Rapidly Measuring Reactivities of Carboxylic Acids to Generate Equireactive Building Block Mixtures:  A Spectrometric Assay

Abstract

The relative reactivity of building blocks is critical for a successful preparation of combinatorial libraries.Here, we present a method for measuring the reactivity of carboxylic acid building blocks in amide-forming reactions. The method involves competitive reactions between a reference and test acid and a tetraphenylporphyrin reaction partner with four reactive sites. Relative reactivities are calculated on the basis of the distribution of substituted porphyrins found in MALDI-TOF mass spectra. Reactivities thus determined were used to prepare reactivity-adjusted building block mixtures. These were reacted with amino-terminal oligonucleotide and peptide scaffolds on solid support, generating small libraries suitable for spectrometrically monitored selection experiments (SMOSE). The rate of building block "drop outs" that fail to couple as expected was not substantially lowered by acquiring spectra from two reactions, performed with different ratios of building blocks, where the effect of a given substituent on the desorption/ionization yield of the porphyrin can be eliminated. Instead, coupling building blocks of similar size together or employing N-hydroxysuccinimide esters rather than activating with a "uronium salt" were found to improve the quality of libraries generated via competitive reactions.

A Novel Method for the Determination of Isokinetic Ratios and Its Application in the Synthesis of Two New Positional Scanning Libraries

“…Several methods have been reported for determining the isokinetic ratio for a variety of different reagent classes such as amino acids, carboxylic acids, and aldehydes {REF 9, REF 10, REF 11, REF 12, REF 13}. These methods include the use of amino acid analysis as well as binary and iterative HPLC analysis, and MALDI-TOF.…”

Positional Scanning Synthesis of a Peptoid Library Yields New Inducers of Apoptosis that Target Karyopherins and Tubulin

“…To assure efficient hit identification from PS‐SCLs, individual building blocks must be equally reactive under the conditions of library synthesis in order to guarantee equal or at least very similar proportions of the different final library members in the generated mixtures. This precondition limits the strategy to transformations in which equal reactivity is assured by appropriate activation techniques6–7 (e.g., peptide synthesis) or in which only building blocks of limited structural variation and reactivity—and, therefore, also diversity—are introduced in each synthesis step 8…”

A Reactivity Test for HBTU-Activated Carboxylic Acids with Low Reactivity and Competitive Coupling ofN-Methylpyrrole Derivatives

“…7 The assay involves competitive coupling reactions between test and reference acid with a porphyrin tetraamine under pseudo-first order conditions and product analysis via MALDI-TOF mass spectra. 6 One class of carboxylic acids, namely the N-methylpyrrole carboxylic acids, has not yet been explored by SMOSE. Methylpyrroles are building blocks for oligopyrroleamides that bind sequence-specifically in the minor groove of DNA duplexes, 8,9 and conjugates of oligonucleotides and oligopyrroleamides show promising binding properties.…”

“…Incomplete activation of the less reactive building block was noted as a possible weakness of earlier assays. 6 The mixture of activated 1 and 5 was reacted with tetrakis[L-alanyl(p-aminophenyl)]porphyrin 6 ( Figure 2), our established reaction partner for reactivity tests, 6 scaling down to one quarter compared to earlier assays. 18 Analysis of the product distribution required the reflectron mode of the MALDI-TOF mass spectrometer, as the mass difference between 1 and acetylleucine (5) is only 3 Daltons.…”

“…For 2, repeated runs gave a relative reactivity of 0.6-1.2 which is in reasonable agreement with the 1.65 calculated from the relative reactivity measured against Ac-Leu-NHS and the reactivity difference between Boc-Val-NHS and AcLeu-NHS (0.05:1). 6 For HBTU-activated 3 and 4, relative reactivities of 0.25-0.46 and 0.26 of that of 13 were measured, respectively.…”