Collection and transportation of SARS-CoV-2 and influenza A virus diagnostic samples: Optimizing the usage of guanidine-based chaotropic salts for enhanced biosafety and viral genome preservation.

Many virus lysis/transport buffers used in molecular diagnostics, including the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, contain guanidine-based chaotropic salts, primarily guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC). Although the virucidal effects of GuHCl and GITC alone against some enveloped viruses have been established, standardized data on their optimum virucidal concentrations against SARS-CoV-2 and effects on viral RNA stability are scarce. Thus, we aimed to determine the optimum virucidal concentrations of GuHCl and GITC against SARS-CoV-2 compared to influenza A virus (IAV), another enveloped respiratory virus. We also evaluated the effectiveness of viral RNA stabilization at the determined optimum virucidal concentrations under high-temperature conditions (35°C) using virus-specific real-time reverse transcription polymerase chain reaction. Both viruses were potently inactivated by 1.0 M GITC and 2.5 M GuHCl, but the GuHCl concentration for efficient SARS-CoV-2 inactivation was slightly higher than that for IAV inactivation. GITC showed better viral RNA stability than GuHCl at the optimum virucidal concentrations. An increased concentration of GuHCl or GITC increased viral RNA degradation at 35°C. Our findings highlight the need to standardize GuHCl and GITC concentrations in virus lysis/transport buffers and the potential application of these guanidine-based salts alone as virus inactivation solutions in SARS-CoV-2 and IAV molecular diagnostics.

Dopamine-Derived Guanidine Alkaloids from a Didemnidae Tunicate: Isolation, Synthesis, and Biological Activities.

Mellpaladines A-C (1-3) and dopargimine (4) are dopamine-derived guanidine alkaloids isolated from a specimen of Palauan Didemnidae tunicate as possible modulators of neuronal receptors. In this study, we isolated the dopargimine derivative 1-carboxydopargimine (5), three additional mellpaladines D-F (6-8), and serotodopalgimine (9), along with a dimer of serotonin, 5,5'-dihydroxy-4,4'-bistryptamine (10). The structures of these compounds were determined based on spectrometric and spectroscopic analyses. Compound 4 and its congeners dopargine (11), nordopargimine (15), and 2-(6,7-dimethoxy-3,4-dihydroisoquinolin-1-yl)ethan-1-amine (16) were synthetically prepared for biological evaluations. The biological activities of all isolated compounds were evaluated in comparison with those of 1-4 using a mouse behavioral assay upon intracerebroventricular injection, revealing key functional groups in the dopargimines and mellpaladines for in vivo behavioral toxicity. Interestingly, these alkaloids also emerged during a screen of our marine natural product library aimed at identifying antiviral activities against dengue virus, SARS-CoV-2, and vesicular stomatitis Indiana virus (VSV) pseudotyped with Ebola virus glycoprotein (VSV-ZGP).

Covalent Organic Frameworks as Nano-Reservoir for Room Temperature RNA Storage.

The emerging role of Ribonucleic acids (RNAs) as therapeutics is alluring. However, RNAs are extremely labile under ambient conditions and typically need to be stored in cryogenic conditions (-20 °C to -80 °C). Hence, storage, stabilization, and transportation of RNA under ambient conditions have been an arduous task and remain an unsolved problem. In this work, a guanidinium-based ionic covalent organic framework (COF), TTGCl with nanotubular morphology, was synthesized and used as nano-reservoirs for room-temperature storage of RNA. To understand the role of the nanotubular morphology and chemical nature of TTGCl in stabilizing the RNA structure and for comparison purposes, a neutral COF, TMT-TT, is synthesized and studied. Further, density functional theory (DFT) studies confirmed non-covalent interaction between the COFs and the RNA nucleobases, facilitating reversible storage of RNA. RNA loaded in COFs was found to be resistant to enzymatic degradation when treated with RNase. Gel electrophoresis and sequencing confirmed the structural integrity of the recovered RNAs and their further processibility.

Highly Concentrated Linear Guanidine Amides from the Marine Sipunculid Phascolosoma granulatum.

