Guanidinium Group Research Articles

Synthesis and antimicrobial properties of new chitosan derivatives containing guanidinium groups

New chitosan derivatives bearing guanidinium functions were synthesized following different synthesis strategies. N-guanidinium chitosan acetate and N-guanidinium chitosan chloride were synthesized by direct reaction between chitosan and cyanamide in the presence of scandium(III) triflate. The synthesis of N-guanidinium chitosan (N,N′-dicyclohexyl) chloride and N-guanidinium chitosan (N-(3-dimethylaminopropyl)-N'-ethyl hydrochloride) chloride involved the reaction of chitosan with carbodiimides in ionic liquid. The chitosan derivatives were characterized by analytical techniques including 13C solid state NMR, FT-IR spectroscopies, thermogravimetry and elemental analysis. The antimicrobial properties of chitosan and the new derivatives were investigated using the minimal inhibitory concentration (MIC) technique. All new guanylated chitosan derivatives displayed high antimicrobial activity in comparison with neat chitosan. The N-guanidinium chitosan acetate reduced the time required for killing to half in comparison with chitosan and recorded MIC values less than 3.125 mg/mL against all assayed microorganisms. This work opens new perspectives for using chitosan derivatives as antimicrobial surfaces.

Structural Superiority of Guanidinium-Rich, Four-Armed Copolypeptides: Role of Multiple Peptide-Membrane Interactions in Enhancing Bacterial Membrane Perturbation and Permeability.

The development of novel antimicrobials is a top priority to address the growing epidemic of multidrug-resistant pathogens. Since cationic nonamphiphilic star-shaped antimicrobials are promising molecular scaffolds that provide a high charge density in binding anionic bacterial bilayers, this research aimed to further increase their membrane perturbation capability by introducing guanidinium groups to the antimicrobials via enhancing membrane insertion. In particular, computational simulation and experimental investigations revealed that our designed guanidinium-rich alternating copolypeptide, four-armed poly(arginine-alt-glycine), can interact with both the headgroups and unsaturated tails of phospholipids in bacterial membranes through multiple interactions, including electrostatic, cation-π, and T-shaped π-π interactions, allowing it to penetrate deeper inside the biologically inaccessible high-energy barrier of the hydrophobic lipid bilayer interior to cause membrane permeabilization and precipitation of the bacterial cytoplasm. Furthermore, glycine was observed to have a unique effect in enhancing the performance of arginine-based copolypeptide. Four-armed poly(arginine-alt-glycine) exhibited broad-spectrum antimicrobial activity, high bactericidal efficiency, and negligible hemolysis. The in vivo antibacterial performance of the copolypeptide was superior to that of doxycycline in a mouse model of Pseudomonas aeruginosa skin infection, accompanied by negligible local and systemic toxicity. Our results demonstrate that this guanidinium-rich, nonamphiphilic, star-shaped structure may promote the development of next-generation antimicrobials.

Harnessing aromaticity and intramolecular hydrogen bonding to tailor organosuperbases by using 2,4,6-cycloheptatriene-1-imine scaffold

The density functional theory (DFT) calculations at B3LYP/6-31 + G(d,p) level were performed for a series of new 2,4,6-cycloheptatriene-1-imine scaffolds which have potential to introduce as organosuperbases. In the present study, we have exploited the 2,4,6-cycloheptatriene-1-imines which strategically substituted at 2 and 7 positions by two potential anchoring arms having lone pair possessing atoms. The designed organic superbases were found to have the higher basicity than the benchmarked molecule, 1,8-bis(dimethylamino)-naphthalene. The proposed superbase B14 bearing guanidine and aniline groups possesses the highest proton affinity value as 1143.8 kJ mol−1. The protonation enthalpy, Gibbs free energy, harmonic oscillator model of aromaticity (HOMA), and nucleus-independent chemical shift (NICS) were used for the basicity comparison. After the protonation of the designed molecules, the positive charge on the conjugate acid is expected to be delocalized in the cycloheptatriene ring and led to its aromatization. The results indicate that the HOMA and NICS values of the ring in the conjugate acid are strongly elevated. On the other hand, very strong intramolecular hydrogen bonding is formed after protonation. These two factors enhance the stability of the conjugate acids and thereby increase the basicity of the designed molecules.

