ABSTRACT
The sperm protein fertilinbeta, a member of the ADAM family of proteins, is implicated in sperm-egg binding in all mammals studied to date. Multivalent inhibitors containing the three amino acid binding sequence of fertilinbeta, ECD, have been shown previously to be more effective inhibitors of fertilization than their monovalent counterparts. Here, we probed sperm-egg interactions with ruthenium-catalyzed ring-opening metathesis polymers that contained from 3 to 70 ECD pharmacophores in densities ranging from 10% to 100%. Evaluation of the polymer potencies, and synthesis of a triblock copolymer from two building blocks, revealed that two multivalent contacts are sufficient for maximal inhibition, and that the distance between ECD pharmacophores required is 7-9 monomers spanning 4-5 nm. We conclude that inhibition requires recruitment of two receptors on the egg surface into an inhibitory complex.
Subject(s)
ADAM Proteins/chemistry , ADAM Proteins/pharmacology , Fertilization/physiology , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/pharmacology , Oligopeptides/chemistry , Oligopeptides/pharmacology , Sperm-Ovum Interactions/drug effects , ADAM Proteins/metabolism , Animals , Catalysis , Female , Fertilins , Male , Membrane Glycoproteins/metabolism , Mice , Oligopeptides/metabolism , Ruthenium/chemistry , Sperm-Ovum Interactions/physiology , Time FactorsABSTRACT
[structure: see text] To probe the activities of sperm ADAM protein (fertilinbeta), we devised a general synthetic strategy to generate fluorescently labeled fertilinbeta oligopeptide polymers. Immunofluorescence studies with these polymers demonstrated that fertilinbeta polymers bind specifically to a protein receptor on the mouse egg plasma membrane.
Subject(s)
Chemistry, Organic/methods , Fluorescent Dyes/chemical synthesis , Membrane Glycoproteins/metabolism , Metalloendopeptidases/chemical synthesis , Oligopeptides/chemical synthesis , Polymers/chemical synthesis , Receptors, Cell Surface/metabolism , ADAM Proteins , Animals , Female , Fertilins , Male , Membrane Glycoproteins/chemical synthesis , Metalloendopeptidases/metabolism , Mice , Microscopy, Fluorescence , Molecular Structure , Oocytes/metabolism , Spermatozoa/metabolism , StereoisomerismSubject(s)
Fertilization in Vitro , Membrane Glycoproteins/pharmacology , Metalloendopeptidases/pharmacology , Polymers/pharmacology , Sperm-Ovum Interactions/drug effects , ADAM Proteins , Animals , Female , Fertilins , Liposomes , Male , Membrane Glycoproteins/chemistry , Metalloendopeptidases/chemistry , Mice , Mice, Inbred ICR , Polymers/chemistryABSTRACT
The ring-opening metathesis polymerization (ROMP) reaction is extraordinarily useful for the preparation of a large variety of polymers. We report that the length (n = 25-50) of high-substituent-density oligopeptide polymers synthesized by ROMP is dramatically improved upon addition of LiCl to reduce polymer and oligopeptide aggregation. This methodology should significantly expand the variety of polymers that may be prepared by ROMP and be of general use with norbornyl oligopeptides of any sequence.
