The elusive phenylethynyl radical and its cation: synthesis, electronic structure, and reactivity.

Alkynyl radicals and cations are crucial reactive intermediates in chemistry, but often evade direct detection. Herein, we report the direct observation of the phenylethynyl radical (C6H5CC˙) and its cation (C6H5CC+), which are two of the most reactive intermediates in organic chemistry. The radical is generated via pyrolysis of (bromoethynyl)benzene at temperatures above 1500 K and is characterized by photoion mass-selected threshold photoelectron spectroscopy (ms-TPES). Photoionization of the phenylethynyl radical yields the phenylethynyl cation, which has never been synthesized due to its extreme electrophilicity. Vibrationally-resolved ms-TPES assisted by ab initio calculations unveiled the complex electronic structure of the phenylethynyl cation, which appears at an adiabatic ionization energy (AIE) of 8.90 ± 0.05 eV and exhibits an uncommon triplet (3B1) ground state, while the closed-shell singlet (1A1) state lies just 2.8 kcal mol-1 (0.12 eV) higher in energy. The reactive phenylethynyl radical abstracts hydrogen to form ethynylbenzene (C6H5CCH) but also isomerizes via H-shift to the o-, m-, and p-ethynylphenyl isomers (C6H4CCH). These radicals are very reactive and undergo ring-opening followed by H-loss to form a mixture of C8H4 triynes, along with low yields of cyclic 3- and 4-ethynylbenzynes (C6H3CCH). At higher temperatures, dehydrogenation from the unbranched C8H4 triynes forms the linear tetraacetylene (C8H2), an astrochemically relevant polyyne.

sBioSITe enables sensitive identification of the cell surface proteome through direct enrichment of biotinylated peptides.

BACKGROUND: Cell surface proteins perform critical functions related to immune response, signal transduction, cell-cell interactions, and cell migration. Expression of specific cell surface proteins can determine cell-type identity, and can be altered in diseases including infections, cancer and genetic disorders. Identification of the cell surface proteome remains a challenge despite several enrichment methods exploiting their biochemical and biophysical properties. METHODS: Here, we report a novel method for enrichment of proteins localized to cell surface. We developed this new approach designated surface Biotinylation Site Identification Technology (sBioSITe) by adapting our previously published method for direct identification of biotinylated peptides. In this strategy, the primary amine groups of lysines on proteins on the surface of live cells are first labeled with biotin, and subsequently, biotinylated peptides are enriched by anti-biotin antibodies and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: By direct detection of biotinylated lysines from PC-3, a prostate cancer cell line, using sBioSITe, we identified 5851 peptides biotinylated on the cell surface that were derived from 1409 proteins. Of these proteins, 533 were previously shown or predicted to be localized to the cell surface or secreted extracellularly. Several of the identified cell surface markers have known associations with prostate cancer and metastasis including CD59, 4F2 cell-surface antigen heavy chain (SLC3A2) and adhesion G protein-coupled receptor E5 (CD97). Importantly, we identified several biotinylated peptides derived from plectin and nucleolin, both of which are not annotated in surface proteome databases but have been shown to have aberrant surface localization in certain cancers highlighting the utility of this method. CONCLUSIONS: Detection of biotinylation sites on cell surface proteins using sBioSITe provides a reliable method for identifying cell surface proteins. This strategy complements existing methods for detection of cell surface expressed proteins especially in discovery-based proteomics approaches.

N-glycoproteomics reveals distinct glycosylation alterations in NGLY1-deficient patient-derived dermal fibroblasts.