Antibacterial AZT derivative regulates metastasis of breast cancer cells

Antimicrobial peptides (AMP) with anticancer activity have drawn remarkable attention in modern treatments. However, long peptide length and protease instability are the most addressing factors, which hampers their further development as therapeutic agents. In view of this, herein, we designed and synthesized a series of AZT-based cationic small molecule incorporating a variety of hydrophobic groups and cationic charges, including amine and guanidine groups to mimic the amphipathic structure of AMPs. These compounds were evaluated for their antibacterial activity against Gram-positive and Gram-negative bacteria. Through an extensive structure activity relationship study (SAR), we identified ADG-2e as the most potent antibacterial agent, which exhibited remarkable potency against drug resistant bacterial strains such as MRSA and MDRPA. Further, ADG-2e was examined for their anti-metastatic ability by investigating the cancer cell migration and invasiveness through scratch wound-healing assay and transwell invasive assay, respectively. In addition, time-lapse cell tracking analysis also performed for analyzing the cell movement pattern. Treatment of ADG-2e against metastatic breast cancer cells (MDA-MB-231) suppressed tumor cell migration by multi-directional lamellipodium formation, indicating their anti-metastatic potential. Thus, our cationic AZT based small molecules may evolve as an appealing class of antibacterial agents with anti-metastasis potential.

Supramolecular Hydrogels Based on Nanoclay and Guanidine-Rich Chitosan: Injectable and Moldable Osteoinductive Carriers.

Supramolecular hydrogels have great potential as biomaterials for tissue engineering applications or vehicles for delivering therapeutic agents. Herein, a self-healing and pro-osteogenic hydrogel system is developed based on the self-assembly of laponite nanosheets and guanidinylated chitosan, where laponite works as a physical crosslinker with osteoinductive properties to form a network structure with a cationic guanidine group on chitosan chains. The hydrogels can be prepared with varying ratios of chitosan to laponite and display self-healing and injectable properties because of supramolecular forces as well as osteoinductive activity due to nanoclay. They enhance cell adhesion and promote osteogenic differentiation of mesenchymal stem cells by activating the Wnt/β-catenin signaling pathway. In addition, the hydrogel is used as a malleable carrier for the demineralized bone matrix (DBM). The loading of the DBM does not affect the self-healing and injectable natures of hydrogels while enhancing the osteogenic capacity, indicating that advanced allograft bone formulations with carriers can facilitate handling and bone healing. This work provides the first demonstration of therapeutic supramolecular design for the treatment of bone defects.

Guanidine-Functionalized Amphiphilic Silica Nanoparticles as a Pickering Interfacial Catalyst for Biodiesel Production

A series of surface-modified SiO2 nanoparticles (NPs) were designed and prepared by combining a guanidine group (1,1,3,3-tetramethylguanidine [TMG]) as the base catalytic functionality and n-alkyl ...

GC-MS and LC-MS/MS pilot studies on the guanidine (NG)-dimethylation in native, asymmetrically and symmetrically NG-dimethylated arginine-vasopressin peptides and proteins in human red blood cells

