Tethered Libraries

FIGURE 2.8 Binding test with tethered peptide library. 

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Enzyme-adduct formation has also been successfully achieved with the Erbl and 2 (receptor tyrosine kinases from EGFR subfamily) [19, 20], suggesting that the tethered library-screening approach would also be amenable to protein kinases. 

Libraries prepared by application of real combinatorial synthetic methods are usually submitted to screening experiments, either as soluble mixtures or as unknown discrete compounds cleaved from, or tethered to individual beads of the solid support. The task in deconvolution is to identify the substance that has a desired property. The deconvolution methods can be classified into two groups deconvolution of mixtures, cleaved from support and deconvolution of tethered libraries. 

Traviglia, S.L., Datwyler, S.A., and Meares, C.F. (1999) Mapping protein-protein interactions with a library of tethered cutting reagents The binding site of sigma (70) on Escherichia coli RNA polymerase. Biochemistry 38, 4259-4265. 

Quinazolines, the benzo derivatives of pyrimidines, were prepared in a variety of ways, from methods analogous to those for synthesizing pyrimidines to vastly different condensation schemes. In analogous fashion to many pyrimidine preparations, Yang and coworkers reported the condensation of polymer-tethered amidines 109 with cyclic anhydrides 110 to yield a library of 2-amino-4(3//)-quinazoIinones 111 after cleavage from the resin <00TL7005>. 

SIP-driven polymer brush library fabrication leverages the fact that the polymerization initiation species are permanently bound to the substrate. Since the initiators are tethered, controlled delivery of monomer solution to different areas of the substrate results in a grafted polymer library. In NIST work, initiators bound via chlorosilane SAMs to silicon substrates were suitable for conducting controlled atom transfer radical polymerization (ATRP) [53] and traditional UV free radical polymerization [54, 55]. Suitable monomers are delivered in solution to the surface via microfluidic channels, which enables control over both the monomer solution composition and the time in which the solution is in contact with the initiating groups. After the polymerization is complete, the microchannel is removed from the substrate (or vice versa). This fabrication scheme, termed microchannel confined SIP ([t-SIP), is shown in Fig. 10. In these illustrations, and in the examples discussed below, the microchannels above the substrate are approximately 1 cm wide, 5 cm long, and 300-500 [tm high. 

By screening compound libraries, selective antagonists for the chemokine CCR3 receptor have been identified these contain arylpiperidine motifs linked by a 3-6 carbon tether to a second aromatic group. Such compounds may have utility in treating chronic inflammatory disorders such as bronchial asthma. 

Tethering of library intermediates to polymer supports requires a functionality devoted to this role hence all library members are typically endowed with the same functional group. 

Fig. 8.1. Generalized selection cycle for in vitro evolution of an RNA catalyst. Random libraries are PCR-amplified, transcribed, modified with a tethered reactant, reacted with a second substrate in solution, and reverse-transcribed. Active RNA/cDNA library constructs are separated from inactive ones so that they can enter the next cycle of selection.

Gel purification is a rapid and efficient way of isolating nucleic acids of the appropriate size from syntheses, PCR reactions, ligations, or tethered product-binding reactions. For preparative separation of random libraries (—150 bases) the following two types of polyacrylamide gels are used ... 

Additional reaction components can be used that may enhance or be essential for catalytic activity in a particular system. These components may serve functions analogous to the cofactors used by protein catalysts (e. g., ATP, NADH, or metal ions). These components can be supplied free in solution or incorporated into the RNA library as previously described. As an example, Figure 8.5 oudines the RNA-catalyzed carbon-carbon bond-forming [4 + 2] cycloadclition reaction between a tethered diene substrate, (2E.4E)-hexa-2,4-clien-l-0-PEG (1), and 1-biotinamido-4-[A -(maleimidomethyl)cyclohexanecarboxamido] butane (BMCC-biotin, 2), a clienophile that is free in solution. The RNA catalyzes the formation of (3), which contains biotin for partitioning purposes. 

Fig. 9. Solid-phase synthesis of an urea-tethered bicyclic guanidine library from resin-bound polyamines.

Obita, T., Muto, T., Endo, T. and Kohda, D. (2003). Peptide library approach with a disulfide tether to refine the Tom20 recognition motif in mitochondrial presequences. J. Mol. Biol. 328, 495-504. 

The fragment library consisted of tacrine and ethidium derivatives with tethers of variable lengths to both azides and alkynes (Figure 10.6). In total, the library included 36 different tacrine-ethidium combinations. Each combination could form two regioisomeric triazoles, so a grand total of 72 different products was possible. 

In the synthesis of a compound library of allosteric Akt kinase inhibitors 39, Lindsley and coworkers employed different HTS techniques (Scheme 24) [54]. A polymer-supported base and a fluorous thiol scavenger were used in the alkylation reaction of 40. F-SPE purified intermediate was then used for microwave-assisted cycloaddition of 41. Similar intermediates have been used for generation of an unnatural canthine alkaloid library 42 by performing cycloaddition reactions with an indo-tethered acyl hy-drazide [55]. 

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