Congenital disorders of glycosylation are genetic disorders that occur due to defects in protein and lipid glycosylation pathways. A deficiency of N-glycanase 1, encoded by the NGLY1 gene, results in a congenital disorder of deglycosylation. The NGLY1 enzyme is mainly involved in cleaving N-glycans from misfolded, retro-translocated glycoproteins in the cytosol from the endoplasmic reticulum before their proteasomal degradation or activation. Despite the essential role of NGLY1 in deglycosylation pathways, the exact consequences of NGLY1 deficiency on global cellular protein glycosylation have not yet been investigated. We undertook a multiplexed tandem mass tags-labeling-based quantitative glycoproteomics and proteomics analysis of fibroblasts from NGLY1-deficient individuals carrying different biallelic pathogenic variants in NGLY1. This quantitative mass spectrometric analysis detected 8041 proteins and defined a proteomic signature of differential expression across affected individuals and controls. Proteins that showed significant differential expression included phospholipid phosphatase 3, stromal cell-derived factor 1, collagen alpha-1 (IV) chain, hyaluronan and proteoglycan link protein 1, and thrombospondin-1. We further detected a total of 3255 N-glycopeptides derived from 550 glycosylation sites of 407 glycoproteins by multiplexed N-glycoproteomics. Several extracellular matrix glycoproteins and adhesion molecules showed altered abundance of N-glycopeptides. Overall, we observed distinct alterations in specific glycoproteins, but our data revealed no global accumulation of glycopeptides in the patient-derived fibroblasts, despite the genetic defect in NGLY1. Our findings highlight new molecular and system-level insights for understanding NGLY1-CDDG.

Photoelectron spectroscopic study of 2-naphthylnitrene and its thermal rearrangement to cyanoindenes.

2-Cyanoindene has recently been identified in the interstellar medium, however current models cannot fully account for its formation pathways. Herein, we identify and characterize 2-naphthylnitrene, which is prone to rearrange to 2- and 3-cyanoindene, in the gas phase using photoion mass-selective threshold photoelectron spectroscopy (ms-TPES). The adiabatic ionization energies (AIE) of triplet nitrene (3A'') to the radical cation in its lowest-energy doublet X̃+(2A') and quartet ã+(4A') electronic states were determined to be 7.72 ± 0.02 and 8.64 ± 0.02 eV, respectively, leading to a doublet-quartet energy splitting (ΔED-Q) of 0.92 eV (88.8 kJ mol-1). A ring-contraction mechanism yields 3-cyanoindene, which is selectively formed under mild pyrolysis conditions (800 K), while the lowest-energy isomer, 2-cyanoindene, is also observed under harsh pyrolysis conditions at 1100 K. The isomer-selective assignment was rationalized by Franck-Condon spectral modeling and by measuring the AIEs at 8.64 ± 0.02 and 8.70 ± 0.02 eV for 2- and 3-cyanoindene, respectively, in good agreement with quantum chemical calculations.

Thermal Decomposition of 2- and 4-Iodobenzyl Iodide Yields Fulvenallene and Ethynylcyclopentadienes: A Joint Threshold Photoelectron and Matrix Isolation Spectroscopic Study.

The thermal decomposition of 2- and 4-iodobenzyl iodide at high temperatures was investigated by mass-selective threshold photoelectron spectroscopy (ms-TPES) in the gas phase, as well as by matrix isolation infrared spectroscopy in cryogenic matrices. Scission of the benzylic C-I bond in the precursors at 850 K affords 2- and 4-iodobenzyl radicals (ortho- and para-IC6H4CH2•), respectively, in high yields. The adiabatic ionization energies of ortho-IC6H4CH2• to the X̃+(1A') and ã+(3A') cation states were determined to be 7.31 ± 0.01 and 8.78 ± 0.01 eV, whereas those of para-IC6H4CH2• were measured to be 7.17 ± 0.01 eV for X̃+(1A1) and 8.98 ± 0.01 eV for ã+(3A1). Vibrational frequencies of the ring breathing mode were measured to be 560 ± 80 and 240 ± 80 cm-1 for the X̃+(1A') and ã+(3A') cation states of ortho-IC6H4CH2•, respectively. At higher temperatures, subsequent aryl C-I cleavage takes place to form α,2- and α,4-didehydrotoluene diradicals, which rapidly undergo ring contraction to a stable product, fulvenallene. Nevertheless, the most intense vibrational bands of the elusive α,2- and α,4-didehydrotoluene diradicals were observed in the Ar matrices. In addition, high-energy and astrochemically relevant C7H6 isomers 1-, 2-, and 5-ethynylcyclopentadiene are observed at even higher pyrolysis temperatures along with fulvenallene. Complementary quantum chemical computations on the C7H6 potential energy surface predict a feasible reaction cascade at high temperatures from the diradicals to fulvenallene, supporting the experimental observations in both the gas phase and cryogenic matrices.