Protein-arginine methyltransferases catalyze the methylation of the guanidine (NG) group of proteinic L-arginine (Arg) to produce monomethyl and dimethylarginine proteins. Their proteolysis releases the free amino acids monomethylarginine (MMA), symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA), respectively. MMA, SDMA and ADMA are inhibitors of the nitric oxide synthase (NOS) activity. High circulating and low urinary concentrations of ADMA and SDMA are considered risk factors in the cardiovascular and renal systems, mainly due to their inhibitory action on NOS activity. Identity, biological activity and concentration of NG-methylated proteins are largely unknown. The present study addressed these issues by using GC-MS and LC-MS/MS approaches. GC-MS was used to quantify free ADMA released by classical HCl-catalyzed hydrolysis of three synthetic Arg-vasopressin (V) peptides and of unknown endogenous NG-dimethylated proteins. The cyclic (c) disulfide forms of Arg-vasopressin analogs, i.e., Arg-vasopressin (cV-Arg-Gly-NH2), asymmetrically NG-dimethylated vasopressin (cV-ADMA-Gly-NH2) and symmetrically NG-dimethylated vasopressin (cV-SDMA-Gly-NH2) were used as model peptides in quantitative GC-MS analyses of ADMA, SDMA and other expected amino acids from the hydrolyzed Arg-vasopressin analogs. cV-ADMA-Gly-NH2 and cV-SDMA-Gly-NH2 were discriminated from cV-Arg-Gly-NH2 by LC-MS and LC-MS/MS, yet they were indistinguishable from each other. The same applies to the respective open (o) reduced and di-S-acetamide forms of oV-ADMA-Gly-NH2, oV-SDMA-Gly-NH2 and oV-Arg-Gly-NH2. Our LC-MS and LC-MS/MS studies suggest that the Arg-vasopressin analogs form [(M−H)]+ and [(M−H)+H]+ in the positive ESI mode and undergo in part conversion of their terminal Gly-NH2 (NH2, 16 Da) group to Gly-OH (OH, 17 Da). The product ion mass spectra of the di-S-acetamide forms are complex and contain several intense mass fragments differing by 1 Da. cV-ADMA-Gly-NH2 and cV-SDMA-Gly-NH2 induced platelet aggregation in platelet-rich human plasma with moderately different initial velocity and maximal aggregation rates compared to cV-Arg-Gly-NH2. Previous studies showed that human red blood cells are rich in large (>50 kDa) ADMA-containing proteins of unknown identity. Our LC-MS/MS proteomic study identified several membrane and cytosolic erythrocytic NG-dimethylated proteins, including spectrin-α (280 kDa), spectrin-β (247 kDa) and protein 4.1 (80 kDa). Being responsible for the stability of the erythrocyte membrane, the newly identified main targets for NG-dimethylation in human erythrocytes should be given a closer look in erythrocytic diseases like hereditary spherocytosis.

Apatite-binding nanoparticulate agonist of hedgehog signaling for bone repair.

The hedgehog signaling pathway plays a critical role in bone development and regeneration. Applications of hedgehog morphogens or small molecular agonists are of interest in bone repair but constrained by low stability, high dose requirement, and nonspecific targeting in vivo. Herein, a nanoparticulate agonist as a new type of hedgehog signaling activator is developed for efficacious bone healing. The shell of nanoparticulate agonist consists of palmitic acid and oxysterol, which could modify hedgehog function and bind with the smoothened receptor to positively modulate hedgehog signaling. Meanwhile, the core is assembled with sonic hedgehog gene/polyethyleneimine complex, which could synergistically enhance hedgehog signaling with oxysterol constituents. Moreover, alendronate is introduced into nanoparticulate agonist to bind with hydroxyapatite for potential bone tissue targeting. Lastly, the nanoparticulate agonist surface is decorated with the guanidine group to overcome cell membrane barriers. The created multifunctional nanoparticulate agonist is successfully integrated onto apatite-coated three-dimensional scaffolds and demonstrates greatly improved osteogenesis in vitro and calvarial bone healing. This work suggests a novel biomaterial design to specifically promote hedgehog signaling for the treatment of bone defects.

Arginine \u03c0-stacking drives binding to fibrils of the Alzheimer protein Tau

Aggregation of the Tau protein into fibrils defines progression of neurodegenerative diseases, including Alzheimer’s Disease. The molecular basis for potentially toxic reactions of Tau aggregates is poorly understood. Here we show that π-stacking by Arginine side-chains drives protein binding to Tau fibrils. We mapped an aggregation-dependent interaction pattern of Tau. Fibrils recruit specifically aberrant interactors characterised by intrinsically disordered regions of atypical sequence features. Arginine residues are key to initiate these aberrant interactions. Crucial for scavenging is the guanidinium group of its side chain, not its charge, indicating a key role of π-stacking chemistry for driving aberrant fibril interactions. Remarkably, despite the non-hydrophobic interaction mode, the molecular chaperone Hsp90 can modulate aberrant fibril binding. Together, our data present a molecular mode of action for derailment of protein-protein interaction by neurotoxic fibrils.

