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Mild transition-metal catalysed cross-couplings enable direct functionalisation of biocatalytically halogenated tryptophans with alkyl iodides, representing a new alternative for late-stage derivatisations of halogenated aromatic amino acids. Moreover, this strategy enables a preparation of (homo)tryptophan regioisomers in a simple two-step synthesis using a Pd-catalysed Negishi cross coupling. This method provides access to non-canonical constitutional surrogates of tryptophan, ready for use in peptide synthesis. © 2021 Wiley-VCH GmbH.

Chiral and enantiopure amines can be produced by enantioselective transaminases via kinetic resolution of amine racemates. This transamination reaction requires stoichiometric amounts of co-substrate. A dual-enzyme recycling system overcomes this limitation: L-amino acid oxidases (LAAO) recycle the accumulating co-product of ( S )-selective transaminases in the kinetic resolution of racemic amines to produce pure ( R )-amines. However, availability of suitable LAAOs is limited. Here we use the heterologously produced, highly active fungal hcLAAO4 with broad substrate spectrum. H 2 O 2 as by-product of hcLAAO4 is detoxified by a catalase. The final system allows using sub-stoichiometric amounts of 1mol% of the transaminase co-substrate as well as the initial application of L-amino acids instead of alpha-keto acids. With an optimized protocol, synthetic potential of this kinetic resolution cascade was proven at the preparative scale (>90mg) by the synthesis of highly enantiomerically pure ( R )-methylbenzylamine (>99%ee) at complete conversion (50%). © 2022 Wiley-VCH GmbH.

In peptidotriazolamers every second peptide bond is replaced by a 1H-1,2,3-triazole. Such foldamers are expected to bridge the gap in molecular weight between small-molecule drugs and protein-based drugs. Amyloid beta (Abeta) aggregates play an important role in Alzheimer's disease. We studied the impact of amide bond replacements by 1,4-disubstituted 1H-1,2,3-triazoles on the inhibitory activity of the aggregation "hot spots" K16 LVFF20 and G39 VVIA42 in Abeta(1-42). We found that peptidotriazolamers act as modulators of the Abeta(1-42) oligomerization. Some peptidotriazolamers are able to interfere with the formation of toxic early Abeta oligomers, depending on the position of the triazoles, which is also supported by computational studies. Preliminary in vitro results demonstrate that a highly active peptidotriazolamer is also able to cross the blood-brain-barrier. © 2021 The Authors. ChemPlusChem published by Wiley-VCH GmbH.

Mild reaction conditions are highly desirable for bio‐orthogonal side chain derivatizations of amino acids, peptides or proteins due to the sensitivity of these substrates. Transition metal catalysed cross‐couplings such as Suzuki–Miyaura reactions are highly versatile, but usually require unfavourable reaction conditions, in particular, when applied with aryl bromides. Ligand‐free solvent‐stabilised Pd‐nanoparticles represent an efficient and sustainable alternative to conventional phosphine‐based catalysts, because the cross‐coupling can be performed at considerably lower temperature. We report on the application of such a highly reactive heterogeneous catalyst for the Suzuki–Miyaura cross‐coupling of brominated tryptophan derivatives. The solvent‐stabilised Pd‐nanoparticles are even more efficient than the literature‐known ADHP‐Pd precatalyst. Interestingly, the latter also leads to the formation of quasi‐homogeneous Pd‐nanoparticles as the catalytic species. One advantage of our approach is the compatibility with aqueous and aerobic conditions at near‐ambient temperatures and short reaction times of only 2 h. The influence of different N α‐protecting groups, boronic acids as well as the impact of different amino acid side chains in bromotryptophan‐containing peptides has been studied. Notably, a surprising acceleration of the catalysis was observed when palladium‐coordinating side chains were present in proximal positions., Gentle conditions: Ligand‐free solvent‐stabilised Pd‐nanoparticles serve as a highly efficient catalyst at ambient temperature for a sustainable and biocompatible Suzuki–Miyaura cross‐coupling between aryl bromides and boronic acids under mild, aerobic and aqueous conditions.

