Structures and function of the amino acid polymerase cyanophycin synthetase.

Cyanophycin is a natural biopolymer produced by a wide range of bacteria, consisting of a chain of poly-L-Asp residues with L-Arg residues attached to the ß-carboxylate sidechains by isopeptide bonds. Cyanophycin is synthesized from ATP, aspartic acid and arginine by a homooligomeric enzyme called cyanophycin synthetase (CphA1). CphA1 has domains that are homologous to glutathione synthetases and muramyl ligases, but no other structural information has been available. Here, we present cryo-electron microscopy and X-ray crystallography structures of cyanophycin synthetases from three different bacteria, including cocomplex structures of CphA1 with ATP and cyanophycin polymer analogs at 2.6 Å resolution. These structures reveal two distinct tetrameric architectures, show the configuration of active sites and polymer-binding regions, indicate dynamic conformational changes and afford insight into catalytic mechanism. Accompanying biochemical interrogation of substrate binding sites, catalytic centers and oligomerization interfaces combine with the structures to provide a holistic understanding of cyanophycin biosynthesis.

A simple and efficient transfection protocol for Cryptosporidium parvum using Polyethylenimine (PEI) and Octaarginine.

The transfection of Cryptosporidium represents a major challenge, and current protocols are based on electroporation of freshly excysted sporozoites using a rather large amount of plasmid DNA which typically has a very poor yield. In this study, we report a fast and simple protocol for transfection of Cryptosporidium parvum that takes advantage of the DNA condensing power of the poly cationic polymer polyethylenimine (PEI) and the gene delivery property of the short cell-penetrating peptide octaarginine. Our novel protocol requires a very low amount of plasmid DNA and does not necessitate special laboratory equipment to be performed. Transfection appears to be more efficient in oocysts just triggered for excystation than the excysted sporozoites. Altogether, the application of octaarginine with PEI allows efficient transfection. To the best of our knowledge, this is the first report on an electroporation-free protocol for transfection of sporozoites of a Cryptosporidium species.

Teruaki Mukaiyama (1927-2018).

Teruaki Mukaiyama, formerly Professor at Tokyo Institute of Technology, Tokyo University, and Tokyo University of Science passed away on November 17, 2018. As one of the most productive organic chemists he has enriched the field of synthetic organic chemistry in 60 years of research. His most important contributions are reviewed herein by a close friend.

Influence of the Membrane Dye R18 and of DMSO on Cell Penetration of Guanidinium-Rich Peptides.

A quantitative analysis by confocal fluorescence microscopy of the entry into HEK293 and MCF-7 cells by fluorescein-labeled octaarginine (1) and by three octa-Adp derivatives (2 - 4, octamers of the ß-Asp-Arg-dipeptide, derived from the biopolymer cyanophycin) is described, including the effects of the membrane dye R18 and of DMSO on cell penetration.

Cell Penetration, Herbicidal Activity, and in-vivo-Toxicity of Oligo-Arginine Derivatives and of Novel Guanidinium-Rich Compounds Derived from the Biopolymer Cyanophycin.

Oligo-arginines are thoroughly studied cell-penetrating peptides (CPPs, Figures 1 and 2). Previous in-vitro investigations with the octaarginine salt of the phosphonate fosmidomycin (herbicide and anti-malaria drug) have shown a 40-fold parasitaemia inhibition with P. falciparum, compared to fosmidomycin alone (Figure 3). We have now tested this salt, as well as the corresponding phosphinate salt of the herbicide glufosinate, for herbicidal activity with whole plants by spray application, hoping for increased activities, i.e. decreased doses. However, both salts showed low herbicidal activity, indicating poor foliar uptake (Table 1). Another pronounced difference between in-vitro and in-vivo activity was demonstrated with various cell-penetrating octaarginine salts of fosmidomycin: intravenous injection to mice caused exitus of the animals within minutes, even at doses as low as 1.4 µmol/kg (Table 2). The results show that use of CPPs for drug delivery, for instance to cancer cells and tissues, must be considered with due care. The biopolymer cyanophycin is a poly-aspartic acid containing argininylated side chains (Figure 4); its building block is the dipeptide H-ßAsp-αArg-OH (H-Adp-OH). To test and compare the biological properties with those of octaarginines we synthesized Adp8-derivatives (Figure 5). Intravenouse injection of H-Adp8-NH2 into the tail vein of mice with doses as high as 45 µmol/kg causes no symptoms whatsoever (Table 3), but H-Adp8-NH2 is not cell penetrating (HEK293 and MCF-7 cells, Figure 6). On the other hand, the fluorescently labeled octamers FAM-(Adp(OMe))8-NH2 and FAM-(Adp(NMe2))8-NH2 with ester and amide groups in the side chains exhibit mediocre to high cell-wall permeability (Figure 6), and are toxic (Table 3). Possible reasons for this behavior are discussed (Figure 7) and corresponding NMR spectra are presented (Figure 8).

