C-Terminally modified peptides via cleavage of the HMBA linker by O-, N- or S-nucleophiles.

A large variety of C-terminally modified peptides was obtained by nucleophilic cleavage of the ester bond in solid phase linked peptide esters of 4-hydroxymethyl benzamide (HMBA). The developed methods provided peptides, C-terminally functionalized as esters, amides and thioesters, with high purity directly from the resin in a single reaction step. A comprehensive screening of the reaction conditions and scope for nucleophilic cleavage of peptides from the HMBA linker was performed.

In vivo imaging of MMP-13 activity in the murine destabilised medial meniscus surgical model of osteoarthritis.

OBJECTIVE: To detect and determine disease severity of osteoarthritis (OA) using a probe activated by matrix metalloproteinase-13 (MMP-13) in vivo in the murine destabilised medial meniscus (DMM) surgical model of OA. DESIGN: We have previously described MMP12ap and MMP13ap, internally quenched fluorescent peptide substrate probes that are activated respectively by MMP-12 and MMP-13. Here we used these probes to follow enzyme activity in vivo in mice knees 4, 6 and 8 weeks following DMM surgery. After in vivo optical imaging, disease severity was determined through traditional histological analysis. The amount of probe activation was analysed for discrimination between DMM, contralateral and sham operated knees, as well as for congruence between activity and histological damage. RESULTS: There was no specific activation of MMP12ap at the time points observed between sham operated and DMM operated, or their respective contralateral joints. The activation of the MMP13ap in the DMM model was highest 6 weeks after surgery, but was only specific compared against sham surgery 8 weeks after surgery (1.5-fold increase). The activation of MMP13ap correlated with histological damage 6 and 8 weeks after surgery, with correlations of 0.484 (P = 0.0032) and 0.478 respectively (P = 0.0049). This correlation dropped to 0.218 (P = 0.011) if all data were considered. CONCLUSION: The current MMP-13 activity probe is suitable for the discrimination between DMM and sham or contralateral knees 8 weeks after surgery, when cartilage loss is typified by the appearance of small fissures up to the tidemark, but not earlier. This activity correlates with the histological damage observed.

Radically altered T cell receptor signaling in glycopeptide-specific T cell hybridoma induced by antigen with minimal differences in the glycan group.

A T cell hybridoma raised against the synthetic glycopeptide T(72)(Tn) was used to study whether the initial TCR signaling events are markedly different when the hybridoma is stimulated with glycopeptides closely related to the cognate glycopeptide antigen. T(72)(Tn) has an alpha-D-GalNAc group O-linked to the central threonine in the decapeptide VITAFTEGLK, and the hybridoma is known to be highly specific for this carbohydrate group. T(72)(Tn)-pulsed APC induced tyrosine phosphorylation of the TCR-zeta 21- and 23-kDa proteins and the downstream p42/44 MAP kinase and strong IL-2 secretion. APC pulsed with T(72)(alpha-D-GlcNAc), which differs from T(72)(Tn) solely by the orientation of a hydroxy group in the carbohydrate structure, completely failed to induce detectable tyrosine phosphorylation and IL-2 secretion. APC pulsed with S(72)(Tn), which differs from T(72)(Tn) by not having a methyl group in the serine amino acid side chain to which the glycan is attached, induced partial tyrosine phosphorylation of the TCR-zeta 21-kDa protein, no tyrosine phosphorylation of the MAP kinases and no IL-2 production. Molecular modeling of the MHC/glycopeptide complex revealed that the dramatic difference between the stimulatory power of T(72)(Tn) and T(72)(alpha-D-GlcNAc) is mainly due to very small differences in the TCR exposed carbohydrate structure.

Combinatorial library of peptide isosters based on Diels-Alder reactions: identification of novel inhibitors against a recombinant cysteine protease from Leishmania mexicana.

A combinatorial split-and-mix library of peptide isosters based on a Diels-Alder reaction was synthesized as a "one-bead-two-compounds" library and encoded by ladder synthesis for facile analysis by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. In the "one-bead-two-compounds" library approach, each bead contains a library member as a putative protease inhibitor along with a fluorescence-quenched substrate for the protease. When the library was screened with CPB2.8 DeltaCTE, a recombinant cysteine protease from L. mexicana, several beads containing compounds with inhibitory activity could be selected from the library and analyzed by MALDI-TOF MS for structure elucidation. Two types of inhibitors were revealed. One novel class of inhibitors had the bicyclic Diels-Alder product isosteric element incorporated internally in a peptide, while the other type was an N-terminal alpha,beta-unsaturated ketone Michael acceptor used as starting material for the Diels-Alder reaction. Selected hit sequences and constructed consensus sequences based on the observed frequencies of amino acids in different subsites were resynthesized and assayed in solution for inhibitor activity and were shown to have IC(50) values in the high nanomolar to low micromolar range.

