Direct screening of solution phase combinatorial libraries encoded with externally sensitized photolabile tags

Abstract

dithiane tag ͉ photoinduced externally sensitized fragmentation ͉ solution library encoding and screening H igh-throughput combinatorial chemistry is critical for modern drug discovery, with the most prolific adaptation being the split-and-mix synthesis of libraries immobilized on polymeric beads (1, 2). These one-bead-one-compound libraries are assayed for binding of biological targets through fluorescenceguided mechanical segregation of the winning beads. The required mechanical manipulations impose a lower limit on the size of the particle used as a solid support. It is further recognized that solution phase combinatorial chemistry holds even greater promise, as it is compatible with both divergent and convergent multistep synthetic schemes, and not constrained to linear synthesis (3). However, its immense potential has not yet been fully realized, partly due to the complexity of assaying solution mixtures for binding. Although various iterative deconvolution methods (4) have been developed to synthesize and screen soluble sublibraries, all of them require redundant synthetic steps and are time consuming and expensive.We have developed a methodology for direct screening of solution phase libraries encoded with photolabile tags that does not depend on mechanical manipulation or iterative deconvolution. Results and DiscussionOur tagging methodology is based on dithiane adducts of carbonyl compounds, which are capable of efficient photoinduced fragmentation, but only in the presence of an external electron transfer (ET) sensitizer. At the core of this approach is the concept we term photolabile scaffolds for molecular recognition: binary molecular systems in which the sensitizer and the dithiane-based photocleavable fragments are each tethered to the respective components of a host-guest molecular recognition pair (5-7). In these pairs, photoinduced fragmentation is contingent on a molecular recognition event, which is necessary to arm the system, making it photolabile. This general concept has now been developed into a methodology for screening of encoded libraries, both unsupported or immobilized on nanosized carriers too small for mechanical handling. Scheme 1 gives a general outline for screening the micelle-solubilized library of ligands L, encoded with the tethered tags T. The receptor R is outfitted with an ET-sensitizer S, and incubated with the library (A). The host-guest binding brings the sensitizer into the vicinity of the adduct (B), which, upon irradiation (C), triggers the release of dithiane tags (D).Our strategy is to encode individual molecules one tag at a time. The kth library member L k can be encoded with a set of tags {T} k, for example, T k1 , T k2 , and T k3 , in the following fashion: one fraction of L k molecules is encoded with T k1 , another fraction is encoded with T k2 , and yet another is encoded with T k3 , such that L k is present in the solution as three subpopulations: L k -tether-T k1 , L k -tether-T k2, and L k -tether-T k3 . In this case, irradiation yields the desired resu...

Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy

“…457 Dithiane-based photolabile tags have also been used for encoding and direct screening of solution-phase combinatorial libraries. 458 …”

Externally sensitized deprotection of PPG-masked carbonyls as a spatial proximity probe in photoamplified detection of binding events

“…Based on this PPG chemistry we have developed a novel binding assay for screening of combinatorial libraries encoded with mass-differentiated dithiane tags, 8 where every successful spatial proximity event releases a unique encoding tag into solution. The tag can then be readily detected by a standard analytical method such as GCMS.…”

Photoactive spatial proximity probes for binding pairs with epigenetic marks

“…Synthesis of 2-alkyl dithianes 14 and dithianyl benzylamine-based tagging pendants 4 20 was previously described.…”

“…14 In these experiments a mini library of compounds, which included biotin, was encoded by tagging each compound with different dithianes. Scheme 2 gives a general outline for screening of such library of ligands (biotin is depicted by a pink "B" pentagon), encoded with the tethered tags (pink circles 1–5).…”

“…So far our dithiane tags-encoding methodology was tested with a model barbiturate-binding artificial receptor 15 and validated for the avidin-biotin binding pair, 14 known to have a very tight K D . In this study we extend this approach onto biological systems exhibiting low micro molar affinities and demonstrate that it can be used to differentiate between very similar epitopes modified via post-translational modifications (PTMs), such as unmodified and methylated lysine.…”