Proteomic Signature of Host Response to SARS-CoV-2 Infection in the Nasopharynx.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has become a global health pandemic. COVID-19 severity ranges from an asymptomatic infection to a severe multiorgan disease. Although the inflammatory response has been implicated in the pathogenesis of COVID-19, the exact nature of dysregulation in signaling pathways has not yet been elucidated, underscoring the need for further molecular characterization of SARS-CoV-2 infection in humans. Here, we characterize the host response directly at the point of viral entry through analysis of nasopharyngeal swabs. Multiplexed high-resolution MS-based proteomic analysis of confirmed COVID-19 cases and negative controls identified 7582 proteins and revealed significant upregulation of interferon-mediated antiviral signaling in addition to multiple other proteins that are not encoded by interferon-stimulated genes or well characterized during viral infections. Downregulation of several proteasomal subunits, E3 ubiquitin ligases, and components of protein synthesis machinery was significant upon SARS-CoV-2 infection. Targeted proteomics to measure abundance levels of MX1, ISG15, STAT1, RIG-I, and CXCL10 detected proteomic signatures of interferon-mediated antiviral signaling that differentiated COVID-19-positive from COVID-19-negative cases. Phosphoproteomic analysis revealed increased phosphorylation of several proteins with known antiviral properties as well as several proteins involved in ciliary function (CEP131 and CFAP57) that have not previously been implicated in the context of coronavirus infections. In addition, decreased phosphorylation levels of AKT and PKC, which have been shown to play varying roles in different viral infections, were observed in infected individuals relative to controls. These data provide novel insights that add depth to our understanding of SARS-CoV-2 infection in the upper airway and establish a proteomic signature for this viral infection.

Proteomic alterations in extracellular vesicles induced by oncogenic PIK3CA mutations.

PIK3CA is one of the most frequently mutated genes in human cancers, with the two most prevalent activating mutations being E545K and H1047R. Although the altered intracellular signaling pathways in these cells have been described, the effect of these mutations on their extracellular vesicles (EVs) has not yet been reported. To study altered cellular physiology and intercellular communication through proteomic analysis of EVs, MCF10A cells and their isogenic mutant versions (PIK3CA E545K and H1047R) were cultured and their EVs enriched by differential ultracentrifugation. Proteins were extracted, digested with trypsin and the peptides labeled with tandem mass tag (TMT) reagents and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Four thousand six hundred and fifty-five peptides were identified from 579 proteins of which 522 proteins have been previously described in EVs. Relative quantitation revealed altered levels of EV proteins including several cell adhesion molecules. Mesothelin, E-cadherin, and epithelial cell adhesion molecule were elevated in both mutant cell-derived EVs. Markers of tumor invasion and progression like galectin-3 and transforming growth factor beta induced protein were increased in both mutants. Overall, activating mutations in PIK3CA result in altered EV composition with characteristic changes associated with these hotspot mutations.

Sorbitol Is a Severity Biomarker for PMM2-CDG with Therapeutic Implications.

OBJECTIVE: Epalrestat, an aldose reductase inhibitor increases phosphomannomutase (PMM) enzyme activity in a PMM2-congenital disorders of glycosylation (CDG) worm model. Epalrestat also decreases sorbitol level in diabetic neuropathy. We evaluated the genetic, biochemical, and clinical characteristics, including the Nijmegen Progression CDG Rating Scale (NPCRS), urine polyol levels and fibroblast glycoproteomics in patients with PMM2-CDG. METHODS: We performed PMM enzyme measurements, multiplexed proteomics, and glycoproteomics in PMM2-deficient fibroblasts before and after epalrestat treatment. Safety and efficacy of 0.8 mg/kg/day oral epalrestat were studied in a child with PMM2-CDG for 12 months. RESULTS: PMM enzyme activity increased post-epalrestat treatment. Compared with controls, 24% of glycopeptides had reduced abundance in PMM2-deficient fibroblasts, 46% of which improved upon treatment. Total protein N-glycosylation improved upon epalrestat treatment bringing overall glycosylation toward the control fibroblasts' glycosylation profile. Sorbitol levels were increased in the urine of 74% of patients with PMM2-CDG and correlated with the presence of peripheral neuropathy, and CDG severity rating scale. In the child with PMM2-CDG on epalrestat treatment, ataxia scores improved together with significant growth improvement. Urinary sorbitol levels nearly normalized in 3 months and blood transferrin glycosylation normalized in 6 months. INTERPRETATION: Epalrestat improved PMM enzyme activity, N-glycosylation, and glycosylation biomarkers in vitro. Leveraging cellular glycoproteome assessment, we provided a systems-level view of treatment efficacy and discovered potential novel biosignatures of therapy response. Epalrestat was well-tolerated and led to significant clinical improvements in the first pediatric patient with PMM2-CDG treated with epalrestat. We also propose urinary sorbitol as a novel biomarker for disease severity and treatment response in future clinical trials in PMM2-CDG. ANN NEUROL 20219999:n/a-n/a.

