Side Chain Carbonyl Research Articles

Highly durable anion exchange membranes with sustainable mitigation of hydroxide attacks for water electrolysis

Low hydroxide conductivity and insufficient alkaline stability are the main problems currently facing anion exchange membranes (AEMs). Conventional quaternary ammonium groups have limited resistance to attack by hydroxide roots. In this paper, quaternary ammonium-type (Cn-PFCO (n = 2, 5)) perfluoroanion exchange polymers with carbonyl side chains were synthesized. The C5–PFCO exhibited an impressive conductivity of 113.4 mS cm−1 at 80 °C, and remarkably, it retained 86.87% of its initial conductivity even after 15 days of immersion in a 1 M KOH aqueous solution. This excellent alkali resistance is due to the generation of intermediate oxygen anions from OH− and carbonyl groups through a reversible reaction. These anions effectively reduce the likelihood of SN2 attack by diminishing the electrostatic potential of the QA groups from Density Functional Theory (DFT) calculations. The electrostatic potential of C2–PFCO is 6.28 eV, while it decreases to 2.69 eV after the formation of intermediate anions. Furthermore, we also used the prepared AEM for pure water electrolysis to explore its performance. The electrochemical performance of AEMWE exhibited remarkable results when supplied with pure water, demonstrating an impressive current density of 262 mA cm−2 at 1.9 V. This study introduces a novel strategy for concurrently enhancing hydroxide conductivity and alkali resistance in AEMs by analyzing the potential for further improvement in AEMs' alkali resistance.

Computational Insights into Protein Aging: Spontaneous Deamidation of Glutamine.

Spontaneous deamidation of amino acids is a physiologically important process, particularly for protein aging and diseases. Despite its widespread occurrence, the mechanism of glutamine deamidation particularly within proteins remains poorly understood. We have used a multiscale computational approach to investigate glutamine deamidation in the tripeptide Glycine-Glutamine-Glycine (Gly-Gln-Gly) and γS-Crystallin protein. Specifically, both the 5- and 6-membered water-assisted deamidation pathways in the tripeptide have been elucidated and compared. Both are found to occur in three stages: iminol formation, cyclization, and deamination. The rate-limiting step in each mechanism is nucleophilic attack of the backbone iminol nitrogen, formed in the first stage, at the glutamine's side-chain carbonyl carbon. For the 6- and 5-membered mechanisms, this occurs with a free energy cost of 136.4 and 179.5 kJ mol-1, respectively. Thus, overall, in the Gly-Gln-Gly tripeptide, the 6-membered pathway is preferred. Furthermore, the free energies for forming cyclic intermediates and products at selected Gln residues (based on experimentally reported % deamidation) in γS-Crystallin have been obtained. It is found that the 5-membered product complex is exergonic at -25.3 kJ mol-1, while the 6-membered product complex is calculated to be endergonic at 90.7 kJ mol-1. Thus, the deamidation pathway in folded and constrained proteins may not exclusively follow the 6-membered route. Molecular dynamics (MD) simulations of γS-Crystallin indicate that deamidation is more likely to occur when two or more water molecules are in the proximity of the glutamine residue. Consequently, significant conformational changes are found to accompany Gln120 deamidation in γS-Crystallin. This in turn can influence water availability at the other Gln residues considered and hence potentially their deamidation. Collectively, these results provide comprehensive insights into spontaneous water-assisted deamidation of glutamine residues in peptides and into the role and impact of Gln deamidation in proteins.

Ru(II)-Catalyzed Carboamination of Olefins with α-Carbonyl Sulfoxonium Ylides.

The first ruthenium-catalyzed carboamination of olefins with α-carbonyl sulfoxonium ylides is reported. The utilization of an inexpensive ruthenium catalyst enables the concise synthesis of pharmaceutically important isoindolin-1-ones, which possess both a stereogenic center and β-carbonyl side chain. This method is mild, efficient, and scalable and allows for the coupling of a wide range of aryl-, heteroaryl-, alkenyl-, and alkyl-substituted sulfoxonium ylides. Moreover, the carbonyl side chain in the resulting product provides a good handle for downstream transformations. For mechanistic studies, a ruthacyle complex is obtained and proven to be the key intermediate in both catalytic and stoichiometric reactions.

Poly(Vinyl Ketones): New Directions in Photodegradable Polymers.

