Maleidride biosynthesis - construction of dimeric anhydrides - more than just heads or tails.

Covering: up to early 2022Maleidrides are a family of polyketide-based dimeric natural products isolated from fungi. Many maleidrides possess significant bioactivities, making them attractive pharmaceutical or agrochemical lead compounds. Their unusual biosynthetic pathways have fascinated scientists for decades, with recent advances in our bioinformatic and enzymatic understanding providing further insights into their construction. However, many intriguing questions remain, including exactly how the enzymatic dimerisation, which creates the diverse core structure of the maleidrides, is controlled. This review will explore the literature from the initial isolation of maleidride compounds in the 1930s, through the first full structural elucidation in the 1960s, to the most recent in vivo, in vitro, and in silico analyses.

NeissLock provides an inducible protein anhydride for covalent targeting of endogenous proteins.

The Neisseria meningitidis protein FrpC contains a self-processing module (SPM) undergoing autoproteolysis via an aspartic anhydride. Herein, we establish NeissLock, using a binding protein genetically fused to SPM. Upon calcium triggering of SPM, the anhydride at the C-terminus of the binding protein allows nucleophilic attack by its target protein, ligating the complex. We establish a computational tool to search the Protein Data Bank, assessing proximity of amines to C-termini. We optimize NeissLock using the Ornithine Decarboxylase/Antizyme complex. Various sites on the target (α-amine or ε-amines) react with the anhydride, but reaction is blocked if the partner does not dock. Ligation is efficient at pH 7.0, with half-time less than 2 min. We arm Transforming Growth Factor-α with SPM, enabling specific covalent coupling to Epidermal Growth Factor Receptor at the cell-surface. NeissLock harnesses distinctive protein chemistry for high-yield covalent targeting of endogenous proteins, advancing the possibilities for molecular engineering.

A Simplified Method of Synthesis to Obtain Zwitterionic Cellulose under Mild Conditions with Active Ionic Moieties.

A simplified procedure to synthesize zwitterionic cellulose by means of N-protected aspartic anhydride under mild conditions was developed. The preparation of modified cellulose samples was carried out under heterogeneous, aqueous conditions by reacting NH4OH-activated cellulose with aspartic anhydrides N-protected with trifluoroacetyl (TFAc) and carbobenzyloxy (Cbz). Modified cellulose samples Cel-Asp-N-TFAc and Cel-Asp-N-Cbz were characterized by Fourier Transform Infrared (FTIR) and 13C solid state Nuclear Magnetic Resonance (NMR) spectroscopy. The functionalization degree of each cellulose sample was determined by the 13C NMR signal integration values corresponding to the cellulose C1 vs. the Cα of the aspartate residue and corroborated by elemental analysis. In agreement, both analytical methods averaged a grafting degree of 20% for Cel-Asp-N-TFAc and 16% for Cel-Asp-N-Cbz. Conveniently, Cel-Asp-N-TFAc was concomitantly partially N-deprotected (65%) as determined by the ninhydrin method. The zwitterion character of this sample was confirmed by a potentiometric titration curve and the availability of these amino acid residues on the cellulose was inspected by adsorption kinetics method with a 100 mg L-1 cotton blue dye solution. In addition, the synthesis reported in the present work involves environmentally related advantages over previous methodologies developed in our group concerning to zwitterionic cellulose preparation.

Mechanism of water extraction from gypsum rock by desert colonizing microorganisms.

Microorganisms, in the most hyperarid deserts around the world, inhabit the inside of rocks as a survival strategy. Water is essential for life, and the ability of a rock substrate to retain water is essential for its habitability. Here we report the mechanism by which gypsum rocks from the Atacama Desert, Chile, provide water for its colonizing microorganisms. We show that the microorganisms can extract water of crystallization (i.e., structurally ordered) from the rock, inducing a phase transformation from gypsum (CaSO4·2H2O) to anhydrite (CaSO4). To investigate and validate the water extraction and phase transformation mechanisms found in the natural geological environment, we cultivated a cyanobacterium isolate on gypsum rock samples under controlled conditions. We found that the cyanobacteria attached onto high surface energy crystal planes ({011}) of gypsum samples generate a thin biofilm that induced mineral dissolution accompanied by water extraction. This process led to a phase transformation to an anhydrous calcium sulfate, anhydrite, which was formed via reprecipitation and subsequent attachment and alignment of nanocrystals. Results in this work not only shed light on how microorganisms can obtain water under severe xeric conditions but also provide insights into potential life in even more extreme environments, such as Mars, as well as offering strategies for advanced water storage methods.