The chemical diversity of annelids, particularly those belonging to the class Sipuncula, remains largely unexplored. However, as part of a Marine Biodiscovery program in Ireland, the peanut worm Phascolosoma granulatum emerged as a promising source of unique metabolites. The purification of the MeOH/CH2Cl2 extract of this species led to the isolation of six new linear guanidine amides, named phascolosomines A-F (1-6). NMR analysis allowed for the elucidation of their structures, all of which feature a terminal guanidine, central amide linkage, and a terminal isobutyl group. Notably, these guanidine amides were present in unusually high concentrations, comprising ∼3% of the dry mass of the organism. The primary concentration of the phascolosomines in the viscera is similar to that previously identified in linear amides from sipunculid worms and marine fireworms. The compounds from sipunculid worms have been hypothesized to be toxins, while those from fireworms are reported to be defensive irritants. However, screening of the newly isolated compounds for inhibitory bioactivity showed no significant inhibition in any of the assays conducted.

Excited States of apo-Guanidine-III Riboswitch Contribute to Guanidinium Binding through Both Conformational and Induced-Fit Mechanisms.

Riboswitches are mRNA segments that regulate gene expression through conformational changes driven by their cognate ligand binding. The ykkC motif forms a riboswitch class that selectively senses a guanidinium ion (Gdm+) and regulates the downstream expression of proteins which aid in the efflux of excess Gdm+ from the cells. The aptamer domain (AD) of the guanidine-III riboswitch forms an H-type pseudoknot with a triple helical domain that binds a Gdm+. We studied the binding of Gdm+ to the AD of the guanidine (ykkC)-III riboswitch using computer simulations to probe the specificity of the riboswitch to Gdm+ binding. We show that Gdm+ binding is a fast process occurring on the nanosecond time scale, with minimal conformational changes to the AD. Using machine learning and Markov-state models, we identified the excited conformational states of the AD, which have a high Gdm+ binding propensity, making the Gdm+ binding landscape complex exhibiting both conformational selection and induced-fit mechanisms. The proposed apo-AD excited states and their role in the ligand-sensing mechanism are amenable to experimental verification. Further, targeting these excited-state conformations in discovering new antibiotics can be explored.

Fork- and Comb-like Lipophilic Structures: Different Chemical Approaches to the Synthesis of Oligonucleotides with Multiple Dodecyl Residues.

Lipophilic oligonucleotide conjugates represent a powerful tool for nucleic acid cellular delivery, and many methods for their synthesis have been developed over the past few decades. In the present study, a number of chemical approaches for the synthesis of different fork- and comb-like dodecyl-containing oligonucleotide structures were performed, including use of non-nucleotide units and different types of phosphate modifications such as alkyl phosphoramidate, phosphoryl guanidine, and triazinyl phosphoramidate. The influence of the number of introduced lipophilic residues, their mutual arrangement, and the type of formed modification backbone on cell penetration was evaluated. The results obtained indicate great potential in the developed chemical approaches, not only for the synthesis of complex oligonucleotide structures but also for the fine-tuning of their properties.

In Situ Observation of Chemically Induced Protein Denaturation at Solvated Interfaces.

Proteins unfold in chaotropic salt solutions, a process that is difficult to observe at the single protein level. The work presented here demonstrates that a liquid-based atomic force microscope and graphene liquid-cell-based scanning transmission electron microscope make it possible to observe chemically induced protein unfolding. To illustrate this capability, ferritin proteins were deposited on a graphene surface, and the concentration-dependent urea- or guanidinium-induced changes of morphology were monitored for holo-ferritin with its ferrihydrite core as well as apo-ferritin without this core. Depending on the chaotropic agent the liquid-based imaging setup captured an unexpected transformation of natively folded holo-ferritin proteins into rings after urea treatment but not after guanidinium treatment. Urea treatment of apo-ferritin did not result in nanorings, confirming that nanorings are a specific signature of denaturation of holo-ferritins after exposture to sufficiently high urea concentrations. Mapping the in situ images with molecular dynamics simulations of ferritin subunits in urea solutions suggests that electrostatic destabilization triggers denaturation of ferritin as urea makes direct contact with the protein and also disrupts the water H-bonding network in the ferritin solvation shell. Our findings deepen the understanding of protein denaturation studied using label-free techniques operating at the solid-liquid interface.

Fluorescent Guanidinium-Azacarbazole for Oxoanion Binding in Water.