The recently described flavin‐dependent halogenase BrvH is able to catalyse both the bromination and chlorination of indole, but shows significantly higher bromination activity. BrvH was annotated as a tryptophan halogenase, but does not accept tryptophan as a substrate. Its native substrate remains unknown. A predictive model with the data available for BrvH was analysed. A training set of compounds tested in vitro was docked into the active site of a complete protein model based on the X‐ray structure of BrvH. The atoms not resolved experimentally were modelled by using molecular mechanics force fields to obtain this protein model. Furthermore, docking poses for the substrates and known non‐substrates have been calculated. Parameters like distance, partial charge and hybridization state were analysed to derive rules for predicting activity. With this model for activity of the BrvH, a virtual screening suggested several structures for potential substrates. Some of the compounds preselected in this way were tested in vitro, and several could be verified as convertible substrates. Based on information on halogenated natural products, a new dataset was created to specifically search for natural products as substrates/products, and virtual screening in this database yielded further hits., To bind or not to bind? That is the question. The generation of a pharmacophore model adapted from a virtual screening methodology frequently employed in medicinal chemistry helped to suggest and identify new substrates for an orphan flavin‐dependent halogenase for which the native substrate is not yet known.

Halogenating enzymes are able to introduce halogen substituents under ambient conditions using non-hazardous reagents with intriguing selectivity, which is highly desired in green chemistry. Although C-H functionalization such as halogenation is a well-known transformation in synthetic chemistry, the selective incorporation of halogens using conventional chemical approaches often remains challenging. Therefore, enzyme-based strategies have been emerging as valuable alternatives in recent years. Inspired by manifold developments of enzymatic halogenation, this review focuses on advances of halogenating enzymes and their application with particular emphasis on FAD-dependent halogenases (FDHs). Catalytic strategies, application scope and engineering of FDHs are outlined pointing to the increasing utility of halogenases as promising biocatalysts. Current limitations as well as potential future developments of their synthetic utility are being discussed.

Pd‐mediated reactions have emerged as a powerful tool for the site‐selective and bioorthogonal late‐stage diversification of amino acids, peptides and related compounds. Indole moieties of tryptophan derivatives are susceptible to C2H‐activation, whereas halogenated aromatic amino acids such as halophenylalanines or halotryptophans provide a broad spectrum of different functionalisations. The compatibility of transition‐metal‐catalysed cross‐couplings with functional groups in peptides, other biologically active compounds and even proteins has been demonstrated. This Review primarily compiles the application of different cross‐coupling reactions to modify halotryptophans, halotryptophan containing peptides or halogenated, biologically active compounds derived from tryptophan. Modern approaches use regio‐ and stereoselective biocatalytic strategies to generate halotryptophans and derivatives on a preparative scale. The combination of bio‐ and chemocatalysis in cascade reactions is given by the biocompatibility and bioorthogonality of Pd‐mediated reactions., Pd‐mediated reactions have emerged as a powerful tool for the site‐selective and bioorthogonal late‐stage diversification of amino acids, peptides and related compounds. Halotryptophans are accessible by biocatalytic approaches opening the application of a variety of cross‐coupling reactions for late‐stage modification of halotryptophan containing biomolecules.

Halogenases are valuable biocatalysts for selective C-H activation, but despite recent efforts to broaden their application scope by means of protein engineering, improvement of thermostability and catalytic efficiency is still desired. A directed evolution campaign aimed at generating a thermostable flavin-dependent tryptophan 6-halogenase with reasonable activity suitable for chemoenzymatic purposes. These characteristics were tackled by combining successive rounds of epPCR along with semi-rational mutagenesis leading to a triple mutant (Thal-GLV) with substantially increased thermostability (T-M=23.5 K) and higher activity at 25 degrees C than the wild type enzyme. Moreover, an active-site mutation has a striking impact on thermostability but also on enantioselectivity. Our data contribute to a detailed understanding of biohalogenation and provide a profound basis for future engineering strategies to facilitate chemoenzymatic application of these attractive biocatalysts.

Flavin-dependent halogenases selectively introduce halogen substituents into (hetero-)aromatic substrates and require only molecular oxygen and halide salts for this regioselective oxidative CH-functionalization. Genomic analysis of Xanthomonas campestris pv. campestris B100 identified three novel putative members of this enzyme class. They were shown to introduce halogen substituents into, e. g., substituted indoles, while preferring bromide over chloride.