Rice Histone Propionylation and Generation of Chemically Derivatized Synthetic H3 and H4 Peptides for Identification of Acetylation Sites and Quantification.

Histone proteins are crucial in the study of chromatin dynamics owing to their wide-ranging implications in the regulation of gene expression. Modifications of histones are integral to these regulatory processes in concert with associated proteins, such as transcription factors and coactivators. One of the biochemical techniques available to enhance analysis of histone proteins is chemical derivatization using propionic anhydride. In this protocol, we describe the use of propionylation to efficiently derivatize acid-extracted histones from rice. We also synthesize H3 and H4 tryptic peptides, thus mimicking the nature of derivatized extracted peptides to aid in identification and quantification using targeted-mass spectrometry. Here we make available the masses of the precursor ions and the retention times (RT) of each synthesized peptide. These provide useful information to facilitate histone data analysis. Lastly, we note that we will distribute these synthetic peptides in nanomolar (nM) concentrations to those who wish to utilize them for assays and further experimental studies.

Enzyme-mimetic self-catalyzed polymerization of polypeptide helices.

Enzymes provide optimal three-dimensional structures for substrate binding and the subsequent accelerated reaction. Such folding-dependent catalytic behaviors, however, are seldom mechanistically explored with reduced structural complexity. Here, we demonstrate that the α-helix, a much simpler structural motif of enzyme, can facilitate its own growth through the self-catalyzed polymerization of N-carboxyanhydride (NCA) in dichloromethane. The reversible binding between the N terminus of α-helical polypeptides and NCAs promotes rate acceleration of the subsequent ring-opening reaction. A two-stage, Michaelis-Menten-type kinetic model is proposed by considering the binding and reaction between the propagating helical chains and the monomers, and is successfully utilized to predict the molecular weights and molecular-weight distributions of the resulting polymers. This work elucidates the mechanism of helix-induced, enzyme-mimetic catalysis, emphasizes the importance of solvent choice in the discovery of new reaction type, and provides a route for rapid production of well-defined synthetic polypeptides by taking advantage of self-accelerated ring-opening polymerizations.

Esterase-Catalyzed Production of Hyperpolarized 13C-Enriched Carbon Dioxide in Tissues for Measuring pH.

13C Magnetic resonance imaging of hyperpolarized (HP) 13C-enriched bicarbonate (H13CO3-) and carbon dioxide (13CO2) is a novel and sensitive technique for tissue pH mapping in vivo. Administration of the HP physiological buffer pair is attractive, but poor polarization and the short T1 of 13C-enriched inorganic bicarbonate salts are major drawbacks for this approach. Here, we report a new class of mixed anhydrides for esterase-catalyzed production of highly polarized 13CO2 and H13CO3- in tissue. A series of precursors with different alkoxy and acyl groups were synthesized and tested for chemical stability and T1. 13C-enriched ethyl acetyl carbonate (13C-EAC) was found to be the most suitable candidate due to the relatively long T1 and good chemical stability. Our results showed that 13C-EAC can be efficiently and rapidly polarized using BDPA. HP 13C-EAC was rapidly hydrolyzed by esterase to 13C-enriched monoacetyl carbonate (13C-MAC), which then decomposed to HP 13CO2. Equilibrium between the newly produced 13CO2 and H13CO3- was quickly established by carbonic anhydrase, producing a physiological buffer pair with 13C NMR signals that can be quantified for pH measurements. Finally, in vivo tissue pH measurements using HP 13C-EAC was successfully demonstrated in the liver of healthy rats. These results suggest that HP 13C-EAC is a novel imaging probe for in vivo pH measurements.

N-Carboxyanhydride Polymerization of Glycopolypeptides That Activate Antigen-Presenting Cells through Dectin-1 and Dectin-2.

