Design,Synthesis And Activity Determination Of QS-21 Analogue As An Immune Adjuvant

QS-21,an immunostimulatory oleanolane-type triterpenoid saponin,is widely used in clinical studies of various vaccines because of its remarkable adjuvant potency.In view of its structural complexity,poor stability,difficulty in purification,dose-limiting toxicity and undesirable hemolytic effects,this project was designed to modify the four structural domains of QS-21,i.e.,triterpene unit,trisaccharide and tetrasaccharide structures,acyl side chain,in order to discover QS-21 analogues with convenient preparation,enhanced immune effect and high efficiency and low toxicity.The main components are as follows:(1)development of universal D/E ring functionalized triterpene unit,and design and synthesis of A fragment by introducing carbonyl and oxime functional groups at C3 position and hydroxyl group at C2 position,respectively,using oleanolic acid as raw material;(2)optimization of branching trisaccharide and bridged linear tetrasaccharide reconstructions by replacing them with inexpensive sugar molecules such as glucose,cellobiose,and maltotriose,and attempting to excise the tetrasaccharide fragment by The synthesis steps were shortened,i.e.,B and C fragments were designed;(3)the complex structure of the multi-chiral central acyl chain was modified,and attempts were made to introduce glucose,cellobiose and maltotriose at the end of the acyl chain and to replace the ester part connected to the sugar unit with an amide bond structure to simplify the structure and improve the stability,i.e.,D fragments were designed;(4)on this basis,each fragment was synthesized by glycosylation and acylation reactions to link The structure of the 24 QS-21 analogues was confirmed by NMR hydrogen spectroscopy,carbon spectroscopy and mass spectrometry.The in vitro hemolysis assay was performed on the designed and synthesized QS-21 analogues,and the concentration-hemolysis relationship curves were plotted by the calculation of hemolysis rate.The study showed that the anti-hemolytic activity of all the QS-21 analogues was superior to that of QS-21,and their concentrations showed a certain quantitative-effect relationship with the hemolysis rate.Among them,the saponin compounds L1-L9 all did not produce hemolysis and showed good hemolysis inhibition;L11-L14 showed high hemolysis rate at a concentration of 500 μg/m L.And the hemolytic effect curves of L12,L13 and L18 all had similar S-shaped relationship curves compared with QS-21,presumably their hemolytic mechanism of action was similar or the same as QS-21.Preliminary conformational relationship studies showed that saponin compounds with unmodified saponin at the C3 position of glycosides,which have good antihemolytic activity,showed no significant differences in their hemolytic effects when different sugar chains were attached at the end of the acyl chain;In contrast,saponins oxidized at the C3 position are more likely to produce erythrocyte hemolysis side effects.