The aldehyde tag is appropriate to selectively label proteins, prepare antibody-drug conjugates or to immobilize enzymes or antibodies for biotechnological and medical applications. The cysteine within the consensus sequence CxPxR of the aldehyde tag is specifically oxidized by the formylglycine-generating enzyme (FGE) to the non-canonical and electrophilic amino acid C-alpha-formylglycine (FGly). Subsequent reductive amination is a common method for site-directed immobilization, which usually results in poor immobilization efficiency due to the reaction conditions. Here, we introduce a new solid support like agarose modified with an aryl substituted pyrazolone (Knoevenagel reagent) that was obtained in a facile and efficient 2-step synthesis. The modified agarose allowed the site-selective and efficient immobilization of aldehyde-containing small molecules, peptides and proteins - in particular enzymes - at physiological pH (6.2-8.2) without any additive or catalyst needed. In comparison to reductive amination, higher loadings and activities were achieved in various buffers at different concentrations and temperatures.

Most anticancer agents are hydrophobic and can easily penetrate the tumor cell membrane by passive diffusion. This may impede the development of highly effective and tumor‐selective treatment options. A hydrophilic β‐glucuronidase‐cleavable linker was used to connect the highly potent antimitotic agent cryptophycin‐55 glycinate with the αvβ3 integrin ligand c(RGDfK). Incorporation of the self‐immolative linker containing glucuronic acid results in lower cytotoxicity than that of the free payload, suggesting that hydrophilic sugar linkers can preclude passive cellular uptake. In vitro drug‐release studies and cytotoxicity assays demonstrated the potential of this small molecule–drug conjugate, providing guidance for the development of therapeutics containing hydrophobic anticancer drugs., Sweet specificity: A β‐glucuronidase‐responsive linker was chosen to minimize the hydrophobicity and connect the potent antimitotic agent cryptophycin‐55 glycinate to the αvβ3 integrin ligand c(RGDfK). The presence of a hydrophilic carbohydrate linker significantly decreased the cytotoxicity of RGD–cryptophycin conjugates relative to the free drug. The conjugate efficiently releases the drug upon linker cleavage by β‐glucuronidase.

An extensive phytochemical study of the aerial parts of Vernonia guineensis Benth. (Asteraceae) led to the isolation of a new flavone, vernoguinoflavone (1) and a naturally isolated glycerol ester, bisarachidicester (2), together with eighteen known secondary metabolites including quercetin (3), luteolin (4), vernopicrin (5), vernomelitensin (6), beta-amyrin (7), oleanolic acid (8), ursolic acid (9), lupeol (10), betulinic acid (11), beta-carotene (12), a mixture of stigmasterol (13) and beta-sitosterol (14), beta-sitosterol-3-O-beta-D-glucoside (15), 2,3-dihydroxypropyl heptacosanoate (16), pentacosanoic acid (17), docosan-1-ol (18), tritriacontan-1-ol (19), and heptatriacontan-1-ol (20). Compounds 3, 4, 7-9, 12 and 16-20 are reported herein for the first time from this species. The structures of these compounds were elucidated based on extensive spectroscopic analyses, particularly 1D and 2D NMR, and HR-ESI-MS and by comparison of their data with those reported in the literature. The crude extract, fractions and some isolated compounds were evaluated for their antibacterial activity against Gram-negative bacteria: Escherichia coli (ATCC 25922), Shigella flexineri (NR 518), Salmonella Muenchen, Salmonella typhimurium and Salmonella typhi (ATCC 19430). All the tested compounds demonstrated inhibitory activities against the tested enteric bacteria with MIC values ranging from 3.12 to 100 g/mL. Flavonoids 1, 3 and 4 isolated from the most active fraction demonstrated the best bioactivities against Escherichia coli, Salmonella Muenchen and Salmonella typhimurium with MIC values ranging from 3.12 to 25 g/mL. © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cryptophycins are a class of 16-membered highly cytotoxic macrocyclic depsipeptides isolated from cyanobacteria. The biological activity is based on their ability to interact with tubulin. They interfere with microtubule dynamics and prevent microtubules from forming correct mitotic spindles, which causes cell-cycle arrest and apoptosis. Their strong antiproliferative activities with 100-fold to 1000-fold potency compared with those of paclitaxel and vinblastine have been observed. Cryptophycins are highly promising drug candidates, as their biological activity is not negatively affected by P-glycoprotein, a drug efflux system commonly found in multidrug-resistant cancer cell lines and solid tumors. Cryptophycin-52 had been investigated in phase II clinical trials but failed because of its high neurotoxicity. Recently, cryptophycin conjugates with peptides and antibodies have been developed for targeted delivery in tumor therapy. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Phytochemical investigation of the methanolic extract of the roots of Zanthoxylum tessmannii Zepernick and Timler (Rutaceae) led to the isolation and characterization of one new aromatic amide named tessmamide (1) along with twelve known compounds, N-benzoyltyramine methyl ether (2), 7,8,9-trimethoxycoumarin (3), 7,8-dimethoxycoumarin (4), integrifoliodiol (5), robustin (6), skimmianine (7), lupeol (8), lupenone (9), a mixture of stigmasterol and beta-sitosterol, and a mixture of their glucosides. The structures of all compounds were determined by comprehensive spectroscopic analyses (1D- and 2D-NMR, EI-MS, and ESI-MS) and comparison with known analogs. The determination of the radical scavenging activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay gave moderate antioxidant values for the crude extracts of the roots of Zanthoxylum tessmannii (IC50 0.8 mg/mL), tessmamide (1; IC50 31.8 mum), and 7,8,9-trimethoxycoumarin (3; IC50 29.3 mum), compared to the standard ascorbic acid (IC50 11.6 mum). © 2019 Wiley-VHCA AG, Zurich, Switzerland.

