Solid-Phase Synthesis and Combinatorial Technologies

484 APPLICATIONS OF SYNTHETIC LIBRARIES

TABLE 9.11 Efficiency of Library Individuals 9.129–9.134 from Screening of the SP Pool Encoded Peptidomimetic Library of Catalysts L34 as Acylation Catalysts

aCalculated after 9 min incubation with the acylation cocktail as an average of three runs. bNot detected.

M3-containing library members was formed. The frequency of 9.130 and 9.131 among the hot beads roughly paralleled their frequency in the pooled library (Fig. 9.48), while 9.132 was spotted only once for unknown reasons. Compound 9.133 belonged to a different chemical class, but its catalytic activity was not reconfirmed when tested as a discrete. Compounds 9.130–9.132 all resulted more active catalysts than 9.129 (Table 9.11), with 9.130 being the most active compound. A less active catalyst from the library, 9.134 (Fig. 9.48), was also reprepared and tested as a discrete (Table 9.11). Its weaker activity was not spotted by IR thermography, confirming the ability of the detection method to select the most active catalysts during the screening.

The pooled encoded format was also used by Boussie and co-workers (179, 180) to prepare and screen Pdand Ni-diimine ligand complexes as olefin polymerization catalytic systems. The successful application of deconvolutive or encoding methods to medium–large SP catalytic system pool libraries should become routine in the future. Careful validation of the synthetic scheme, the screening, and the detection methods will ensure the applicability of HTS of SP catalytic pool libraries to each specific transformation investigated.

9.5 APPLICATIONS TO MOLECULAR RECOGNITION

9.5.1 General Considerations

The existence of biomolecules, such as proteins, peptides, carbohydrates, and nucleotides, able to recognize a ligand with extremely high selectivity and specificity through a series of noncovalent interactions has long been a challenge for synthetic chemists. The access, via synthetic routes, to artificial receptors able to tightly bind specific substrates for various applications would produce, among others, artificial catalytic systems to catalyze known chemical transformations, synthetic target mimicks for setting up relevant pharmaceutical screening assays, and synthetic affinity receptors useful for the isolation, purification, and/or identification of active principles. All these applications would largely benefit from the higher stability and tractability of synthetic

9.5 APPLICATIONS TO MOLECULAR RECOGNITION 485

recognition systems when compared to their biological counterparts, as well as from the possibility to tailor their structure and to reduce extremely complex biomolecules to smaller and simpler, yet efficient artificial models. Another advantage would be the lower cost and ease of an assessed synthetic process to produce a synthetic supramolecular recognition element, rather than overproducing and isolating large quantities of the corresponding biomolecule. Even more importantly, a full understanding of supramolecular phenomena, and transfer of this knowledge to synthetic chemistry routes, would allow the design and synthesis of artificial elements, with no biological counterpart, to catalyze new reactions, isolate new compounds, and set up novel relevant assays.

Implications and applications of supramolecular chemistry have been reviewed recently (181–183). Synthetic receptors derived from cyclodextrins (184), calixarenes (185), fullerenes (186), crown ethers (187), and dendrimers (188, 189) have been reported. Unfortunately, the elusive balance of obtaining high affinity by means of a large number of weak, noncovalent interactions, as between biomolecules and their ligands, has proved extremely difficult to translate onto synthetic counterparts, and synthetic receptors matching the prowess of enzymes in detecting their substrates and processing them are still far from reality.

Combinatorial technologies can help the search for supramolecular entities, and several significant reviews have thoroughly illustrated the applications of libraries in molecular recognition (190–196). Synthetic peptide libraries have been used to refine the selectivity of synthetic receptors and to determine the tolerability for substitutions in a given position of diand tripeptide structures (197–203), speeding up the characterization of these entities. Even more importantly, libraries of synthetic receptors have been prepared using rigid scaffolds decorated with amino acidic building blocks to modulate their recognition properties. Examples from Clark Still and co-workers (204, 205) and from Nestler and co-workers (206, 207) presented different peptidosteroid-based rigid libraries and more flexible pentamethylene linker libraries to bind enkephalin-related peptides. Fessmann and Kilburn (208) reported a 2197member receptor library based on a trisubstituted pyridine scaffold tested in an on-bead fluorescent assay for their binding with a dansylated tripeptide. Goodman et al. (209) dealt with metal-template strategies to assemble synthetic receptor libraries (209), in analogy to template-assisted library synthesis, which was discussed in Section 8.6. Jung et al. (210) reported an application for cyclopeptide libraries as synthetic receptors for enantiomeric resolution of racemic amino acid mixtures. The next section introduces an example reporting both the synthesis and screening of a ligand library to determine the specificity of a synthetic receptor and then the use of this information to design a synthetic receptor library to optimize the recognition properties.

