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.

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).

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.

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.

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).

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.

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.

Helical content of a ß(3)-octapeptide in methanol: molecular dynamics simulations explain a seeming discrepancy between conclusions derived from CD and NMR data.

Connecting experimental observables with the underlying conformational ensemble is a long-standing problem in the structure determination of biomolecules. The simulations described in this article attempt to resolve a seeming discrepancy between the conformational features derived from measured NOE intensities, (3)J-coupling constants, and circular dichroism (CD) spectra for two ß-peptides differing in a linker between two side-chains. Although both peptides are very similar in terms of the r(-6) averaged distances between atom pairs involved in the observed NOEs, the molecular dynamics trajectories suggest why the CD spectra show a greater 3(14)-helical propensity for the linked, cyclic peptide than for the linear one, whereas slightly more NMR NOE peaks are observed and assigned for the latter. The nine 100 ns unrestrained simulations show better agreement with the observed experimental data than the single conformations derived from the published NMR structures by additional energy minimization with the GROMOS force field. They show why the seemingly contradictory quantities obtained by NMR and CD spectroscopy can arise from a single conformational ensemble.

Substrate profiling of IGF-1R and InsR: identification of a potent pentamer substrate.

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. As part of a study on the substrate requirements of Insulin-like Growth Factor 1 Receptor (IGF-1R) and Insulin Receptor (InsR), we evaluated and applied a universal assay system able to monitor the phosphorylation of unlabelled peptides of any length in real time. In contrast to already reported profiling methodologies, we were able to assess the k(cat)/K(M) ratio of peptides as short as tetramers. Notably, we were able to identify an efficient pentamer substrate that exhibited kinetic properties close to those of a 250-amino acid protein derived from IRS-1, a natural substrate of IGF-1R and InsR.

On the mechanism of eukaryotic cell penetration by α- and ß-oligoarginines--targeting infected erythrocytes.

Fluorescein-labeled α- and ß-octaarginine amides were synthesized. The route, by which these oligoarginine (OA) derivatives enter cells (hepatocytes, fibroblasts, macrophages), was investigated by confocal fluorescence microscopy. Comparisons (by co-localization experiments) with compounds of known penetration modes revealed that the ß-octaarginine amide also uses multiple pathways to enter cells. There was no difference between the α- and the ß-OAs. Like other cell-penetrating peptides (CPPs), the ß-octaarginine eventually winds up in the nucleoli of the cell nuclei (cf. Chem. Biodiversity, 2004, 1, 65). Surprisingly, there was no entry of α- or ß-OA into intact and healthy human erythrocytes (which do not possess a nucleus). Blood cells infected by Plasmodium falciparum (malaria parasite) were, however, entered readily, and the OAs went all the way through a couple of membranes into the parasite. The potential of these results for delivering specific antimalarial drugs directly into the parasite is discussed.

On the terminal homologation of physiologically active peptides as a means of increasing stability in human serum--neurotensin, opiorphin, B27-KK10 epitope, NPY.

The terminal homologation by CH(2) insertion into the peptides mentioned in the title is described. This involves replacement of the N-terminal amino acid residue by a ß(2) - and of the C-terminal amino acid residue by a ß(3) -homo-amino acid moiety (ß(2) hXaa and ß(3) hXaa, resp.; Fig. 1). In this way, the structure of the peptide chain from the N-terminal to the C-terminal stereogenic center is identical, and the modified peptide is protected against cleavage by exopeptidases (Figs. 2 and 3). Neurotensin (NT; 1) and its C-terminal fragment NT(8-13) are ligands of the G-protein-coupled receptors (GPCR) NT1, NT2, NT3, and NT analogs are promising tools to be used in cancer diagnostics and therapy. The affinities of homologated NT analogs, 2b-2e, for NT1 and NT2 receptors were determined by using cell homogenates and tumor tissues (Table 1); in the latter experiments, the affinities for the NT1 receptor are more or less the same as those of NT (0.5-1.3 vs. 0.6 nM). At the same time, one of the homologated NT analogs, 2c, survives in human plasma for 7 days at 37° (Fig. 6). An NMR analysis of NT(8-13) (Tables 2 and 4, and Fig. 8) reveals that this N-terminal NT fragment folds to a turn in CD(3) OH. - In the case of the human analgesic opiorphin (3a), a pentapeptide, and of the HIV-derived B27-KK10 (4a), a decapeptide, terminal homologation (→3b and 4b, resp.) led to a 7- and 70-fold half-life increase in plasma (Fig. 9). With N-terminally homologated NPY, 5c, we were not able to determine serum stability; the peptide consisting of 36 amino acid residues is subject to cleavage by endopetidases. Three of the homologated compounds, 2b, 2c, and 5c, were shown to be agonists (Fig. 7 and 11). A comparison of terminal homologation with other stability-increasing terminal modifications of peptides is performed (Fig. 5), and possible applications of the neurotensin analogs, described herein, are discussed.

Generation of secondary, tertiary, and quaternary centers by geminal disubstitution of carbonyl oxygens.

Methods for replacing the carbonyl oxygen by two new substituents (C=O→CR(1)R(2)) are discussed in this Minireview, whereby R may be H, NR(2), alkyl, allyl, benzyl, vinyl, alkynyl, aryl, heteroaryl, or acyl groups. The most frequently used starting materials for geminal disubstitution with the formation of two C-C bonds (R(1),R(2)≠H, NR(2)) are amides and thioamides, which react with organometallic nucleophiles R-M (M=Li, MgX, CeX(2), TiX(3), ZrX(3)) to give tertiary sec- and tert-alkylamines. Quaternary centers can be built directly from ketones by treatment with Me(3)Al, MeTiCl(3), or Me(2)TiCl(2) (R(1)R(2)C=O→R(1)R(2)CMe(2)). The scope and limitations of the various methods and mechanistic models are briefly discussed. The remarkable variety and diversity of structures thus accessible are demonstrated by numerous examples.