The sperm proteins fertilinβ and cyritestin, members of the ADAM family of proteins, are critical for mammalian sperm-oocyte binding. Multivalent probes containing the three-amino acid binding sequence of fertilinβ, ECD, and cyritestin, QCD have been designed, synthesized, and tested to investigate gamete interactions.

To improve the binding affinity of monomeric peptide ligands for the oocyte surface, we developed synthetic multivalent polymers to mimic the multivalent display of fertilinβ on the sperm surface. We employed ruthenium-catalyzed ring opening metathesis polymerization (ROMP) to prepare these mimics. Tricyclohexylphosphine substituted ruthenium catalyst 2, [(H₂IMes)(PCy ₃)(Cl)₂Ru=CHPh] did not initiate efficiently with norbornenyl monomers, and the resulting polymers had uncontrolled and broadly distributed molecular weights. However, 3-bromo-pyridine substituted ruthenium catalyst 3, [(H₂IMes)(3-Br-pyr)(Cl)₂Ru=CHPh] initiates more rapidly than propagation. Therefore, the controlled living ROMP of norbornenyl polymers was achieved.

Fertilinβ mimic polymers 9₁₀₀-[3] synthesized with catalyst 3 were compared to previously synthesized fertilinβ mimic polymers. In addition, the inhibition potencies of multivalent cyritestin mimic polymers 18_n-[3] were tested. Tri-block copolymers 9₂-13₉₆-9₂-[3] and 18₂ -13₉₆-18₂-[3] containing both ECD and QCD ligands were assayed to explore the relationship and degree of identity between fertilinβ and cyritestin receptors. These block copolymers were the same length as their corresponding homopolymers, but they had a lower density of fertilinβ and cyritestin peptides. The homo and block polymers had the same potencies, and there was no synergy of inhibition between fertilinβ and cyritestin mimics. Both polymers require the oocyte β₁ integrin for inhibition. We conclude that QCD and ECD mimics bind in the same complex of proteins and may bind to the same receptors.

The structural requirements defined were incorporated into new linear polymer systems that include tagging elements for probing the identity of oocyte surface receptors for fertilinβ and cyritestin. Bivalent homopolymers were used to minimize non-specific interactions. Fluorescent fertilinβ and cyritestin mimic polymers labeled the oolemma of the wild-type mouse oocytes, but not the oolemma of β₁ integrin knockout mouse oocytes. Thus, β₁ integrins are required for binding of fertilinβ and cyritestin mimic polymers. The polymer synthesis was further developed to enable photo cross-linking and identification of additional members of the integrin receptor complex.