Guanidino Group Research Articles

Changes in conformational dynamics of basic side chains upon protein-DNA association.

Basic side chains play major roles in recognition of nucleic acids by proteins. However, dynamic properties of these positively charged side chains are not well understood. In this work, we studied changes in conformational dynamics of basic side chains upon protein–DNA association for the zinc-finger protein Egr-1. By nuclear magnetic resonance (NMR) spectroscopy, we characterized the dynamics of all side-chain cationic groups in the free protein and in the complex with target DNA. Our NMR order parameters indicate that the arginine guanidino groups interacting with DNA bases are strongly immobilized, forming rigid interfaces. Despite the strong short-range electrostatic interactions, the majority of the basic side chains interacting with the DNA phosphates exhibited high mobility, forming dynamic interfaces. In particular, the lysine side-chain amino groups exhibited only small changes in the order parameters upon DNA-binding. We found a similar trend in the molecular dynamics (MD) simulations for the free Egr-1 and the Egr-1–DNA complex. Using the MD trajectories, we also analyzed side-chain conformational entropy. The interfacial arginine side chains exhibited substantial entropic loss upon binding to DNA, whereas the interfacial lysine side chains showed relatively small changes in conformational entropy. These data illustrate different dynamic characteristics of the interfacial arginine and lysine side chains.

Biochemical characterization of the substrate specificity of two unique members of the mammalian protein arginine methyltransferase family, PRMT7 and PRMT9

Protein arginine methylation is a widespread and important posttranslational modification in eukaryotic cells, shown to be involved in the activation or repression of transcription, modification of the splicing machinery, signaling, and DNA repair. Mammalian protein arginine methyltransferases include a family of nine sequence‐related enzymes that transfer one or two methyl groups onto the terminal guanidino groups on arginine residues, producing monomethylarginine only (MMA, type III), symmetric dimethylarginine (SDMA) and MMA (Type II), or asymmetric dimethylarginine (ADMA) and MMA (Type I). While PRMT1, 2, 3, 4, 6, and 8 have been characterized as type I enzymes, and PRMT5 as a type II enzyme, the role and activity types of the two final members of this family of enzymes, PRMT7 and PRMT9, had been unclear due to conflicting results in the literature, and the substrates for these enzymes had been elusive. Both PRMT7 and PRMT9 are distinct members of the family with two methyltransferase or methyltransferase‐like domains and containing acidic residues in otherwise well‐conserved substrate double E binding motif, features not seen in the other PRMT enzymes. Recent work in our laboratory confirmed PRMT7 as the only type III MMA‐forming enzyme in the group, with a unusual low temperature optimum for activity, and a heretofore not seen preference for a basic stretch of residues in an R‐X‐R sequence for methylation. Mutations of the acidic residues in the substrate‐binding motif results in a loss of the specific R‐X‐R activity and the appearance of a G‐R‐G specificity typical of many of the other PRMTs. The physiological substrate of PRMT7 has yet to be confirmed, although histone H2B is an effective in vitro substrate. PRMT9, on the other hand, had no reported activity, until immunoprecipitation from HeLa cells showed it pulled down two splicing factors, SF3B2 and SF3B4, in a complex. Amino acid analysis showed that PRMT9 methylates SF3B2 to produce both MMA and SDMA, thus making it the second type II enzyme in mammals. PRMT9 knockdown results in modulation of alternative splicing events. This enzyme appears to be relatively specific for the SF3B2 protein; a peptide containing the methylatable arginine residue was not found to be a substrate, and typical substrates of other PRMTs are not recognized by PRMT9. We found that the position of the methylated arginine residue in SF3B2 is important, and the acidic residues in the substrate‐binding motif also play an important role in substrate recognition. Thus, PRMT7 and PRMT9 represent unique members of the mammalian PRMT family.Support or Funding InformationNIH grants GM026020 to S.G.C., DK062248 to M.T.B. and GM007185, a Ruth L. Kirschstein National Research Service Award to A.H.

Small-Molecule CD4 Mimics Containing Mono-cyclohexyl Moieties as HIV Entry Inhibitors.

