PEGA Resin Research Articles

Divergent Pathway for the Solid-Phase Conversion of Aromatic Acetylenes to Carboxylic Acids, α-Ketocarboxylic Acids, and Methyl Ketones

An efficient divergent pathway for the selective and quantitative solid-phase conversion of aromatic acetylenes to the corresponding carboxylic acids, alpha-keto-carboxylic acids, and methyl ketones is presented. A range of aromatic trimethylsilyl-protected acetylene building blocks was synthesized in excellent yields using a Sonogashira cross-coupling protocol and used in solid-phase synthesis on PEGA resin. Dependent on the selection of conditions, the same solid-supported alkyne could be quantitatively converted to an aromatic carboxylic acid, an aromatic alpha-ketocarboxylic acid, or an aromatic methyl ketone. The conversion to carboxylic acid involved an OsO4/NaIO4/HMTA-mediated oxidative cleavage of the silyl-deprotected alkyne to provide the aromatic carboxylate in excellent yield. The alpha-ketocarboxylic acids were obtained by direct treatment of the trimethylsilyl-protected alkyne with OsO4/NMO/HMTA, while the ketones were obtained by simple acid-mediated hydration of the alkyne using aqueous TFA. In general, all products were obtained in excellent purities, typically above 90%. In addition, it was shown that the alkyne-containing building blocks could easily be incorporated into resin-bound peptides and after chemoselective conversion of the alkyne the new functional groups could be used for further derivatization into peptidomimetic compounds.

Enzymatic Release and Macrolactonization of Cryptophycins from a Safety-Catch Solid Support

Thioesters need not apply: Cryptophycin thioesterase (Crp TE) cleaves and macrolactonizes linear cryptophycin substrates bound to activated safety-catch PEGA resin. This novel enzymatic solid-phase approach was used to further investigate the tolerance of Crp TE for structural variations of substrates.

Microwave-Assisted Solid-Phase Synthesis of 2,5-Diketopiperazines: Solvent and Resin Dependence

Solid-phase synthesis of diketopiperazines (DKPs) was preformed using various combinations of resins (polystyrene, TentaGel, ArgoGel, and PEGA) and solvents (toluene, tert-butyl alcohol, water, and toluene/2-butanol (1:4, v/v). The DKPs were synthesized from solid-phase bound dipeptides via intramolecular aminolysis. Both thermal and microwave-assisted solid-phase synthesis of DKPs gave high yields of products independently of resin and organic solvent used; however, only the PEGA resin resulted in high yields of DKPs in water independent of heating method. The short reaction times, high yields, and the possibility to run reactions in water when an appropriate resin is used makes the microwave-assisted solid-phase synthesis the method of choice. The method should be suitable for solid-phase synthesis of diketopiperazine-based libraries.

Practical syntheses of [1,3,5]‐triazine dendritic molecules on solid supports

AbstractA practical and divergent synthesis of supported [1,3,5]‐triazine dendritic molecules on Wang resin, PEGA resin, SynPhase™ Lanterns, and silica gel is described. The alkylamine linkers used allow derivatization with functionality for both synthetic (e.g., supported reagent and scavenger activity) and chemical biology applications. The use of supported intermediates allows differentiation of symmetric linkers without the need for protecting group chemistry. The synthetic route uses inexpensive, readily available starting materials in a straightforward and scaleable strategy. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 2248–2259, 2006

Conformational Studies of Resin-Bound Vancomycin and the Complex of Vancomycin and Ac2-l-Lys-d-Ala-d-Ala

The molecular target of vancomycin, a commonly used glycopeptide antibiotic, is the D-Ala-D-Ala dipeptide subunit on the bacterial cell wall. The molecular basis of interaction between vancomycin and D-Ala-D-Ala in solution is well-known. However, there is no structural data on vancomycin, and its interaction with D-Ala-D-Ala when the drug is tethered to a solid support. In this Article, vancomycin was directly coupled onto TentaGel or PEGA resin through its C terminus. High-resolution magic angle spinning NMR studies indicated that conformation of PEGA bead-bound vancomycin is identical to that of the free drug. Broadening and shifts of the same proton resonances were observed in solution-phase vancomycin or PEGA-bound vancomycin when complexed with Ac(2)-L-Lys-D-Ala-D-Ala. This study demonstrates that bead-bound molecules can behave the same as solution-phase molecules in terms of molecular interaction with its target molecule, thus validating the on-bead screening approach of the "one-bead-one-compound" combinatorial library method.

