Spontaneous Determinants of Protein Aging Mini Review

The universal chirality is the commonly accepted view of nature. Biological chirality is the distinct part of the more general phenomena. Following this view, all living organisms are characterized by the non-equilibrium state of their molecular constituents. From the thermodynamic perspective, the non-equilibrium state of biomolecular ensemble holds inevitable consequences being the substrate of spontaneous reactions directed to equilibrium (not associated with life) state. At the protein level, spontaneous biological reactions represent the natural part of proteins' post-translational modifications (PTMs). The essential contribution to the origin and maintenance of the non-equilibrium state belongs to prevalent bio-molecular chirality. Correspondently, spontaneous PTMs such as racemization and glycation, working against life-supporting prevalent chirality, are known as the significant determinants of protein misfolding, dysfunctions, and aggregation. Accumulation of aberrant protein during life-span allows consideration of time-dependent spontaneous racemization and glycation as protein aging. Spontaneous PTMs of proteins is occurring in the interaction with other forms of enzymatic and non-enzymatic PTMs. In this review, we are considering the contribution of spontaneous racemization and non-enzymatic glycosylation to protein aging.

Synthesis, Analytical Characterization and Anti-Diabetic Activity of Some Heterocycles of Quinazolin-4(3H)one

Aim: Novel quinazolin-4(3H)-one heterocycles were synthesized and assessed for their anti-diabetic activity. Non-enzymatic glycosylation of haemoglobin assay was carried out to identify their potential as anti-diabetic. The cyclization of quinazolinone-4(3H)-one heterocycles was achieved, whereas carbon-carbon cross coupling reactions were carried out using Sonogashira and Suzuki-Miyaura reaction conditions and characterized with analysis. This synthesis method afforded corresponding 2, 3 and 6 substituted quinazolin-4(3H)-ones (3a to 3m) with excellent yields. Methods: 2-Amino-6-bromobenzoic acid was used as a substrate which was converted to corresponding benzamide derivatives (1a-1b) by reaction with benzylamine or cyclohexylamine using acid-amine reaction, followed by cyclization and oxidation using suitable aldehyde in DMSO under microwave condition to give bromo substituted quinazolin-4(3H)-ones (2a-2c), which were cross coupled to suitable terminal alkyne with palladium catalyst as well as copper co-catalyst using Sonogashira condition to obtain desired (3a-3h) and suitable boronic acid with palladium catalyst using Suzuki-Miyaura condition to obtain desired (3i-3m). All synthesized compounds were characterized by FTIR, proton NMR, LC-MS analysis and evaluated for in vitro anti-diabetic activity using non-enzymatic glycosylation of haemoglobin assay. Results: Compounds 3m showed good inhibition of glycosylation of haemoglobin which in turn suggest good anti-oxidant potential on metabolism of glucose and hence lower glucose concentration. It showed IC50 value of 35.91±0.82 µg/mL which was comparable to the standard alpha tocopherol (34.47±0.87µg/mL). Conclusion: In-vitro non-enzymatic glycosylation of haemoglobin method is one of important assays to judge the control of diabetes. The haemoglobin present in RBCs has an affinity to bind with glucose. The greater the glucose level in blood, more amount of glucose-bound (called glycosylated) haemoglobin will be formed. Accordingly, presence of lower concentration of glycosylated haemoglobin is a sure guide to the lower concentration of glucose in the blood. Synthesized compounds (3a-3m) lower the blood glucose level and 3m has highest potential among those which can be further developed as potent anti-diabetic.

Inhibitory effect of oleanolic acid on non-enzymatic glycation and glycometabolism in insulin resistant HepG2 cells

In this paper, we studied the inhibitory effect of oleanolic acid (OA) on non-enzymatic glycosylation and the improvement of glycometabolism in insulin resistant (IR) human liver tumour (HepG2) cells. The anti-glycosylation activity of OA was determined by bovine serum albumin (BSA) fructose model. The results showed that OA moderately inhibited the formation of the intermediates of non-enzymatic glycosylation, fructosamine and α-dicarbonyl compounds, and strongly inhibited the formation of advanced glycation end products (AGEs). In addition, we analysed the effect of OA on glycometabolism induced by palmitic acid (PA) in HepG2 cells. The results showed that OA had almost no impact on HepG2 cell viability at concentrations lower than 30 µM. With the increase of OA concentration, glucose production in IR HepG2 cells decreased, while glycogen content increased. Meanwhile, OA has a significant inhibitory effect on reactive oxygen species (ROS) levels in IR-HepG2 cells. Those results suggested that OA could be a promising natural blood glucose decreasing substance in the pharmaceutical and functional food industries.

