SCREENING SOLUTION-PHASE COMBINATORIAL LIBRARIES USING PULSED ULTRAFILTRATION ELECTROSPRAY MASS-SPECTROMETRY

A met bud Is described whereby a family of homologues is synthesized i n a one-pot reaction, without isolation or purification, and the react ion mixture is screened using a competitive binding assay based on pul sed ultrafiltration/electrospray mass spectrometry (PUF/ESMS) to tenta tively identify those derivatives hatting the highest affinity for a t arget receptor. As a model system to test this approach, a synthetic s cheme designed to prepare a series of analogues of the adenosine deami nase inhibitor erythro-9(2-hydroxy-3-nonyl)adenine (EHNA), as dia-ster eomeric mixtures, was carried out. Pulsed ultrafiltration screening of the crude reaction mixture against controls without protein detected protonated molecules corresponding to EHNA-type derivatives and three of its linear, alkyl homologues but did not show protonated molecules for an isobutyl or benzylic EHNA derivative, suggesting the latter was inactive. To verify this conclusion, we prepared E/THNA, the linear h omologues, and the benzylic derivative (each as a diastereomeric mixtu re) and bioassayed them for their adenosine deaminase inhibition index ([I]/[S](0.5)). The bioassay results for the individually synthesized analogues were in good agreement with that predicted by the observed relative ion enhancement in the PUF experiments. Thus, the PUF protoco l might be used as a general method to quickly provide direction to th e chemist in search of drug candidates.