Combinatorial Functionalization of Metalbinding Core Structures

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Cyclen, a cyclic tetramine, its derivatives, and several other related macrocylic ligands possess affinity to metal ions and have been widely investigated and used as synthetic transmembrane ionophores and fluorescent sensors [22, 23]. Still and coworkers synthesized libraries of up to 105 peptide-modified macrocyclic cyclens [24]. After attaching one of the ring nitrogen atoms of the cyclen core to a poly-(ethylene)glycol-grafted polystyrene resin (PEG-PS-resin), the remaining three secondary nitrogen atoms of the cyclen scaffold were then functionalized using a "split-and-pool" protocol. Employing fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis methodology, peptidic side arms derived from a pool of 19 side-chain-protected amino acids (AA)n, were linked via aminoethyl spacers. Four dif-

Library 1: R = (CH2)2NHAA1NH2 (19) Library 2: R = (CH2)2NHAA1AA2NH2 (361) Library 3: R = (CH2)2NHAA1AA2AA3NH2 (6859) Library 4: R = (CH2)2NHAA1AA2AA3AA4NH2 (130321)

^^ = PEG-grafted Polystyrene Resin

Fig. 32.2. Libraries of peptidic cyclen-based metal Cu(II)-binding agents synthesized on solid support. The numbers in parenthesis represent the appropriate library size. AA, = amino acids.

ferent cyclen libraries were synthesized, differing in both length and nature of the peptidic side arms (Fig. 32.2).

The side-chain-protected and -deprotected resins were agitated with diluted aqueous Cu(II) and Co(II) ion solutions. Selective metal binding was screened visually in a pooled assay identifying binding to Cu(II) and Co(II) by characteristic bright blue and red colors. Deconvolution of the chemically encoded polymer beads allowed the identification of ligand structures that were responsible for the selective binding of the metal ions [25]. The peptidic sequences generally differed for Cu(II) and Co(II) ions, and the length and chemical nature of the peptidic appendages in both protected and unprotected amino acid forms significantly influenced the affinity of the new peptidic functionalized tetraamines for Cu(II) and Co(II) ions relative to the corresponding unfunctionalized or alkylated cyclen core.

In a related combinatorial procedure, the development of fluorescent sensors for nanomolar aqueous copper was reported by Sames and coworkers [26]. A library of ionophoric ligands was generated based on three different N-containing macro-cyclic or tridentate scaffold using a split-and-pool strategy and an orthogonal tert-butyloxycarbonyl (Boc)/Fmoc protecting group protocol. The library was assayed visually by incubating the solid support-bound ligands with aqueous Cu(II) solution followed by reaction with a copper-selective staining reagent (blue color). All identified ionophores contained a carboxylic group as well as an aromatic nitrogen heterocycle. A pyrazine-containing ligand was selected as a lead structure for further development of a fluorescent sensor bearing a covalently linked dansyl fluo-rophore (Fig. 32.3).


Fluorescent Sensor

= Tentagel

= Microsphere

Fig. 32.3. Pyrazine-containing lead structure obtained from visual primary screening of the solid-phase-bound ionophore library. Structure of the identified sensitive and selective copper ion chemosensor bearing a covalently linked dansyl fluorophore.

After covalent attachment of the fluorescent sensor to polymeric microspheres (3.1 mm), the microsphere sensors were examined in the presence of 50 nM buffered CuCl2 solution using fluorescent optical microscopy equipped with a custom fiber optic [27]. Affinity (Kd = 10 nM) and binding selectivity (Cu > Co > Ni > Fe) were assessed by fluorescence quenching and competition experiments.

The X-ray structure of metalloprotein recombinant isopenicillin N synthase (IPNS), which is involved in the biosynthesis of isopenicillin N, has served as a lead structure for library design. IPNS possesses at its active site two iron-binding peptides, Ile-His-Arg and Trp-His-Glu-Asp-Val, which are linked by a short peptidic sequence [28]. Hoping to discover potentially new catalysts, Baldwin, Wood, and coworkers combinatorially explored various versions of resin-bound peptide libraries of the general structure resin-Arg-His-Ile-(AAi)n-Val-Asp-Glu-His-Trp-Ac (AAi = amino acid; n = 1-3), and examined their metal-binding abilities (Fig. 32.4) [29].