Acceptor-mediated regioselective enzyme catalyzed sialylation: chemoenzymatic synthesis of GAA-7 ganglioside glycan

The glycan of GAA-7 was facilely synthesized by enzymatic glycosylation through changing the nonreducing acceptor into GalNH2 or GalAz to successfully switch the regioselectivity of PmST1.

2’-Deoxyribose Mediated Glycation Leads to Alterations in BSA Structure Via Generation of Carbonyl Species

The non-enzymatic glycosylation is a very common phenomenon in the physiological conditions which is mediated by distinct chemical entities containing reactive carbonyl species (RCS) and participates in the modification of various macromolecules particularly proteins. To date, various carbonyl species, i.e., glucose, fructose, D-ribose and methylglyoxal have been used frequently to assess the in-vitro non-enzymatic glycosylation. Similarly, 2'-Deoxyribose is one of the most abundant reducing sugar of the living organisms which forms the part of deoxyribonucleic acid and may react with proteins leading to the production of glycation intermediates, advanced glycation end products (AGEs) and highly reactive RCS. Thymidine phosphorylase derived degradation of thymidine contributes to the formation of 2'-Deoxyribose, therefore, acting as a major source of cellular 2'- Deoxyribose. Since albumin is a major serum protein which plays various roles including binding and transporting endogenous and exogenous ligands, it is more prone to be modified through different physiological modifiers; therefore, it may serve as a model protein for in-vitro experiments to study the effect of 2’Deoxyribose mediated modifications in the protein. In this study, Bovine Serum Albumin (BSA) was glycated with 50 and 100 mM 2'-Deoxyribose followed by examining secondary and tertiary structural modifications in BSA as compared to its native (unmodified) form by using various physicochemical techniques. We evident a significant modification in 2'-Deoxyribose-glycated BSA which was confirmed through increased hyperchromicity, keto amine moieties, carbonyl and hydroxymethylfurfural content, fluorescent AGEs, altered secondary structure conformers (α helix and β sheets), band shift in the amide-I region and diminished free lysine and free arginine content. These modifications were reported to be higher in 100 mM 2'-Deoxyribose-glycated BSA than 50 mM 2'- Deoxyribose-glycated BSA. Our findings also demonstrated that the rate of glycation is positively affected by the increased concentration of 2'-Deoxyribose. The results of the performed study can be implied to uncover the phenomenon of serum protein damage caused by 2'-Deoxyribose leading towards diabetic complications and the number of AGE-related diseases.

Non-enzymatic Stereoselective S-glycosylation of Polypeptides and Proteins

Glycosylation (1-4) is an essential and powerful technique that Nature employs to regulate the properties and functions of proteins and polypeptides. Our capacity to emulate Nature’s power, however, is limited by the methods available (5) to perform glycosylation on these complex biomolecules. So far, very few glycosylation reactions could operate under the conditions tolerated by biomolecules (e.g., aqueous media, mild pH, and ambient temperature), and the need to install glycosyl groups in a stereo-controlled fashion poses additional, significant challenges. Here we report a non-enzymatic glycosylation reaction that builds axial S-glycosidic bonds under biorelevant conditions. Our strategy exploits the exceptional functional group tolerance of radical processes, and is enabled by the design and use of allyl glycosyl sulfones as precursors to glycosyl radicals. Our method could introduce a variety of glycosyl units to the cysteine residues of polypeptides in a highly selective fashion. The power of this method is further demonstrated in the direct glycosylation of bioexpressed proteins. Computational and experimental studies provide insights into the reaction mechanism.