Solid-phase Library Research Articles

The Application of Quantitative Analytical Constructs for Chemistry Optimization, Monomer Rehearsal and Reactivity Prediction in Solid Phase Library Synthesis

The use of quantitative dual linker analytical constructs for the high-throughput generation of solid phase reaction data is described. This methodology can be applied to chemistry optimization, monomer rehearsal and monomer reactivity prediction as a tool to aid the production of solid phase compound libraries. Keywords: analytical methods, combinatorial chemistry, mass spectrometry, solid-phase synthesis

A solid phase library synthesis of hydroxyindoline-derived tricyclic derivatives by Mitsunobu approach.

Hydroxyindoline-derived scaffold, 9, was synthesized with the goal of generating a library of indoline-based natural product-like tricyclic derivatives to be utilized as small-molecule chemical probes. The tricyclic ring was obtained by a Mitsunobu reaction of the N-nosyl amino acid conjugate with the primary hydroxyl group. The solid-phase synthesis was achieved by immobilizing scaffold 9 onto the solid support giving a compound, 15. This was then subjected to a series of reactions on solid phase, including the Mitsunobu reaction, leading to the desired indoline-derived tricyclic derivative. The final product has two diversity sites: (i) amino acid as the first diversity and (ii) amidation of the secondary amine for the second diversity. These two diversity sites were utilized in the library generation by IRORI split-and-mix approach.

Construction of 6-Amino-2,2-Dimethyl-3,4,6-Trisubstituted-2H-1-Benzopyran Library by Solid Phase Synthesis

We report the solid-phase library construction of 2000 analogues of 6-amino-2,2-dimethyl-3,4,6-trisubstituted-2H-1-benzopyran. The polymer-bound hydroxyalkoxychromanes 5, produced by nucleophilic reactions with various alcohols on epoxides generated in situ, served as key intermediates for subsequent diversification. Further reactions on these hydroxyalkoxychromanes 5 with various electrophiles, such as alkyl halides and acid halides, produced the desired 6-amino-2,2-dimethyl-3,4,6-trisubstituted-2H-1-benzopyran analogues 9, 10, and 11. The progress of reactions could be monitored as solid-bound intermediates by ATR-FTIR or HR-MAS-NMR spectroscopy. The final compounds, obtained in good yields and high purity upon cleavage from the resins, were characterized by LC/MS, HRMS, (1)H NMR, and (13)C NMR spectroscopy.

Solid-phase library synthesis of reversed-statine type inhibitors of the malarial aspartyl proteases plasmepsin I and II

With the aim to develop inhibitors of the plasmepsin I and II aspartic proteases of the malaria parasite Plasmodium falciparum, we have synthesized sets of libraries from novel reversed-statine isosteres, using a combination of solution phase and solid phase chemistry. The synthetic strategy furnishes the library compounds in good to high overall yields and with excellent stereochemical control throughout the developed route. The products were evaluated for their plasmepsin I and II inhibiting properties and were found to exhibit modest but promising activity. The best inhibitor exhibits an in vitro activity of 28% inhibition of plasmepsin II at an inhibitor concentration of 0.5 μM ( K i for Plm II=5.4 μM).

Novel chemistry of alpha-tosyloxy ketones: applications to the solution- and solid-phase synthesis of privileged heterocycle and enediyne libraries.

New synthetic technologies for the preparation and elaboration of alpha-tosyloxy ketones in solution- and on solid-phase are described. Both olefins and ketones serve as precursors to these relatively stable chemical entities: olefins via a novel one-pot epoxidation, arylsulfonic acid displacement, and oxidation sequence, and ketones by direct exposure to arylsulfonic acids in the presence of diacetoxy iodobenzene. Reaction of these substrates with O-, S-, or N-centered nucleophiles leads to incorporation of the nucleophile with concomitant expulsion of the sulfonate, while exposure to bis-functional nucleophiles furnishes annulated heterocyclic systems. In addition, the reactions of carbon-centered nucleophiles with alpha-tosylyloxy ketones are also explored. The collated data for all these nucleophiles provide compelling evidence for the proposal that different reaction pathways are followed when alpha-tosyloxy ketones are engaged by "hard" versus "soft" nucleophiles. The accessibility and site-selectivity of the chemistry demonstrated herein offer the promise of an expanded use for this moiety in solid-phase library construction, in particular, and in the field of organic synthesis, in general.