Diffusion of reagents in macrobeads.

A simple and effective method of measuring diffusion rates of various acylating reagents in macro beads (in this work diameters greater than 570 microm) of amino-functionalized resins is presented. The extent of diffusion at various points of time was determined by treating beads with a staining reagent ("chloranil", 2,3,5,6-tetrachloro-1,4-benzoquinone) that colored the regions of the beads that had not yet been permeated by reagent (the central part of the beads). The volume of unstained resin (permeated part) was compared with the volume of the stained part, and diffusion rate constants were calculated. Factors influencing diffusion such as reagent concentration, solvent, temperature, resin type, and mechanical effects were investigated. The results showed that diffusion was promoted by increased temperature, good swelling of the resin, small reagents, and high concentrations but not by sonication or mechanical agitation.

Solid-phase glycosylation of peptide templates and on-bead MAS-NMR analysis: perspectives for glycopeptide libraries.

The efficient solid-phase glycosylation of amino acid side chains (serine (Ser), threonine (Thr), and tyrosine (Tyr)) in peptides was demonstrated with a variety of glycosyl trichloroacetimidate donors in high yields and purities. A novel photolabile linker, with no chiral centre, was introduced to facilitate analysis by both matrix-assisted laser desorption ionisation time of flight (MALDI-TOF) mass spectrometry and nanoprobe magic angle spinning (MAS) NMR spectroscopy. Product analysis by nanoprobe MAS NMR spectroscopy, LC-MS and MALDI-TOF mass spectrometry of the glycosylation reactions indicated that the reactivity order of the hydroxy side-chain functions of amino acids in peptides on the solid-phase was Tyr>Ser>Thr. The nearly quantitative glycosylation yields and the efficient on-bead product analysis by nanoprobe MAS NMR spectroscopy have made a truly solid-phase approach for the synthesis and analysis of glycopeptide libraries possible.

alpha-Ketocarbonyl peptides: a general approach to reactive resin-bound intermediates in the synthesis of peptide isosteres for protease inhibitor screening on solid support.

alpha-Ketocarbonyl peptides were generated from peptide precursors on solid support via a metal-ion-catalyzed transamination. The reaction proceeded to completion within 2 h with glyoxylate as electrophile and copper(II) ions as catalyst in an aqueous acetate buffer at pH 5.5-6.0. The variety of naturally occurring alpha-amino acid substrates gave rise to a diverse set of differentially functionalized ketones. The highly reactive terminal ketocarbonyls were prone to aldol-type dimerization and could be transferred into stable moieties by oxime formation, reduction to the alcohol, or reductive amination, respectively. The alpha-ketocarbonyl peptides were efficient in nucleophilic addition of C-nucleophiles such as phosphono-ylides and allylsilanes.

Substrate specificity of recombinant cysteine proteinase, CPB, of Leishmania mexicana.

The primary S(1) subsite specificity of a recombinant cysteine proteinase, CPB2.8 Delta CTE, of Leishmania mexicana was investigated in a systematic way using a series of peptides derived from Abz-KLRFSKQ-EDDnp in which Arg was substituted by all natural amino acids (where Abz is ortho-amino-benzoyl and EDDnp is N-[2,4-dinitrophenyl]-ethylenediamine). The peptides from this series with charged side chain amino acids, Cys, Cys(SBzl), and Thr(OBzl) were well hydrolysed. All other substitutions resulted in peptides that were resistant or hydrolysed very slowly and inhibited the enzyme with K(i) values in the range of 9--400 nM. Looking for natural substrates for CPB2.8, we observed that the recombinant enzyme failed to release kinin from human kininogen, an activity earlier observed with cruzipain from Trypanosoma cruzi (Del Nery et al., J. Biol. Chem. 272 (1997) 25713.). This lack of activity seems to be a result of the resistance to hydrolysis of the sequence at the N-terminal site of bradykinin in the human kininogen. The preferences for the S(3), S(2) and S(1)'-S(3)' for some amino acids were also examined using substrates derived from Abz-KLRFSKQ-EDDnp with variations at Lys, Leu, Phe, Ser and Lys, using the amino acids Ala, Phe, Leu, His or Pro. Peptides with Phe at P(1)' presented the highest affinity to the leishmanial enzyme. For comparison, some of the obtained peptides were also assayed with recombinant human cathepsin L and cruzain. The best substrates for CPB2.8 Delta CTE were also well hydrolysed by cathepsin L, however, the best inhibitors of the parasite enzyme have low affinity to cathepsin L. These promising data provide leads for the design of anti-parasitic drugs directed against the leishmanial enzyme.