The Guanidine Pseudoalkaloids 10-Methoxy-Leonurine and Leonurine Act as Competitive Inhibitors of Tyrosinase

Tyrosinase plays a key role in the production of melanin. A variety of industrial fields have shown interest in the development of tyrosinase inhibitors from plants. In this study, compounds 1–5 derived from Leonurus japonicas were evaluated to determine their ability to inhibit tyrosinase. Of these, 10-methoxy-leonurine (1) and leonurine (2) exhibited IC50 values of 7.4 ± 0.4 and 12.4 ± 0.8 μM, respectively, and acted as competitive inhibitors of tyrosinase, with Ki values in the micromolar range. In silico modeling revealed a guanidine group located in the inner cavity and a benzene ring docked within the active site of these compounds. These guanidine pseudoalkaloids show potential not only as tyrosinase inhibitors but also as lead compounds in new scaffolds for the development of novel inhibitors.

Unexpected aqueous UCST behavior of a cationic comb polymer with pentaarginine side chains

Thermoresponsive polymers, undergoing a reversible chemical or physical change using temperature as stimulus, attract increasing interest in particular as adaptable biomaterials. Except for zwitterionic polymers, fully charged polymers require the presence of specific ions to exhibit an upper critical solution temperature (UCST) in water. Herein, we report the discovery of an UCST in pure water for fully cationic comb polymers based on oligoarginine pendent grafts. These polymers were prepared using an original strategy based on solid-phase peptide synthesis of pentaarginine methacrylate-based macromonomer and its polymerization through reversible addition-fragmentation chain transfer. Despite their cationic nature, guanidinium groups from the arginine have the ability to self-associate at low temperature through hydrophobic interactions into stacked pair configuration defying the expected Coulomb interactions. These results pave the way to biomedical applications such as antimicrobial materials and drug delivery systems through the tuning of the polymer structure.

Site-specific functionalization with amino, guanidinium, and imidazolyl groups enabling the activation of 10–23 DNAzyme

10–23 DNAzyme has been extensively explored as a therapeutic and biotechnological tool, as well as in DNA computing. Faster cleavage or transformation is always needed. The present research displays a rational modification approach for a more efficient DNAzyme. In the catalytic core, amino, guanidinium and imidazolyl groups were introduced for its chemical activation through the adenine base. Among the six adenine residues, A9 is the unique residue that realizes all the positive effects; the 6-amino and 8-position of adenine and the 7-position of 8-aza-7-deaza-adenine could be used for the introduction of the functional groups. A12 is a new choice for catalytic improvement with an 8-substituent. Therefore, more active DNAzymes could be expected by this nucleobase-modified activation approach.

The role of water and influence of hydrogen bonding on the self-assembly aggregation induced emission of an anthracene-guanidine-derivative

We report a luminescent anthracene-guanidine derivative that forms rare T-shape dimers, resulting in an excimer with a quantum yield approaching one. Water plays a fundamental role through H-bonding guiding the self-assembly. These results establish a new framework for environmentally friendly aggregation-induced emission luminogens.

Prevention of DNA multimerization using phosphoryl guanidine primers during isothermal amplification with Bst exo- DNA polymerase

Over the last two decades, isothermal amplification of nucleic acids has gained more attention due to a number of advantages over the widely used polymerase chain reaction. For isothermal amplification, DNA polymerases with strand-displacement activity are needed, and Bst exo- polymerase is one of the most commonly used. Unfortunately, Bst exo- causes nonspecific DNA amplification (so-called multimerization) under isothermal conditions that results in undesirable products (multimers) consisting of tandem nucleotide repeats. Multimerization occurs only for short ssDNA or primer dimers, and the efficiency of multimerization depends significantly on the reaction conditions, but slightly depends on the sequence of DNA templates. In this study we report the prevention of DNA multimerization using a new type of modified oligonucleotide primers with internucleosidic phosphates containing 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups). Primers with one, two or three PG groups located at the 3′- or 5′-ends or in the middle of the primers were designed. It turned out, such bulky groups interfere with the moving of Bst exo- polymerase along DNA chains. However, one modified phosphate does not notably affect the efficiency of polymerization, and the elongation is completely inhibited only when three contiguous modifications occur. Multimerization of the linear ssDNA templates is blocked by three modifications in the middle of both primers whereas specific amplification of the circular ssDNA by rolling circle amplification is not inhibited. Thus, incorporation of three PG groups is sufficient to prevent multimerization and allows to create improved primers for reliable isothermal amplification with Bst exo- DNA polymerase.