Solid-phase synthesis of trisubsituted guanidines.

The solid-phase library synthesis of trisubstituted guanidines was accomplished. Amines were loaded onto the 4-formyl-3,5-dimethoxyphenoxymethyl linker via reductive amination. Subsequent acylation with Fmoc-4-aminomethylbenzoic acid followed by Fmoc deprotection gave solid-supported primary amines. Alternatively, sulfonylation of resin-bound secondary amines with 4-cyanobenzenesulfonyl chloride followed by borane reduction also gave solid-supported primary amines. Both resins were acylated with isocyanates to furnish solid-supported ureas. Dehydration of ureas with p-toluenesulfonyl chloride in pyridine gave solid-supported carbodiimides. Nucleophilic addition of amines to the carbodiimide bond followed by cleavage off the solid support gave trisubstituted guanidines.

The one-bead two-compound assay for solid phase screening of combinatorial libraries.

Fluorescent quenched substrate libraries are a very powerful tool for investigation of protease activity and specificity. Particularly, libraries where the fluorescent resonance energy transfer (FRET) pair is 3-nitrotyrosine and 2-amino-benzamide are easy to prepare by split and combine synthesis to yield a one-bead one-compound library format. The solid support is critical for the successful hydrolysis of the resin-bound substrates. For this purpose, a range of highly porous poly(ethylene glycol) (PEG)-based resins have been developed. Active substrates yield highly fluorescent beads and these are selected under a fluorescence microscope or isolated on a bead sorter. Edman sequence analysis yields the substrate sequence, the cleavage point, and the degree of conversion. The method gives a complete map of the substrate specificity, and substrates with high affinity for the active site can be selected. These may in turn be used as inhibition indicators in a second solid phase library assay for enzyme inhibition where each single bead is transformed into an assay container. The substrate is attached to temporarily shielded functional groups after completion of inhibitor library synthesis. By using a photolabile linker and ladder synthesis, the active inhibitors may be rapidly identified by mass spectrometry. In each bead, the putative inhibitor competes with the substrate attached for binding to the enzyme, and when the inhibitor binds strongly, the substrate remains intact and quenched. Thus dark beads indicate inhibitors, and these may be isolated using a bead-sorter and the structure determined by mass spectrometry. A selection of the best substrates and inhibitors should always be resynthesized for solution kinetics and confirmation of the results obtained on solid support. The inhibitor assay is almost free from false positives, which is a consequence of combining the binding of the protease to the inhibitor with observation of activity toward a FRET substrate. The K(i) of the identified inhibitors are typically in the nM range.

Virtual screening of combinatorial libraries across a gene family: in search of inhibitors of Giardia lamblia guanine phosphoribosyltransferase.

Parasitic protozoa lack the ability to synthesize purine nucleotides de novo, relying instead on purine salvage enzymes for their survival. Guanine phosphoribosyltransferase (GPRT) from the protozoan parasite Giardia lamblia is a potential target for rational antiparasitic drug design, based on the experimental evidence, which indicates the lack of interconversion between adenine and guanine nucleotide pools. The present study is a continuation of our efforts to use three-dimensional structures of parasitic phosphoribosyltransferases (PRTs) to design novel antiparasitic agents. Two micromolar phthalimide-based GPRT inhibitors were identified by screening the in-house phthalimide library. A combination of structure-based scaffold selection using virtual library screening across the PRT gene family and solid phase library synthesis led to identification of smaller (molecular weight, <300) ligands with moderate to low specificity for GPRT; the best inhibitors, GP3 and GP5, had K(i) values in the 23 to 25 microM range. These results represent significant progress toward the goal of designing potent inhibitors of purine salvage in Giardia parasites. As a second step in this process, altering the phthalimide moiety to optimize interactions in the guanine-binding pocket of GPRT is expected to lead to compounds with promising activity against G. lamblia PRT.