Light-controlled shape-changing azomacrocycles exhibiting reversible modulation of pyrene fluorescence emission.

We report the design, synthesis, and study of light-induced shape-changing azomacrocycles. These systems have been incorporated with azobenzene photoswitches using alkoxy tethers and triazole units to afford flexibility and binding. We envision that such azomacrocycles are capable of reversibly binding with the guest molecule. Remarkably, we have demonstrated fully light-controlled fluorescence quenching and enhancement in the monomeric emission of pyrene (guest). Such modulations have been achieved by the photoisomerization of the azomacrocycle and, in turn, host-guest interactions. Also, the azomacrocycles tend to aggregate and can also be controlled by light or heat. We uncovered such phenomena using spectroscopic, microscopic, and isothermal titration calorimetry (ITC) studies and computations.

Photochemistry of 3,6-Didehydropyridazine Biradical─An Untraceable Para Benzyne Analogue.

We report matrix isolation infrared spectroscopic studies to characterize 3,6-didehydropyridazine 6, a heterocyclic analogue of para benzyne, combined with computations. In this regard, we have utilized 3,6-diiodopyridazine 11 as a photolytic precursor. The experiments toward the generation of the biradical are carried out in argon and nitrogen matrices at 4 K. Instead of the elusive biradical, we have observed a ring-opening product maleonitrile (Z)-7 upon irradiation at 254 nm. In contrast, prolonged irradiation at 254 nm leads only to Z-E isomerization, forming fumaronitrile (E)-7. The mechanistic aspects of ring-opening, product selectivity, and Z-E photoisomerization steps have been investigated in detail using high-level ab initio computations. These studies have found that 3,6-didehydropyridazine 6 is an untraceable intermediate, and the ring-opening step leading to maleonitrile is barrierless. In addition, we have proposed the involvement of the S1 (π-π*) state via conical intersection in the Z-E photoisomerization of maleonitrile.

DIA-Based Proteome Profiling of Nasopharyngeal Swabs from COVID-19 Patients.

Since the recent outbreak of COVID-19, there have been intense efforts to understand viral pathogenesis and host immune response to combat SARS-CoV-2. It has become evident that different host alterations can be identified in SARS-CoV-2 infection based on whether infected cells, animal models or clinical samples are studied. Although nasopharyngeal swabs are routinely collected for SARS-CoV-2 detection by RT-PCR testing, host alterations in the nasopharynx at the proteomic level have not been systematically investigated. Thus, we sought to characterize the host response through global proteome profiling of nasopharyngeal swab specimens. A mass spectrometer combining trapped ion mobility spectrometry (TIMS) and high-resolution QTOF mass spectrometer with parallel accumulation-serial fragmentation (PASEF) was deployed for unbiased proteome profiling. First, deep proteome profiling of pooled nasopharyngeal swab samples was performed in the PASEF enabled DDA mode, which identified 7723 proteins that were then used to generate a spectral library. This approach provided peptide level evidence of five missing proteins for which MS/MS spectrum and mobilograms were validated with synthetic peptides. Subsequently, quantitative proteomic profiling was carried out for 90 individual nasopharyngeal swab samples (45 positive and 45 negative) in DIA combined with PASEF, termed as diaPASEF mode, which resulted in a total of 5023 protein identifications. Of these, 577 proteins were found to be upregulated in SARS-CoV-2 positive samples. Functional analysis of these upregulated proteins revealed alterations in several biological processes including innate immune response, viral protein assembly, and exocytosis. To the best of our knowledge, this study is the first to deploy diaPASEF for quantitative proteomic profiling of clinical samples and shows the feasibility of adopting such an approach to understand mechanisms and pathways altered in diseases.

