Secondary Amine Binary Coding

In 1996, Gallop and coworkers developed another robust encoding strategy [11, 12], based on secondary amine tags, which are incorporated into a polyamide backbone. The secondary amine binary coding scheme utilizes an amine-based polymeric resin that is differentially functionalized with sites for both ligand synthesis and tag addition (Scheme 5.5). The ligand synthesis site is derivatized with a N-Fmoc-protected photocleavable linker group [13]. This linker allows for the release of the ligand from the resin by exposure to ultraviolet (UV) light. The amino group of the tag site is protected with orthogonal to the Fmoc group functionalities (e.g. Boc or Alloc). The tag site occupies only 10% of the total number of amino groups on the resin. Each 130-mm-diameter bead of the TentaGel S resin, which is recommended by the authors of this methodology, contains about 300 pmol of amino groups. Therefore, the theoretical yield of the ligand is 270 pmol per bead. Such nh2 nh2

PG-Gly (10%) Coupling reagent

1. Piperidine

2. Couple

Fmoc'

Fmoc'

PG O

Y^if

OMe no

NH

Ligand Site --

Fmoc-[Photolinker]

Fmoc

J2 PG= Boc, Alloc

Scheme 5.5. Resin construct for secondary amine binary coding [11,12].

quantities being photo-released in 100 ml of a solvent would be adequate for the concentration of the ligand in most biological assays.

The tags are a set of relatively hydrophobic amines, such as HN(Et)(Bu), HNMe(C6H13), HNBu2, HNMe(C7H15), and HN[CH2CH(Et)C4H9]2. The set is selected to ensure reliable separation of the dansyl derivatives of the amines by reversed-phase high-performance liquid chromatography (HPLC). The tagging monomer units are synthesized by reaction of an N-protected iminodiacetic anhydride with a secondary amine from the set (Scheme 5.6).

Scheme 5.6. Preparation of tag monomers for secondary amine coding.

The resulting N-protected N-[(dialkylcarbamoyl)methyl]glycines are assembled into binary mixtures, which are incorporated into the tag sites of the resin beads by using HATU or other peptide-coupling reagents (Scheme 5.7). Addition of each new building block at the ligand synthesis site in the course of a "split-and-pool" combinatorial synthesis is accompanied by the incorporation of the preselected mixture of the monomer units at the tag addition site. Selection of the protecting groups allows for the addition of the tags either before or after the addition of the building block to the ligand. The ability to choose different protecting groups for the N-protected tag monomers helps to resolve potential chemical compatibility issues.

Scheme 5.7. Coupling of secondary amine tag constructs to solid support.

Upon completion of the library synthesis, each bead is distributed into a separate well of a microtiter plate. The ligands are released from the beads into the assay medium by exposure to UV light at 365 nm. The tag residues remain coval-ently attached to the beads. After screening, beads from the wells containing active compounds are collected for decoding. The decoding process is shown schematically in Scheme 5.8, and begins with the acid hydrolysis of the beads in 6 N hydrochloric acid. Under these conditions all amide bonds are hydrolyzed, releasing free secondary amine tags into the solution. After evaporation of the HCl, the amines are converted into the corresponding dansyl derivatives by treatment with dansyl chloride. Analysis of the resulting mixture of dansylated tags is carried out

6NHCI O PG O

Dns-CI

NMe,

NMe,

NMe,

HPLC Analysis with

Fluorescence Detector

NMe,

Scheme 5.8. The decoding process for secondary amine binary coding.

by reversed-phase HPLC on a microbore column. Fluorescence detection allows for the reliable analysis of 20-30 fmol of a dansylated tag. Only 2-5% of the entire dansylated hydrolyzate from a single bead is sufficient to obtain unambiguous results.

The secondary amine encoding method was used in the synthesis of a library of pyrrolidine-based inhibitors of angiotensin converting enzyme (ACE) via [2 + 3] cycloaddition [14].

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