Phosphinic peptide inhibitors of macrophage metalloelastase (MMP-12). Selectivity and mechanism of binding.

Pseudopeptide inhibitors of MMP-12 with a phosphinic dipeptide G psi[PO(2)H-CH(2)]L covering the P1-P1'- positions originating from a combinatorial solid phase library have been identified and kinetically analysed with respect to binding mechanism and selectivity towards MMP-7, MMP-9, MMP-13 and MMP-14. One compound with a low nanomolar dissociation constant for MMP-12 showed significantly lower affinity towards all other MMPs tested compared to MMP-12. Two compounds showed selectivity against MMP-9, MMP-13 and MMP-14. One additional compound showed selectivity against MMP-7. The selectivity of these compounds could partly be rationalized by analysis of homology models of the enzymes. Truncated versions of one inhibitor spanning P2 to P2', P3 to P2' or P2 to P3' showed that interactions on both the prime and the non-prime side are important for binding. A two-step binding mechanism, with a rate limiting second step, was shown for binding of a tryptophane containing inhibitor to MMP-12 by transient state analysis, using the tryptophane residue of the inhibitor as fluorescent probe.

Application of base cleavable safety catch linkers to solid phase library production.

We have used sulfide "Safety Catch" linkers to anchor typical medicinal chemistry functional groups to amine resins. Compounds are loaded as the ester, carbamate, or amine. At the end of the synthesis, the linker is activated by peracid. The sulfone resins are then cleaved by beta-elimination in the gas phase or in solution by secondary amines to produce acids and primary, secondary, or tertiary amines. Comparison of cleavage rates to other sulfone resins including SEM showed significantly faster cleavage for this system with conditions similar to Fmoc deprotection. Application of this strategy to a medicinal chemistry library gives good yields and purities of the resulting compounds.

DNA-binding peptides searched from the solid-phase combinatorial library with the use of the magnetic beads attaching the target duplex DNA

We have exhibited successful and rapid screening of DNA-binding peptide ligands from solid-phase library beads with the use of the target DNA-conjugated magnetic beads. The target duplex DNA (3) has a polyether linker between two complementary sequences (T4A3G-ether linker-CT3A4) and is stable in the duplex form during the selection procedure. Finally, 71 pentapeptide sequences were identified from the solid-phase pentapeptide library. From an analysis of the peptide sequences identified in this study, it has been revealed that peptide ligands contain hydrophobic amino acids as the major component. The synthetic peptides with identified sequences and a combination of the major components have exhibited moderate to high binding affinity to the duplex DNA in competition experiments with ethidium–DNA complexes.

Single-bead structure elucidation. Requirements for analysis of combinatorial solid-phase libraries by Nanoprobe MAS-NMR spectroscopy

A complete NMR structure analysis of an eight-residue peptide, covalently bound to a single-bead of a poly(ethylene glycol) based (POEPOP 1500) resin, is described. Well resolved 1D and 2D 1H NMR spectra were obtained using magic angle spinning (MAS) Nanoprobe NMR spectroscopy at 500 MHz. The quantity of peptide on individual beads was determined after each NMR experiment by cleaving the peptide from the resin, and subsequent measurement of the fluorescence from the Abz-containing peptide in solution. It was found that about 1.6 nmol of peptide was the minimum amount needed to obtain a high resolution 1D 1H NMR spectrum in less than 20 minutes. For a complete structure elucidation by 2D 1H NMR a loading of about 6 nmol of peptide on a single-bead was required. It has been demonstrated that, with a sufficient loading on PEG-based resins, a complete structure elucidation of a resin bound octapeptide on a single bead can be achieved. This observation may be useful for screening and analysis of ‘one-bead, one-compound’ libraries.