Expanding the clinical and metabolic phenotype of DPM2 deficient congenital disorders of glycosylation.

Pathogenic alterations in the DPM2 gene have been previously described in patients with hypotonia, progressive muscle weakness, absent psychomotor development, intractable seizures, and early death. We identified biallelic DPM2 variants in a 23-year-old male with truncal hypotonia, hypertonicity, congenital heart defects, intellectual disability, and generalized muscle wasting. His clinical presentation was much less severe than that of the three previously described patients. This is the second report on this ultra-rare disorder. Here we review the characteristics of previously reported individuals with a defect in the DPM complex while expanding the clinical phenotype of DPM2-Congenital Disorders of Glycosylation. In addition, we offer further insights into the pathomechanism of DPM2-CDG disorder by introducing glycomics and lipidomics analysis.

PASS-DIA: A Data-Independent Acquisition Approach for Discovery Studies.

A data-independent acquisition (DIA) approach is being increasingly adopted as a promising strategy for identification and quantitation of proteomes. As most DIA data sets are acquired with wide isolation windows, highly complex MS/MS spectra are generated, which negatively impacts obtaining peptide information through classical protein database searches. Therefore, the analysis of DIA data mainly relies on the evidence of the existence of peptides from prebuilt spectral libraries. Consequently, one major weakness of this method is that it does not account for peptides that are not included in the spectral library, precluding the use of DIA for discovery studies. Here, we present a strategy termed Precursor ion And Small Slice-DIA (PASS-DIA) in which MS/MS spectra are acquired with small isolation windows (slices) and MS/MS spectra are interpreted with accurately determined precursor ion masses. This method enables the direct application of conventional spectrum-centric analysis pipelines for peptide identification and precursor ion-based quantitation. The performance of PASS-DIA was observed to be superior to both data-dependent acquisition (DDA) and conventional DIA experiments with 69 and 48% additional protein identifications, respectively. Application of PASS-DIA for the analysis of post-translationally modified peptides again highlighted its superior performance in characterizing phosphopeptides (77% more), N-terminal acetylated peptides (56% more), and N-glycopeptides (83% more) as compared to DDA alone. Finally, the use of PASS-DIA to characterize a rare proteome of human fallopian tube organoids enabled 34% additional protein identifications than DDA alone and revealed biologically relevant pathways including low abundance proteins. Overall, PASS-DIA is a novel DIA approach for use as a discovery tool that outperforms both conventional DDA and DIA experiments to provide additional protein information. We believe that the PASS-DIA method is an important strategy for discovery-type studies when deeper proteome characterization is required.

Label-free proteomics reveals serum proteins whose levels differ between pancreatic ductal adenocarcinoma patients with short or long survival.

Pancreatic ductal adenocarcinoma is the most common and aggressive type of pancreatic cancer, with a 5-year survival rate that is less than 10%. New biomarkers to aid in predicting the prognosis of pancreatic ductal adenocarcinoma patients are needed. Previous proteomic studies have to a great extent focused on finding proteins of value for the diagnosis of pancreatic ductal adenocarcinoma. There is a lack of studies that have profiled the serum or plasma proteome in order to discover candidates for new prognostic biomarkers. In this study, we have used ultra-performance liquid chromatography-ultra-definition mass spectrometry to analyze the serum samples of 21 pancreatic ductal adenocarcinoma patients with short or long survival. Statistical analysis discovered 31 proteins whose expression differed significantly between pancreatic ductal adenocarcinoma patients with short or long survival. Pathway analysis discovered multiple canonical pathways enriched in this data set, with several pathways having roles in inflammation and lipid metabolism. The serum proteins identified here, which include complement components and several enzymes, could be of value as candidates for new noninvasive prognostic markers.

Preoperative Radiotherapy Leads to Significant Differences in the Plasma Protein Profile of Rectal Cancer Patients.