Poly(vinyl ketones) (PVKs) have received considerable attention over the past few decades due to their unique photochemistry and photodegradation properties under ultraviolet (UV) light. Many PVKs rapidly undergo photodegradation under UV light. The side-chain carbonyl moieties of PVKs permit photolysis through Norrish type I or Norrish type II reaction mechanisms and can also be readily modified by nucleophilic addition reactions. These unique properties lead to this class of polymers serving as versatile scaffolds for generating functional materials. This review captures the evolution of synthetic routes to access well-defined PVKs, along with their photochemistry and photo-degradation pathways, and discusses recent and potential applications of these photodegradable materials.

Difference in Catalytic Loop Repositioning Leads to GMP Variation between Two Human GBP Homologues.

Interferon-gamma-inducible human large GTPases, hGBP1 and hGBP2, have a distinctive feature of hydrolyzing GTP to GDP and GMP through successive phosphate cleavages. In hGBP1, GMP is the major product, which is essential for its anti-pathogenic activities. However, its close homologue hGBP2 produces significantly less GMP, despite having a similar active site architecture. The molecular basis for less GMP formation and catalytic residue(s) in hGBP2 are not fully explored. To address these issues, we performed systematic biochemical, biophysical, and microsecond simulation studies. Our data suggest that the less GMP formation in hGBP2 is due to the lack of H-bond formation between the W79 side-chain (located near the active site) and main-chain carbonyl of K76 (present in the catalytic loop) in the substrate-bound hGBP2. The absence of this H-bond could not redirect the catalytic loop toward the beta phosphate after the cleavage of gamma-phosphate, a step essential for enhanced GMP formation. Furthermore, based on the mutational and structural analyses, this study for the first time indicates that the same residue, T75, mediates both phosphate cleavages in hGBP2 and hGBP1. This suggests the conservation of the catalytic residue in hGBP homologues. These findings emphasize the indispensable role of correct catalytic loop repositioning for efficient beta phosphate cleavage. This led us to propose a new substrate hydrolysis mechanism by hGBP1 and hGBP2, which may also be helpful to understand the GTP hydrolysis in other hGBP homologues. Overall, the study could provide insight into how these two close homologues play crucial roles in host-mediated immunity through different mechanisms.

Synthesis and characteristics of some new polythiophene containing α,β‐unsaturated carbonyl groups

AbstractSome new polythiophenes with α,β‐unsaturated carbonyl groups were synthesized using a chemical oxidative coupling reaction. IR, 1H‐NMR, UV‐Vis and Raman spectra were used to confirm the expected structure of polymer which is suitable for the suggested synthetic route. Two of all synthesized polythiophenes were soluble well in DMSO and CHCl3 solvent. The morphology, thermal stability, photoluminescence properties and conductivity of polymers were studied. The trans‐configuration of α,β‐unsaturated carbonyl side chain improve featured properties of these polymers.

Molecular Docking, Synthesis, and Tyrosinase Inhibition Activity of Acetophenone Amide: Potential Inhibitor of Melanogenesis.

Tyrosinase and its related proteins are responsible for pigmentation disorders, and inhibiting tyrosinase is an established strategy to treat hyperpigmentation. The carbonyl scaffolds can be effective inhibitors of tyrosinase activity, and the fact that both benzoic and cinnamic acids are safe natural substances with such a scaffolded structure, it was speculated that hydroxyl-substituted benzoic and cinnamic acid derivatives may exhibit potent tyrosinase inhibitory activity. These moieties were incorporated into new chemotypes that displayed in vitro inhibitory effect against mushroom tyrosinase with a view to explore antimelanogenic ingredients. The most active compound, 2-((3-acetylphenyl)amino)-2-oxoethyl(E)-3-(2,4-dihydroxyphenyl)acrylate (5c), inhibited mushroom tyrosinase with an IC50 of 0.0020 ± 0.0002 μM, while 2-((3-acetylphenyl)amino)-2-oxoethyl 2,4-dihydroxybenzoate (3c) had an IC50 of 27.35 ± 3.6 μM in comparison to the positive control arbutin and kojic acid with a tyrosinase inhibitory activity of IC50 of 191.17 ± 5.5 μM and IC50 of 16.69 ± 2.8 μM, respectively. Analysis of enzyme kinetics revealed that 5c is a competitive and reversible inhibitor with dissociation constant (Ki) value 0.0072 μM. In silico docking studies with mushroom tyrosinase (PDB ID 2Y9X) predicted possible binding modes in the enzymatic pocket for these compounds. The orthohydroxyl of the cinnamic acid moiety of 5c is predicted to form hydrogen bond with the active site side chain carbonyl of Asn 260 (2.16 Å) closer to the catalytic site Cu ions. The acetyl carbonyl is picking up another hydrogen bond with Asn 81 (1.90 Å). The inhibitor 5c passed the panassay interference (PAINS) alerts. This study presents the potential of hydroxyl-substituted benzoic and cinnamic acids and could be beneficial for various cosmetic formulations.