Synthesis of monofunctionalized gold nanoparticles by fmoc solid-phase reactions.

In this communication, solid-phase reactions for the synthesis of Lys-monofunctionalized gold nanoparticles are described. A controlled and selective fabrication of linear nanoparticle arrays can be achieved through peptide linkage systems, and therefore it is essential to prepare Fmoc amino acid nanoparticle building blocks susceptible to Fmoc solid-phase peptide synthesis. Gold nanoparticles containing carboxylic acids (2) in the organic shell were covalently ligated to Lys on solid supports through amide bond coupling reactions. We employed Fmoc-Lys-substituted polymer resins such as Fmoc-Lys-Wang or Fmoc-Lys-HMPA-PEGA. The low density of Lys on the matrix enabled 2 nm-sized gold nanoparticles to react with Lys in a 1:1 ratio. Subsequent cleavage reactions using 60% TFA reagent resulted in Lys transfer from the solid matrix to gold nanoparticles, and the Fmoc-Lys-monofunctionalized gold nanoparticles (5) were obtained with 3-15% yield. Synthesis using HMPA-PEGA resin increased productivity due to the superior swelling properties of PEGA resin in DMF. Monofunctionalization of nanoparticles was microscopically characterized using TEM for the ethylenediamine-bridged nanoparticle dimers (6). By counting the number of 6, we found that at least 60% of cleaved nanoparticles were monofunctionalized by Lys. This method is highly selective and efficient for the preparation of monofunctionalized nanoparticles.

Introduction of permanently charged groups into PEGA resins leads to improved biotransformations on solid support

The application of biotransformations in solid phase synthesis is an attractive alternative to chemical methodologies. An important issue that needs to be addressed in this context is accessibility of functional groups within a porous polymer to the biocatalyst. This paper shows that such accessibility can be improved for penicillin G amidase by introducing permanent charges into the polymer. The effect appears to be due to better swelling of the polymer in buffer and to electrostatic interactions between polymer and enzyme.

Solid-phase synthesis for the identification of high-affinity bivalent lectin ligands.

The development of carbohydrate-based therapeutics has been frustrated by the low affinities that characterize protein-carbohydrate complexation. Because of the oligomeric nature of most lectins, the use of multivalency may offer a successful strategy for the creation of high-affinity ligands. The solid-phase evaluation of libraries of peptide-linked multivalent ligands facilitates rapid examination of a large fraction of linker structure space. If such solid-phase assays are to replicate solution binding behavior, the potential for intermolecular bivalent binding on bead surfaces must be eliminated. Here we report the solid-phase synthesis and analysis of peptide-linked, spatially segregated mono- and bivalent ligands for the legume lectin concanavalin A. Bead shaving protocols were used for the creation of beads displaying spatially segregated binding sequences on the surface of Tentagel resins. The same ligands were also synthesized on PEGA resin to determine the effect of ligand presentation on solid-phase binding. While we set out to determine the lower limit of assay sensitivity, the unexpected observation that intermolecular bivalent ligand binding is enhanced for bivalent ligands relative to monovalent ligands allowed direct observation of the level of surface blocking required to prevent intermolecular bivalent ligand binding. For a protein with binding sites separated by 65 A, approximately 99.9% of Tentagel(1) surface sites and 99.99% of the total sites on a PEGA bead must be blocked to prevent intermolecular bivalent binding. We also report agglutination and calorimetric solution-phase binding studies of mono- and bivalent peptide-linked ligands.

Improved biotransformations on charged PEGA supports.

PEGA supports functionalised with permanent charges show superior swelling properties in aqueous media when compared to neutral PEGA; a novel positively charged PEGA resin significantly improves penicillin G amidase (PGA) catalysed biotransformation on solid support, by favouring accessibility of the negatively charged enzyme.