Halogenierte Arene stellen wichtige Bausteine in Medizinalchemie und Agrochemie dar. Chemische elektrophile aromatische Halogenierungen verwenden elementare Halogene, wahrend FAD-abhangige Halogenasen lediglich Halogenidsalze und O2 bei Raumtemperatur in wassrigem Milieu fur die regioselektive Halogenierung benotigen. Enzymatische Halogenierungen galten als ineffizient aufgrund der mangelnden Stabilitat der Halogenasen, was die praparative Anwendung limitierte. Wir konnten zeigen, dass die Langzeitstabilitat und damit die Effizienz der Tryptophan-7-Halogenase RebH signifikant durch die simultane Immobilisierung von RebH und allen Hilfsenzymen zur Cofaktorregenerierung erhoht werden kann. Durch die Verwendung von RebH-combiCLEAs als multifunktioneller wiederverwendbarer Biokatalysator haben wir eine einfache Methode zur Halogenierung von Tryptophan und dessen Derivaten im Gramm-Masstab entwickelt, die vermutlich ebenso fur andere FAD-abhangige Halogenasen anwendbar ist.

Gliadin, a protein present in wheat, rye, and barley, undergoes incomplete enzymatic degradation during digestion, producing an immunogenic 33-mer peptide, LQLQPF(PQPQLPY)3 PQPQPF. The special features of 33-mer that provoke a break in its tolerance leading to gliadin sensitivity and celiac disease remains elusive. Herein, it is reported that 33-mer gliadin peptide was not only able to fold into polyproline II secondary structure but also depending on concentration resulted in conformational transition and self-assembly under aqueous condition, pH 7.0. A 33-mer dimer is presented as one initial possible step in the self-assembling process obtained by partial electrostatics charge distribution calculation and molecular dynamics. In addition, electron microscopy experiments revealed supramolecular organization of 33-mer into colloidal nanospheres. In the presence of 1 mM sodium citrate, 1 mM sodium borate, 1 mM sodium phosphate buffer, 15 mM NaCl, the nanospheres were stabilized, whereas in water, a linear organization and formation of fibrils were observed. It is hypothesized that the self-assembling process could be the result of the combination of hydrophobic effect, intramolecular hydrogen bonding, and electrostatic complementarity due to 33-mer's high content of proline and glutamine amino acids and its calculated nonionic amphiphilic character. Although, performed in vitro, these experiments have revealed new features of the 33-mer gliadin peptide that could represent an important and unprecedented event in the early stage of 33-mer interaction with the gut mucosa prior to onset of inflammation. Moreover, these findings may open new perspectives for the understanding and treatment of gliadin intolerance disorders.

Two novel acridone-quinoline alkaloids, acriquinoline A (1) and acriquinoline B (2), together with twenty-two known compounds were isolated from the methanol extract of the root of Citrus reticulata Blanco. The structures of all compounds were determined by comprehensive analyses of their 1D and 2D NMR and mass spectral (EI and ESI) data. The possible biosynthesis for the formation of above compounds is proposed, based on close examination of their structures. Compounds 1, 2, 6, 10 and 14-17 exhibited strong suppressive effect on phagocytosis response upon activation with serum opsonized zymosan in the range of IC50 0.2-10.5μM, which was tested in vitro for oxidative burst studies of whole blood. However, compounds displayed low cytotoxic activity against the human Caucasian prostate adenocarcinoma cell line PC-3, with IC50 between 30.8 and 60.5μM compared to the standard doxorubicin with IC50 0.9μM. These compounds, tested against bacteria, fungi and plant pathogen oomycetes by the paper disk agar diffusion assay, resulting in missing to low activities corresponding with MICs>1mg/mL.