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.

Identification of peptides inhibitory to recombinant cysteine proteinase, CPB, of Leishmania mexicana.

We have identified peptides that are relatively resistant to hydrolysis by a recombinant cysteine proteinase, CPB2.8DeltaCTE, of Leishmania mexicana, and yet exhibit inhibition constant (K(i)) values in the nanomolar range. Common to these peptides is a basic-hydrophobic-hydrophobic motif in the P3-P1 sites, which is also present in the pro-region of the enzyme. A nine-amino acid stretch, FAARYLNGA, which has good homology to the pro-region of mammalian cathepsin L was identified as the part of the pro-region most likely to interact with the active site of the parasite enzyme. This peptide is not hydrolyzed by CPB2.8DeltaCTE and inhibited it with a K(i) of 4 microM. Extension of this sequence at both the N- and C-termini and the introduction of ortho-aminobenzoic acid at the N-terminal site reduced the K(i) value to 30 nM. The best substrate for CPB2.8DeltaCTE was also well hydrolyzed by cathepsin L, however the best inhibitor of the parasite enzyme inhibit poorly cathepsin L, with K(i) value two order of magnitude higher than against the parasite enzyme. These promising data provide insights into the peculiar specificity of the parasite enzyme and will aid the design of antiparasitic drugs directed against the leishmanial enzyme.

Surfactant Mediated Cationic and Anionic Suspension Polymerization of PEG-Based Resins in Silicon Oil: Beaded SPOCC 1500 and POEPOP 1500.

A novel surfactant has been synthesized for use in cationic and anionic ring-opening suspension polymerization of PEG-based macromonomers in silicon oil. A polymer of acrylate esters containing pentamethyldisiloxane and PEG was prepared by radical polymerization. The surfactant can stabilize an emulsion of PEG-based macromonomers, initiator, and solvent in silicon oil such that polymer beads are obtained by ring-opening polymerization, initiated either by a Lewis acid (cationic ring opening) or potassium tert-butoxide (anionic ring opening). The average bead size could be controlled by varying the stirring rate and the amount of surfactant and solvent. The surfactant does not interfere with the polymerization and can be removed together with residual silicon oil by a simple washing procedure.

Synthesis of aldehyde building blocks protected as acid labile N-Boc N,O-acetals: toward combinatorial solid phase synthesis of novel peptide isosteres.

The synthesis of (RS)-3'-tert-butoxycarbonyl-perhydro-1', 3'-oxazine-2'-yl acetic acid and the syntheses of the simple and C-2 substituted 3'-tert-butoxycarbonyl-perhydro-1',3'-oxazine-2'(RS)-yl propionic acids from simple starting materials are presented. The simple compounds were prepared from 1,3-propanediol and 1, 4-butanediol, respectively, via a short series of facile steps, in 70% overall yield in both cases. For the syntheses of the C-2 substituted compounds of the longer homologue, (RS)-3'-tert-butoxycarbonyl-perhydro-1',3'-oxazine-2'-yl propionic acid, a malonic ester route was selected, thus allowing easy incorporation of various side chains. The stability of the novel aldehyde protection group, the N-Boc N,O-acetal moiety, under various acidic conditions was investigated, and it was found to cleanly and rapidly yield the aldehyde under strong acidic conditions or, if desired, slower under less harsh conditions. As a demonstration of the use of the building blocks, one building block was coupled to a solid support and, after unmasking of the aldehyde, submitted to three different types of nucleophilic reactions (Pictet-Spengler condensation, reductive amination, Horner-Wadsworth-Emmons olefination) followed by further chemical modification, and the identity of the structures were verified after cleavage from the resin.

N-Terminal peptide aldehydes as electrophiles in combinatorial solid phase synthesis of novel peptide isosteres.

