ABSTRACT
Methods for chemical modification of native proteins in a controlled fashion are in high demand. Here, a novel protocol that exploits bifunctional reagents for transient targeting of solvent exposed disulphides to direct the introduction of a single exogenous reactive thiol handle at a lysine side chain has been developed. The protocol has successfully been applied to functionalize six different Fabs and human growth hormone.
Subject(s)
Disulfides/chemistry , Growth Hormone/chemistry , Humans , Lysine/chemistry , Molecular StructureABSTRACT
Fatty acyl analogues of muramyldipeptide (MDP) (abbreviated N-L18 norAbuGMDP, N-B30 norAbuGMDP, norAbuMDP-Lys(L18), norAbuMDP-Lys(B30), norAbuGMDP-Lys(L18), norAbuGMDP-Lys(B30), B30 norAbuMDP, L18 norAbuMDP) are designed and synthesized comprising the normuramyl-l-α-aminobutanoyl (norAbu) structural moiety. All new analogues show depressed pyrogenicity in both free (micellar) state and in liposomal formulations when tested in rabbits in vivo (sc and iv application). New analogues are also shown to be selective activators of NOD2 and NLRP3 (inflammasome) in vitro but not NOD1. Potencies of NOD2 and NLRP3 stimulation are found comparable with free MDP and other positive controls. Analogues are also demonstrated to be effective in stimulating cellular proliferation when the sera from mice are injected sc with individual liposome-loaded analogues, causing proliferation of bone marrow-derived GM-progenitors cells. Importantly, vaccination nanoparticles prepared from metallochelation liposomes, His-tagged antigen rOspA from Borrelia burgdorferi, and lipophilic analogue norAbuMDP-Lys(B30) as adjuvant, are shown to provoke OspA-specific antibody responses with a strong Th1-bias (dominance of IgG2a response). In contrast, the adjuvant effects of Alum or parent MDP show a strong Th2-bias (dominance of IgG1 response).
Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/analogs & derivatives , Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Adjuvants, Immunologic/pharmacology , Antigens, Surface/pharmacology , Bacterial Outer Membrane Proteins/pharmacology , Bacterial Vaccines/pharmacology , Borrelia burgdorferi/immunology , Lipoproteins/pharmacology , Acetylmuramyl-Alanyl-Isoglutamine/chemistry , Acetylmuramyl-Alanyl-Isoglutamine/immunology , Adjuvants, Immunologic/chemistry , Animals , Antibody Formation , Antigens, Surface/chemistry , Antigens, Surface/immunology , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines/chemistry , Bacterial Vaccines/immunology , Female , HEK293 Cells , Humans , Immunization , Lipoproteins/chemistry , Lipoproteins/immunology , Lyme Disease/immunology , Lyme Disease/microbiology , Mice , Mice, Inbred BALB C , NLR Family, Pyrin Domain-Containing 3 Protein/agonists , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , RAW 264.7 CellsABSTRACT
A simple and scalable synthesis of 2,14-dithiacalix[4]arene with alternating bridges (-CH2- and -S-) is reported. Proper selection of the bisphenol-based starting building blocks can provide not only the title compound (58%) but also yet unreported homooxa analogues possessing three different bridging units (-CH2-, -S- and -CH2-O-CH2-) in the molecule. These systems exhibit interesting conformational behaviour allowing for the study of flip-flop motion of the circular hydrogen bond arrays using dynamic NMR techniques.
