Mass Encoding

For unambiguous results, the set of building blocks used at the second step of the library synthesis should not contain any compounds with the same mass as building block B. 

The code block can also consists of equimolar mixtures built from a set of isotopically labeled dipeptides Gly-Gly, Gly-[13C]Gly, Gly-[13C]2Gly, [l3( ]Gly-[13C]Gly, [13C]Gly-[I3C]2Gly, [13C]2Gly-[13C]2Gly. These dipeptides incorporate equally to their content in the 

In ratio encoding strategy, the encoded information is derived from just two peaks in the mass spectrum. A mixture of a reagent, common to all members of the library, is prepared from different ratios of the isotopic isomers of the reagent. The resulting mass spectrum will reveal two distinct peaks corresponding to each of the isomers. The relative heights of the peaks carry the encoded information. 

The mass encoding strategy was used by Wagner and co-workers [33] for the synthesis of a peptoid library. A set of 20 14N 15N ratio-encoded imidazoles was synthesized to investigate pharmacokinetic applications of isotopic labeling . 

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Fig. 3.3 Synthetic scheme for mass-encoded library NGL127A443 containing isobaric positional isomers 1 and 19. Reprinted from [40] with permission from Elsevier.

Next, custom software is used to interrogate the deconvoluted data set to identify the protein s mass and the intensity of the peak, determine any potential modification above a user-defined intensity threshold and, if there is a hit, calculate the mass and the relative conjugation of the fragment. In fact, the percent conjugation is used as a measure of relative affinities of the fragment hits. Since the library is mass encoded (all compounds in a well have a unique mass), the calculated mass of any hits are queried into a database to identify their structures. 

Geysen HM, Wagner CD, Bodnar WM, Markworth CJ, Parke GJ, Schoenen FJ, Wagner DS, Kinder DS, Isotope or mass encoding of combinatorial libraries, Chemistry Biology, 3 679-688, 1996. 

Mass spectrometry and isotope techniques can be used effectively for the encoding/decoding of pooled libraries. Two of these techniques are mass encoding [44] and stable isotope encoding [44,45]. Because stable isotope encoding is more direct and lends itself more readily to combinatorial library screening with mass spectrometry, it is discussed below. 

Annis, A. Genome-Scale Drug Discovery by Affinity Selection-Mass Spectrometry-Based Screening of Mass-Encoded Small Molecule Libraries, ... 

FIGURE 8.9 An outline of the standard library production scheme with four 96-well reaction blocks. Each of the 384 wells is preloaded with beads with a unique mass-encoded analytical constructed resin. Normally 16 monomer As are added across the rows and 24 monomer Bs down the columns. After a mix and split procedure, 96 monomer Cs are reacted in individual wells to generate 36,864 compounds. 

Library production starts with the addition of the first position of diversity (monomer A). Four beads from each of the 384 wells are used for the QC assessment to ensure homogeneity in the coupUng reaction across all beads in a given well and identify any automated synthesizer errors in the delivery of reagents. In cases where specific monomers do not couple properly to the resin or fail the QC analysis, these monomers are eliminated from the rest of the library. After completion of the second step (monomer B), there is maximally 384 unique compounds spatially and mass encoded in the reaction plates. Again, four beads from each well are used in the QC process. 

Wagner, D.S. Geysen, H.M. Markwortli, C.J. Schoenen, RJ. Mass Encoding of Combinatorial Libraries, Asilmar Conference in Mass Spectrometry, 1997. 

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