Three different peptide libraries with a total of 7240 individual oligomers, each differing in length and amino acid content, were synthesized using Fmoc peptide-coupling chemistry in a split-and-pool protocol. Up to three variable amino acids were incorporated in between the conserved peptide sequences Ile-His-Arg and Val-Asp-Glu-His-AcTrp. Selective ion binding was assayed by agitating the resin beads with aqueous solutions of Cu(II), Fe(II), and Co(II) sources. Significant metal binding was accompanied by a color change of the beads. Co(II), for which the color changed to pink/purple, bound most selectively. No encoding/deconvolution strategy was implemented in order to characterize the ligand structure that gave

Fig. 32.4. A peptide based on isopenicillin N-synthase. Schematic diagram of the active site. The wavy lines represent the remainder of the protein. A peptidic library designed to mimic the metal-binding region. n = 0-3; AA = amino acid; Ac = acetyl.

rise to colored metal complexes from the pooled assay, and catalytic activity of the new metal complexes in the ring closure of the Arnstein tripeptide (ACV) to isopenicilline N was not addressed.

Metalloproteins, synthetically or biosynthetically appended to proteins or other biomolecules, are finding increasing utility in the biochemical analysis of non-covalent protein-nucleic acid and protein-protein interactions. The amino terminal Cu(II)- or Ni(II)-binding (ATCUN) motif is a structural feature of several naturally occurring proteins such as certain types of albumins (e.g. human serum albumin (HSA), bovine serum albumin (BSA), and rabbit serum albumin (RSA) and neuromedins C and K, among several others [30]. Generally, this domain is described as Ni(II)-H2N-AA1 -AA2-His. Long and coworkers employed a ''positional scanning'' combinatorial protocol to optimize the desoxyribose-based cleavage of B-form DNA by Ni(II)-H2N-AA1 -AA2-His metallopeptides [31]. Using a standard Boc protocol and ''split-and-mix'' technique on methylbenzhydrylamine (mBHA) resin, two libraries were generated from a selection of L-amino acids in which the first (AA1) and the second position (AA2) of the peptide ligand were varied within the H2N-AA1 -AA2-His sequence. The libraries were assayed after cleavage from the solid support for increased direct DNA cleavage relative to Ni(II)-H2N-Gly-Gly-His after incubation with a Ni(II) source and oxidative activation of the metal complex with KHSO5 or magnesium monoperoxophthalate (MMPP). Increased catalytic activity was found when the amino-terminal peptide position contained a hydrophobic amino acid and the second peptide position contained an ionic or polar amino acid. The optimized and resynthesized metal-lotripeptide domain Ni(II)-HN-Pro-Lys-His was found to oxidatively cleave DNA



Fig. 32.4. A peptide based on isopenicillin N-synthase. Schematic diagram of the active site. The wavy lines represent the remainder of the protein. A peptidic library designed to mimic the metal-binding region. n = 0-3; AA = amino acid; Ac = acetyl.

^^ = PEG-grafted polystyrene resin

^^ = PEG-grafted polystyrene resin an order of magnitude faster than the reference Ni(II)-H2N-Gly-Gly-His. DNA-binding affinity was slightly increased relative to Ni(II)-H2N-Gly-Gly-His, but metal complexation and the A/T-rich site DNA-binding selectivity were not altered.

Another approach to generate large libraries of metal-binding proteins is to take advantage of the immune system. Janda and coworkers identified antibodies that bind unique metals with excellent affinity by combining a metallo-panning agent with the high-throughput screening of a combinatorial antibody library [32]. The single-chain antibody (scFv) library was constructed from the blood of 50 healthy volunteers, the resulting phage scFv antibody library was estimated to be 1 x 109 in diversity. This library was then screened against three metal pool mixtures and an immobilized phosphorodithioate metallo-panning reagent that binds metals, leaving them coordinatively unsaturated. Two single-chain antibodies were identified and isolated that bind lanthanum and yttrium in the mM range. Inductively coupled plasma mass spectroscopy analysis suggests that the selected antibodies contain a single metal ion binding site.

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