Another example described in Section 9.5.3 reports the synthesis and screening of a synthetic receptor library aimed at transition metal binding. Other similar examples have been reported recently. Burger and Clark Still (211) prepared ionophoric, cyclenbased libraries decorated by amino acid units and screened them for their ability to complex copper and cobalt ions; Malin et al. (212) identified novel hexapeptidic technetium-binding sequences from the screening of cellulose-bound libraries; and

486 APPLICATIONS OF SYNTHETIC LIBRARIES

Shibata et al. (213) prepared and screened an SP oligopeptide library for its ability to complex cobalt ions, isolating several high-binding sequences.

Finally, libraries aimed to chiral resolution of racemates will be covered here; in particular, the use of chiral stationary phases (CSPs) has recently been reported for the identification of materials to be used for chiral separation of racemates by HPLC. The group of Frechet reported the selection of two macroporous polymethacrylate-supported 4-aryl-1,4-dihydropyrimidines (DHPs) as CSPs for the separation of amino acid, anti-inflammatory drugs, and DHP racemates from an 140-member discrete DHP library (214, 215) as well as a deconvolutive approach for the identification of the best selector phase from a 36-member pool library of macroporous polymethacrylategrafted amino acid anilides (216, 217). Welch and co-workers (218, 219) reported the selection of the best CSP for the separation of a racemic amino acid amide from a 50-member discrete dipeptide N-3,5-dinitrobenzoyl amide library and the follow-up, focused 71-member library (220). Wang and Li (221) reported the synthesis and the Circular Dichroism- (CD) based screening of a 16-member library of CSPs for the HPLC resolution of a leucine ester. Welch et al. recently reviewed the field of combinatorial libraries for the discovery of novel CSPs (222). Dyer et al. (223) reported an automated synthetic and screening procedure based on Differential Scanning Calorimetry (DSC) for the selection of chiral diastereomeric salts to resolve racemic mixtures by crystallization. Clark Still reported another example which is discussed in detail in Section 9.5.4.

9.5.2 An Example: Ligand and Receptor Libraries Based on Guanidinium Tweezer Receptors

Davies et al. (224) reported the synthesis of a 1000-member SP autoencoded, cyclic peptide library L35 (Fig. 9.49) using a recently reported synthetic strategy and standard mix-and-split peptide conditions. Each monomer set M1–M3 was composed of ten α-amino acids (Fig. 9.49). Such a library could expose the C-terminus inverted tripeptide library (L35c, 80% of sites, screening portion) by mild acidic cleavage (step a, Fig. 9.49), allowing an on-bead screening protocol (vide infra) and selection of positives (step b). A subsequent strong acidic cleavage (step c, Fig. 9.49) released the tripeptide library L35r in solution while exposing the N-terminus sequenceable portion of the same resin-bound library (L35s, 20% of sites, on-bead Edman sequencing of positive beads, step d).

The C-exposed library L35c was screened for its binding affinity for the guanidinium-based, fluorescent tweezer receptor 9.135 (225), whose structure is reported in Fig. 9.50. The dansylated chains should have assured the fluorescent detection of beads containing ligand tripeptidic sequences, and a series of control experiments ruled out the possibility of significant, nonspecific interaction of the beads with the receptor or with the biological assay constituents. The assay conditions were carefully optimized, and around 7000 beads (seven library equivalents, complete representation of the library individuals) were incubated with 9.135.

Microscope observation of the beads after incubation spotted the brilliantly fluorescent ones from the background, containing both nonfluorescent and slightly fluo-

AA

1 COOH

L35r

a:1% TFA in DCM, rt; b: on-bead screening, positive beads selection and withdrawn

c:100% TFA, rt; d: Edman sequencing.

M1-M3 = AA1-AA3 = L-Ala, Gly, L-Glu(OtBu), L-Leu, L-Met,

L-Phe, D-Phgly, L-Pro, L-Ser(OtBu), L-Val.

