Arpad Furka

Combinatorial chemistry: 20 years on…

Abstract ‘The mass of a protein library...would exceed that of the universe by more than two hundred orders of magnitude.’

Redistribution in Combinatorial Synthesis. A Theoretical Approach

In modified versions of the split-mix method, the conventional solid support resin was replaced by labeled macroscopic support units in order to produce individual compounds in multi milligram quantities while the high productivity of the original procedure is preserved. Recently it has also been shown that tagging the units is unnecessary. Omission of the tags is possible if (i) the unlabeled support units are arranged into spatially ordered groups and the relative spatial arrangement of the units is maintained during the chemical reactions (ii) the support units are redistributed between the reaction steps according to a predetermined pattern permitted by the combinatorial redistribution rule and (iii) the sorting process is simulated by a computer that can trace the synthetic history of each support unit. Different kinds of solid support units, formation of spatially ordered groups, sorting devices and basic redistribution patterns (serial, semi-parallel and parallel) are discussed. It is also shown that particularly the semi-parallel and the parallel redistribution assure fast sorting.

Coloured peptides: synthesis, properties and use in preparation of peptide sub-library kits.

Several methods were developed for the solid‐phase synthesis (SPPS) of coloured peptides and peptide libraries. At first a bifunctional red compound, 4‐(4‐(N‐ethyl‐N‐(3‐(tert‐butyloxycarbonyl)aminopropyl)amino)phenylazo)benzoic acid (Boc‐EPAB), was coupled with chloromethyl resin to obtain a new solid support suitable for SPPS using Boc chemistry. Peptides synthesized on this coloured resin had the chromophore at their C‐termini. N‐terminally coloured peptides were synthesized on a traditional solid support, coupled with chromophoric carboxylic acid before cleavage. A model pentapeptide, Phe‐Ala‐Val‐Leu‐Gly, and its ten derivatives were synthesized and their properties studied. It was found that the presence of chromophores decreases the water solubility of peptides. However, insertion of solubilizing tags (penta‐lysine sequences or polyoxyethyl chains) into the molecule of any coloured derivative resulted in enhancement of the solubility. The RP‐HPLC hydrophobicity indexes (φ0) of the coloured peptides were also determined because φ0 values are closely related to their water solubility. A coloured pentapeptide library was synthesized using the portioning‐mixing method. Each component of this library contained the red azo dye (EPAB) and the penta‐lysine tag. Before the last coupling step the samples were not mixed. All of the 19 sub‐libraries obtained after cleavage were readily soluble in water, giving intense red solutions.

An improved method for isolation of the C-terminal fragment of proteins

An efficient and easily realizable method for the isolation of the C-terminal fragment is described. Proteins are esterified by methanolic HCl and subsequently digested with pepsin. The peptide mixture is submitted to paper electrophoresis in pH 2.1 buffer. The identification of the C-terminal peptide is performed by preparing a guide peptide map, using pH 5.5 buffer in the second dimension. The C-terminal fragment appears as an on-diagonal spot. It can be isolated by a pH 5.5 run of the corresponding band from the first (pH 2.1) electrophoretogram. Since the C-terminal peptide is the fastest moving component, there is no need for its further purification. The expected yield is about 40%.

Preparation of Cherry-Picked Combinatorial Libraries by String Synthesis

String synthesis [1-3] is an efficient and cheap manual method for preparation of combinatorial libraries by using macroscopic solid support units. Sorting the units between two synthetic steps is an important operation of the procedure. The software developed to guide sorting can be used only when complete combinatorial libraries are prepared. Since very often only selected components of the full libraries are needed, new software was constructed that guides sorting in preparation of non-complete combinatorial libraries. Application of the software is described in details.

Deconvolution by omission libraries.

Omission libraries, synthesized by omitting one amino acid in all coupling positions, are very efficient tools for the rapid identification of the amino acid components of bioactive peptides. Based on the determined amino acids, an occurrence library can be defined and prepared which is much less complex than the full one while still comprising the bioactive peptide.

Split-mix synthesis using macroscopic solid support units.

Publisher Summary This chapter discusses the split-mix synthesis using macroscopic solid support units. Split-mix synthesis made it possible to prepare new compounds and that procedure was based on the solid-phase method, in which each coupling cycle was replaced by the operations: (1) dividing the solid support into equal portions, (2) coupling each portion individually with a different building block, and (3) mixing the portions. Repetition of these operation steps resulted in an exponential increase in the number of synthesized compounds. The products could be used either as mixtures or as unidentified individual compounds formed on microscopic polymer beads. Any combinatorial synthetic method carried out with macroscopic solid support units, even if coding is omitted, has to ensure that the route of every unit in the entire multistep synthetic process can be traced. This chapter presents a description of the various steps to be followed in string synthesis.

Relative energy of organic compounds IV. Radicals, carbocations, and carbanions

The structure-dependent energies of organic radicals, cations, and anions are deduced from their calculated relative enthalpies and are compared to the relative enthalpies of their parent compounds. The use of relative enthalpies to express the relative energies of organic radicals, cations, and anions proved to be as fruitful as in the case of their parent organic compounds. The same energy-determining structural factors may have stronger, weaker, or even opposite effects in the radicals, cations, or anions than those in their parent molecules.