Orthogonal pooling

Primary screens were performed in duplicate on mixtures of 80 compounds (1 pM per compound, 5 pM protein), where samples in each mixture were orthogonally pooled so that no two compounds that are in one well are also together in another well. Primary hits were achieved when the same compound in two wells were observed. The GPC spin-column eluates were partially resolved using a reversed-phase C18 HPLC column (Waters Xterra 2.1 x 20.0 mm) with a 1-min ballistic gradient and total cycle time of 2 min. The HPLC eluates were analyzed with a Micromass eight-way MUX interfaced to an LCT Tof ESI-MS system. Achieved throughput was 100 000 compounds per day. 

Pooling experiments are of two basic types, simple or orthogonal. In simple pooling, each individual appears in exactly one pool see Figure 1(a), where each circle in the box represents an individual, and individuals in the same column are in the same pool. An active pool response must be followed by individual testing to determine which specific individuals in the pool are active. In orthogonal pooling... 

Figure 1. (a) Simple pooling Individuals, represented by circles, are pooled according to their column location each individual appears in exactly one pool, (b) Two-way orthogonal pooling Individuals are pooled according to both their column and row locations each individual appears in exactly two pools. 

Orthogonal pooling. The term arthoganal means perpendicular or uncorrelaled. In this type of prxiling. we disirihuic the... 

The synthetic advantages of preparing mixtures instead of individual compounds are offset by the need to deconvolute mixture libraries following bioassay. Deconvolution shortcuts, such as synthesis of multiple libraries containing orthogonal pools (36), are sometimes useful but can only be relied on when the activity of the mixture results from a single highly active compound. Keep-... 

Orthogonal gradients, when combined with a large pool of fimctional polymers, offer powerful combinatorial platforms for studying complex phenomena they can be tailored to serve as separation devices or as templates for organization of nonpolymeric materials, hi the following section, we will elaborate further on some of these apphcations. 

The earliest report of a solution-phase combinatorial library of small drug-like molecules was by Smith et al. [2], The reaction of 40 individual acid chlorides with an equimolar mixture of 40 nucleophiles (alcohols or amines) and of the individual nucleophiles with an equimolar mixture of the acid chlorides produced a library of 1600 amides and esters as pools of 40 compounds. The orthogonal design strategy determined that each possible product should appear in a unique pair of samples to allow the rapid identification of lead compounds after screening. 

Screening large pools of compounds has several disadvantages, and often results in missed or false activities. To obviate such limitations, different pooling and structure identification strategies such as orthogonal libraries and binary encoded libraries have been developed and reported to provide examples of bioactive molecules. 

---