, which can be used in many areas of the biological and medical sciences, particularly for targeted proteolytic drug design. For this reason, parameters, including the free energy of binding, the chemical nature of amino acid residues, secondary structures, and electrostatic potentials, of Zr(W5)2-OVA and Zr(W5)2-OVA-surfactant were analyzed by molecular docking simulations. The investigations showed that the presence of surfactants decreases the binding affinity of Zr(W5)2 for OVA amino acids, and hydrogen bonds and van der Waals interactions are formed between Zr(W5)2 and OVA amino acids. Additionally, GROMACS further illustrated the significance of SDS and CTAB surfactants in influencing the conformational changes of the OVA that lead to selective protein hydrolysis. In agreement with molecular dynamics simulation results, the experimental analysis showed more protein hydrolysis for the Zr(W5)2-OVA-surfactant systems. For instance, circular dichroism spectroscopy indicated that Zr(W5)2-OVA-CTAB and Zr(W5)2-OVA-TX-100 were more hydrolytically efficient due to the increased level of ß-structures rather than α-chains, which showed that surfactants can facilitate the accessibility of Zr(W5)2 to the cleavage sites by inducing partial unfolding of the OVA structure.

Enhancing osteoblast proliferation and bone regeneration by poly (amino acid)/selenium-doped hydroxyapatite.

Among various biomaterials employed for bone repair, composites with good biocompatibility and osteogenic ability had received increasing attention from biomedical applications. In this study, we doped selenium (Se) into hydroxyapatite (Se-HA) by the precipitation method, and prepared different amounts of Se-HA-loaded poly (amino acid)/Se-HA (PAA/Se-HA) composites (0, 10 wt%, 20 wt%, 30 wt%) byin-situmelting polycondensation. The physical and chemical properties of PAA/Se-HA composites were characterized by x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and their mechanical properties. XRD and FT-IR results showed that PAA/Se-HA composites contained characteristic peaks of PAA and Se-HA with amide linkage and HA structures. DSC and TGA results specified the PAA/Se-HA30 composite crystallization, melting, and maximum weight loss temperatures at 203.33 °C, 162.54 °C, and 468.92 °C, respectively, which implied good thermal stability. SEM results showed that Se-HA was uniformly dispersed in PAA. The mechanical properties of PAA/Se-HA30 composites included bending, compressive, and yield strengths at 83.07 ± 0.57, 106.56 ± 0.46, and 99.17 ± 1.11 MPa, respectively. The cellular responses of PAA/Se-HA compositesin vitrowere studied using bone marrow mesenchymal stem cells (BMSCs) by cell counting kit-8 assay, and results showed that PAA/Se-HA30 composites significantly promoted the proliferation of BMSCs at the concentration of 2 mg ml-1. The alkaline phosphatase activity (ALP) and alizarin red staining results showed that the introduction of Se-HA into PAA enhanced ALP activity and formation of calcium nodule. Western blotting and Real-time polymerase chain reaction results showed that the introduction of Se-HA into PAA could promoted the expression of osteogenic-related proteins and mRNA (integrin-binding sialoprotein, osteopontin, runt-related transcription factor 2 and Osterix) in BMSCs. A muscle defect at the back and a bone defect at the femoral condyle of New Zealand white rabbits were introduced for evaluating the enhancement of bone regeneration of PAA and PAA/Se-HA30 composites. The implantation of muscle tissue revealed good biocompatibility of PAA and PAA/Se-HA30 composites. The implantation of bone defect showed that PAA/Se-HA30 composites enhanced bone formation at the defect site (8 weeks), exhibiting good bone conductivity. Therefore, the PAA-based composite was a promising candidate material for bone tissue regeneration.

Efficient and economic protein labeling for NMR in mammalian expression systems: Application to a preT-cell and T-cell receptor protein.

Protein nuclear magnetic resonance (NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium-substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli-based expression systems. One such class of proteins includes T-cell receptors and their related preT-cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the ß subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid-specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 µM from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.

General Synthesis of Conformationally Constrained Noncanonical Amino Acids with C(sp3)-Rich Benzene Bioisosteres.

Recent years have seen novel modalities emerge for the treatment of human diseases resulting in an increase in beyond rule of 5 (bRo5) chemical matter. As a result, synthetic innovations aiming to enable rapid access to complex bRo5 molecular entities have become increasingly valuable for medicinal chemists' toolkits. Herein, we report the general synthesis of a new class of noncanonical amino acids (ncAA) with a cyclopropyl backbone to achieve conformational constraint and bearing C(sp3)-rich benzene bioisosteres. We also demonstrate preliminary studies toward utilities of these ncAA as building blocks for medicinal chemistry research.