INTRODUCTION: Colorectal cancer (CRC) is the third most common cancer worldwide, accounting for 10% of the global cancer burden. Rectal cancer accounts for around 30% of CRC cases, and patients with resectable rectal cancer are often given preoperative radiotherapy (PRT) to reduce the rate of local recurrence. The human plasma proteome is an exceptionally complex proteome and ideal to study due to its ability to reflect the presence of diseases such as cancer and the ease of obtaining blood samples. Previous proteomic studies involving rectal cancer patients have mostly focused on the identification of proteins involved in resistance to radiotherapy. OBJECTIVE: The aim of this study was to investigate the overall effects of PRT on plasma protein expression in rectal cancer patients, as there is a lack of such studies. METHODS: Here, we have used mass spectrometry and subsequent statistical analyses to analyze the plasma samples of 30 rectal cancer patients according to PRT status (positive or negative) and tumor stage (II or III). RESULTS AND CONCLUSIONS: We discovered 42 proteins whose levels differed significantly between stage II and III rectal cancer patients who did or did not receive PRT. This study shows that PRT, although localized to the pelvis, leads to measurable, tumor stage-specific changes in plasma protein expression. Future studies of plasma proteins should, when relevant, take this into account and be aware of the widespread effects that PRT has on the plasma proteome.

Human Spermatozoa Quantitative Proteomic Signature Classifies Normo- and Asthenozoospermia.

Scarcely understood defects lead to asthenozoospermia, which results in poor fertility outcomes. Incomplete knowledge of these defects hinders the development of new therapies and reliance on interventional therapies, such as in vitro fertilization, increases. Sperm cells, being transcriptionally and translationally silent, necessitate the proteomic approach to study the sperm function. We have performed a differential proteomics analysis of human sperm and seminal plasma and identified and quantified 667 proteins in sperm and 429 proteins in seminal plasma data set, which were used for further analysis. Statistical and mathematical analysis combined with pathway analysis and self-organizing maps clustering and correlation was performed on the data set.It was found that sperm proteomic signature combined with statistical analysis as opposed to the seminal plasma proteomic signature can differentiate the normozoospermic versus the asthenozoospermic sperm samples. This is despite the results that some of the seminal plasma proteins have big fold changes among classes but they fall short of statistical significance. S-Plot of the sperm proteomic data set generated some high confidence targets, which might be implicated in sperm motility pathways. These proteins also had the area under the curve value of 0.9 or 1 in ROC curve analysis.Various pathways were either enriched in these proteomic data sets by pathway analysis or they were searched by their constituent proteins. Some of these pathways were axoneme activation and focal adhesion assembly, glycolysis, gluconeogenesis, cellular response to stress and nucleosome assembly among others. The mass spectrometric data is available via ProteomeXchange with identifier PXD004098.

Patients with early-stage oropharyngeal cancer can be identified with label-free serum proteomics.

BACKGROUND: The increasing incidence of oropharyngeal squamous cell carcinoma (OPSCC) is mainly related to human papillomavirus (HPV) infection. As OPSCCs are often diagnosed at an advanced stage, mortality and morbidity remain high. There are no diagnostic biomarkers for early detection of OPSCC. METHODS: Serum from 25 patients with stage I-II OPSCC, and 12 healthy controls, was studied with quantitative label-free proteomics using ultra-definition MSE. Statistical analyses were performed to identify the proteins most reliably distinguishing early-stage OPSCCs from controls. P16 was used as a surrogate marker for HPV. P16-positive and P16-negative tumours were analysed separately. RESULTS: With two or more unique proteins per identification, 176 proteins were quantified. A clear separation between patients with early-stage tumours and controls was seen in principal component analysis. Latent structures discriminant analysis identified 96 proteins, most reliably differentiating OPSCC patients from controls, with 13 upregulated and 83 downregulated proteins in study cases. The set of proteins was studied further with network, pathway and protein-protein interaction analyses, and found to participate in lipid metabolism, for example. CONCLUSIONS: We found a set of serum proteins distinguishing early-stage OPSCC from healthy individuals, and suggest a protein set for further evaluation as a diagnostic biomarker panel for OPSCC.

Evaluation of Substituent Effect in Z-Isomer Stability of Arylazo-1 H-3,5-dimethylpyrazoles: Interplay of Steric, Electronic Effects and Hydrogen Bonding.

