Protein phosphorylation database and prediction tools.

Protein phosphorylation, one of the main protein post-translational modifications, is required for regulating various life activities. Kinases and phosphatases that regulate protein phosphorylation in humans have been targeted to treat various diseases, particularly cancer. High-throughput experimental methods to discover protein phosphosites are laborious and time-consuming. The burgeoning databases and predictors provide essential infrastructure to the research community. To date, >60 publicly available phosphorylation databases and predictors each have been developed. In this review, we have comprehensively summarized the status and applicability of major online phosphorylation databases and predictors, thereby helping researchers rapidly select tools that are most suitable for their projects. Moreover, the organizational strategies and limitations of these databases and predictors have been highlighted, which may facilitate the development of better protein phosphorylation predictors in silico.

Comprehensive Urinary Proteome Profiling Analysis Identifies Diagnosis and Relapse Surveillance Biomarkers for Bladder Cancer.

Bladder cancer (BCa) is the predominant malignancy of the urinary system. Herein, a comprehensive urine proteomic feature was initially established for the noninvasive diagnosis and recurrence monitoring of bladder cancer. 279 cases (63 primary BCa, 87 nontumor controls (NT), 73 relapsed BCa (BCR), and 56 nonrelapsed BCa (BCNR)) were collected to screen urinary protein biomarkers. 4761 and 3668 proteins were qualified and quantified by DDA and sequential window acquisition of all theoretical mass spectra (SWATH-MS) analysis in two discovery sets, respectively. Upregulated proteins were validated by multiple reaction monitoring (MRM) in two independent combined sets. Using the multi-support vector machine-recursive feature elimination (mSVM-RFE) algorithm, a model comprising 13 proteins exhibited good performance between BCa and NT with an AUC of 0.821 (95% CI: 0.675-0.967), 90.9% sensitivity (95% CI: 72.7-100%), and 73.3% specificity (95% CI: 53.3-93.3%) in the diagnosis test set. Meanwhile, an 11-marker classifier significantly distinguished BCR from BCNR with 75.0% sensitivity (95% CI: 50.0-100%), 81.8% specificity (95% CI: 54.5-100%), and an AUC of 0.784 (95% CI: 0.609-0.959) in the test cohort for relapse surveillance. Notably, six proteins (SPR, AK1, CD2AP, ADGRF1, GMPS, and C8A) of 24 markers were newly reported. This paper reveals novel urinary protein biomarkers for BCa and offers new theoretical insights into the pathogenesis of bladder cancer (data identifier PXD044896).

6-Plex Tandem Phosphorus Tags (TPT) for Accurate Quantitative Proteomics.

Isobaric chemical labeling is a widely used strategy for high-throughput quantitative proteomics based on mass spectrometry. However, commercially available reagents have high costs in applications as well as the sensitivity limitations for detection of the trace protein samples. Previously, we developed a 2-plex isobaric labeling strategy based on phosphorus chemistry for ultrasensitive proteome quantification with high accuracy. In this work, 6-plex tandem phosphorus tags (TPT) were developed with 3-fold increase in the multiplexing quantitative capacity compared to the 2-plex isobaric phosphorus reagents introduced previously. High isotope enrichment of 18O labeling was incorporated into the phosphoryl group with three exchangeable oxygen atoms by using commercially available H218O. The combinational incorporations of 18O atom in reporter ions and balance group set up the low-cost foundation for development of multiplex TPT reagents. The novel 6-plex TPT reagents could produce phosphoramidate as unique reporter ions with approximately 1 Da mass difference and thus enable 6-plex quantitative analysis in high-resolution ESI-MS/MS analysis. Using HeLa cell tryptic peptides, we concluded that 6-plex TPT reagents could facilitate large-scale accurate quantitative proteomics with very high labeling efficiency.

NaOAc-Assisted Aerobic Oxidation Protocol for the Synthesis of Pentacoordinate Chalcogenyl Spirophosphoranes with P-Se/P-S Bonds under Open Air.