Red-Emissive Guanylated Polyene-Functionalized Carbon Dots Arm Oral Epithelia against Invasive Fungal Infections.

Oral candidiasis as a highly prevalent and recurrent infection in medically compromised individuals is mainly caused by the opportunistic fungal pathogen Candida albicans. This epithelial infection, if not controlled effectively, can progress to life-threatening systemic conditions and complications. The efficacy of current frontline antifungals is limited due to their poor bioavailability and systemic toxicity. As such, an efficient intervention is essential for controlling disease progression and recurrence. Herein, a theranostic nanoplatform (CD-Gu+-AmB) was developed to track the penetration of antifungals and perturb the invasion of C. albicans at oral epithelial tissues, via decorating the homemade red-emissive carbon dots (CD) with positively charged guanidine groups (Gu+) followed by conjugation with antifungal polyene (amphotericin B, AmB) in a reacting site-controllable manner. The generated CD-Gu+-AmB favorably gathered within the Candida cells and exhibited potent antifungal effects in both planktonic and biofilm forms. It selectively accumulated in the nuclei of human oral keratinocytes and exhibited undetectable toxicity to the host cells. Moreover, we reported for the first time the penetration and exfoliation profiles of CD in a three-dimensional organotypic model of human oral epithelial tissues, demonstrating that the extra- and intracellular accumulation of CD-Gu+-AmB effectively resisted the invasion of C. albicans by forming a "shielding" layer throughout the entire tissue. This study establishes a multifunctional CD-based theranostic nanoplatform functioning as a traceable and topically applied antifungal to arm oral epithelia, thereby shedding light on early intervention of mucosal candidiasis for oral and general health.

Retention of Coiled-Coil Dimer Formation in the Absence of Ion Pairing at Positions Flanking the Hydrophobic Core.

Hydrophobic interactions govern how proteins fold and interact with other molecules, but the impact of nearby polar functionality on the effective hydrophobicity of nonpolar surfaces remains unclear. Here we use a common protein quaternary structure motif, the parallel coiled-coil dimer, to ask whether the identity of basic residues (arginine vs lysine; guanidinium vs ammonium) arrayed along one side of the constituent α-helices influences the favorability of dimerization driven by burial of hydrophobic surface on the other side of each helix. Significant sequence redesign was necessary to achieve the desired juxtaposition of nonpolar and cationic functionality, because we needed to eliminate charged side chains from positions flanking the nonpolar helix surface. Natural and designed sequences that form coiled coils are almost universally rich in acidic and basic residues at these flanking positions. Our arginine coiled-coil dimer was moderately more stable than the lysine analogue, which contrasts with behavior previously observed with helical β-amino acid oligomers bearing guanidinium versus ammonium groups. We attribute this backbone-dependent difference to variations in the extent to which the helical propensities of α- and β-residues can be modulated by design. These findings highlight the challenge of identifying noncovalent forces that direct structure formed by a flexible backbone.

Conformational Studies on Two FtsZ Targeting Cyclic Peptides

Two FtsZ targeting cyclic peptides 1 (Ac-[Orn-Leu-Met-Asp]-Ala-Phe-Arg-Ser-NH2) and 2 (Ac-Ser-Leu-Met-[Asp-Ala-Phe-Arg-Orn]-NH2) were found to be inhibitors of FtsZ polymerization, that makes them excellent starting point for the future development of a new class of antimicrobials. We investigated their solution structure by means of nuclear magnetic resonance (NMR) and molecular dynamic simulations (MD). Deep analysis of 2D NMR spectra (COSY, TOCSY and NOESY), recorded in DMSO-d6, allowed the assignments of all peptide signals and suggested the presence of strong turn structures. We also noticed that the guanidine group of Arg significantly affects the spectral properties and the chromatographic behavior of these peptides depending on whether it makes part of the cycle or not. MD simulations allowed to investigate the conformational preference of the two cyclic peptides and to associate diversity in their structure and dynamics to their different behavior. In particular, peptide 1 showed enhanced flexibility and structural variance with respect to peptide 2.