The C-type lectins dectin-1 and dectin-2 contribute to innate immunity against microbial pathogens by recognizing their foreign glycan structures. These receptors are promising targets for vaccine development and cancer immunotherapy. However, currently available agonists are heterogeneous glycoconjugates and polysaccharides from natural sources. Herein, we designed and synthesized the first chemically defined ligands for dectin-1 and dectin-2. They comprised glycopolypeptides bearing mono-, di-, and trisaccharides and were built through polymerization of glycosylated N-carboxyanhydrides. Through this approach, we achieved glycopolypeptides with high molecular weights and low dispersities. We identified structures that elicit a pro-inflammatory response through dectin-1 or dectin-2 in antigen-presenting cells. With their native proteinaceous backbones and natural glycosidic linkages, these agonists are attractive for translational applications.

A Class of Reactive Acyl-CoA Species Reveals the Non-enzymatic Origins of Protein Acylation.

The mechanisms underlying the formation of acyl protein modifications remain poorly understood. By investigating the reactivity of endogenous acyl-CoA metabolites, we found a class of acyl-CoAs that undergo intramolecular catalysis to form reactive intermediates that non-enzymatically modify proteins. Based on this mechanism, we predicted, validated, and characterized a protein modification: 3-hydroxy-3-methylglutaryl(HMG)-lysine. In a model of altered HMG-CoA metabolism, we found evidence of two additional protein modifications: 3-methylglutaconyl(MGc)-lysine and 3-methylglutaryl(MG)-lysine. Using quantitative proteomics, we compared the "acylomes" of two reactive acyl-CoA species, namely HMG-CoA and glutaryl-CoA, which are generated in different pathways. We found proteins that are uniquely modified by each reactive metabolite, as well as common proteins and pathways. We identified the tricarboxylic acid cycle as a pathway commonly regulated by acylation and validated malate dehydrogenase as a key target. These data uncover a fundamental relationship between reactive acyl-CoA species and proteins and define a new regulatory paradigm in metabolism.

N-Carboxyanhydride-Mediated Fatty Acylation of Amino Acids and Peptides for Functionalization of Protocell Membranes.

Early protocells are likely to have arisen from the self-assembly of RNA, peptide, and lipid molecules that were generated and concentrated within geologically favorable environments on the early Earth. The reactivity of these components in a prebiotic environment that supplied sources of chemical energy could have produced additional species with properties favorable to the emergence of protocells. The geochemically plausible activation of amino acids by carbonyl sulfide has been shown to generate short peptides via the formation of cyclic amino acid N-carboxyanhydrides (NCAs). Here, we show that the polymerization of valine-NCA in the presence of fatty acids yields acylated amino acids and peptides via a mixed anhydride intermediate. Notably, Nα-oleoylarginine, a product of the reaction between arginine and oleic acid in the presence of valine-NCA, partitions spontaneously into vesicle membranes and mediates the association of RNA with the vesicles. Our results suggest a potential mechanism by which activated amino acids could diversify the chemical functionality of fatty acid membranes and colocalize RNA with vesicles during the formation of early protocells.

Quantitative assessment of chemical artefacts produced by propionylation of histones prior to mass spectrometry analysis.

Histone PTMs play a crucial role in regulating chromatin structure and function, with impact on gene expression. MS is nowadays widely applied to study histone PTMs systematically. Because histones are rich in arginine and lysine, classical shot-gun approaches based on trypsin digestion are typically not employed for histone modifications mapping. Instead, different protocols of chemical derivatization of lysines in combination with trypsin have been implemented to obtain "Arg-C like" digestion products that are more suitable for LC-MS/MS analysis. Although widespread, these strategies have been recently described to cause various side reactions that result in chemical modifications prone to be misinterpreted as native histone marks. These artefacts can also interfere with the quantification process, causing errors in histone PTMs profiling. The work of Paternoster V. et al. is a quantitative assessment of methyl-esterification and other side reactions occurring on histones after chemical derivatization of lysines with propionic anhydride [Proteomics 2016, 16, 2059-2063]. The authors estimate the effect of different solvents, incubation times, and pH on the extent of these side reactions. The results collected indicate that the replacement of methanol with isopropanol or ACN not only blocks methyl-esterification, but also significantly reduces other undesired unspecific reactions. Carefully titrating the pH after propionic anhydride addition is another way to keep methyl-esterification under control. Overall, the authors describe a set of experimental conditions that allow reducing the generation of various artefacts during histone propionylation.