Solid-phase library synthesis of triazolopyridazines via [4+2] cycloadditions

The solid-phase library synthesis of triazolopyridazines was accomplished using a [4+2] cycloaddition strategy. The Horner-Wadsworth-Emmons (HWE) reagent diethylphosphonoacetic acid was attached to the N-terminus of amino acids on Rink amide solid-phase peptide synthesis support. The HWE reaction with a variety of α,β-unsaturated aldehydes gave high conversion to diene amides. Subsequent [4+2] cycloaddition with 4-substituted urazines gave triazolopyridazines upon cleavage from resin. An alternate [4+2] cycloaddition method with urazine followed by a Mitsunobu reaction also provided triazolopyridazines.

Synthesis and screening of an indexed motif-library containing non-proteinogenic amino acids.

In an effort to increase the probability of finding novel peptides in resin-bound combinatorial libraries displaying affinity to various macromolecular targets, we increased the diversity of a solid-phase library considerably by synthesizing multiple structures on each bead - a motif-library - including 45 building blocks. The building blocks consist of L-aa, D-aa and eight hydrophobic non-proteinogenic alpha-amino acids. A library with the format O-Z0-1-O-Z0-1-O-XX-resin was synthesized giving the four motifs OOOXX, OZOOXX, OOZOXX, OZOZOXX corresponding to 364.500 different motifs (45(3) x 4 theoretical combinations). The positions O are defined amino acids while Z represents three mixtures pi, omega, phi, where pi is a mixture of polar and charged residues, omega is a mixture of aliphatic residues and phi is a mixture of aromatic residues. X represents a mixture of all 45 residues. The library was screened with the macromolecular target streptavidin which served as a model receptor. Binding peptides were sequenced by microsequencing. We included small amounts of norvaline and norleucine in the library, which served as index residues to be able to distinguish between LD-amino acids and other residues with the same retention time in the HPLC system. Beads that interact with the receptor were found, and the binding motifs that appeared had no homology to known binding motifs found in either L-aa or D-aa libraries, instead motifs with the non-proteinogenic residues L-phenylglycine, O-benzyl-L-hydroxyproline and O-benzyl-L-tyrosine dominated. The novel peptides inhibit binding of biotin to streptavidin but do not bind to avidin, and the affinity is higher than the peptides found in linear all L-aa peptide libraries.

PEGA supports for combinatorial peptide synthesis and solid-phase enzymatic library assays.

Permeable resins cross-linked with long PEG chains were synthesized for use in solid-phase enzyme library assays. High molecular weight bis-amino-polyethylene glycol (PEG) 4000, 6000, 8000 were synthesized by a three-step reaction starting from PEG-bis-OH. Macromonomers were synthesized by partial or di-acryloylation of bis-amino-PEG derivatives. Bis/mono-acrylamido-PEG were copolymerized along with acrylamide by inverse suspension copolymerization to yield a less cross-linked resin (Type I, compounds 6-9). Furthermore, acryloyl-sarcosin ethyl ester was co-polymerized along with bis-acrylamido PEG to obtain more crosslinked capacity resin (Type II, compounds 13-19). N,N-Dimethylacrylamide was used as a co-monomer in some cases. The polymer was usually obtained in a well-defined beaded form and was easy to handle under both wet and dry conditions. The supports showed good mechanical properties and were characterized by studying the swelling properties, size distribution of beads, and by estimating the amino group capacity. Depending on the PEG chain length, the monomer composition and the degree of cross-linking the PEGA supports showed a high degree of swelling in a broad range of solvents, including water, dichloromethane, DMF, acetonitril, THF and toluene: no swelling was observed in diethyl ether. The PEGA resins (Type I) with an amino acid group capacity between 0.07 and 1.0 mmol/g could be obtained by variation of the monomer composition in the polymerization mixture. Fluorescent quenched peptide libraries were synthesized on the new polymer using a multiple column library synthesizer and incubated with the matrix metalloproteinase MMP-9 after it had been activated by 4-aminophenyl mercuric acetate resulting in 67/83 kDa active enzyme. The bright beads were separated manually under a fluorescence microscope and sequenced to obtain peptide substrates for MMP-9. After treatment with ethylene diamine, high-loaded resins (Type II) have been employed in continuous flow peptide synthesis to yield peptides in excellent yield and purity.