CD4 mimics are small molecules that inhibit the protein-protein interaction between gp120 and CD4, which is a key interaction for the entry of human immunodeficiency virus (HIV) into host immune cells. In the present study, mono-cyclohexyl-type CD4 mimics were designed to form hydrophobic and electrostatic interactions with Val430 and Asp368 located in the entrance of the Phe43 cavity of gp120, the interaction site of CD4. YIR-329, a novel 1-azaspiro[5.5]undecane derivative with a cyclohexyl ring attached to the piperidine ring, exhibited only slightly weaker anti-HIV activity than a previously described lead HAR-171, and modeling results indicated the formation of advantageous interactions by the para-chlorophenyl moiety of YIR-329. To introduce an electrostatic interaction with Asp368, derivatives with a guanidino group on the piperidine nitrogen atom were synthesized. Mono-cyclohexyl-type CD4 mimics with a guanidino group, such as YIR-819 (N(1) -(4-chlorophenyl)-N(2) -(1-(2-(N-(amidino)glycinamide)ethyl)-2-cyclohexylpiperidin-4-yl)oxalamide) and YIR-821 (1-(2-(5-guanidinovaleramide)ethyl derivative of YIR-819), were identified that exhibit approximately fivefold more potent anti-HIV activity than YIR-329. In combination with a neutralizing antibody, their anti-HIV activities were augmenting. Modeling results suggest that these compounds interact effectively with Val430 and either Asp368 or Asp474 in the gp120 Phe43 cavity. YIR-819 and YIR-821 represent useful lead compounds for the further development of HIV-1 entry inhibitors and could potentially be useful for co-administration with neutralizing antibodies for the treatment of HIV infection and AIDS.

Simple approach for predicting the heats of formation of high nitrogen content materials

Heat of formation is an important thermochemical parameter, which determines the combustion and detonation performances of energetic materials and nitrogen-rich compounds can be considered as high-performance energetic materials. A novel method is introduced to predict the heats of formation of high-nitrogen content materials (N > 50%) including different derivatives of triazole, tetrazole, triazine, tetrazine, furazan, and some of organic nitrogen-containing chains. It is based on elemental composition and some of the molecular moieties, such as azido, azo, azoxy, guanidino, carbonyl, hydroxide and amino groups. High reliability of the new model has been proved in comparison with two recent quantitative structure-property relationships (QSPR) and group additivity methods as well as different quantum mechanical approaches. For 96 high nitrogen content compounds, the values of standard deviation (SD) are 61.6, 131.1 and 110.1 kJ/mol for the new method, QSPR method and group additivity method. The values of SD are 92.8 and 103.3 kJ/mol for the new and quantum mechanical methods, respectively, where the calculated data of different quantum mechanical approaches were available.

Benzylic rearrangement stable isotope labeling for quantitation of guanidino and ureido compounds in thyroid tissues by liquid chromatography-electrospray ionization mass spectrometry

Benzylic rearrangement stable isotope labeling (BRSIL) was explored to quantify the guanidino and ureido compounds (GCs and UCs). This method employed a common reagent, benzil, to label the guanidino and ureido groups through nucleophilic attacking then benzylic migrating. The use of BRSIL was investigated in the analysis of five GCs (creatine, l-arginine, homoarginine, 4-guanidinobutyric acid, and methylguanidine) and two UCs (urea and citrulline). The labeling was found simple and specific. The introduction of bi-phenyl group and the generation of nitrogen heterocyclic ring in the benzil-d0/d5 labeled GCs and UCs improved the retention behaviors in liquid chromatography (LC) and increased the sensitivity of electrospray ionization mass spectrometry (ESI MS) detection. The fragment ion pairs of m/z 182/187 and m/z 210/215 from the benzil-d0/d5 tags facilitated the discovery of potential GCs and UCs candidates residing in biological matrices. The use of BRSIL combined with LC-ESI MS was applied for simultaneously quantitation of GCs and UCs in thyroid tissues. It was demonstrated that nine GCs and UCs were detected, six of which were further quantified based on corresponding standards. It was concluded that five GCs and UCs (l-arginine, homoarginine, 4-guanidinobutyric acid, methylguanidine, and citrulline) were statistically significantly different (p < 0.05) between the para-carcinoma and carcinoma thyroid tissue samples.