The efrapeptin family of peptide antibiotics produced by the fungus Tolypocladium niveum, and the neo-efrapeptins from the fungus Geotrichum candidumare inhibitors of F(1)-ATPase with promising antitumor, antimalaria, and insecticidal activity. They are rich in C(α)-dialkyl amino acids (Aib, Iva, Acc) and contain one β-alanine and several pipecolic acid residues. The C-terminus bears an unusual heterocyclic cationic cap. The efrapeptins C-G and three analogues of efrapeptin C were synthesized using α-azido carboxylic acids as masked amino acid derivatives. All compounds display inhibitory activity toward F(1)-ATPase. The conformation in solution of the peptides was investigated with electronic CD spectroscopy, FT-IR spectroscopy, and VCD spectroscopy. All efrapeptins and most efrapeptin analogues were shown to adopt helical conformations in solution. In the case of efrapeptin C, VCD spectra proved that a 3(10)-helix prevails. In addition, efrapeptin C was conformationally studied in detail with NMR and molecular modeling. Besides NOE distance restraints, residual dipolar couplings (RDC) observed upon partial alignment with stretched PDMS gels were used for the conformational analysis and confirmed the 3(10)-helical conformation.

Novel efficient and robust affinity chromatography material: There are several strategies known for the purification of integrins by affinity chromatography, but the disadvantages of common strategies like insufficient selectivity or compelling conditions for the elution still require alternatives. A new strategy, based on the immobilized C-terminally modified peptide Ac-Gly-Ala-c-(CysSS-Arg-Arg-Glu-Thr-Ala-Trp-Ala-CysSS)-Gly-Ala-O(CH2CH2O)2CH2CH2-NH2 allows for the affinity purification of the integrin α5β1. While RGD peptides have been proven in the past to be inappropriate for selective purification of integrins by affinity chromatography, the new peptide can be efficiently used for selective enrichment of the integrin α5β1. It is a specific ligand of the target protein, but does not contain an RGD sequence. The application of well-characterized affinity chromatography material with a site-specifically immobilized peptide allows to obtain integrin α5β1 in a single chromatography step without contamination by other integrins. This process combines the advantages of a selective and monospecific protein-ligand recognition with mild elution conditions and a low sensitivity of the immobilized ligand with respect to column regeneration.

Synthesis, structural and biological studies of pentapeptides containing two Delta Phe residues (Z and E isomers) in position 2 and 4 in peptide chain were performed. All the investigated peptides adopted bent conformation and majority of them could exist as two different. conformers in solution. Only pentapeptides. containing free N-termini appeared to act as weak inhibitors of cathepsin C with the slow-binding, competitive mechanism of inhibition. free acids being bound slightly better than their methyl esters. Results of Molecular modeling suggested significant difference between peptides, depending of the type of amino acid residue in position 5 in peptide chain. Dehydropeptides containing GIN, residue in this position may act as competitive slow-reacting substrates and therefore exhibit inhibitory-like properties. Copyright (C) 2008 European Peptide Society and John Wiley & Sons, Ltd.

Objective: The outcome of peripheral nerve repair is often unsatisfactory, and efficient therapies are not available. We tested the therapeutic potential of functional mimics of the human natural killer cell glycan (3-sulfoglucuronyl beta 1-3 galactoside) (HNK-1) epitope, a carbohydrate indicated to favor specificity of motor reinnervation in mice. Methods: We applied a linear HNK-1 mimic peptide, scrambled peptide, or vehicle substances in polyethylene cuffs used to reconstruct the severed femoral nerves of adult mice. We used video-based motion analysis and morphological and tracing techniques to monitor the outcome of nerve repair. Results: After glycomimetic application, quadriceps muscle function recovered to 93% of normal within 3 months. Restoration of function was less complete (71-76%) in control groups. Better functional recovery was associated with larger motoneuron somata, better axonal myelination in the quadriceps nerve, and enhanced precision of target reinnervation. Lesion-induced death of motoneurons was reduced by 20 to 25%. The glycomimetic enhanced survival and neurite outgrowth of both mouse and human motoneurons in vitro by 30 to 75%. Application of a novel cyclic glycomimetic also enhanced functional recovery in vivo. Interpretation: The improved outcome of nerve repair after glycomimetic application may be attributed to neurotrophic effects. Our results hold promise for therapeutic use in humans.