Oxoanions such as carboxylates, phosphates, and sulfates play important roles in both chemistry and biology and are abundant on the cell surface. We report on the synthesis and properties of a rationally designed guanidinium-containing oxoanion binder, 1-guanidino-8-amino-2,7-diazacarbazole (GADAC). GADAC binds to a carboxylate, phosphate, and sulfate in pure water with affinities of 3.6 × 104, 1.1 × 103, and 4.2 × 103 M-1, respectively. Like 2-azacarbazole, which is a natural product that enables scorpions to fluoresce, GADAC is fluorescent in water (λabs = 356 nm, λem = 403 nm, ε = 13,400 M-1 cm-1). The quantum yield of GADAC is pH-sensitive, increasing from Φ = 0.12 at pH 7.4 to Φ = 0.53 at pH 4.0 as a result of the protonation of the aminopyridine moiety. The uptake of GADAC into live human melanoma cells is detectable in the DAPI channel at low micromolar concentrations. Its properties make GADAC a promising candidate for applications in oxoanion binding and fluorescence labeling in biological (e.g., the delivery of cargo into cells) and other contexts.

Guanidine-modified albumin-MMAE conjugates with enhanced endocytosis ability.

Aiming to address the insufficient endocytosis ability of traditional albumin drug conjugates, this paper reports elegant guanidine modification to improve efficacy for the first time. A series of modified albumin drug conjugates were designed and synthesized with different structures, including guanidine (GA), biguanides (BGA) and phenyl (BA), and different quantities of modifications. Then, the endocytosis ability and in vitro/vivo potency of albumin drug conjugates were systematically studied. Finally, a preferred conjugate A4 was screened, which contained 15 BGA modifications. Conjugate A4 maintains spatial stability similar to that of the unmodified conjugate AVM and could significantly enhance endocytosis ability (p*** = 0.0009) compared with the unmodified conjugate AVM. Additionally, the in vitro potency of conjugate A4 (EC50 = 71.78 nmol in SKOV3 cells) was greatly enhanced (approximately 4 times) compared with that of the unmodified conjugate AVM (EC50 = 286.00 nmol in SKOV3 cells). The in vivo efficacy of conjugate A4 completely eliminated 50% of tumors at 33 mg/kg, which was significantly better than the efficacy of conjugate AVM at the same dose (P** = 0.0026). In addition, theranostic albumin drug conjugate A8 was designed to intuitively realize drug release and maintain antitumor activity similar to conjugate A4. In summary, the guanidine modification strategy could provide new ideas for the development of new generational albumin drug conjugates.

Asymmetric [3+2]-cyclization of α-imino amide surrogates to construct 3,4-diaminopyrrolidine-2,5-diones.

Using newly designed α-imino amide surrogates and azlactones as amphiphilic reactants, catalyzed by a chiral bifunctional guanidine, the construction of chiral 3,4-diaminopyrrolidine-2,5-diones and their derivatives was realized via formal [3+2]-cyclization. The role of guanidine as a multiple hydrogen bond donor was demonstrated by DFT calculations.

Disruption of Spatial Proximities among Charged Groups in Equilibrium-Denatured States of Proteins Tracked Using Protein Charge Transfer Spectra.

The absorption and luminescence originating from protein charge transfer spectra (ProCharTS) depend on the proximity between multiple charged groups in a protein. This makes ProCharTS absorbance/luminescence intensity a sensitive probe for detecting changes in the protein structure, which alter the proximity among charged groups in the protein. In this work, ProCharTS absorbance of charge-rich proteins like human serum albumin (HSA), α3C, and α3W was used to monitor structural changes upon chemical denaturant-induced protein unfolding under equilibrium conditions. The denaturation midpoints were estimated using nonlinear regression analysis. For HSA, absorbance at 325 and 340 nm estimated the GdnHCl-induced denaturation midpoints to be 0.80 and 0.61 M, respectively. A similar analysis of α3C and α3W ProCharTS absorbance yielded denaturation midpoints of 0.88 and 0.86 M at 325 nm and 0.96 and 0.66 M at 340 nm, respectively. A previously reported molten globule-like state in the GdnHCl-induced HSA unfolding pathway was detected by the increase in HSA ProCharTS absorbance at 0.5 M GdnHCl. To validate the above results, protein unfolding was additionally monitored using conventional methods like circular dichroism (CD), Trp, and dansyl fluorescence. Our results suggest that disruption of charged amino acid sidechain contacts as revealed by ProCharTS occurs at lower denaturant concentrations compared to the loss of secondary/folded structure monitored by CD and fluorescence. Further, HSA ProCharTS absorbance at 315-340 nm revealed that tertiary contacts among charged residues were disrupted at lower GdnHCl concentrations compared to sequence adjacent contacts. Our data underscore the utility of ProCharTS as a novel label-free tool to track unfolding in charge-rich proteins.