The NaOAc-assisted aerobic oxidation reaction of pentacoordinate hydrospirophosphoranes and dichalcogenyl compounds with open air as a green oxidant has been developed under mild conditions. A series of novel pentacoordinate spirophosphoranes with P-Se/P-S bonds were synthesized in excellent yields. The reaction mechanism was determined by 31P nuclear magnetic resonance tracing experiments, high-resolution mass spectrometry tracing experiments, and X-ray diffraction analysis. The method features a broad substrate scope, good functional group tolerance, and a high degree of atomic utilization and is meaningful for the synthesis of bioactive chalcogenphosphate compounds with chalcogen and phosphorus moieties.

piSTING: A Pocket-Independent Agonist Based on Multivalency-Driven STING Oligomerization.

The stimulator of interferon genes (STING) pathway is a potent therapeutic target for innate immunity. Despite the efforts to develop pocket-dependent small-molecule STING agonists that mimic the endogenous STING ligand, cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), most of these agonists showed disappointing results in clinical trials owing to the limitations of the STING pocket. In this study, we developed novel pocket-independent STING-activating agonists (piSTINGs), which act through multivalency-driven oligomerization to activate STING. Additionally, a piSTING-adjuvanted vaccine elicited a significant antibody response and inhibited tumour growth in therapeutic models. Moreover, a piSTING-based vaccine combination with aPD-1 showed remarkable potential to enhance the effectiveness of immune checkpoint blockade (ICB) immunotherapy. In particular, piSTING can strengthen the impact of STING pathway in immunotherapy and accelerate the clinical translation of STING agonists.

Brønsted Acids Promote Olefin Oxidations by Bioinspired Nonheme CoIII(PhIO)(OH) Complexes: A Role for Low-Barrier Hydrogen Bonds.

Introduction of Brønsted acids into biomimetic nonheme reactions promotes the oxidative ability of metal-oxygen complexes significantly. However, the molecular machinery of the promoted effects is missing. Herein, a comprehensive investigation of styrene oxidation by a cobalt(III)-iodosylbenzene complex, [(TQA)CoIII(OIPh)(OH)]2+ (1, TQA = tris(2-quinolylmethyl)amine), in the presence and absence of triflic acid (HOTf) was performed using density functional theory calculations. Results revealed for the first time that there is a low-barrier hydrogen bond (LBHB) between HOTf and the hydroxyl ligand of 1, which forms two valence-resonance structures [(TQA)CoIII(OIPh)(HO---HOTf)]2+ (1LBHB) and [(TQA)CoIII(OIPh)(H2O--OTf-)]2+ (1'LBHB). Due to the oxo-wall, these complexes (1LBHB and 1'LBHB) cannot convert to high-valent cobalt-oxyl species. Instead, styrene oxidation by these oxidants (1LBHB and 1'LBHB) shows novel spin-state selectivity, i.e., on the ground closed-shell singlet state, styrene is oxidized to an epoxide, whereas on the excited triplet and quintet states, an aldehyde product, phenylacetaldehyde, is formed. The preferred pathway is styrene oxidation by 1'LBHB, which is initiated by a rate-limiting bond-formation-coupled electron transfer process with an energy barrier of 12.2 kcal mol-1. The nascent PhIO-styrene-radical-cation intermediate undergoes an intramolecular rearrangement to produce an aldehyde. The halogen bond between the OH-/H2O ligand and the iodine of PhIO modulates the activity of the cobalt-iodosylarene complexes 1LBHB and 1'LBHB. These new mechanistic findings enrich our knowledge of nonheme chemistry and hypervalent iodine chemistry and will play a positive role in the rational design of new catalysts.

A Facile and High-Sensitivity Method for Determining Proteinogenic Amino Acid Enantiomers by Integrating Chiral Phosphinate Derivatizing, 31P NMR and Parallel Reaction Monitoring.

Here, we have documented a new protocol to determine d/l-amino acids by derivatizing amino acids via a chiral phosphinate. (RP)-l-Menthyl phenylphosphinate was able to bond both primary and secondary amines, as well as improve the sensitivity of analytes in MS. Eighteen pairs of amino acids were successfully labeled except for Cys which has a thiol group on the side chain, and the chirality of amino acids can be discriminated by 31P NMR. Seventeen pairs of amino acids were separated by a C18 column within 45 min of elution, and resolution values ranged from 2.01 to 10.76. The lowest limit of detection was 10 pM acquired at parallel reaction monitoring, in which two factors collectively contributed that the ability of protonation of phosphine oxide and the sensitivity of parallel reaction monitoring. Chiral phosphine oxides might be a promising tool in future chiral metabolomics.