Visible-Light Microscopic Discovery of Up to 150 µm Long Helical Amyloid Fibrils Built of the Dodecapeptide H-(Val-Ala-Leu)4 -OH and of Decapeptides Derived from Insulin.

In the formation of amyloid fibrils from small peptides, the appearance of superhelices of (P)- or (M)-helicity has been observed for the first time; high concentrations of the peptides and extended periods of incubation at physiological pH appear to be important for this phenomenon. In view of the general importance of peptide and protein aggregation, we give a brief overview with selected examples for demonstration.

Interaction of ß(3) /ß(2) -peptides, consisting of Val-Ala-Leu segments, with POPC giant unilamellar vesicles (GUVs) and white blood cancer cells (U937)--a new type of cell-penetrating peptides, and a surprising chain-length dependence of their vesicle- and cell-lysing activity.

Many years ago, ß(2) /ß(3) -peptides, consisting of alternatively arranged ß(2) - and ß(3) h-amino-acid residues, have been found to undergo folding to a unique type of helix, the 10/12-helix, and to exhibit non-polar, lipophilic properties (Helv. Chim. Acta 1997, 80, 2033). We have now synthesized such 'mixed' hexa-, nona-, dodeca-, and octadecapeptides, consisting of Val-Ala-Leu triads, with N-terminal fluorescein (FAM) labels, i.e., 1-4, and studied their interactions with POPC (=1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) giant unilamellar vesicles (GUVs) and with human white blood cancer cells U937. The methods used were microfluidic technology, fluorescence correlation spectroscopy (FCS), a flow-cytometry assay, a membrane-toxicity assay with the dehydrogenase G6PDH as enzymatic reporter, and visual microscopy observations. All ß(3) /ß(2) -peptide derivatives penetrate the GUVs and/or the cells. As shown with the isomeric ß(3) /ß(2) -, ß(3) -, and ß(2) -nonamers, 2, 5, and 6, respectively, the derivatives 5 and 6 consisting exclusively of ß(3) - or ß(2) -amino-acid residues, respectively, interact neither with the vesicles nor with the cells. Depending on the method of investigation and on the pretreatment of the cells, the ß(3) /ß(2) -nonamer and/or the ß(3) /ß(2) -dodecamer derivative, 2 and/or 3, respectively, cause a surprising disintegration or lysis of the GUVs and cells, comparable with the action of tensides, viral fusion peptides, and host-defense antimicrobial peptides. Possible sources of the chain-length-dependent destructive potential of the ß(3) /ß(2) -nona- and ß(3) /ß(2) -dodecapeptide derivatives, and a possible relationship with the phosphate-to-phosphate and hydrocarbon thicknesses of GUVs, and eukaryotic cells are discussed. Further investigations with other types of GUVs and of eukaryotic or prokaryotic cells will be necessary to elucidate the mechanism(s) of interaction of 'mixed' ß(3) /ß(2) -peptides with membranes and to evaluate possible biomedical applications.

Antibacterial activity of enrofloxacin and ciprofloxacin derivatives of ß-octaarginine.

ß(3) -Octaarginine chains were attached to the functional groups NH and CO2 H of the antibacterial fluoroquinolones ciprofloxacin (→1) and enrofloxacin (→2), respectively, in order to find out whether the activity increases by attachment of the polycationic, cell-penetrating peptide (CPP) moiety. For comparison, simple amides, 3-5, of the two antimicrobial compounds and ß(3) -octaarginine amide (ßR8 ) were included in the antibacterial susceptibility tests to clarify the impact of chemical modification on the microbiological activity of either scaffold (Table).

Structure-based evolution of subtype-selective neurotensin receptor ligands.