Probing the Molecular Interactions between CXC Chemokine Receptor 4 (CXCR4) and an Arginine-Based Tripeptidomimetic Antagonist (KRH-1636).

We here report an experimentally verified binding mode for the known tripeptidomimetic CXCR4 antagonist KRH-1636 (1). A limited SAR study based on the three functionalities of 1 was first conducted, followed by site-directed mutagenesis studies. The receptor mapping showed that both the potency and affinity of 1 were dependent on the transmembrane residues His(113), Asp(171), Asp(262), and His(281) and also suggested the involvement of Tyr(45) and Gln(200) (potency) and Tyr(116) and Glu(288) (affinity). Molecular docking of 1 to an X-ray structure of CXCR4 showed that the l-Arg guanidino group of 1 forms polar interactions with His(113) and Asp(171) and the (pyridin-2-ylmethyl)amino moiety is anchored by Asp(262) and His(281), whereas the naphthalene ring is tightly packed in a hydrophobic subpocket formed by the aromatic side chains of Trp(94), Tyr(45), and Tyr(116). The detailed picture of ligand-receptor interactions provided here will assist in structure-based design and further development of small-molecule peptidomimetic CXCR4 antagonists.

Can Nitriles Be Stronger Bases Than Proton Sponges in the Gas Phase? A Computational Analysis

DFT calculations have been performed for a series of push-pull nitriles [(R2N)n(X═Y)iC≡N, where i = 0, 1, or 2, n = 1, 2, or 3, R2N = H2N, Me2N, or C4H8N, X = CH, N, or P, Y = CH or N]. The possible protonation N-sites (N-cyano, N-imino, and N-amino) have been examined and their proton affinities (PA) estimated. For all compounds in the series, even for those containing the guanidino, phosphazeno, and diphosphazeno pushing groups, the N-cyano atom is the favored site of protonation. The n-π conjugation strongly decreases the PA value of the pushing amino group in favor of the pulling cyano one. Nitriles with the phosphazeno groups [(R2N)3P═N-P(R2N)2═N and (R2N)3P═N] exhibit the strongest basicity in the series. Some of them (with PA > 1000 kJ mol(-1)) are stronger bases than DMAN, the so-called "proton sponge". Nitriles bearing the guanidino group [(R2N)2C═N] are less basic than those with the phosphazeno group [(R2N)3P═N] but more basic than those with the formamidino group (R2N-CH═N) containing the same substituent R. The N-imino atoms, present in the transmitter group (X═N, X = CH, N, or P), display PA values lower than those of the N-cyano site by more than 30 kJ mol(-1). When proceeding from the unsubstituted derivatives (R = H) to the methylated ones (R = Me), the Me groups at the N-amino atom increase the PA value of the N-cyano site for Me2N-X═Y-C≡N (X, Y = CH or N) by ca. 30-60 kJ mol(-1). For the guanidino and phosphazeno derivatives containing two and three amino groups, respectively, this effect is not additive. The four Me groups for (Me2N)2C═N-C≡N and the six Me groups for (Me2N)3P═N-C≡N increase the PA(N-cyano) values by only 30-50 kJ mol(-1). The C≡N bond lengths of the neutral forms are well correlated with the PA(N-cyano) values.

Cyanamide is biosynthesized from L-canavanine in plants.

Cyanamide had long been recognized as a synthetic compound but more recently has been found as a natural product from several leguminous plants. This compound’s biosynthetic pathway, as yet unelaborated, has attracted attention because of its utility in many domains, such as agriculture, chemistry, and medicine. We noticed that the distribution of L-canavanine in the plant kingdom appeared to include that of cyanamide and that the guanidino group structure in L-canavanine contained the cyanamide skeleton. Here, quantification of these compounds in Vicia species suggested that cyanamide was biosynthesized from L-canavanine. Subsequent experiments involving L-[guanidineimino-15N2]canavanine addition to young Vicia villosa seedlings resulted in significant incorporation of 15N-label into cyanamide, verifying its presumed biosynthetic pathway.

Residue Asn277 affects the stability and substrate specificity of the SMG1 lipase from Malassezia globosa.