Figure 9.49 On-bead screening and decoding protocol for the inverted SP peptide library of artificial receptors L35.

rescent beads. Around 3% of beads were positive, corresponding to around 200 of the 7000 beads tested, or to around 30 active library individuals (if all active beads were reliably spotted by the screening). Twenty beads were manually removed, cleaved with strong acid conditions, and sequenced. Fifteen different sequences were determined, showing a strong conservation in the C-terminus position (two residues, Val strongly preferred with 95% recurrence), some preferences in the N-terminus position (five

9.5 APPLICATIONS TO MOLECULAR RECOGNITION 489

encoded SP pool library

a:TFA/DCM 1/4, rt; b: DIPEA; c: coupling with M1; d: piperidine, DMF, rt; e: coupling with M2;

f:coupling with M3; g: (Boc)2O, DIPEA, rt.

M1-M3 = L-Ala, Gly, L-Phe, L-Ser(OtBu), L-Val

9.138

Figure 9.51 Synthesis of the SP-encoded pool, guanidinium-based tweezer receptor library L36 and structure of the selection substrate 9.138.

same synthetic strategy used for the synthesis of 9.135. Compound 9.135 was included in the library structure as a positive control. The library was incubated with the tripeptide 9.138 (Fig. 9.51), selected among the active sequences obtained from the previous screening, but no brilliantly fluorescent beads were obtained even after prolonged incubation times. Probably the transition from a strongly basic, unprotected guanidinium group (as in 9.135) to a weakly basic, supported sulfonylated

490 APPLICATIONS OF SYNTHETIC LIBRARIES

guanidinium group (as for L36 individuals) affected the binding affinity of the synthetic tweezers. The same library should be made with a different linker, preserving the guanidinium basicity. This could determine the importance of steric hindrance (represented by the resin bead) around the core guanidinium, with eventual testing of the library after release into solution to cross-check the on-bead screening results.

9.5.3 An Example: Synthesis and Screening of a Capped Peptidomimetic Library for Metal Binding Activity

Francis et al. (227) reported the synthesis of a >10,000-member encoded SP pool library L37 characterized by a turn element (monomers M2, prepared using simple synthetic routes) surrounded by two α-amino acids (monomers M1 and M3) and capped on the N-terminus (monomers M4). The structure of the library and the monomer sets are reported in Fig. 9.52. Mix-and-split protocols and a popular encoding method (177) were used to prepare and encode the library.

Library L37 was tested on-bead for its coordination of Ni2+ and Fe3+ ions, and suitable experimental incubation protocols were set up for both metal ion solutions (Figs. 9.53 and 9.54, respectively). As the preliminary screenings at high metal concentration detected a large number of stained beads, two library equivalents (around 24,000 beads, representing >90% of the library population) were screened in the presence of decreasing metal ion concentration until only a small number of stained beads remained. With a 2.5 × 10–4 M Ni2+ concentration, only six beads were positive, and their isolation and decoding produced compounds 9.139–9.142 (Fig. 9.53). Protected histidine as M1 and M3 and only two M2 and M4 monomers were observed, indicating marked structural preferences for Ni2+ coordination. Solution synthesis and screening of 9.139 and 9.140 confirmed their coordination with Ni2+. With a 5 × 10–6 M Fe3+ concentration 64 beads were spotted, isolated, and decoded. All of them had a fixed monomer in positions M3 and M4 (see generic structure 9.143, Fig. 9.54), half of them had L-methionine as preferred M1 monomer, but no preference was detectable for the turn element. Distinct preferences for Ni2+ and Fe3+ coordination were observed, and simultaneous on-bead library screening with solutions of Ni2+ and Fe3+ confirmed this tendency. Other metal ions (Cu2+, Pt4+, Sn4+, Pd2+) were also used as screening probes, producing results that, albeit less rationalizable, again highlighted the different preferences of various metal salts toward L37 individuals.

9.5.4 An Example: Selection of an Enantioselective Resolving Resin Using On-Bead Screening

Weingarten et al. (228) reported the synthesis of an encoded SP 60-member pool library L38 that was screened to find the best chiral selector for the resolution of racemate mixtures of dye-containing amino acid Pfp esters 9.144a (L-enantiomer, blue dye) and 9.144b (D-enantiomer, red dye, Fig. 9.55). The structures of the library L38 and of the three monomer sets M1 (15 representatives, N-Fmoc α-amino acids), M2

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Figure 9.53 Screening of the SP encoded primary peptidomimetic library of metal complexing agents L37 for Ni2+ complexation and structures of the hits9.139–9.142 obtained from its screening.

a: 5x10-6 FeCl3, AcOH, NaOAc, MeOH, rt; b: KSCN, AcOH, MeOH, rt.

O R2

9.143

HN R3

O

MeO N

O O

Figure 9.54 Screening of the SP encoded primary peptidomimetic library of metal complexing agents L37 for Fe3+ complexation and structures of the hit 9.143 obtained from its screening.