Subtype-selective agonists of the neurotensin receptor NTS2 represent a promising option for the treatment of neuropathic pain, as NTS2 is involved in the mediation of µ-opioid-independent anti-nociceptive effects. Based on the crystal structure of the subtype NTS1 and previous structure-activity relationships (SARs) indicating a potential role for the sub-pocket around Tyr11 of NT(8-13) in subtype-specific ligand recognition, we have developed new NTS2-selective ligands. Starting from NT(8-13), we replaced the tyrosine unit by ß(2)-amino acids (type 1), by heterocyclic tyrosine bioisosteres (type 2) and peptoid analogues (type 3). We were able to evolve an asymmetric synthesis of a 5-substituted azaindolylalanine and its application as a bioisostere of tyrosine capable of enhancing NTS2 selectivity. The S-configured test compound 2 a, [(S)-3-(pyrazolo[1,5-a]pyridine-5-yl)-propionyl(11)]NT(8-13), exhibits substantial NTS2 affinity (4.8 nm) and has a nearly 30-fold NTS2 selectivity over NTS1. The (R)-epimer 2 b showed lower NTS2 affinity but more than 600-fold selectivity over NTS1.

A theoretical and experimental study of the effects of silyl substituents in enantioselective reactions catalyzed by diphenylprolinol silyl ether.

The effect of silyl substituents in diphenylprolinol silyl ether catalysts was investigated. Mechanistically, reactions catalyzed by diphenylprolinol silyl ether can be categorized into three types: two that involve an iminium ion intermediate, such as for the Michael-type reaction (type A) and the cycloaddition reaction (type B), and one that proceeds via an enamine intermediate (type C). In the Michael-type reaction via iminium ions (type A), excellent enantioselectivity is realized when the catalyst with a bulky silyl moiety is employed, in which efficient shielding of a diastereotopic face of the iminium ion is directed by the bulky silyl moiety. In the cycloaddition reaction of iminium ions (type B) and reactions via enamines (type C), excellent enantioselectivity is obtained even when the silyl group is less bulky and, in this case, too much bulk reduces the reaction rate. In other cases, the yield increases when diphenylprolinol silyl ethers with bulky substituents are employed, presumably by suppressing side reactions between the nucleophilic catalyst and the reagent. The conformational behaviors of the iminium and enamine species have been determined by theoretical calculations. These data explain the effect of the bulkiness of the silyl substituent on the enantioselectivity and reactivity of the catalysts.

Joy and flustration with organofluorine compounds - a fluorous autobiography.

An overview is given about our work on fluoro-organic compounds, published or described in PhD theses between 1977 and 2013. After a discussion of structural F-effects and F-tagging applications the material is ordered by the various areas of our research, in which we have used and/or prepared F-derivatives: Li- and Ti-organic compounds and reagents, polylithiated hydroxy-esters and nitroalkanes, the enantiopure trifluoro-lactic, -Roche, and -3-hydroxy-butanoic acids as toolbox for the preparation of numerous F3C-substituted compounds, including natural products and dendrimers, and fluoro-α-, -ß-, and -δ-amino acids, as well as peptides with back-bond-bound fluorine. The strong influence on ß-peptide folding by fluoro-substituents in the α-position of ß-amino-acid residues is discussed in terms of the α-fluoro-amide conformational effect. Finally, some cases of totally unexpected effects on reactivity and structure exerted by fluoro-substitution are presented and taken as examples for our use of the terms flustrate and flustration in connection with organo-fluorine chemistry.

Syntheses, receptor bindings, in vitro and in vivo stabilities and biodistributions of DOTA-neurotensin(8-13) derivatives containing ß-amino acid residues - a lesson about the importance of animal experiments.

Neurotensin(8-13) (NTS(8-13)) analogs with C- and/or N-terminal ß-amino acid residues and three DOTA derivatives thereof have been synthesized (i.e., 1-6). A virtual docking experiment showed almost perfect fit of one of the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives, 6a, into a crystallographically identified receptor NTSR1 (Fig.1). The affinities for the receptors of the NTS analogs and derivatives are low, when determined with cell-membrane homogenates, while, with NTSR1-exhibiting cancer tissues, affinities in the single-digit nanomolar range can be observed (Table 2). Most of the ß-amino acid-containing NTS(8-13) analogs (Table 1 and Fig.2), including the (68) Ga complexes of the DOTA-substituted ones (6; Figs.2 and 5), are stable for ca. 1 h in human serum and plasma, and in murine plasma. The biodistributions of two (68) Ga complexes (of 6a and 6b) in HT29 tumor-bearing nude mice, in the absence and in the presence of a blocking compound, after 10, 30, and 60 min (Figs. 3 and 4) lead to the conclusion that the amount of specifically bound radioligand is rather low. This was confirmed by PET-imaging experiments with the tumor-bearing mice (Fig.6). Comparison of the in vitro plasma stability (after 1 h) with the ex vivo blood content (after 10-15 min) of the two (68) Ga complexes shows that they are rapidly cleaved in the animals (Fig.5).