Thermostability and substrate specificity are important characteristics of enzymes for industrial application, which can be improved by protein engineering. SMG1 lipase from Malassezia globosa is a mono- and diacylglycerol lipase (MDL) that shows activity toward mono- and diacylglycerols, but no activity toward triacylglycerols. SMG1 lipase is considered a potential biocatalyst applied in oil/fat modification and its crystal structure revealed that an interesting residue-Asn277 may contribute to stabilize loop 273–278 and the 3104 helix which are important to enzyme characterization. In this study, to explore its role in affecting the stability and catalytic activity, mutagenesis of N277 with Asp (D), Val (V), Leu (L) and Phe (F) was conducted. Circular dichroism (CD) spectral analysis and half-life measurement showed that the N277D mutant has better thermostability. The melting temperature and half-life of the N277D mutant were 56.6 °C and 187 min, respectively, while that was 54.6 °C and 121 min for SMG1 wild type (WT). Biochemical characterization of SMG1 mutants were carried out to test whether catalytic properties were affected by mutagenesis. N277D had similar enzymatic properties as SMG1 WT, but N277F showed a different substrate selectivity profile as compared to other SMG1 mutants. Analysis of the SMG1 3D model suggested that N277D formed a salt bridge via its negative charged carboxyl group with a positively charged guanidino group of R227, which might contribute to confer N277D higher temperature stability. These findings not only provide some clues to understand the molecular basis of the lipase structure/function relationship but also lay the framework for engineering suitable MDL lipases for industrial applications.

Specific tandem mass spectrometric detection of AGE-modified arginine residues in peptides.

Glycation is a non-enzymatic reaction of protein amino and guanidino groups with reducing sugars or dicarbonyl products of their oxidative degradation. Modification of arginine residues by dicarbonyls such as glyoxal and methylglyoxal results in formation of advanced glycation end-products (AGEs). In mammals, these modifications impact in diabetes mellitus, uremia, atherosclerosis and ageing. However, due to the low abundance of individual AGE-peptides in enzymatic digests, these species cannot be efficiently detected by LC-ESI-MS-based data-dependent acquisition (DDA) experiments. Here we report an analytical workflow that overcomes this limitation. We describe fragmentation patterns of synthetic AGE-peptides and assignment of modification-specific signals required for unambiguous structure retrieval. Most intense signals were those corresponding to unique fragment ions with m/z 152.1 and 166.1, observed in the tandem mass spectra of peptides, containing glyoxal- and methylglyoxal-derived hydroimidazolone AGEs, respectively. To detect such peptides, specific and sensitive precursor ion scanning methods were established for these signals. Further, these precursor ion scans were incorporated in conventional bottom-up proteomic approach based on data-dependent acquisition (DDA) LC-MS/MS experiments. The method was successfully applied for the analysis of human serum albumin (HSA) and human plasma protein tryptic digest with subsequent structure confirmation by targeted LC-MS/MS (DDA). Altogether 44 hydroimidazolone- and dihydroxyimidazolidine-derived peptides representing 42 AGE-modified proteins were identified in plasma digests obtained from type 2 diabetes mellitus (T2DM) patients.

Molecular dynamics study of the recognition of dimethylated CpG sites by MBD1 protein.

The cell is able to regulate which genes to express via chemical marks on the DNA and on the histone proteins. In all vertebrates, the modification on the DNA is methylation at position 5 of the two cytosines present in the dinucleotide sequence CpG. The information encoded by these chemical marks on the DNA is processed by a family of protein factors containing a conserved methyl-CpG binding domain (MBD). Essential to their function, the MBD proteins are able to bind DNA containing dimethylated CpG sites, whereas binding to unmethylated sites is not observed. In this paper, we perform molecular dynamics simulations to investigate the mechanism by which the mCpG binding domain of MBD1 is able to bind specifically dimethylated CpG sites. We find that the binding affinity of MBD1 to a DNA containing dimethylated CpG site is stronger by 26.4 kJ/mol relative to binding the same DNA but with an unmethylated CpG site. The contribution of each of the methyl groups to the change in free energy is very similar and additive. Therefore, this binding affinity (to a dimethylated DNA) is halved when considered relative to binding a hemimethylated DNA, a result that is also supported by experimental observations. Despite their equal contributions, the two methyl groups are recognized differently by MBD1. In one case, demethylation induces conformational changes in which the hydrophobic patch formed by the conserved residues Val20, Arg22, and Tyr34 moves away from the (methyl)cytosine, weakening the DNA-protein interactions. This is accompanied by an intrusion of a bulk water into the binding site at the protein-DNA interface. As a consequence, there is a reduction and rearrangements of the protein-DNA hydrogen bonds including a loss of a crucial hydrogen bond between Tyr34 and the (methyl)cytosine. The methylcytosine on the opposite strand is recognized by conformational changes of the surrounding conserved hydrophobic residues, Arg44 and Ser45, in which Arg44 participate in the 5mC-Arg-G triad. More specifically, the hydrogens of the methyl group form weak hydrogen bonds with the guanidino group and backbone carbonyl of the conserved Arg44, interactions that are absent when the cytosine is unmethylated. The results presented in this paper contribute to our knowledge of the different ways the chemical mark on the DNA is recognized by the epigenetic machinery.