N-Terminal peptide aldehydes were synthesized on a solid support and utilized as electrophiles in nucleophilic reactions in order to furnish novel and diverse peptide isosteres. The aldehyde moiety of the peptide was synthesized by coupling a protected aldehyde building block to the peptide and deprotecting it quantitatively in less than 3 min. It was found that protection of the two succeeding amide nitrogens was necessary in order to avoid reaction between the aldehyde and backbone amides. The N-terminal peptide aldehydes were successfully reacted in the following way: (a) reductive amination with a large variety of amines, leading to N-alkyl-gamma-aminobutyric peptide isosteres positioned centrally in the peptide; (b) reductive amination with amino esters, leading to N-terminal 2,5-diketopiperazine peptides; (c) Horner-Wadsworth-Emmons olefination, leading to unsaturated peptide isosteres positioned centrally in the peptide; and (d) Pictet-Spengler condensations, leading to tetrahydro-beta-carbolines either positioned centrally in a peptide or fused with a diketopiperazine ring in the N-terminus of the peptide.

Solid-phase synthesis of chemotactic peptides using alpha-azido acids.

Four chemotactic peptides, For-Met-Xxx-Phe-OMe, with an alpha,alpha-disubstituted amino acid at position 2 have been synthesized by the azido acid method [Meldal M, Juliano MA, Jansson AM. 1997. Azido acids in a novel method of solid-phase peptide synthesis. Tetrahedron Lett. 38: 2531-2534] on solid-phase, and were tested for biological activity. Dipropylglycine in the central position (Xxx) was found to be as active as the natural chemotactic peptide for chemotactic activity toward human neutrophils. Higher yields were obtained than previously reported solution-phase syntheses of chemotactic peptides, and EEDQ was used successfully for the difficult solid-phase formylation of amino groups.

Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8.

Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P(4), P(1) and P(1)' subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S(4) subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S(1) and S(1)' subsites that flank the scissile peptide bond. The S(1) subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P(1)' position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P(1)' subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.

Hydrolysis by cathepsin B of fluorescent peptides derived from human prorenin.

Cathepsin B is a lysosomal thiolprotease that, because of its colocalization with renin and its ability to activate prorenin, has been proposed as a prorenin processing enzyme. To characterize the biochemical aspect of this potential cathepsin B activity in more detail, we synthesized and assayed with human cathepsin B the internally quenched fluorescent peptide Abz-FSQPMKRLTLGNTTQ-EDDnp (Abz, ortho-aminobenzoic acid fluorescent group and EDDnp, N-¿2, 4-dinitrophenyl-ethylenediamine quencher group) that contains 7 amino acids for each side of the R-L bond that is the processing site of human prorenin. Human cathepsin B hydrolyzed this peptide at the correct site (R-L bond), with k(cat)/K(m)=75 mmol/L(-1) s(-1). Analogues of this peptide obtained by Ala scanning at positions P(5) to P(5)' were also synthesized and assayed as substrates for human cathepsin B. The obtained specificity constant (k(cat)/K(m)) values have a significant parallel with the previous data of prorenin activation by AtT-20 cells and in vitro by cathepsin B. In addition, we demonstrated the presence of cathepsin B-like activity in rat mesangial cells and the ability of its whole soluble fraction lysates, as well as that of purified cloned rat cathepsin B, to hydrolyze Abz-IKKSSF-EDDnp at the K-S bond, which contains 6 amino acids of rat prorenin processing site. The specificity data of cathepsin B toward peptides derived from prorenin processing site support the view that human or rodent cathepsin B could be involved in the intracellular processing of prorenin that is locally synthesized or taken up from the extracellular compartment.

Glycopeptide and Oligosaccharide Libraries.

Despite the burgeoning interest in the various biological functions and consequent therapeutic potential of the vast number of oligosaccharides found in nature on glycoproteins and cell surfaces, the development of combinatorial carbohydrate chemistry has not progressed as rapidly as expected. The reason for this imbalance is rooted in the difficulty of oligosaccharide assembly and analysis that renders synthesis a rather cumbersome endeavor. Parallel approaches that generate series of analogous compounds rather than real libraries have therefore typically been used. Since generally low affinity is obtained for interactions between carbohydrate receptors and modified oligosaccharides designed as mimetics of natural carbohydrate ligands, glycopeptides have been explored as alternative mimics. Glycopeptides have been proven in many cases to be superior ligands with higher affinity for a receptor than the natural carbohydrate ligand. High-affinity glycopeptide ligands have been found for several types of receptors including the E-, P-, and L-selectins, toxins, glycohydrolases, bacterial adhesins, and the mannose-6-phosphate receptor. Furthermore, the assembly of glycopeptides is considerably more facile than that of oligosaccharides and the process can be adapted to combinatorial synthesis with either glycosylated amino acid building blocks or by direct glycosylation of peptide templates. The application of the split and combine approach using ladder synthesis has allowed the generation of very large numbers of compounds which could be analyzed and screened for binding of receptors on solid phase. This powerful technique can be used generally for the identification and analysis of the complex interaction between the carbohydrates and their receptors.