Polyvalent binding to carbohydrates immobilized on an insoluble resin.

Numerous studies have established that polyvalency is a critical feature of cell surface carbohydrate recognition. Nevertheless, carbohydrate-protein interactions are typically evaluated by using assays that focus on the behavior of monovalent carbohydrate ligands in solution. It has generally been assumed that the relative affinities of monovalent carbohydrate ligands in solution correlate with their polyvalent avidities. In this paper we show that carbohydrate ligands synthesized directly on TentaGel beads interact with carbohydrate-binding proteins in a polyvalent manner. The carbohydrate-derivatized beads can, therefore, be used as model systems for cell surfaces to evaluate polyvalent carbohydrate-protein interactions. By using a combinatorial approach to synthesize solid-phase libraries of polyvalent carbohydrates, one can rapidly address key issues in the area of cell surface carbohydrate recognition. For example, studies reported herein demonstrate that there is an unanticipated degree of specificity in recognition processes involving polyvalent carbohydrates. However, the correlation between polyvalent avidities and solution affinities is poor. Apparently, the presentation of carbohydrates on the polymer surface has a profound influence on the interaction of the ligand with the protein receptor. These findings have implications for how carbohydrates function as recognition signals in nature, as well as for how polyvalent carbohydrate-protein interactions should be studied.

Peptide mimics of a conformationally constrained protective epitopes of respiratory syncytial virus fusion protein

Aims: To identify peptides that mimic (mimotopes) conformational and protective epitopes of RSV fusion protein and to assess their efficacy as immunogens and potential vaccines. Material and methods: An 8-mer solid-phase (TG resin) library was screened with a neutralising and protective RSV fusion protein specific monoclonal antibodies (Mab-19). After selection of positive beads, reactive sequences were identified by microsequencing and 8-mer peptides were synthesised. Improvement of binding was analysed by amino acid replacement using the SPOTs method. Results: Mabs were not able to bind to the free and soluble peptides, nor did these peptides induce anti-RSV specific antibodies. However, several peptides re-synthesised on a TG resin (to produce de-protected 8-mer peptides linked to the resin) or as SPOTs reacted specifically. Therefore it was critical to be able to reproduce this conformation in order to use these mimotopes as immunogens and potential vaccines. Using C-terminal constrained versions of the mimotopes, strong binding of one of the Mabs to the peptides was demonstrated by surface-plasmon resonance. Immunisation of Balb/c mice with these peptide-mimics produced anti-sera that: (1) reacted specifically with RSV; (2) inhibited the binding of the Mab to the virus; (3) neutralised RSV in vitro with high titres (range: 80–640); and (4) reduce significantly the viral load in the lungs of mice challenged with RSV ( P<0.01). Conclusions: This report demonstrates for the first time that: (1) a protective epitope of the conserved RSV fusion protein can be mimicked by synthetic peptides; and (2) immunisations with these mimotopes induced specific anti-RSV neutralising antibodies and reduced viral load in vivo. These results represent a novel concept for the development of a vaccine against RSV.

Characterization of the substrate specificity of the major cysteine protease (cruzipain) from Trypanosoma cruzi using a portion-mixing combinatorial library and fluorogenic peptides.