Glycopeptides as Oligosaccharide Mimics: High Affinity Sialopeptide Ligands for Sialoadhesin from Combinatorial Libraries

Two different sialic acid containing glycopeptide (sialopeptide) libraries were synthesized using the portion mixing method and ladder synthesis. The libraries were attached via an IMP spacer and a photolabile linker to PEGA(1900) resin in order to facilitate rapid and unambiguous structural analysis of hits by MALDI-TOFMS. One library contained a lactamized sialic acid moiety at the N terminus of a pentapeptide, while a second library displayed a sialic acid residue at the center of a heptapeptide. The sialopeptide libraries were screened against the recombinant binding domain (SnD1) of a sialic acid binding Ig-like protein, sialoadhesin (Siglec-1). No ligands were identified from the lactamized sialic acid library, underscoring the importance of the carboxylic acid moiety for binding. Screening of the second gave few distinct hits (approximately 0.03% of library) with a high consensus. The high-affinity ligands contained, in most cases, a WG motif following the sialylated Thr. The strength of binding of selected ligands was determined by surface plasmon resonance. The best sialopeptide ligand, WLLT(Sa)WGT, exhibited micromolar affinity of SnD1; >10 times the affinity of SnD1 to 3'-sialyl lactose.

Solid-phase oligosaccharide and glycopeptide synthesis using glycosynthases.

Enzymatic approaches for the preparation of oligosaccharides are interesting alternatives to traditional chemical synthesis, the main advantage being the regio- and stereoselectivity offered without the need for protecting groups. The use of solid-phase techniques offers easy workup procedures and the prospect of automatability. Here, we report the first application of glycosynthases to solid-phase oligosaccharide synthesis by use of the 51 kDa serine and glycine mutants of Agrobacterium sp. beta-glucosidase, Abg E358S and E358G. Acceptors were linked to PEGA resin through a backbone amide linker (BAL), and using these mutated enzymes, a galactose moiety was transferred from a donor sugar, alpha-D-galactosyl fluoride, with high efficiency (>90%) together with excellent recovery of material. Furthermore, it was demonstrated that a resin-bound model glycopeptide was also an acceptor for the glycosynthase.

Dynamic and magnetic susceptibility effects on the MAS NMR linewidth of a tetrapeptide bound to different resins

AbstractUnder magic angle spinning, the NMR spectrum of the tetrapeptide Ala‐Ile‐Gly‐Met bound to a Wang resin, and swollen in DMF, exhibits proton and carbon linewidths that are sharp enough to allow the complete characterization of the peptide using classical liquid‐state NMR methods. The proton linewidths of the bound peptide remain, however, about three times larger than those of the free peptide in solution. The residual NMR linewidth originates essentially from incompletely averaged magnetic susceptibility effects due to the Wang resin. Replacing the aromatic Wang resin with a PEGA or POEPOP resin removes this effect. To investigate the contribution to line broadening of the peptide dynamics, relaxation studies were performed on the peptide bound to Wang and POEPOP resins. Copyright © 2001 John Wiley & Sons, Ltd.

Synthesis of tumor associated sialyl-T-glycopeptides and their immunogenicity.

Sialyl-T-glycopeptides were synthesized by solid-phase techniques, using a PEGA resin as the solid support. An appropriately protected building block containing alpha-Neu5Ac-(2 --> 3)-beta-Gal-(1 --> 3)-alpha-GalN3-(1-->) attached to Fmoc-Thr/Ser-OPfp was employed in a solid phase glycopeptide assembly of a 10-mer glycopeptide, using a general Fmoc/OPfp-ester strategy. Reduction of the azido group of the GalN3 residue was effected on solid-phase, using DTT and DBU. After acidolytic cleavage from the resin, the methyl ester of the sialic acid residue and acetyl groups were removed with 30% NaOMe/MeOH in MeOH and water pH 14, at -30 degrees C for 2 h. At this low temperature, the highly basic conditions did not result in any detectable beta-elimination. However, one O-acetyl group, located at the 2-position of the Gal was resistant to hydrolysis. To remove this remaining acetyl group, reaction with hydrazine hydrate in CHCl3 and MeOH at room temperature for 2.5 h was successful. The two target sequences of sialyl-T-glycopeptides were obtained in good yield. In contrast to the the analogs carrying the T-antigen, the Sial-T-glycopeptides were nonimmunogenic, supporting the idea that the sialylation is a method of circumventing the recognition by the immune system.

Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity.