Solvent Effects Used for Optimal Simultaneous Analysis of Amino Acids via 19F NMR Spectroscopy.

19F NMR has been extensively used in simultaneous analysis of multicomponent due to its 100% natural isotope abundance, high NMR-sensitivity, and wide-range chemical shifts. The solvent effects are usually observed in NMR spectroscopy and cause large changes in 19F chemical shifts. Herein, we propose that the simultaneous analysis of a complex mixture can be achieved using solvent effects via 19F NMR spectroscopy, such as a mixture solution of amino acids (AAs). AAs are not only cell-signaling molecules, but are also considered as biomarkers of some diseases. Hence, the analysis of AAs is important for human health and the diagnosis of diseases. In this work, the key to the success of sensing 19 biogenic AAs is the use of 2-fluorobenzaldehyde (2FBA) as a highly sensitive derivatizing agent and solvent effects to produce distinguishable 19F NMR signals. As a result, the resolution of 19F NMR spectroscopy of multiple 2FBA-labeled AAs is obviously higher than other methods based on 19F NMR. Moreover, 14 and 18 AAs can be satisfactorily differentiated and unambiguously identified in different complicated media supporting the growth of mammalian cells. Furthermore, quantification of the concentration of AAs can be made, and the limit of detection reaches 10 µM. Our work provides new insights into the simultaneous analysis of a multicomponent mixture based on solvent effects by 19F NMR spectroscopy.

Comprehensive Profiling of Amine-Containing Metabolite Isomers with Chiral Phosphorus Reagents.

Metabolite isomers play diverse and crucial roles in various metabolic processes. However, in untargeted metabolomics analysis, it remains a great challenge to distinguish between the constitutional isomers and enantiomers of amine-containing metabolites due to their similar chemical structures and physicochemical properties. In this work, the triplex stable isotope N-phosphoryl amino acids labeling (SIPAL) is developed to identify and relatively quantify the amine-containing metabolites and their isomers by using chiral phosphorus reagents coupled with high-resolution tandem mass spectroscopy. The constitutional isomers could be effectively distinguished with stereo isomers by using the diagnosis ions in MS/MS spectra. The in-house software MS-Isomerism has been parallelly developed for high-throughput screening and quantification. The proposed strategy enables the unbiased detection and relative quantification of isomers of amine-containing metabolites. Based on the characteristic triplet peaks with SIPAL tags, a total of 854 feature peaks with 154 isomer groups are successfully recognized as amine-containing metabolites in liver cells, in which 37 amine-containing metabolites, including amino acids, polyamines, and small peptides, are found to be significantly different between liver cancer cells and normal cells. Notably, it is the first time to identify S-acetyl-glutathione as an endogenous metabolite in liver cells. The SIPAL strategy could provide spectacular insight into the chemical structures and biological functions of the fascinating amine-containing metabolite isomers. The feasibility of SIPAL in isomeric metabolomics analysis may reach a deeper understanding of the mirror-chemistry in life and further advance the discovery of novel biomarkers for disease diagnosis.

Database of space life investigations and information on spaceflight plant biology.