The electronic and steric effects of aryl substituents and the influence of hydrogen bonding in Z-isomer stability of phenylazopyrazole derivatives have been investigated. In this regard, 38 substituted phenylazopyrazole derivatives and 6 N-methyl phenylazopyrazoles (with meta substitutions) have been synthesized. Their photoswitching behavior, photostationary states (PSS), and kinetics of thermal reverse isomerization were evaluated experimentally using UV-vis and NMR spectroscopic techniques. Furthermore, density functional theory (DFT) computations have been performed for more detailed insights. Despite the presence of substantial substituent effects inferred through Taft and Hammett relationships, the concentration dependency in controlling the isomerization rates has also been observed. Kinetics studies at different concentrations, solvent effects, and computations have confirmed the decisive role of hydrogen bonding and solvent-assisted tautomerism in this regard. Through this study, a complex interplay of steric, electronic effects and hydrogen bonding as factors in dictating the stability of Z-isomers in arylazo-1 H-3,5-dimethylpyrazoles has been demonstrated.

Through bond and through space interactions in dehydro-diazine radicals: a case study of 3c-5e interactions.

Owing to the 3c-5e (3-centred-5-electrons) interactions between two nitrogen lone pairs and a radical electron, the dehydrodiazine radical isomers are very interesting from the fundamental point of view. Among them, pyrimidine has three (1a-1c), pyridazine has two (2a and 2b) and pyrazine has only one (3a) radical isomer. Based on quantum chemical calculations at the (U)B3LYP, (U)M06-2X, (U)BLYP, CBS-QB3 and (U)CCSD(T) levels with cc-pVTZ as the basis set, we found the 4-dehydropyrimidine (1b) radical to be the most stable isomer among the three pyrimidine radicals, followed by the 2-dehydropyrimidine (1a) and 5-dehydropyrimidine (1c) radical isomers. In the case of pyridazine, 3-dehydro radical isomer (2a) is more stable than 4-dehydropyridazine (2b). Bond dissociation energy (BDE) calculations and estimation of radical stabilization energies (RSE) using isodesmic reactions revealed the stability order among the six isomeric diazine radicals as 1c < 2b < 2a < 1a < 3a < 1b. Spin densities at each radical centre and non-zero values at nitrogen centres provided information about the extent of delocalization of radical electrons, which was consistent with the relative stability order of all the isomers. The multiconfigurational CASSCF and natural bond orbital (NBO) calculations suggested the presence of direct through space interactions (between lone pairs and a radical, TS) that play a dominant role over the through bond (through intervening bonds, TB) interactions in deciding the stability order. To confirm these results, we have also estimated the proton affinities (PAs) for each nitrogen atom and compared them with their respective parent diazines, where lowering of the PA values convincingly envisaged the extent and strength of interactions between the nitrogen and radical centre. Atoms-in-molecules (AIM) analysis and estimation of hyperfine coupling constants have also been performed to verify these results. All these results showed that the through space interaction between the lone pair and the radical electron is very important for the electronic structural and stability aspects in dehydrodiazine radicals.

Oral squamous cell carcinoma patients can be differentiated from healthy individuals with label-free serum proteomics.

BACKGROUND: No blood biomarkers to detect early oral cavity squamous cell carcinoma (OSCC) without clinical signs exist - diagnosis is solely based on histology of a visible tumour. Most OSCC patients are diagnosed at advanced stage, which leads to significant morbidity and poor survival. Our aim was to find the serum screening or detection biomarkers in OSCC. METHODS: Serum samples from patients with OSCC treated at the Department of Otorhinolaryngology - Head and Neck Surgery, Helsinki University Hospital (Finland) were collected. Age- and gender-matched healthy individuals served as controls. Quantitative label-free proteomics in high definition MSE mode(HDMSE) was performed on 13 patients and 12 healthy samples. Various statistical analyses were performed on quantitative proteomics data to obtain the most influential proteins, which classify the patients vs healthy samples. RESULTS: In quantitative proteomic analysis (HDMSE), 388 proteins were quantified in our pilot study. A complete separation between cases and controls was seen in supervised and unsupervised classification techniques such as orthogonal projections on latent structure-discriminant analysis (OPLS-DA) and self-organising maps. Using OPLS-DA S-plot, we identified a set of eight proteins that completely separated OSCC patients from healthy individuals. CONCLUSIONS: Although the tumour stages varied from I to IVa, these potential biomarkers were able to identify all OSCCs demonstrating their sensitivity to detect tumours of all stages. We are the first to suggest a set of serum biomarkers in our pilot study to be evaluated further as a diagnostic panel to detect preclinical OSCC in risk patients.