Labdane and isopimarane diterpenoids from Rosmarinus officinalis solid wastes: MS/MS spectrometric fragmentations and neuroprotective effect

The aim of this study was to purify and identify new types of secondary products from Rosmarinus officinalis solid wastes after essential oil been extracted, further study their MS/MS spectrometric fragmentations and evaluate their cytoprotective effects against H2O2-induced oxidative damage in SH-SY5Y cells. Six labdane diterpenoids, five isopimarane diterpenoids and two ferulic acid esters were obtained in which three labdane diterpenoids and four isopimarane diterpenoids were undescribed compounds. The losses of hydroxyl, tigloyl, 2-methy-butanoyl, acetyl and carbonyl groups or side chains were the main elimination modes of the new compounds in MS/MS analyses. Compounds 1, 4, 6, 10 and 12 showed the ability to protect the cells against oxidative injure whose cell viability rates were more than 65% in the concentration of 1 or 25 µM respectively. These studies broadened the understanding of secondary products of rosemary and promoted the deep developments and utilizations of rosemary after essential oil been extracted.

Influence of a Hydroxyl Group on the Deamidation and Dehydration Reactions of Protonated Asparagine-Serine Investigated by Combined Spectroscopic, Guided Ion Beam, and Theoretical Approaches.

Deamidation of asparaginyl (Asn) peptides is a spontaneous post-translational modification that plays a significant role in degenerative diseases and other biological processes under physiological conditions. In the gas phase, deamidation of protonated peptides is a major fragmentation channel upon activation by collision-induced dissociation. Here, we present a full description of the deamidation process from protonated asparagine-serine, [AsnSer+H]+, via infrared (IR) action spectroscopy and threshold collision-induced dissociation (TCID) experiments in combination with theoretical calculations. The IR results demonstrate that deamidation proceeds via bifurcating reaction pathways leading to furanone- and succinimide-type product ion structures, with a population analysis indicating the latter product dominates. Theory demonstrates that nucleophilic attack of the peptidyl amide oxygen onto the Asn side chain leads to furanone formation, whereas nucleophilic attack by the peptidyl amide nitrogen onto the Asn side-chain carbonyl carbon leads to the formation of the succinimide product structure. TCID experiments find that furanone formation has a threshold energy of 145 ± 12 kJ/mol and succinimide formation occurs with a threshold energy of 131 ± 12 kJ/mol, consistent with theoretical energies and with the spectroscopic results indicating that succinimide dominates. The results provide information regarding the inductive and steric effects of the Ser side chain on the deamidation process. The other major channel observed in the TCID experiments of [AsnSer+H]+ is dehydration, where a threshold energy of 104 ± 10 kJ/mol is determined. A complete IR and theoretical analysis of this pathway is also provided. As for deamidation, a bifurcating pathway is found with both dominant oxazoline and minor diketopiperazine products identified. Here, the Ser side chain is directly involved in both pathways.

Solid-State NMR Reveals the Structural Transformation of the TDP-43 Amyloidogenic Region upon Fibrillation.

TDP-43 is a primary pathological hallmark protein of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, which may exist in the form of amyloid inclusions in the cells of patients. In addition to serving as a biomarker for these diseases, TDP-43 can also directly trigger neurodegeneration. We previously determined the amyloidogenic core region of TDP-43 (residues 311-360) and showed by solution NMR that this region includes two α-helices [(321-330) and (335-343)] in solution. We suggested that the helix-to-sheet structural transformation initiates TDP-43 aggregation. In the present study, X-ray diffraction shows that TDP-43 (311-360) aggregates adopt a cross-β structure. Thioredoxin (Trx)-fused TDP-43 (311-360) can undergo liquid-liquid phase separation (LLPS) before fibrillation, suggesting that phase separation is an intermediate step before amyloid formation. Solid-state NMR (SSNMR), carried out to elucidate the structural changes of TDP-43 (311-360) at the atomic level, indicates five β-strands of the amyloids formed, with the major two β-strands contributed by the first helical region in the solution structure. The NMR evidence is also in support of the fibril having a parallel in-register conformation, implying a mechanism in which the helix-helix interactions in LLPS are converted into β-strand parallel lateral association upon fibrillation. Our studies have assigned many key interresidue interactions that contribute to the stability of the fibril, including F316 with I318 and Q327 and W334 with A325, A326, A329, and S332. SSNMR with 1H detection reveals a unique close interaction between the indole Nε1-Hε1 of W334 and the side-chain carbonyl of Q343. This interaction could be a very important factor in initiating TDP-43 (311-360) folding/misfolding in LLPS.