Physical properties of poly(ethylene glycol) (PEG)-based resins for combinatorial solid phase organic chemistry: a comparison of PEG-cross-linked and PEG-grafted resins.

Three series of poly(ethylene glycol) (PEG)-based polymers were synthesized and characterized with respect to their physical properties. Polyoxyethylene-polyoxypropylene (POEPOP), polyoxyethylene-polyoxetane (SPOCC), and polyoxyethylene-polystyrene (POEPS-3) were synthesized respectively by anion polymerization, cation polymerization, and radical polymerization. Both bulk and suspension modes were used to synthesize the polymers from derivatized PEG monomers (PEG 400, PEG 900, and PEG 1500). The three supports were compared with two commercially available PEG-grafted supports (TentaGel S OH, ArgoGel-OH) and two polystyrene supports (aminomethylated polystyrene [PS-NH2] and macroporous aminomethylated polystyrene [PLAMS]) with respect to their swelling properties, loading, NMR spectral quality, as well as solvent and reagent accessibility. Loadings of 0.3-0.7 mmol/g were obtained for the PEG-based resins. Swelling of the PEG-based resins was determined to be higher than that of the PEG-grafted resins and polystyrene supports. The PEG-based resins gave better resolved high-resolution NMR spectra than the PEG-grafted resins when examined by magic angle spinning nanoprobe (MAS) NMR spectroscopy. Moreover, fluorescence quenching of polymer bound 2-amino-benzoate by protonation with p-toluenesulfonic acid showed moderate to fast diffusion through the polymer depending on the solvent and the polymer matrix.

Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana.

A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8DeltaCTE). Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages. Maximum enzyme activity accompanied removal of the entire pro-region. This was facilitated by acidification. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L. mexicana lysates, and had the same N-terminal sequence as the native enzyme. The procedure yielded >3.5 mg of active enzyme per litre of E. coli culture.

The substrate specificity of a recombinant cysteine protease from Leishmania mexicana: application of a combinatorial peptide library approach.

The substrate specificity of CPB2.8DeltaCTE, a recombinant cysteine protease from Leishmania mexicana, was mapped by screening a fluorescence-quenched combinatorial peptide library. Results from library screening indicated a preference for Arg or Lys in the S(3) subsite and for hydrophobic residues, both aliphatic and aromatic, in S(2). The S(1) subsite exhibited a specificity for the basic residues Arg and Lys. Generally, the specificity of the primed subsites was less strict compared with the non-primed side which showed preference for Arg, Lys and Ala in S'(1), Arg, Pro and Gly in S'(2) and Lys, Arg and Ser in S'(4). By contrast, a strict preference for the basic residues Arg and Lys was found for S'(3). Overall, there was a trend for basic residues in alternating subsites and smaller residues in the primed sites compared with the non-primed sites. In addition, there were strict requirements for the amino acids in subsites S(3)--S(1). Fluorescence-quenched peptides from the library with the highest on-resin cleavage were resynthesised and their kinetics of hydrolysis by CPB2.8DeltaCTE assessed in solution phase assays. Several good substrates containing the quintessential dipeptide particular to cathepsin-L-like enzymes, -F-R/K-, in P(2) and P(1) were identified (e.g. Y(NO(2))-EKFR down arrow RGK-K(Abz)G, Abz=2-aminobenzoyl; k(cat)K(m)(-1)=4298 mM(-1)s(-1)). However, novel substrates containing the dipeptide -L/I-Q- in P(2) and P(1) were also well hydrolysed (e.g. Y(NO(2))-YLQ down arrow GIQK-K(Abz)G; k(cat)K(m)(-1)=2583 mM(-1)s(-1)). The effect of utilising different fluorescent donor--quencher pairs on the value of k(cat)K(m)(-1) was examined. Generally, the use of the Abz/Q-EDDnp donor--quencher pair (EDDnp=N-(2,4-dinitrophenyl)ethylenediamine) instead of K(Abz)/Y(NO(2)) resulted in higher k(cat)K(m)(-1) values for analogous substrates.