Improved efficacy of fosmidomycin against Plasmodium and Mycobacterium species by combination with the cell-penetrating peptide octaarginine.

Cellular drug delivery can improve efficacy and render intracellular pathogens susceptible to compounds that cannot permeate cells. The transport of physiologically active compounds across membranes into target cells can be facilitated by cell-penetrating peptides (CPPs), such as oligoarginines. Here, we investigated whether intracellular delivery of the drug fosmidomycin can be improved by combination with the CPP octaarginine. Fosmidomycin is an antibiotic that inhibits the second reaction in the nonmevalonate pathway of isoprenoid biosynthesis, an essential pathway for many obligate intracellular pathogens, including mycobacteria and apicomplexan parasites. We observed a strict correlation between octaarginine host cell permeability and its ability to improve the efficacy of fosmidomycin. Plasmodium berghei liver-stage parasites were only partially susceptible to an octaarginine-fosmidomycin complex. Similarly, Toxoplasma gondii was only susceptible during the brief extracellular stages. In marked contrast, a salt complex of octaarginine and fosmidomycin greatly enhanced efficacy against blood-stage Plasmodium falciparum. This complex and a covalently linked conjugate of octaarginine and fosmidomycin also reverted resistance of Mycobacteria to fosmidomycin. These findings provide chemical genetic evidence for vital roles of the nonmevalonate pathway of isoprenoid biosynthesis in a number of medically relevant pathogens. Our results warrant further investigation of octaarginine as a delivery vehicle and alternative fosmidomycin formulations for malaria and tuberculosis drug development.

Enantiomeric and diastereoisomeric (mixed) L/ D-octaarginine derivatives - a simple way of modulating the properties of cell-penetrating peptides.

Cell-penetrating peptides (CPPs) are promising vehicles for delivery of drugs, antibiotics, proteins, nucleic acid derivatives, etc. into eukaryotic and prokaryotic target cells. To prevent premature degradation, CPPs consisting of D- or ß-amino acid residues have been used. We present simple models for the various modes of delivery of physiologically active cargoes by CPPs, depending on the nature of their conjugation (Fig. 1), and we describe the plasma stability of oligoarginines (OAs) 1-4, the most common unnatural CPPs. Fluorescein-labeled L-octaarginine 1 was found to have a half-life (t1/2 ) of <0.5 min, the D-enantiomer (2) of >7 d (Fig. 2). For possible medicinal applications, the former type of derivative would be too unstable, and the latter one undesirably persistent. Thus, seven of the 256 possible 'mixed' Flua-L/D-octaarginine amides, 4a-4g, were synthesized and shown to have half-lives in heparine-stabilized human plasma between 8 min and 5.5 h (Figs. 3 and 4). The cell penetration of the new OAs was investigated with 'healthy' and with apoptotic HEK cells (Figs. 5-8), and their interactions with phospholipid bilayers were studied, using anionic lipid vesicles (Figs. 9 and 10). There are surprisingly large differences in the rates of cell penetration and binding to vesicle walls between the various stereoisomeric octaarginine derivatives 1, 2, and 4a-4g (Figs. 5 and 7). - The role of D-amino acids and D-peptides in nature and in drug design is briefly discussed and referenced.

Permeation through phospholipid bilayers, skin-cell penetration, plasma stability, and CD spectra of α- and ß-oligoproline derivatives.

After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell-penetrating properties of polycationic proline-containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002, 124, 8876) is acknowledged, according to which fluorescein-labeled tetradecaproline is slowly taken up by rat kidney cells (NRK-49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004, 1, 1111) observation that a hexa-ß(3)-Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo-L- and oligo-D-α-prolines, as well as of oligo-ß(2)h- and oligo-ß(3)h-prolines without and with fluorescence labels (1-8; Fig. 1). Permeation through protein-free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2-4). This methodology is applied for the first time for quantitative determination of translocation rates of cell-penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6-8, resp.). The stabilities of oligoprolines in heparin-stabilized human plasma increase with decreasing chain lengths (Figs. 9-11). Time- and solvent-dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.

