Development Of A Ratiometric Fluorescent Sensor For Methylglyoxal

Reactive carbonyl species(RCS)are a family of molecules with highly reactive carbonyl groups that are continuous produced via oxidation of carbohydrates,lipids and amino acids in various living organism from bacterial to human.Representative examples are 4-hydroxy-trans-2-noneal,4-oxo-trans-2-nonenal,acrolein,acetaldehyde,methylglyoxal,glyoxal,malondialdehyde,glycolaldehyde,ribosome,glucose,3-deoxyglucose and formaldehyde.So far many of these species have been demonstrated to play important roles in physiological and pathological processes.Among these species,methylglyoxal(MGO)has received increasing attention,because it has been suggested to be a major precursor of advanced glycation end products(AGEs).MGO is endogenous generated from glycolysis,lipid peroxidation and protein amino acid metabolism and can react with free amino groups of lysine or arginine.It can also react with thiol groups of cysteine to form AGEs.The MGO-mediated AGEs may induce protein dysfunction,activation of membrane receptors and proinflammatory signaling,exerting multiple and diverse effects in cellular oxidative stress,inflammation,and endothelial cells dysfunction.Literature published in the past several years also demonstrate that elevated levels of MGO are associated with pathologies of serious human diseases such as obesity,cardiovascular disease,hyperalgesia,kidney disease,metabolic syndrome,colorectal cancer,and diabetes.The elevation of MGO levels in diabetic human blood and urine is widely observed.MGO has also been reported to be involved in the mechanisms which may contribute to the development and progression of diabetes.It indicates that the long-used diagnostic makers blood glucose and glycated hemoglobin(HbAic)are insufficient to predict the development of diabetic complication.Owing to the correlation and association between MGO-induced AGEs levels and clinic features of various diabetic complications such as diabetic nephropathy,retinopathy and cardiovascular,MGO is suggested to be a potential maker for diabetic complication.One of critical debates in this field is biologically relevant levels of MGO as current reports spanning over 103-fold concentration range.Accurate and reliable measurement of MGO concentration in biological samples is needed for better understanding of it's roles in cellular process and pathologies of related diseases.Currently,the major methods for MGO detection are electrochemical and absorbance-based assays,HPLC and LC-MS approaches.These methods often require complicated post-mortem processing and destruction of tissues or cell samples.Fluorescence imaging is a powerful tool for real-time visualization of biomolecules in living systems because of its high sensitivity,non-invasiveness and convenience.To the best of our knowledge,only one example of MGO fluorescent probe MBO for cell imaging,it has been reported based on the MGO-induce cyclization of o-phenylenediamine(OPD)moiety by Spiegel et al.Nevertheless,the OPD of MBO can react with another important biological small molecular NO,displaying significant response to NO which may interfere with MGO imaging in biological systems.On the other hand,as a turn-on fluorescent probe,the detection signal of MBO relies on single emission intensity change,which can be distinctly affected by instrument efficiency,light scattering,local probe concentration and microenvironment.In contrast the ratiometric fluorescent probes of MGO,employing the ratio of the emission intensity at two different wavelengths,could provide a self-calibration effect to reduce most of aforementioned interference,and thus enable more accurately quantitative tracking of MGO in biologic samples.Therefore,the development of ideal ratiometric fluorescent probe for the detection of endogenous MGO with good selectivity is still required.Herein,we find 2-amino-2-phenylacetamide can react with MGO through model reactions with high specificity.So,we present the first ratiometric fluorescent probe with good selectivity over other biologic relevant species(including NO and FA)for the detection of endogenous MGO in living cells,named WHL45.The ESIPT process will be inhibited after WHL45 interacting with MGO.In addition,WHL45 can be used to investigate the formation approach of endogenous MGO in living cells.Moreover,we also demonstrate its' application in real clinic samples such as the whole blood cells from normal diabetes and diabetes complication patients.Our results indicate that WHL45 could be potentially used not only for basic science research but also for clinic practice.