α-Actinin-1 promotes activity of the L-type Ca2+ channel Cav 1.2.

The L-type Ca2+ channel CaV 1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of CaV 1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV 1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646-1,664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The CaV 1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single-channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits CaV 1.2 to defined surface regions and simultaneously boosts its open probability so that CaV 1.2 is mostly active when appropriately localized.

<p>Isolation, pharmacological evaluation and molecular docking studies of bioactive compounds from Grewia optiva</p>

BackgroundTraditionally, Grewia optiva is widely used for the treatment of many diseases like dysentery, fever, typhoid, diarrhea, eczema, smallpox, malaria and cough.MethodsShade-dried roots of G. optiva were extracted with methanol. Based on HPLC results, chloroform and ethyl acetate fractions were subjected to silica column isolation and four compounds: glutaric acid (V), 3,5 dihydroxy phenyl acrylic acid (VI), (2,5 dihydroxy phenyl) 3',6',8'-trihydroxyl-4H chromen-4'-one (VII) and hexanedioic acid (VIII) were isolated in pure form. Ellman’s assay was used to determine the anticholinesterase potential of isolated compounds while their antioxidant potential was estimated by DPPH and ABTS scavenging assays.ResultsAmongst the isolated compounds, VI and VII exhibited excellent percent inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) (83.23±1.11, 82.72±2.20 and 82.11±2.11, 82.23±1.21, respectively, at 1000 µg/mL) with IC50 of 76, 90, 78 and 92 µg/mL, respectively. Highest percent radicals scavenging against DPPH and ABTS (87.41±1.20 and 86.13±2.31) with IC50 of 64 and 65 µg/mL, respectively, were observed for compound VII. Molecular docking studies also supported the binding of compound VI and VII with the target enzyme. The para-hydroxyl group of the phenolic moiety is formed hydrogen bonds with the active site water molecule and the side chain carbonyl and hydroxyl residues of enzyme.ConclusionThe isolated compounds inhibited the DPPH and ABTS-free radicals, and AChE and BChE enzymes. It was concluded that these compounds could be used in relieving the oxidative stress and pathological symptoms associated with excessive hydrolysis of acetyl and butyryl choline. The results of the study were supported by docking studies for compounds VI and VII.

Dimethylformamide-mediated synthesis and characterization of novel pyrazole- and pyrimidine-based 3,4-dihydropyrimidine-2(1H)-thione derivatives

Pyrimidine, an essential component of nucleic acid is currently reported for its potential application in Acquired Immune Deficiency Syndrome (AIDS) chemotherapy. Also, pyrazole nucleus, a versatile heterocyclic compound is gaining more attention in drug designs owing to its pharmacological therapeutic potentials. Hence, this present study deals with cost effective synthesis of 6-methyl-4-phenyl-5-(substituted-5-phenyl-4H-pyrazol-3-yl)-3,4-dihydropyrimidine-2(1H)-thione derivatives which are concisely known as pyrazole-based pyrimidine scaffolds. The multicomponent reaction of benzaldehyde, acetyl acetone and thiourea in the presence of catalytic amount of hydrochloric acid (HCl)ab initio produced 5-aceto-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine, 1. Later, room temperature Claisen-Schmidt condensation of precursor 1 with diverse aromatic aldehydes which were benzaldehyde derivatives led to the formation of α,β-unsaturated carbonyl side chain, 2a-h. Finally, the thermal annellation through synthetic cyclization furnished crude products which were purified by recrystallization to afford 6-methyl-4-phenyl-5-(substituted-5-phenyl-4H-pyrazol-3-yl)-3,4-dihydropyrimidine-2(1H)-thione derivatives 3a-h in a cheap condition. The chemical structures were authenticated using IR, UV, 1H-NMR and 13C-NMR as well as analytical data. The final products 3a-h possessed good candidature for further investigation regarding their biological activities and pharmacological potential for new drug discovery.