The substrate specificity of the major cysteinyl proteinase of the parasitic protozoan Trypanosoma cruzi (cruzipain) was investigated, by combinatorial replacement of amino acid residues at positions P5-P'5, using a fluorescent quenched solid-phase library assay. Positively charged residues appear to be a general preference in the P5-P3 and the P'5-P'3 positions, while a hydrophobic residue was always required at the P2 position. A broad range of amino acids could be accepted at the P'1 position. A clear difference in terms of specificity between cruzipain and human cathepsin L was observed for the accommodation of Pro at the P2 position. The P1 specificity was investigated by a more detailed enzyme kinetic analysis using peptidyl-MCA (where MCA is methylcoumarin amide) and Abz-peptidyl-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] as substrates, and the results were compared with those obtained using human cathepsin L. Cruzipain showed a clear preference for benzyl-Cys or Arg at the P1 position. Human cathepsin L presented similar behaviour to that of cruzipain for the hydrolysis of the epsilon-NH2-Cap-Leu-Xaa-MCA (where Cap is epsilon-aminocaproyl) and Abz-Lys-Leu-Xaa-Phe-Ser-Lys-Gln-EDDnp series, whereas the mammalian enzyme was able to tolerate large P1 residues, such as phenylalanine, better than cruzipain in the latter series.

Screening a Random Pentapeptide Library, Composed of 14 D-Amino Acids, against the COOH-terminal Sequence of Fructose-1,6-bisphosphate Aldolase from Trypanosoma brucei

A random pentapeptide library composed of 14 D-amino acids, including two unusual amino acids, thus representing 537,824 different peptide sequences anchored on polystyrene beads was created with each bead bearing a single pentapeptide sequence. This library was used for affinity screening against the fructose-1, 6-bisphosphate aldolase of Trypanosoma brucei labeled with biotin as well as versus the COOH-terminal labeled with fluorescein isothiocyanate. The thus selected peptide beads were identified and the appropriate sequences synthesized as peptide amides and evaluated for enzyme activity inhibition. Screening against the whole enzyme did not result in selection of an enzyme inhibitor. However, we demonstrate here that screening against a part of the enzyme involved in the catalytic activity may lead to the discovery of an enzyme inhibitor as well as an enzyme activator. Two low affinity inhibitors, RRVKF-NH2 and KThiKAR-NH2, with an IC50 of approximately 1 mM and approximately 0.2 mM, respectively, were identified. Two other pentapeptides with the sequence SWChaKK-NH2 and SKChaKM-NH2 are able to activate the enzyme fructose-1, 6-bisphosphate aldolase. Thus, successful screening of solid phase libraries can be accomplished using selected sequences of the target enzyme.

Synthesis and Screening of an Indexed Motif-Library Containing Non-proteinogenic Amino Acids

In an effort to increase the probability of finding novel peptides in resin-bound combinatorial libraries displaying affinity to various macromolecular targets, we increased the diversity of a solid-phase library considerably by synthesizing multiple structures on each bead – a motif-library – including 45 building blocks. The building blocks consist of L-aa, D-aa and eight hydrophobic non-proteinogenic α-amino acids. A library with the format O-Z0–1-O-Z0–1-O-XX-resin was synthesized giving the four motifs OOOXX, OZOOXX, OOZOXX, OZOZOXX corresponding to 364.500 different motifs (453×4 theoretical combinations). The positions O are defined amino acids while Z represents three mixtures Π, Ω, ϖ, where Π is a mixture of polar and charged residues, Ω is a mixture of aliphatic residues and ϖ is a mixture of aromatic residues. X represents a mixture of all 45 residues. The library was screened with the macromolecular target streptavidin which served as a model receptor. Binding peptides were sequenced by microsequencing. We included small amounts of norvaline and norleucine in the library, which served as index residues to be able to distinguish between LD-amino acids and other residues with the same retention time in the HPLC system. Beads that interact with the receptor were found, and the binding motifs that appeared had no homology to known binding motifs found in either L-aa or D-aa libraries, instead motifs with the non-proteinogenic residues L-phenylglycine, O-benzyl-L-hydroxyproline and O-benzyl-L-tyrosine dominated. The novel peptides inhibit binding of biotin to streptavidin but do not bind to avidin, and the affinity is higher than the peptides found in linear all L-aa peptide libraries. © 1997 European Peptide Society and John Wiley & Sons, Ltd