Impact of dual-enzyme treatment on the octenylsuccinic anhydride esterification of soluble starch nanoparticle.

The hypothesis of improving the esterification of sugary maize soluble starch through dual-enzyme pretreatment was investigated. Native starch nanoparticle (NSP) was enzymatically pretreated using ß-amylase and transglucosidase (ESP) and then esterified with octenylsuccinic anhydride (OSA). The degree of substitution (DS), reaction efficiency (RE), molecular weight (Mw), molecular density (ρ) and in vitro digestibility were determined. Fourier transform infrared spectroscopy and confocal laser scanning microscopy were used to analyze starch particle and its OS derivatives. The emulsification properties of OS-NSP and OS-ESP were also compared. The results showed that dual-enzyme modification increased the DS and RE of OSA modified starch particle compared with the control. Enzymatic modification had a thinning effect at the surface of starch particle, resulting in lower Mw. The extent of reduction in ρ of OS-ESP was greater than that of OS-NSP. At equivalent DS, OSA modification of EPS was more effective than that of NPS in reducing digestibility. Also, there was brighter fluorescence spheres of OS-ESP in comparison to OS-NSP at equivalent DS, suggesting more OS groups were substituted on the chains near the branch points at less density areas. OS-ESP with higher DS (0.0197) had lower zeta-potential and average particle size for superior emulsion stabilization properties with high stability. The results revealed the OS-starch prepared under dual-enzyme pretreatment was a Pickering particle stabilizer for potential application in encapsulation and delivery of bioactive components.

Tackling aspecific side reactions during histone propionylation: The promise of reversing overpropionylation.

Histone proteins are essential elements for DNA packaging. Moreover, the PTMs that are extremely abundant on these proteins, contribute in modeling chromatin structure and recruiting enzymes involved in gene regulation, DNA repair and chromosome condensation. This fundamental aspect, together with the epigenetic inheritance of histone PTMs, underlines the importance of having biochemical techniques for their characterization. Over the past two decades, significant improvements in mass accuracy and resolution of mass spectrometers have made LC-coupled MS the strategy of choice for accurate identification and quantification of protein PTMs. Nevertheless, in previous work we disclosed the limitations and biases of the most widely adopted sample preparation protocols for histone propionylation, required prior to bottom-up MS analysis. In this work, however, we put forward a new specific and efficient propionylation strategy by means of propionic anhydride. In this method, aspecific overpropionylation at serine (S), threonine (T) and tyrosine (Y) is reversed by adding hydroxylamine (HA). We recommend using this method for future analysis of histones through bottom-up MS.

Quantitative assessment of methyl-esterification and other side reactions in a standard propionylation protocol for detection of histone modifications.

Histone modifications play an important role in regulating chromatin stability and gene expression, but to date, investigating them remains challenging. In order to obtain peptides suitable for MS-based analysis, chemical derivatization of N-terminus and lysine residues by propionic anhydride is commonly performed. Several side reactions (methyl-esterification, amidation, solvolysis, overpropionylation, and missed propionylation) during propionylation protocols have been described, yet their relative abundances remain vague. Because methyl-esterification could interfere with correct interpretation of the modification pattern, it is essential to take measures to avoid it. Here we present in-depth quantitative analyses of methyl-esterification and the other side reactions in a standard propionylation protocol containing methanol, and when replacing methanol with isopropanol or acetonitrile. We show that the use of alternative solvents can eliminate methyl-esterification and that even though other side reactions are not prevented, their contribution can be kept relatively small. We also show that replacing methanol can be of importance also in other proteomics methods, such as mixed cation exchange, using methanol under acidic conditions.

Multifunctional novel Diallyl disulfide (DADS) derivatives with ß-amyloid-reducing, cholinergic, antioxidant and metal chelating properties for the treatment of Alzheimer's disease.