Structure of N-formimino-L-glutamate iminohydrolase from Pseudomonas aeruginosa.

N-Formimino-l-glutamate iminohydrolase (HutF), from Pseudomonas aeruginosa with a locus tag of Pa5106 (gi|15600299), is a member of the amidohydrolase superfamily. This enzyme catalyzes the deamination of N-formimino-l-glutamate to N-formyl-l-glutamate and ammonia in the histidine degradation pathway. The crystal structure of Pa5106 was determined in the presence of the inhibitors N-formimino-l-aspartate and N-guanidino-l-glutaric acid at resolutions of 1.9 and 1.4 Å, respectively. The structure of an individual subunit is composed of two domains with the larger domain folding as a distorted (β/α)8-barrel. The (β/α)8-barrel domain is composed of eight β-strands flanked by 11 α-helices, whereas the smaller domain is made up of eight β-strands. The active site of Pa5106 contains a single zinc atom that is coordinated by His-56, His-58, His-232, and Asp-320. The nucleophilic solvent water molecule coordinates with the zinc atom at a distance of 2.0 Å and is hydrogen bonded to Asp-320 and His-269. The α-carboxylate groups of both inhibitors are hydrogen bonded to the imidazole moiety of His-206, the hydroxyl group of Tyr-121, and the side chain amide group of Gln-61. The side chain carboxylate groups of the two inhibitors are ion-paired with the guanidino groups of Arg-209 and Arg-82. Computational docking of high-energy tetrahedral intermediate forms of the substrate, N-formimino-l-glutamate, to the three-dimensional structure of Pa5106 suggests that this compound likely undergoes a re-faced nucleophilic attack at the formimino group by the metal-bound hydroxide. A catalytic mechanism of the reaction catalyzed by Pa5106 is proposed.

Synthesis and transmembrane anion/cation symport activity of a rigid bis(choloyl) conjugate functionalized with guanidino groups

A rigid bis(choloyl) conjugate functionalized with guanidino groups was synthesized and fully characterized on the basis of NMR (1H and 13C) and ESI MS (LR and HR) data. Its transmembrane ionophoric activity across egg-yolk l-α-phosphatidylcholine-based liposomal membranes was investigated by means of chloride ion selective electrode technique and pH discharge assay. The data indicate that under the assay conditions, this conjugate was capable of promoting the transport of anions, presumably via a cation/anion symport process. A Hill analysis reveals that two molecules of this compound are assembled into the transport-active species.

Exceptional proton affinities of push–pull nitriles substituted by the guanidino and phosphazeno groups

The G2(MP2)-calculated PA(N-cyano) for (H2N)2CN–CN and (H2N)3PN–CN are larger than that of HCN by 186 and 250 kJ mol−1, respectively.