Synthetic and natural guanidine derivatives as antitumor and antimicrobial agents: A review.

Guanidines are fascinating small nitrogen-rich organic compounds, which have been frequently associated with a wide range of biological activities. This is mainly due to their interesting chemical features. For these reasons, for the past decades, researchers have been synthesizing and evaluating guanidine derivatives. In fact, there are currently on the market several guanidine-bearing drugs. Given the broad panoply of pharmacological activities displayed by guanidine compounds, in this review, we chose to focus on antitumor, antibacterial, antiviral, antifungal, and antiprotozoal activities presented by several natural and synthetic guanidine derivatives, which are undergoing preclinical and clinical studies from January 2010 to January 2023. Moreover, we also present guanidine-containing drugs currently in the market for the treatment of cancer and several infectious diseases. In the preclinical and clinical setting, most of the synthesized and natural guanidine derivatives are being evaluated as antitumor and antibacterial agents. Even though DNA is the most known target of this type of compounds, their cytotoxicity also involves several other different mechanisms, such as interference with bacterial cell membranes, reactive oxygen species (ROS) formation, mitochondrial-mediated apoptosis, mediated-Rac1 inhibition, among others. As for the compounds already used as pharmacological drugs, their main application is in the treatment of different types of cancer, such as breast, lung, prostate, and leukemia. Guanidine-containing drugs are also being used for the treatment of bacterial, antiprotozoal, antiviral infections and, recently, have been proposed for the treatment of COVID-19. To conclude, the guanidine group is a privileged scaffold in drug design. Its remarkable cytotoxic activities, especially in the field of oncology, still make it suitable for a deeper investigation to afford more efficient and target-specific drugs.

Synthesis and evaluation of a deltic guanidinium analogue of a cyclic RGD peptide.

A guanidine group is abundantly found in natural products and drugs. Guanidine has the highest basicity among many common functional groups in nature. Because of its high basicity, it generally exists as a protonated guanidinium and functions as a cationic hydrogen bond donor. Finding an appropriate bioisostere of guanidinium is challenging because of its high basicity and unique trigonal planar shape. In this study, we explored the possibility of "deltic guanidinium" as a bioisostere of guanidinium using a cyclic arginine-glycine-aspartic acid (RGD) peptide as a parent compound. We synthesized c(deltic RGDyK), in which a guanidinium group of an arginine residue in c(RGDyK) is replaced with deltic guanidinium. A target binding assay, biodistribution study, and metabolic stability assay were conducted with c(deltic RGDyK) and its radioiodinated variant. The deltic guanidinium analog peptides exhibited similar biological properties to the parent peptides and improved in vivo stability, indicating that deltic guanidinium could work as a unique bioisostere of guanidinium.

Nanopore single-molecule biosensor in protein denaturation analysis.

Unclear issues in protein studies include but not limited to the stability and denaturation mechanism in the presence of denaturants. Herein, we report a dynamic monitoring approach based on nanopore single-molecule biosensor, which can detect the protein's folding and unfolding transitions by recording a nanopore ionic current. When gradually increasing the concentration of denaturant guanidine hydrochloride (GdmCl), sensitive responses were observed with lysozyme unfolding. The emergence of the featured biphasic-pulse demonstrated the existence of a stable intermediate. It was the first time to experimentally confirm the dynamic equilibrium between the intermediate and the native states at single molecule level, therefore consolidating the standpoint of lysozyme denaturation process following the three-state model. Additionally, we got more insights into the conformation about the intermediate as globular-like structure, larger gyration radius, and enhanced positive charge density. We considered that the manner of denaturant toward lysozyme adopts the "direct" model based on stronger electrostatic and van der Waals forces. Nanopore biosensor exhibited excellent sensitivity with a low detection concentration of 280 pM and reproducibility in analysing the folding intermediate of lysozyme.

A variant of guanidine-IV riboswitches exhibits evidence of a distinct ligand specificity.