A series of novel Diallyl disulfide (DADS) derivatives were designed, synthesized and evaluated as chemical agents, which target and modulate multiple facets of Alzheimer's disease (AD). The results showed that the target compounds 5a-l and 7e-m exhibited significant anti-Aß aggregation activity, considerable acetylcholinesterase (AChE) inhibition, high selectivity towards AChE over butyrylcholinesterase (BuChE), potential antioxidant and metal chelating activities. Specifically, compounds 7k and 7l exhibited highest potency towards self-induced Aß aggregation (74% and 71.4%, 25 µM) and metal chelating ability. Furthermore, compounds 7k and 7l disaggregated Aß fibrils generated by Cu(2+)-induced Aß aggregation by 80.9% and 78.5%, later confirmed by transmission electron microscope (TEM) analysis. Besides, 7k and 7l had the strongest AChE inhibitory activity with IC50 values of 0.056 µM and 0.121 µM, respectively. Furthermore, molecular modelling studies showed that these compounds were capable of binding simultaneously to catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. All the target compounds displayed moderate to excellent antioxidant activity with ORAC-FL values in the range 0.546-5.86Trolox equivalents. In addition, absorption, distribution, metabolism and excretion (ADME) profile and toxicity prediction (TOPKAT) of best compounds 7k and 7l revealed that they have drug like properties and possess very low toxic effects. Collectively, the results strongly support our assertion that these compounds could provide good templates for developing new multifunctional agents for AD treatment.

Pyridoxylamine reactivity kinetics as an amine based nucleophile for screening electrophilic dermal sensitizers.

Chemical allergens bind directly, or after metabolic or abiotic activation, to endogenous proteins to become allergenic. Assessment of this initial binding has been suggested as a target for development of assays to screen chemicals for their allergenic potential. Recently we reported a nitrobenzenethiol (NBT) based method for screening thiol reactive skin sensitizers, however, amine selective sensitizers are not detected by this assay. In the present study we describe an amine (pyridoxylamine (PDA)) based kinetic assay to complement the NBT assay for identification of amine-selective and non-selective skin sensitizers. UV-Vis spectrophotometry and fluorescence were used to measure PDA reactivity for 57 chemicals including anhydrides, aldehydes, and quinones where reaction rates ranged from 116 to 6.2 × 10(-6) M(-1) s(-1) for extreme to weak sensitizers, respectively. No reactivity towards PDA was observed with the thiol-selective sensitizers, non-sensitizers and prohaptens. The PDA rate constants correlated significantly with their respective murine local lymph node assay (LLNA) threshold EC3 values (R(2) = 0.76). The use of PDA serves as a simple, inexpensive amine based method that shows promise as a preliminary screening tool for electrophilic, amine-selective skin sensitizers.

Synthesis and in vitro biocompatibility assessment of a poly(amic acid) derived from ethylenediaminetetraacetic dianhydride.

Poly(amic acid) (PAA) derived from ethylenediaminetetracetic dianhydride shows great potential as a biomaterial suitable for biomedical applications. To evaluate this polymer class further, in vitro cell toxicity (WST-1/ECS, ELISA based) and cell compatibility (cell adhesion and cell proliferation) tests were conducted to establish structure-toxicity relationships. PAAs with a number-average molecular weight ranging between 100 to 200 kg/mol were synthesized at 37°C after 24 h. Porcine radial artery cells (RACs) and descending aorta endothelial cells (ECs) were seeded independently in a 96-well cell culture plate at a cell density of 5000 cells/cm(2) to observe toxic effects. Similarly, RACs and ECs were seeded independently onto PAA coated and uncoated cover slips at a cell density of 7000 cells/cm(2) to observe growth patterns. Our results showed no toxicity after 96 h of incubation and in addition, both RACs and ECs adhered and proliferated on the PAA films, preserving their phenotype during this time. The tested synthetic material seems promising as a future biomaterial and should elicit a desired cellular response upon implantation.

Poly(ester anhydride)/mPEG amphiphilic block co-polymer nanoparticles as delivery devices for paclitaxel.