Extensive spaceflight life investigations (SLIs) have revealed observable space effects on plants, particularly their growth, nutrition yield, and secondary metabolite production. Knowledge of these effects not only facilitates space agricultural and biopharmaceutical technology development but also provides unique perspectives to ground-based investigations. SLIs are specialized experimental protocols and notable biological phenomena. These require specialized databases, leading to the development of the NASA Science Data Archive, Erasmus Experiment Archive, and NASA GeneLab. The increasing interests of SLIs across diverse fields demand resources with comprehensive content, convenient search facilities, and friendly information presentation. A new database SpaceLID (Space Life Investigation Database http://bidd.group/spacelid/ ) was developed with detailed menu search tools and categorized contents about the phenomena, protocols, and outcomes of 459 SLIs (including 106 plant investigations) of 92 species, where 236 SLIs and 57 plant investigations are uncovered by the existing databases. The usefulness of SpaceLID as an SLI information source is illustrated by the literature-reported analysis of metabolite, nutrition, and symbiosis variations of spaceflight plants. In conclusion, this study extensively investigated the impact of the space environment on plant biology, utilizing SpaceLID as an information source and examining various plant species, including Arabidopsis thaliana, Brassica rapa L., and Glycyrrhiza uralensis Fisch. The findings provide valuable insights into the effects of space conditions on plant physiology and metabolism.

Protocell Self-Assembly Driven by Sodium Trimetaphosphate.

The co-evolution of peptide formation and membrane self-assembly is considered an essential step in the origin of life. However, more research is required on both processes, particularly on the interaction between prebiotic simple fatty-acid membranes and peptide synthesis. In this study, the sodium trimetaphosphate (P3 m)-activated peptide formation reaction of phenylalanine (Phe) in an alkaline decanoic acid-decanol vesicle system was systematically investigated. The experimental results showed that peptide formation could competitively occur with N-acyl amino acid (NAA) formation. NAA formation did not follow the traditional P3 m-activated peptide formation reaction involving the intermediate cyclic acylphosphoramidate (CAPA). Decanoic acid was activated by P3 m to form a mixed anhydride, which then reacted with an amino acid to form the amide NAA. As a kind of membrane-forming amphiphile, NAA can form vesicles independently and reduce the critical vesicle concentration of the fatty-acid vesicles. Moreover, 11 other representative amino acids, namely alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), isoleucine (Ile), leucine (Leu), proline (Pro), serine (Ser), threonine (Thr), valine (Val), and arginine (Arg), were selected for investigation. All of them reacted with decanoic acid to form NAA via the activation effect of P3 m. The abovementioned mechanism involving P3 m-activated carboxylic acid has not been reported in the literature. Our experimental results indicate that the participation of decanoic acid in the P3 m activation-based peptide formation reaction system plays a significant role in the emergence of functionalized protocells. The P3 m activation effect can provide diversified raw membrane materials to form and stabilize protocell membranes; moreover, the small peptides, such as Phe-Leu, formed in the same reaction system can induce the amplification of primitive cells. This implies that synergistic symbiosis between membrane and peptide can be realized via the P3 m activation effect.

Alkoxyphosphorane/Borane Cooperative Alkylations: A Frustrated Lewis Pair Version of the Mitsunobu Reaction.

The combination of the alkoxyphosphoranes, Ph2 P(OR)(O2 C6 Cl4 ) and the borane B(C6 F5 )3 generates the zwitterions 3 which act as FLP to effect the alkylation of several nucleophiles affording C-C, C-N, C-H and C-Cl coupling products. A DFT study shows the reaction proceeds via an FLP activation pathway generating an alkoxyphosphonium intermediate which effects the alkylation of the nucleophiles, akin to the Mitsunobu reaction.

Investigation of the Asymmetric Addition Reactions Induced by Pentacoordinated Hydrospirophosphorane Substrate.

Pentacoordinated bisaminoacyl hydrospirophosphoranes were first found to induce the asymmetric addition reactions as a novel chiral organic framework. Asymmetric addition reactions of bisaminoacyl hydrospirophosphoranes with aromatic aldehyde and in situ generated imine were investigated, and the corresponding α-hydroxyspirophosphonates and α-amino spirophosphonates were obtained. The addition reaction of hydrospirophosphoranes with ΔP configuration showed better stereoselectivity than that with ΛP configuration, not only for the addition reaction to aromatic aldehyde but also to in situ generated imine. Furthermore, the stereochemical mechanisms of asymmetric addition reactions induced by pentacoordinated hydrospirophosphorane were proposed by 31P NMR tracing experiment and X-ray diffraction analysis.

Cp2Fe-Mediated Electrochemical Synthesis of Phosphorylated Oxindoles and Indolo[2,1-a]isoquinolin-6(5H)-ones.