To map the substrate specificity of cysteine proteases, two combinatorial peptide libraries were synthesized and screened using the archetypal protease, papain. The use of PEGA resin as the solid support for library synthesis facilitated the application of an on-resin fluorescence-quenched assay. Results from the screening of library 2 indicated a preference for Pro or Val in the S3 subsite and hydrophobic residues in S2; the most prevalent residue not being Phe but Val. The S1 subsite exhibited a dual specificity for both small, nonpolar residues, Ala or Gly, as well as larger, Gln, and charged residues, Arg. Small residues predominated in the S1'-S4' subsites. Active peptides from the libraries and variations thereof were resynthesized and their kinetics of hydrolysis by papain assessed in solution phase assays. Generally, there was a good correlation between the extent of substrate cleavage on solid phase and the kcat/KM's obtained in solution phase assays. Several good substrates for papain were obtained, the best substrates being Y(NO2)PMPPLCTSMK(Abz) (kcat/KM = 2109 (mM s)-1), Y(NO2)PYAVQSPQK(Abz) (kcat/KM = 1524 (mM s)-1), and Y(NO2)PVLRQQRSK(Abz) (kcat/KM = 1450 (mM s)-1). These results were interpreted in structural terms by the use of molecular dynamics (MD). These MD calculations indicated two different modes for the binding of substrates in the narrow enzyme cleft.

A new linker for the synthesis of C-terminal peptide α-oxo-aldehydes

A tartaric acid-based linker was elaborated on an amino PEGA resin for the synthesis of C-terminal α-oxo-aldehydes. A solid phase periodic oxidation allowed the formation of the glyoxylyl moiety and the separation of the final product from the solid support.

Comparison of resin and solution screening methodologies in combinatorial chemistry and the identification of a 100 nM inhibitor of trypanothione reductase.

Two identical polyamine peptide conjugate libraries were screened against the parasitic enzyme trypanothione reductase. One of these libraries was in a solution format, while the other was resin-based and was used in two resin-based screens (a diminution assay and a direct bead screening). Potent inhibitors (100 nM) of trypanothione reductase were identified both in the solution screen and in the resin-based screens when using the PEGA resin of Meldal. Resin screening of both types failed to work with TentaGel resin. Importantly there was excellent agreement between the solution and resin-based assays, suggesting both methods are reliable for the screening of combinatorial 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.

Evaluation of resins for on-bead screening: A study of papain and chymotrypsin specificity using pega-bound combinatorial peptide libraries

TentaGel, ArgoGel and PEGA resins were evaluated for on-bead biological screening, using a fluorescently-labelled peptide attached to each and assayed for papain activity. Peptide attached to PEGA was cleaved in near quantitative yield at the expected sites, whilst an identical sequence on TentaGel and ArgoGel beads was hydrolysed in very low yields and nonspecifically on ArgoGel. The compatibility of PEGA with enzymes was further demonstrated by the determination of subsite specificities of papain and chymotrypsin using PEGA-bound peptide libraries, which proved to be similar to those observed in free solution.

Synthesis and characterization of polyethylene glycol polyacrylamide copolymer (PEGA) resins containing carbohydrate ligands. Evaluation as supports for affinity chromatography

The PEGA resin, a beaded polyethylene glycol dimethylacrylamide copolymer, was evaluated as an affinity support for the purification of carbohydrate-binding macromolecules, namely, the cation-independent mannosyl phosphate receptor (CI-MPR) and a polyclonal antibody directed against a Streptococcus Group A oligosaccharide. Two polyethylene glycol (PEG) derivatives, a di-acryloylated PEG1900derivative or a longer di-acryloylated PEG4000derivative, were used as cross-linkers. The longer cross-linker was synthesized in four steps from polyethylene glycol 4000. The mannosyl 6-phosphate (M6P)-containing immunoaffinity columns were prepared through the inverse suspension radical copolymerization of the corresponding allyl glycoside with acrylamide and the PEG cross-linker. The resin with the shorter cross-linker (PEG1900derivative) had a 6.3% molar cross-linking while that with the longer cross-linker (PEG4000derivative) had a 3.8% molar cross-linking. For the Streptococcus Group A trisaccharide-containing immunoaffinity columns, three PEGA affinity supports bearing free amino groups were prepared and subsequently substituted with a trisaccharide activated as its squarate adduct. While one resin contained the shorter cross-linker PEG1900and had a 3% molar cross-linking, the other two resins contained the longer cross-linker PEG4000 with a molar cross-linking of 5% and 3%, respectively. In affinity chromatographic studies, the M6P-containing columns were ineffective in retaining the cation-independent mannosyl phosphate receptor (CI-MPR, ~ 215kDa), whereas antibody (~ 150kDa) retention was observed with two of the three Streptococcus Group A trisaccharide-containing immunoaffinity columns. Key words: PEGA resins, immunoaffinity supports, carbohydrate ligands, antibody purification.

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. © 1998 European Peptide Society and John Wiley & Sons, Ltd.