Tetraguanidino-functionalized phenazine and fluorene dyes: synthesis, optical properties and metal coordination

In this work the first phenazine derivatives with guanidino substituents were prepared and their structural and electronic properties studied in detail. The guanidino groups decrease the HOMO-LUMO gap, massively increase the quantum yield for fluorescence and offer sites for metal coordination. The yellow-orange colored 2,3,7,8-tetraguanidino-substituted phenazine shows intense fluorescence. The wavelength of the fluorescence signal is strongly solvent dependent, covering a region from 515 nm in Et2O solution (with a record quantum yield of 0.39 in Et2O) to 640 nm in water. 2,3-Bisguanidino-substituted phenazine is less fluorescent (maximum quantum yield of 0.17 in THF), but exhibits extremely large Stokes shifts. In contrast, guanidino-functionalized fluorenes emit only very weakly. Subsequently, the influence of coordination on the electronic properties and especially the fluorescence of the phenazine system was analysed. Coordination first takes place at the guanidino groups, and leads to a blue shift of the luminescence signal as well as a massive decrease of the luminescence lifetime. Luminescence is almost quenched completely upon Cu(I) coordination. On the other hand, in the case of Zn(II) coordination the fluorescence signal remains strong (quantum yield of 0.36 in CH3CN). In the case of strong zinc Lewis acids, an excess of metal compound leads to additional coordination at the phenazine N atoms. This is accompanied by significant red-shifts of the lowest-energy transition in the absorption and fluorescence spectra. Pentanuclear complexes with two phenazine units were isolated and structurally characterized, and further aggregation leads to chain polymers.

Mechanisms and Factors Influencing Adsorption of Microcystin-LR on Biochars

The adsorption of microcystin-LR (MCLR) by biochar has never been well understood. For the first time, the unconventional adsorption of hydrophilic MCLR on wood-based biochars was comprehensively investigated as a function of biochar properties, environmental temperature, solution pH, coexisting dissolved organic matter (DOM), and polar organic competitors. High-temperature-prepared biochar from 700 °C (BC-700) and low-temperature-prepared biochar from 300 °C (BC-300) were characterized with significantly different surface areas but similar alkaline nature. Despite a very low surface area, BC-300 exhibited very high adsorption capacity, which implies the important contribution of surface groups to biochar. MCLR adsorption on biochars was pH dependent and was strongly reduced by macromolecular DOM. Polycarboxylic aliphatic acids and 2-(2-hydroxyethyl) guanidinium cation, which are similar to specific structural groups in MCLR, exhibited an evident competitive effect. The results indicated that both carboxylic and guanidino groups of MCLR serve significant functions in MCLR adsorption to biochar. The adsorption mechanisms may be primarily related to the columbic attractions and the hydrogen bonding interactions between MCLR and biochar surface. In particular, the irreversible adsorption enhancement of MCLR was observed on BC-700, which suggests that biochar amendment can aid in immobilizing MCLR from water to sediment, thereby prolonging MCLR environmental fate in biochar-amended sediment.

Roles of basic amino acid residues in the activity of μ-conotoxin GIIIA and GIIIB, peptide blockers of muscle sodium channels.

To study in detail the roles of basic amino acid residues in the activity of μ-conotoxin GIIIA (μ-GIIIA) and GIIIB (μ-GIIIB), specific blockers of muscle sodium channels, seven analogs of μ-GIIIA, and two analogs of μ-GIIIB were synthesized. μ-GIIIA analogs were synthesized by replacing systematically the three Arg residues (Arg1, Arg13, and Arg19) with one, two, and three Lys residues. μ-GIIIB analogs were synthesized by replacing simultaneously all four Lys residues (Lys9, Lys11, Lys16, and Lys19) with Arg residues and further replacement of acidic Asp residues with neutral Ala residues. Circular dichroism spectra of the synthesized analogs suggested that the replacement did not affect the three dimensional structure. The inhibitory effects on the twitch contractions of the rat diaphragm showed that the side chain guanidino group of Arg13 of μ-GIIIA was important for the activity, whereas that of Arg19 had little role for biological activity. Although [Arg9,11,16,19]μ-GIIIB showed higher activity than native μ-GIIIB, highly basic [Ala2,12, Arg9,11,16,19]μ-GIIIB showed lower activity, suggesting that there was an appropriate molecular basicity for the maximum activity.

Trinuclear complexes and coordination polymers of redox-active guanidino-functionalized aromatic (GFA) compounds with a triphenylene core.