An efficient and environmentally friendly electrochemical synthesis of phosphorylated oxindoles and indolo[2,1-a]isoquinolin-6(5H)-ones mediated by readily available Cp2Fe has been developed, which illustrated a broad substrate scope and diverse functional group compatibility. This protocol featured an external oxidant-free process and was at room temperature, which was proposed to be driven by the anodic oxidation of Cp2Fe.

19F NMR enantiodiscrimination and diastereomeric purity determination of amino acids, dipeptides, and amines.

Chiral amino-group compounds are of significance for human health, such as biogenic amino acids (AAs), dipeptides, and even various drugs. Enantiospecific discrimination of these chiral compounds is vital in diagnosing diseases, identifying pathological biomarkers and enhancing pharmaceutical chemistry research. Here, we report a simple and rapid 19F NMR-based strategy to differentiate chiral AAs, dipeptides, and amines, that were derivatized with (R)-2-(2-fluorophenyl)-2-hydroxyacetic acid ((R)-2FHA). As a result, 19 proteinogenic AAs (37 isomers) as well as Gly could be concurrently resolved. Moreover, various mirror-image dipeptides, such as Ser-His, Leu-Leu, and Ala-Ala, were commendably recognized. Intriguingly, we found that the absolute configuration of AAs in the N-terminus of dipeptides decided the relative 19F chemical shifts between two enantiomers. Besides, the ability of this method for enantiodiscrimination was further demonstrated by non-AA amines, including aromatic and aliphatic amines, and even amines having chiral centers several carbons away from the amino-group. The structurally similar antibiotics, amoxicillin and ampicillin, were well discriminated. Furthermore, this method accurately determines the de or dr values of non-racemic mixtures. Therefore, our strategy provides an effective approach for 19F NMR-based enantiodiscrimination and diastereomeric purity determination of amino-group compounds.

Elusive Active Intermediates and Reaction Mechanisms of ortho-/ipso-Hydroxylation of Benzoic Acid by Hydrogen Peroxide Mediated by Bioinspired Iron(II) Catalysts.

Aromatic hydroxylation of benzoic acids (BzOH) to salicylates and phenolates is fundamentally interesting in industrial chemistry. However, key mechanistic uncertainties and dichotomies remain after decades of effort. Herein, the elusive mechanism of the competitive ortho-/ipso-hydroxylation of BzOH by H2O2 mediated by a nonheme iron(II) catalyst was comprehensively investigated using density functional theory calculations. Results revealed that the long-postulated FeV(O)(anti-BzO) oxidant is an FeIV(O)(anti-BzO•) species 2 (anti- and syn- are defined by the orientation of the carboxyl oxygen of BzO to the oxo), which rules out the noted two-oxidant mechanism proposed previously. We propose a new mechanism in which, following the formation of an FeV(O)(syn-BzO) species (3) and its electromer FeIV(O)(syn-BzO•) (3'), 3/3' either converts to salicylate and phenolate via intramolecular self-hydroxylation (route A) or acts as an oxidant to oxygenate another BzOH to generate the same products (route B). In route A, the rotation of the BzO group along the C-O bond forms 2, in which the BzO group is orientated by π-π stacking interactions. An electrophilic ipso-addition forms a phenolate by concomitant decarboxylation or an ortho-attack forms a cationic complex, which readily undergoes an NIH shift and a BzOH-assisted proton shift to form a salicylate. In route B, 3 oxidizes an additional BzOH molecule directed by hydrogen bonding and π-π stacking interactions. In both routes, selectivity is determined by the chemical property of the BzO ring. These mechanistic findings provide a clear mechanistic scenario and enrich the knowledge of hydroxylation of aromatic acids.

Potential Active Marine Peptides as Anti-Aging Drugs or Drug Candidates.

