Glycosylation Modification,anti-inflammatory Activity,and Interaction With PDIA6 Protein Of Benzophenone Polyphenols

Objective:Recombinant human protein disulfide-isomerase A6（PDIA6）,involved in endoplasmic reticulum stress,is closely related to inflammation and oxidative stress.In our previous research work,polyphenol compounds 5,2’-dibromo-2,4’,5’-trihydroxydiphenylmethanone（LF1）,5,2’-dibromo-2-hydroxy-4’,5’-dimethoxylbenzophenone（LF2）,3,4-dihydroxy-4’-tert-butylbenzophenone（LF3）,and2-bromo-3,4-dihydroxy-4’-tert-butylbenzophenone（LF4）were obtained with the strong anti-inflammatory and antioxidant activities.However,its high lipid solubility may reduce the drug-likeness of compound.A large number of glycoprotein and glycolipid receptor molecules exist in the cell envelope,introducing the glycosyl groups in lipid-soluble molecules can improve water solubility and even increase targeting ability.In the present study,we aimed to synthesize a series of glycosyl derivatives by introducing the glycosyl groups to the lead compounds LF1,LF2,LF3,and LF4,respectively,investigate their interaction with PDIA6 by Biolayer interferometry（BLI）technology,and further evaluate their anti-inflammatory abilities,to improve the water solubility and obtain the“hit”compounds with the better druggability.Methods:（1）A series of glycosyl derivatives were synthesized starting fromα-bromo acetyl glycosyl by catalytic etherification and deacetylation with LF1,LF2,LF3,LF4 as the lead compounds,and their structures were confirmed by ESI-MS,¹H NMR,¹³C NMR spectra,even ¹H-¹H NOESY.（2）The dynamical process of the combination between the target compound and PDIA6 was monitored to detect the binding specificity by BLI technology.（3）The LPS-induced inflammation model on RAW264.7 cells was constructed,and the cell viabilities were measured by CCK-8 assay.The levels of IL-1β,TNF-α,IL-6,and IL-10 were also detected with ELISA kit,the anti-inflammatory abilities of target compounds were therefore evaluated.Results:（1）Twenty-five target compounds were synthesized,and all compounds were not been reported.Their structures were confirmed by ESI-MS,¹H NMR,and ¹³C NMR spectra.（2）Eight target compounds can combine with PDIA6 in a concentration dependent manner,which also verified the specific binding.The equilibrium dissociation constant（K_D）values of 4b,8b,17b,24b,and 25b were ranged from 8.70×10^-4 to 3.71×10^-5mol/L,which were equivalent to the lead compound LF1 with a K_D value of 6.56×10^-4mol/L.The K_D value of 10b was 7.74×10^-5 mol/L,which were equivalent to the lead compound LF2 with a K_D value of 4.82×10^-5 mol/L.The K_D value of 18b and 23 b were 1.45×10^-4 mol/L and 2.69×10^-4 mol/L respectively,which were lower than the lead compound LF4 with a K_D value of 1.37×10^-5 mol/L.（3）At 10μM,all target compounds presented the significant inhibitory effects on LPS-induced IL-1βproduction in RAW264.7 cells,target compounds 4b,5b,6b,7b,9b,10b,12b,13b,14b,15b,16b,17b,18b,19b,and 25b can inhibit the expression of IL-6,compound 1b can inhibit the expression of TNF-α,and compounds 2b,19b,24b and 25b can promote the production of IL-10.Conclusion:（1）Twenty-five glycosyl derivatives were obtained,and all compounds were not been reported.（2）Target compounds exhibited the strong inhibitory effects on LPS-induced inflammation in RAW264.7 cells,of which,compounds 4b,10b,17b,18b,and 25b not only specifically combined with PDIA6,but also significantly inhibited the levels of IL-1βand IL-6,and 25b can also promote the production of IL-10.