This work focused on the preparation and characterization of a novel amphiphilic block co-polymer and paclitaxel-loaded co-polymer nanoparticles (NPs) and in vitro evaluation of the release of paclitaxel and cytotoxicity of NPs. mPEG-b-P(OA-DLLA)-b-mPEG was prepared via melt polycondensation of methoxy poly(ethylene glycol) (mPEG), octadecanedioic acid (OA) and D,L-lactic acid (DLLA) and characterized by FT-IR, (1)H-NMR, (13)C-NMR, GPC, DSC and XRD. The paclitaxel-loaded mPEG-b-P(OA-DLLA)-b-mPEG NPs were prepared by nanoprecipitation and then characterized by LPSA, TEM and (1)H-NMR. In vitro release behaviors of the paclitaxel-loaded NPs were investigated by HPLC. In vitro cytotoxicity of NPs was evaluated by MTT assay with normal mouse lung fibroblast cells (L929) as model cells. The composition of mPEG-b-P(OA-DLLA)-b-mPEG is consistent with that of the designed co-polymer. The paclitaxel-loaded NPs are of spherical shape with core/shell structure and size smaller than 300 nm. Paclitaxel can be continuously released from the paclitaxel-loaded NPs and the in vitro release rate of paclitaxel decreases with increasing the content of the P(OA-DLLA) segments in the co-polymer. The mPEG-b-P(OA-DLLA)-b-mPEG NPs are non-toxic to L929. The results suggest that mPEG-b-P(OA-DLLA)-b-mPEG NPs are a potential candidate carrier material for the controlled delivery of paclitaxel and other hydrophobic compounds.

Elucidation of the active conformation of cinchona alkaloid catalyst and chemical mechanism of alcoholysis of meso anhydrides.

Complementary to enantioselective transformations of planar functionalities, catalytic desymmetrization of meso compounds is another fundamentally important strategy for asymmetric synthesis. However, experimentally established stereochemical models on how a chiral catalyst discriminates between two enantiotopic functional groups in the desymmetrization of a meso substrate are particularly lacking. This article describes our endeavor to elucidate the chemical mechanism and characterization of the active conformation of the cinchona alkaloid-derived catalyst for a desymmetrization of meso cyclic anhydrides via asymmetric alcoholysis. First, our kinetic studies indicate that the cinchona alkaloid-catalyzed alcoholysis proceeds by a general base catalysis mechanism. Furthermore, the active conformer of the cinchona alkaloid-derived catalyst DHQD-PHN was clarified by catalyst conformation studies with a designed, rigid cinchona alkaloid derivative as a probe. These key mechanistic insights enabled us to construct a stereochemical model to rationalize how DHQD-PHN differentiates the two enantiotopic carbonyl groups in the transition state of the asymmetric alcoholysis of meso cyclic anhydrides. This model not only is consistent with the sense of asymmetric induction of the asymmetric alcoholysis but also provides a rationale on how the catalyst tolerates a broad range of cyclic anhydrides. These mechanistic insights further guided us to develop a novel practical catalyst for the enantioselective alcoholysis of meso cyclic anhydrides.

Immobilization of modified papain with anhydride groups on activated cotton fabric.

Papain (EC 3.4.22.2) has been chemically modified using two novel reagents including different anhydrides of 1,2,4-benzenetricarboxylic and pyromellitic acids. Then, the modified papain was immobilized on the activated cotton fabric by a two-step method. The number of free amino groups in the modified protein was investigated through the 2,4,6-trinitrobenzenesulfonic acid method. Energy dispersive spectrometer was used to characterize papain immobilization. Some parameters of both modified and native papain immobilized on cotton fabric, such as optimum temperature, optimum pH, and the stabilities for reservation in various detergents were studied and compared. The resultant papain had its optimum pH shifted from 6.0 to 9.0. Compared with immobilized native papain, the thermal stability and the resistance to alkali and washing detergent of immobilized modified enzyme were improved considerably. When the concentration of detergent was 20 mg/ml, the activity of the immobilized pyromellitic papain retained about 40% of its original activity, whereas the native papain was almost inhibited. This work demonstrated that the cotton fabric immobilized modified papain has potential applications in the functional textiles field.