24 organische Farbstoffe andern auf einem Chip ihren Farbton in Abhangigkeit vom Analyten, ein „Nanocar” rollt durch Beruhrung einer STM-Spitze, ein Geiseltierchen produziert einen 66-gliedrigen Rekord-Makrocyclus, Organokatalyse bewahrt sich in der Synthese von Kohlenhydraten und spontane chemische, polymerasefreie Primerverlangerung gelingt mit gewohnlichen Nucleosiden — kurz, die organische Chemie bluht und gedeiht.

The new compound banganxanthone C (I) is isolated from the leaves of Garcinia polyantha together with five known compounds.

Die Genexpression in Eukaryoten wird auf der Ebene der Transkription durch die spezifische Bindung von Transkriptionsfaktoren an definierte DNA-Sequenzen gesteuert. Auf diese Weise werden Wachstum, Entwicklung und Differenzierung der Zellen reguliert. Die M glichkeit, mithilfe modifizierter synthetischer Transkriptionsaktivatoren steuernd in den Stoffwechsel eingreifen zu k nnen, bietet interessante Perspektiven f r die molekulare Zellbiologie im Rahmen der Biomimetik und synthetischen Biologie. 6] Zum Design biologisch aktiver synthetischer Enzyme und neuer Proteinbasierter Materialien bietet sich die Kombination von bioorganischer Bottom-up-Synthese und Einzelmolek l-Affinit tsspektroskopie an. Gegenstand solcher Untersuchungen sind beispielsweise der Beitrag einer einzelnen erkennenden Helix zur Spezifit t der DNA-Bindung eines kompletten Proteins, der Einfluss einer einzelnen punktmutierten Aminos ure auf die biologische Spezifit t und Affinit t, die minimale Sequenzl nge eines Peptids, die Bindungsspezifit t gew hrleistet, sowie das Design k nstlicher Proteine, die ein synthetisches Helix-Turn-Helix(HTH)-Bindemotiv enthalten. Eine interessante Fragestellung ist es hierbei, die Spezifit t der DNA-Bindung von synthetischen Peptiden mit einer der Bindedom ne eines Transkriptionsfaktors hnlichen Sequenz aufzukl ren. Wir untersuchten ein 20-Peptid mit der nativen Sequenz des bindenden Epitops aus dem Transkriptionsaktivator PhoB (E. coli) sowie drei Punktmutanten dieses Peptids.

Katalyse, Peptidchemie und Nanostrukturen bargen einige der Perlen aus organisch-chemischer Forschung: Kombinatorische Ligandenoptimierung, H-Verbruckung einzahniger zu zweizahnigen Liganden, Diels-Alderase- Aktivitaten im molekularen Detail, ein 36-er Peptid zur Behandlung von Aids, totalsynthetisch in 106 Stufen hergestellt, ein 21-er Peptid mit Lasso-Struktur, Kohlenstoffnanorohren ah elektromechanische Lager.

By screening a combinatorial pentapeptide amide collection in an inhibition assay, we systematically evaluated the potential of 19 proteinogenic amino acids and seven nonproteinogenic amino acids to serve as building blocks for inhibitors of human cathepsin L. Particularly efficient were aromatic, bulky, hydrophobic amino-acid residues, especially leucine, and positively charged residues, especially arginine. Building blocks for potential inhibitory peptides were combined by random selection from their activity pattern. This random approach for the design of inhibitors was introduced to compensate for the inaccuracy induced by shifted docking of combinatorial compound collections at the active center of cathepsin L. Thereby, we obtained structurally defined pentapeptide amides which inhibited human cathepsin L at nanomolar concentrations. Among the most potent novel inhibitors, one peptide, RKLLW-NH2, shares the amphiphilic character of the nonamer fragment VMNGLQNRK of the autoinhibitory, substrate-like, but reverse-binding prosegment of human cathepsin L which blocks the active center of the enzyme. Obviously, RKLLW-NH2 carries the functions that are important for enzyme-peptide interaction in a condensed form. This hypothesis was confirmed by structure-activity studies using truncated and modified pentapeptides.