Functionalization of 8-17 DNAzymes modulates catalytic efficiency and divalent metal ion preference.

8-17 and 17E DNAzyme are being explored as biosensors for metal ions and RNA motifs of interest, more sensitive and efficient DNAzymes are required to meet the practical applications. Their similarity in the catalytic cores and differences in catalytic efficiency and metal ion dependence initiated great interest about the contribution of the catalytic residues. Functionalization of four adenine residues in the catalytic cores of 8-17 DNAzyme and 17E was conducted with amino, guanidinium, and imidazolyl groups. In the bulge loops of 8-17 and 17E, N6-(3-aminopropyl)-2'-deoxyadenosine (residue 1) at A15 led to new DNAzymes 8-17DZ-A15-1 and 17E-A15-1, with much more efficient cleavage ability in the Ca2+-mediated reaction and the greater preference for Ca2+ over Mg2+ than 8-17 DNAzyme and 17E, respectively, especially with a concentration-dependent increase of the selectivity, which is different from most DNAzymes with the similar dependence on both Mg2+ and Ca2+. With this kind of post-selection modification on 8-17 DNAzymes, for the first time, the catalytic efficiency and metal ion selectivity could be positively modulated. It is also helpful for the catalyic mechanistic studies of these DNAzymes, especially, the role of the unconserved A15 should be emphasized.

Guanidine-Containing Polyhydroxyl Macrolides: Chemistry, Biology, and Structure-Activity Relationship.

Antimicrobial resistance has been seriously threatening human health, and discovering new antimicrobial agents from the natural resource is still an important pathway among various strategies to prevent resistance. Guanidine-containing polyhydroxyl macrolides, containing a polyhydroxyl lactone ring and a guanidyl side chain, can be produced by many actinomycetes and have been proved to possess many bioactivities, especially broad-spectrum antibacterial and antifungal activities. To explore the potential of these compounds to be developed into new antimicrobial agents, a review on their structural diversities, spectroscopic characterizations, bioactivities, acute toxicities, antimicrobial mechanisms, and the structure-activity relationship was first performed based on the summaries and analyses of related publications from 1959 to 2019. A total of 63 guanidine-containing polyhydroxyl macrolides were reported, including 46 prototype compounds isolated from 33 marine and terrestrial actinomycetes and 17 structural derivatives. Combining with their antimicrobial mechanisms, structure-activity relationship analyses indicated that the terminal guanidine group and lactone ring of these compounds are vital for their antibacterial and antifungal activities. Further, based on their bioactivities and toxicity analyses, the discovery of guanidyl side-chain targeting to lipoteichoic acid of Staphylococcus aureus indicated that these compounds have a great potency to be developed into antimicrobial and anti-inflammatory drugs.

Side-Chain Protonation States of a Fluorescent Arginine.

An arginine derivative with a fluorescent side-chain, Boc-Arg(Nap)-OH, was prepared by palladium(0)-catalyzed coupling of Boc-Arg-OH with a 4-bromonaphthalimide. The presence of the fluorophore lowers the pKa of the side-chain guanidinium group by several orders of magnitude, to 9.0 (±0.1), allowing the derivative to access an electrically neutral protonation state that is not generally available to arginine itself. Computational modeling (DFT) predicts that protonation takes place at the side-chain C═N atom that bears the fluorophore. Calculated electronic absorptions for the protonated (356 nm) and neutral species (440 nm) are in good agreement with experiment. When irradiated with light, excited-state proton transfer (ESPT) occurs from cationic side-chains to suitably basic solvents, resulting in fluorescence emission from the neutral species. Arg(Nap) can be incorporated into peptides with sterically accessible N-termini using specially adapted conditions of solid-phase peptide synthesis.