Aging is an irreversible physiological process in the human body, and the aging characteristics of the body that accompany this process also lead to many other chronic diseases, such as neurodegenerative diseases represented by Alzheimer's disease and Parkinson's disease, cardiovascular diseases, hypertension, obesity, cancer, and so on. The marine environment is highly biodiverse, the natural active products of these organisms constitute a vast treasure trove of marine drugs or drug candidates that play an essential role in disease prevention and treatment, and the active peptide products among them have received special attention because of their unique chemical properties. Therefore, the development of marine peptide compounds as anti-aging drugs is emerging as an important research area. This review highlights the currently available data on marine bioactive peptides with anti-aging potential from 2000 to 2022 by analyzing the prevalent aging mechanisms, critical aging metabolic pathways and well-established multi-omics aging characteristics, as well as grouping different bioactive and biological species lines of peptides from marine organisms and discussing their research modalities and functional characteristics. Active marine peptides is a promising topic to explore and to develop their potential as anti-aging drugs or drug candidates. We expect this review to be instructive for future marine drug development and to reveal new directions for future biopharmaceuticals.

Screening of Oral Potential Angiotensin-Converting Enzyme Inhibitory Peptides from Zizyphus jujuba Proteins Based on Gastrointestinal Digestion In Vivo.

Plant proteins are a good source of active peptides, which can exert physiological effects on the body. Predicting the possible activity of plant proteins and obtaining active peptides with oral potential are challenging. In this study, the potential activity of peptides from Zizyphus jujuba proteins after in silico simulated gastrointestinal digestion was predicted using the BIOPEP-UWM™ database. The ACE-inhibitory activity needs to be further investigated. The actual peptides in mouse intestines after the oral administration of Zizyphus jujuba protein were collected and analyzed, 113 Zizyphus jujuba peptides were identified, and 3D-QSAR models of the ACE-inhibitory activity were created and validated using a training set (34 peptides) and a test set (12 peptides). Three peptides, RLPHV, TVKPGL and KALVAP, were screened using the 3D-QSAR model and were found to bind to the active sites of the ACE enzyme, and their IC50 values were determined. Their values were 6.01, 3.81, and 17.06 µM, respectively. The in vitro digestion stabilities of the RLPHV, TVKPGL, and KALVAP peptides were 82%, 90%, and 78%. This article provides an integrated method for studying bioactive peptides derived from plant proteins.

A Practical Method for Amino Acid Analysis by LC-MS Using Precolumn Derivatization with Urea.

Amino acid (AA) analysis is important in biochemistry, food science, and clinical medicine. However, due to intrinsic limitations, AAs usually require derivatization to improve their separation and determination. Here, we present a liquid chromatography-mass spectrometry (LC-MS) method for the derivatization of AAs using the simple agent urea. The reactions proceed quantitatively under a wide range of conditions without any pretreatment steps. Urea-derivatized products (carbamoyl amino acids) of 20 AAs exhibit better separation on reversed-phase columns and increased response in a UV detector compared to underivatized ones. We applied this approach to AA analysis in complex samples using a cell culture media as a model, and it showed potential for the determination of oligopeptides. This fast, simple, and inexpensive method should be useful for AA analysis in complex samples.

Engaging Isatins and Amino Acids in Multicomponent One-Pot 1,3-Dipolar Cycloaddition Reactions-Easy Access to Structural Diversity.

Multicomponent reactions (MCRs) have undoubtedly emerged as the most indispensable tool for organic chemists worldwide, finding extensive utility in the synthesis of intricate natural products, heterocyclic molecules with significant bioactivity, and pharmaceutical agents. The multicomponent one-pot 1,3-dipolar cycloaddition reactions, which were initially conceptualized by Rolf Huisgen in 1960, find extensive application in contemporary heterocyclic chemistry. In terms of green synthesis, the multicomponent 1,3-dipolar cycloaddition is highly favored owing to its numerous advantages, including high step- and atom-economies, remarkable product diversity, as well as excellent efficiency and diastereoselectivity. Among the numerous pieces of research, the most fascinating reaction involves the utilization of azomethine ylides generated from isatins and amino acids that can be captured by various dipolarophiles. This approach offers a highly efficient and convenient method for constructing spiro-pyrrolidine oxindole scaffolds, which are crucial building blocks in biologically active molecules. Consequently, this review delves deeper into the dipolarophiles utilized in the 1,3-dipolar cycloaddition of isatins and amino acids over the past six years.