Analytical constructs

Fig. 1. 20 The design of resins containing analytical constructs for MS analysis.

We would like to describe in this section the very beautiful idea proposed by L.H. Kauffman for the analytical construction of powerful polynomial invariants of knots and links. 

McKeown, S.C. Watson, S.P. Carr, R.A.E. Marshall, P.A. Photolabile Carbamate Based Dual Linker Analytical Construct for Facile Monitoring of Solid Phase Chemistry TLC for Solid Phase Tetrahedron Lett. 40, 2407-2410 (1999). 

Two of the difficulties encountered with solid-phase synthesis is quantitation and identihcation of all the products that result from resin-based solid-phase methods. Analyhcal constructs were developed to facilitate rapid quanhta-hve and quahtahve analyhcal measurements during compound synthesis and Ubrary produchon [10,11]. The analytical construct designed by Diversity Sciences possesses mulhple funchons. The construct serves as the physical link between the solid support and the site of synthesis, promotes the analyhcal analysis of a library synthesis at each step of the procedure, and also provides a facile method of bead encoding. 

FIGURE 8.1 (A) A display of the block diagram of the analytical construct designed by... 

I he standard analytical construct used for library production in Diversity Sciences at GlaxoSmithKline is shown in Figure 8.1. The analytical construct uses the common Knorr acid cleavable linkers at the first and second linker positions. The terminal linker is photocleavable and is released upon exposure to 350-nM light [12-15]. The construct contains the mass-code block and uses isotopically labeled Gly as a peak splitting element to facilitate compound identification by mass spectrometry. Also, the construct has two lysine amino acid groups to aid the ionization process of both the code block and the ligand... 

The initial concept and prachcal use of analytical constructs was designed by Diversity Sciences to facilitate mass-spectral analysis during combinatorial processes [10]. Several internal and external groups adopted the original concept and published similar results [16-20]. Each element of the analyhcal construct developed by Diversity Science is outlined below in more depth in the following sections. 

It is often difficult to determine the degree to which the chemistry proceeded on the entire library population and whether peaks in a mass spectrum are due to the product, side reactions, reagents, solvents, or impurities. Diversity Sciences developed mass-spectral methods to distinguish all components that are cleaved from a solid support and implemented the method into the analytical construct. While early studies demonstrated promising results for fragmentation methods with tandem mass spectrometry (MS/MS), stable isotopes were routinely implemented as signature peaks for the identification of compounds that are produced from solid-phase reactions [27]. 

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. 

Diversity Sciences developed a library synthesis strategy that combines the simplicity of parallel synthesis and the power of resin-mixing techniques. The general format is four 96-well plates that give rise to 384 synthetic wells, as shown in Figure 8.9. The layout of the synthesis blocks enables 16 unique monomers in monomer position A (across rows) and 24 unique monomers in monomer position B (down the columns). All of the 384 wells are preloaded with off-the-shelf resin where each well has a unique binary code embedded in the analytical construct. The first two points of diversity (monomer A and monomer B) is added in all possible combinations by parallel synthesis. Each spatial location has a unique binary-mass code that encodes for a particular combination of monomer A and monomer B. For example, binary code number 8 represents monomer Al and monomer B8. After the addition of monomer B, the resin from all 384 wells is mixed together and split into 96 identical pools, to which monomer C is added. The third monomer, monomer C, is spatially encoded, since the 96 pools are not mixed after the last step and screened as pools. Upon decoding, the identification of the binary code reveals the combination of monomer A and monomer B on each bead. 

Williams, G.M. Carr, R.A.E. Congreve, M.S. Kay, C. McKeown, S.C. Murray, P.J. Scicinski, J.J. Watson, S.P. Analysis of Solid-phase Reactions Product Identification and Quantification by Use of UV-Chromophore-Containing Dual-Linker Analytical Constructs, Angew. Chem., Intern. Ed. Eng. 39, 3293-3296 (2000). 

Congreve, M.S. Ley, S.V. Scicinski, J.J. Analytical Construct Resins for Analysis of Solid-phase Chemistry, Chem. Europ. J. 8,1768-1776 (2002). 

Despite these efforts, an exact determination of the relative amounts of compounds was not possible. This led to the development of analytical constructs of type 181 (Fig. 16) containing chromophores that are suited for the facile detection and quantification of compounds in mixtures [202, 203]. Commonly used chromophores for this purpose are dansyl and anthracenyl units. Both of these chromophores allow the detection of products at the single-bead level. 

Recently, a convenient preparation of an analytical construct of type 182 (Fig. 17) has been reported. The synthesis of 182 is possible without a need for extensive chromatography. Furthermore, the deuterium mass tag can be incorporated vhth D2O in a cost-effective manner [204]. 

Another useful application of analytical constructs is in the evaluation of the stabilities of linkers towards different reaction conditions [205]. Cleaving of the analytical construct (at linker 1) after subjecting the linkers to the reaction conditions allows insight into the extent of linker modification under these conditions. 

The indole safety-catch linker outlined in Section 2.5 was also evaluated employing a similar analytical construct [207]. 

Sohd supports incorporating analytical constructs are not commercially available as yet Once this is the case, they will certainly find widespread apphcation. 

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