A new method for the preparation of tetraalkyl ethenylidenebis(phosphonates) has been developed. This two-step procedure involves the base-catalyzed reaction of a methylenebis(phosphonate) ester with paraformaldehyde followed by acid-catalyzed elimination of methanol. The effecta of variations in the reaction conditions of the first step of this process are described. Ethenylidenebis(phosph0nate) esters can be converted to the free acid by reaction with bromotrimethylsilane.Ethenylidenebis(phosph0nic acid) (1) and its esters have found utility as sequestering agents,' in the development of polymeric flame retardants,2 and in certain pharmaceutical application^.^ Currently, ethenylidenebis(ph0s-Registry No.

Echistatin is a 49-amino-acid protein from Echis carinatus venom. It contains four disulfide bonds. Since the disulfide bonding is critical for biological activity; it is very important to assign the disulfide linkage in this protein. Echistatin was incubated in 250 mM oxalic acid at 100 degrees C for 4 hr under nitrogen. Under these conditions, many overlapping disulfide-containing peptides were identified by ionspray mass spectrometry. Ionspray MS/MS data indicate that the four disulfide bonds are Cys 2-Cys 11, Cys 7-Cys 32, Cys 8-Cys 37, and Cys 20-Cys 39. To our knowledge, this is the first time all four disulfide bonds in echistatin have been assigned in one experiment without disulfide bond exchange. This approach, which combines oxalic acid hydrolysis and ionspray MS/MS, may be very useful for assigning disulfide bridges in other proteins from the disintegrin family.

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