A journey from the pool of chiral synthetic building blocks to cell-penetrating peptides, to a novel type of enzyme - and back.

The roles of polyhydroxy-butyrates/alkanoates (PHB/PHA) in biology, for the preparation of chiral building blocks, and as a source of inspiration for the discovery of ß- and γ-peptides are discussed. The syntheses and structures of ß-peptides are outlined. The prerequisites for mimicking peptide/protein interactions with ß-peptides and two examples are presented. Single terminal ß-amino-acid residues can lead to stabilization of peptides (cf. NTS(8-13)) in plasma. Cell-penetrating α-L-, α-D-, mixed α-L/D- and ß-oligoarginines (OAs) and -oligoprolines, as well as the mechanism(s) of internalization are compared. Recent studies show that infected erythrocytes, parasitic organisms and mycobacteria are entered by OA-derivatives, which have been employed as transporters of the antibiotic fosmidomycin. While ß-peptides are generally enzymatically stable (for days in mammals), a microorganism (S. xenopeptidilytica) with an Ntn enzyme (3-2W4 BapA) was discovered that cleaves only ß-peptides, and that was applied in preparations of (enantiopure) ß-amino acids and ß-peptides.

The Substrate-Activity-Screening methodology applied to receptor tyrosine kinases: a proof-of-concept study.

Protein kinases are widely recognized as important therapeutic targets due to their involvement in signal transduction pathways. These pathways are tightly controlled and regulated, notably by the ability of kinases to selectively phosphorylate a defined set of substrates. A wide variety of disorders can arise as a consequence of abnormal kinase-mediated phosphorylation and numerous kinase inhibitors have earned their place as key components of the modern pharmacopeia. Although "traditional" kinase inhibitors typically act by preventing the interaction between the kinase and ATP, thus stopping substrate phosphorylation, an alternative approach consists in disrupting the protein-protein interaction between the kinase and its downstream partners. In order to facilitate the identification of potential chemical starting points for substrate-site inhibition approaches, we desired to investigate the application of Substrate Activity Screening to kinases. We herein report a proof-of-concept study demonstrating, on a model tyrosine kinase, that the key requirements of this methodology can be met. Namely, using peptides as model substrates, we show that a simple ADP-accumulation assay can be used to monitor substrate efficiency and that efficiency can be optimized in a modular manner. More importantly, we demonstrate that structure-efficiency relationships translate into structure-activity relationships upon conversion of the substrates into inhibitors.

Crystal structures of BapA complexes with ß-lactam-derived inhibitors illustrate substrate specificity and enantioselectivity of ß-aminopeptidases.

ß-Aminopeptidases have exclusive biocatalytic potential because they react with peptides composed of ß-amino acids, which serve as building blocks for the design of non-natural peptidomimetics. We have identified the ß-lactam antibiotic ampicillin and the ampicillin-derived penicilloic acid as novel inhibitors of the ß-aminopeptidase BapA from Sphingosinicella xenopeptidilytica (K(i) values of 0.69 and 0.74 mM, respectively). We report high-resolution crystal structures of BapA in noncovalent complexes with these inhibitors and with the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride. All three inhibitors showed similar binding characteristics; the aromatic moiety extended into a hydrophobic binding pocket of the active site, and the free amino group formed a salt bridge with Glu133 of BapA. The exact position of the inhibitors and structural details of the ligand binding pocket illustrate the specificity and the enantioselectivity of BapA-catalyzed reactions with ß-peptide substrates.

Autoproteolytic and catalytic mechanisms for the ß-aminopeptidase BapA--a member of the Ntn hydrolase family.

The ß-aminopeptidase BapA from Sphingosinicella xenopeptidilytica belongs to the N-terminal nucleophile (Ntn) hydrolases of the DmpA-like family and has the unprecedented property of cleaving N-terminal ß-amino acid residues from peptides. We determined the crystal structures of the native (αß)4 heterooctamer and of the 153 kDa precursor homotetramer at a resolution of 1.45 and 1.8 Å, respectively. These structures together with mutational analyses strongly support mechanisms for autoproteolysis and catalysis that involve residues Ser250, Ser288, and Glu290. The autoproteolytic mechanism is different from the one so far described for Ntn hydrolases. The structures together with functional data also provide insight into the discriminating features of the active site cleft that determine substrate specificity.