Structure-dependent catalytic properties of mesoporous cobalt oxides in furfural hydrogenation

As the development of noble metal free catalysts became important in the biomass conversion, catalytic hydrogenation of furfural (FAL) is investigated over ordered mesoporous cobalt oxide (m-Co3O4). When m-Co3O4 is reduced at 350 and 500 °C in hydrogen, the original crystal structure of Co3O4 is changed to CoO and Co, respectively. Here we examine the effect of the structure, porosity, and oxidation state of m-Co3O4 to identify catalytically active species for hydrogenation of FAL. Among cobalt oxide catalysts having different crystal structures and symmetry, m-CoO having p6mm symmetry exhibits the highest activity. In product selectivity, the CoO phase induces FAL hydrogenolysis by selective production of 2-methyl furan (MF), while the Co3O4 and Co phases promote preferential hydrogenation of side chain (carbonyl group) of FAL to furfuryl alcohol. Density functional theory calculations also reveal that the adsorption of FAL on CoO(111) is higher than Co(111). Overall, these studies demonstrate that CoO as the most active phase is responsible for the high FAL conversion and the distinct pathway of FAL to MF.

Nitrogen heterocycles form peptide nucleic acid precursors in complex prebiotic mixtures

The ability to store information is believed to have been crucial for the origin and evolution of life; however, little is known about the genetic polymers relevant to abiogenesis. Nitrogen heterocycles (N-heterocycles) are plausible components of such polymers as they may have been readily available on early Earth and are the means by which the extant genetic macromolecules RNA and DNA store information. Here, we report the reactivity of numerous N-heterocycles in highly complex mixtures, which were generated using a Miller-Urey spark discharge apparatus with either a reducing or neutral atmosphere, to investigate how N-heterocycles are modified under plausible prebiotic conditions. High throughput mass spectrometry was used to identify N-heterocycle adducts. Additionally, tandem mass spectrometry and nuclear magnetic resonance spectroscopy were used to elucidate reaction pathways for select reactions. Remarkably, we found that the majority of N-heterocycles, including the canonical nucleobases, gain short carbonyl side chains in our complex mixtures via a Strecker-like synthesis or Michael addition. These types of N-heterocycle adducts are subunits of the proposed RNA precursor, peptide nucleic acids (PNAs). The ease with which these carbonylated heterocycles form under both reducing and neutral atmospheres is suggestive that PNAs could be prebiotically feasible on early Earth.

A highly efficient step-wise biotransformation strategy for direct conversion of phytosterol to boldenone

Collaborative microbial communities are ubiquitous in nature and exhibit appealing functions for enhanced production of natural products, which provides new possibility for biotechnology development. In this study, we bridged Mycobacterium neoaurum with Pichia pastoris to establish a step-wise biotransformation strategy for efficient biosynthesis of boldenone (BD) from phytosterol (PS). Firstly, the producing strains were rationally designed with overexpression of 3-ketosteroid-Δ1-dehydrogenase (KsdD) and 17β-hydroxysteroid dehydrogenase (17βHSD) in M. neoaurum and P. pastoris, respectively. Then, to shorten the total biotransformation process and provide reducing power, semi-batch fermentation strategy and glucose supplementation strategy were introduced at side-chain degradation stage and carbonyl reduction stage, respectively. Under the optimal transformation conditions, the productivity of BD was increased from 10% to 76% and the total biotransformation process was shortened by 41.7%, which is the shortest among the ever reported. Our results demonstrated an excellent biological strategy for production of many other valuable microbial products from bioresources.

Degradation of Aqueous Polycyclic Musk Tonalide by Ultraviolet-Activated Free Chlorine

Chlorine-incorporating ultraviolet (UV) provides a multiple barrier for drinking water disinfection. Meanwhile, post-UV employment can promote the degradation of micropollutants by radical production from chlorine residual photolysis. This work studied the degradation of one such chemical, tonalide (AHTN), by low-pressure UV-activated free chlorine (FC) under typical UV disinfection dosage of <200 mJ·cm−2 and water matrix of filtered tank effluent. AHTN was rapidly degraded by UV/FC in accordance with pseudo-first-order kinetics. The reaction rate constants of AHTN with reactive chlorine species and hydroxyl radical (HO•) were estimated. Mechanistic exploration evidenced that under UV/FC, AHTN degradation was attributable to direct photolysis, ClO•, and HO•. The carbonyl side chain of AHTN served as an important attack site for radicals. Water matrices, such as natural organic matter (NOM), HCO 3 − , Cu 2 + , PO 4 3 − , and Fe 2 + , showed noticeable influence on the UV/FC process with an order of NOM > HCO 3 − > Cu 2 + > PO 4 3 − > Fe 2 + . Reaction product analysis showed ignorable formation of chlorinated intermediates and disinfection byproducts.