Herein, we report on the synthesis, redox activity, and coordination chemistry of 2,3,6,7,10,11-hexakis(tetramethylguanidino)triphenylene. CV measurements indicated that the new compound could be oxidized in three separate reversible two-electron oxidation events. The HOMO and LUMO energies were estimated from the oxidation wave and the onset of absorption in the UV/vis spectrum. Our discussion also includes the related new compound 2,3,6,7,10,11-hexakis(N,N'-dimethylethyleneguanidino)triphenylene. Then trinuclear Cu(I) and Cu(II) complexes of the new triphenylene ligands were characterized, and their electronic properties are discussed. In contrast to previously studied redox-active GFA ligands, oxidation of trinuclear copper(I) iodide complexes with I2 leads to copper instead of ligand oxidation. In the tetra-coordinated Cu(II) complexes, the coordination mode is intermediate between tetrahedral and square planar. The optical properties of the complexes were studied, and low-energy electronic transitions were assigned to ligand-to-metal charge-transfer (LMCT) excitations. We then extended our analysis to trinuclear Ni(II) and Co(II) complexes. The magnetic coupling mediated through the triphenylene ligand in the trinuclear Cu(II) and Co(II) complexes was studied by SQUID magnetometry, revealing ferromagnetic coupling of the spin centers and different degrees of spin delocalization into the guanidino groups. Finally, we show that the GFA ligands could be linked to one- or two-dimensional coordination polymers and porous materials with a layer structure by reaction with silver halides.

Time- and NADPH-dependent inhibition of cytochrome P450 3A4 by the cyclopentapeptide cilengitide: significance of the guanidine group and accompanying spectral changes.

Cilengitide is a stable cyclic pentapeptide containing an Arg-Gly-Asp motif responsible for selective binding to αVβ3 and αVβ5 integrins. The candidate drug showed unexpected inhibition of cytochrome P450 (P450) 3A4 at high concentrations, that is, a 15-mM concentration caused attenuation of P450 3A4 activity (depending on the probe substrate): 15-19% direct inhibition, 10-23% time-dependent inhibition (30-minute preincubation), and 54-60% metabolism-dependent inhibition (30-minute preincubation). The inactivation efficiency determined with human liver microsomes was 0.003 ± 0.001 min(-1) mM(-1) and was 0.04 ± 0.01 min(-1) mM(-1) with baculovirus-based microsomes containing recombinant P450 3A4. Neither heme loss nor covalent binding to apoprotein could explain the observed reductions in residual activity. Slowly forming type II difference spectra were observed, with maximum spectral changes after 2 hours. Binding to both reduced and oxidized P450 3A4 was observed, with apparent Kd values of 0.66 μM and 6 μM. The significance of the guanidine group in inhibition was demonstrated using ligand binding spectral changes and inactivation assays with guanidine analogs (debrisoquine, N-acetylarginine-O-methyl ester) and the acetylated ornithine derivative of cilengitide. The observed inhibition could be explained by direct inhibition, plus by formation of stable complexes with both ferric and ferrous forms of heme iron and to some extent by the formation of reactive species capable to react to the protein or heme. Formation of the complex required time and NADPH and is attributed to the guanidino group. Thus, the NADPH-dependent inhibition is considered to be mainly due to the formation of a stable complex rather than the formation of reactive species.

Guanidino groups greatly enhance the action of antimicrobial peptidomimetics against bacterial cytoplasmic membranes

Antimicrobial peptides or their synthetic mimics are a promising class of potential new antibiotics. Herein we assess the effect of the type of cationic side chain (i.e., guanidino vs. amino groups) on the membrane perturbing mechanism of antimicrobial α-peptide–β-peptoid chimeras. Langmuir monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG) were used to model cytoplasmic membranes of both Gram-positive and Gram-negative bacteria, while lipopolysaccharide Kdo2-lipid A monolayers were mimicking the outer membrane of Gram-negative species. We report the results of the measurements using an array of techniques, including high-resolution synchrotron surface X-ray scattering, epifluorescence microscopy, and in vitro antimicrobial activity to study the molecular mechanisms of peptidomimetic interaction with bacterial membranes. We found guanidino group-containing chimeras to exhibit greater disruptive activity on DPPG monolayers than the amino group-containing analogues. However, this effect was not observed for lipopolysaccharide monolayers where the difference was negligible. Furthermore, the addition of the nitrobenzoxadiazole fluorophore did not reduce the insertion activity of these antimicrobials into both model membrane systems examined, which may be useful for future cellular localization studies.