Anticholinesterase, antioxidant potentials, and molecular docking studies of isolated bioactive compounds from Grewia optiva

ABSTRACTGrewia optiva Drummond ex Burret (Tiliceae) is extensively applied for the treatment of typhoid, diarrhea, malaria, etc., in traditional medication. In this study, four bioactive compounds I (β- sitosterol), II (1, 2, 3 benzene triol), III (7-O-methyl cathachin) and VI (betulinic acid) were isolated from the root of G. optiva. The compounds were characterized through UV, FTIR, NMR, and mass spectroscopic techniques. The isolated compounds were tested for the inhibition of synthetic-free radicals: ABTS and DPPH, and anticholinesterases: AChE and BChE. Compound III demonstrated highest percent free radicals scavenging activities against DPPH and ABTS (86.11 ± 2.20, 85.23 ± 1.61, respectively, for 1000 µg/ml concentrations) with IC50 of 63 µg/ml which were comparable to the IC50 value (35 µg/ml) of ascorbic acid. All isolated compounds also showed excellent inhibitory potential against AChE and BChE. However, β-sitosterol amongst them exhibited highest percent inhibition (87.66 ± 1.93, 85.71 ± 1.17, respectively, at 1000 µg/ml concentration) with IC50 of 62 µg/ml, which was close to the standard galanthamine IC50 values (40 µg/ml). To support the findings of the study silico docking model was applied that predicted possible binding modes of β-sitosterol with AChE and BChE. The para hydroxyl group of the phenolic moiety interacted with the active site water molecule and the side chain carbonyl residues through H-bonding. The remaining part of the active compound packed in a shallow pocket through H-bond.

Characterizing Novel Modifications of a Therapeutic Protein Using Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry, Sedimentation Velocity Analytical Ultracentrifugation, and Structural Modeling.

Heterogeneity of biopharmaceutical products is common due to various co- and post-translational modifications and degradation events that occur during the biological production process and throughout the shelf life. Product-related variants resulting from these modifications potentially affect a product's biological activity and safety, and thus, their detailed structure characterization is of great importance for successful development of protein therapeutics. Specifically, in this study, two novel low-level product variants in a recombinant therapeutic protein were characterized via chromatographic enrichment followed by proteolytic digestion and analysis using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). One of the variants was identified to be the therapeutic protein missing a 61-amino-acid fragment from its N-terminus. Consequently, the other variant was found to be the therapeutic protein carrying the 61-amino-acid long peptide. Furthermore, detailed structure at the modification site of the latter variant was determined as that amino group from the protein's N-terminus linked to side chain carbonyl carbon at Asp 61 residue of the peptide, based on the complementary information from collision induced dissociation and electron transfer dissociation MS/MS analysis. Results from sedimentation velocity analytical ultracentrifugation and computational structural modeling supported the hypothesis that formation of these two variants was a result of protein self-association. In dimeric state, the head-to-toe stacking conformation of two therapeutic protein molecules allowed spatial closeness between the N-terminus of one molecule and the 61st amino acid of the other molecule, resulting in a novel peptide transfer between the two protein molecules.

Interaction Enthalpy of Side Chain and Backbone Amides in Polyglutamine Solution Monomers and Fibrils.

We determined an empirical correlation that relates the amide I vibrational band frequencies of the glutamine (Q) side chain to the strength of hydrogen bonding, van der Waals, and Lewis acid-base interactions of its primary amide carbonyl. We used this correlation to determine the Q side chain carbonyl interaction enthalpy (Δ Hint) in monomeric and amyloid-like fibril conformations of D2Q10K2 (Q10). We independently verified these Δ Hint values through molecular dynamics simulations that showed excellent agreement with experiments. We found that side chain-side chain and side chain-peptide backbone interactions in fibrils and monomers are more enthalpically favorable than are Q side chain-water interactions. Q10 fibrils also showed a more favorable Δ Hint for side chain-side chain interactions compared to backbone-backbone interactions. This work experimentally demonstrates that interamide side chain interactions are important in the formation and stabilization of polyQ fibrils.