Riboswitches are regulatory RNAs that specifically bind a small molecule or ion. Like metabolite-binding proteins, riboswitches can evolve new ligand specificities, and some examples of this phenomenon have been validated. As part of work based on comparative genomics to discover novel riboswitches, we encountered a candidate riboswitch with striking similarities to the recently identified guanidine-IV riboswitch. This candidate riboswitch, the Gd4v motif, is predicted in four distinct bacterial phyla, thus almost as widespread as the guanidine-IV riboswitch. Bioinformatic and experimental analysis suggest that the Gd4v motif is a riboswitch that binds a ligand other than guanidine. It is found associated with gene classes that differ from genes regulated by confirmed guanidine riboswitches. In inline-probing assays, we showed that free guanidine binds only weakly to one of the tested sequences of the variant. Further tested compounds did not show binding, attenuation of transcription termination, or activation of a genetic reporter construct. We characterized an N-acetyltransferase frequently associated with the Gd4v motif and compared its substrate preference to an N-acetyltransferase that occurs under control of guanidine-IV riboswitches. The substrates of this Gd4v-motif-associated enzyme did not show activity for Gd4v RNA binding or transcription termination. Hence, the ligand of the candidate riboswitch motif remains unidentified. The variant RNA motif is predominantly found in gut metagenome sequences, hinting at a ligand that is highly relevant in this environment. This finding is a first step to determining the identity of this unknown ligand, and understanding how guanidine-IV-riboswitch-like structures can evolve to bind different ligands.

Geometrical benchmarking and analysis of redox potentials of copper(I/II) guanidine-quinoline complexes: Comparison of semi-empirical tight-binding and DFT methods and the challenge of describing the entatic state (part III).

Copper guanidine-quinoline complexes are an important class of bioinorganic complexes that find utilization in electron and atom transfer processes. By substitution of functional groups on the quinoline moiety the electron transfer abilities of these complexes can be tuned. In order to explore the full substitution space by simulations, the accurate theoretical description of the effect of functional groups is essential. In this study, we compare three different methods for the theoretical description of the structures. We use the semi-empirical tight-binding method GFN2-xTB, the density functional TPSSh and the double-hybrid functional B2PLYP. We evaluate the methods on five different complex pairs (Cu(I) and Cu(II) complexes), and compare how well calculated energies can predict the redox potentials. We find even though B2PLYP and TPSSh yield better accordance with the experimental structures. GFN2-xTB performs surprisingly well in the geometry optimization at a fraction of the computational cost. TPSSh offers a good compromise between computational cost and accuracy of the redox potential for real-life complexes.

Anion-Controlled Synthesis of Novel Guanidine-Substituted Oxanorbornanes.

The cycloaddition of simple alkyl-substituted guanidine derivatives is an interesting approach toward polycyclic superbases and guanidine-based organocatalysts. Due to the high nucleophilicity of guanidines, an aza-Michael reaction with dienophiles is more common and presents a huge obstacle in achieving the desired synthetic goal. Our preliminary investigations indicated that the proton could act as a suitable protecting group to regulate the directionality of the reaction. To investigate the role of the protonation state and type of anion, the reactivity of furfuryl guanidines with dimethyl acetylenedicarboxylate was explored. Furfuryl guanidines showed a strong reaction dependence on the nucleophilicity of the counterion and the structure of guanidine. While the reaction of DMAD with the guanidinium halides provided products of an aza-Michael addition, Diels-Alder cycloaddition occurred if non-nucleophilic hexafluorophosphate salts were used. Depending on the structure and the reaction conditions, oxanorbornadiene products underwent subsequent intramolecular cyclization. A tendency toward intramolecular cyclization was interpreted in terms of the pKa of different positions of the guanidine functionality in oxanorbornadienes. New polycyclic guanidines had a slightly decreased pKa in acetonitrile and well-defined geometry suitable for the buildup of selective sensors.

New Guanidine Alkaloids Batzelladines O and P from the Marine Sponge Monanchora pulchra Induce Apoptosis and Autophagy in Prostate Cancer Cells.

Two new guanidine alkaloids, batzelladines O (1) and P (2), were isolated from the deep-water marine sponge Monanchora pulchra. The structures of these metabolites were determined by NMR spectroscopy, mass spectrometry, and ECD. The isolated compounds exhibited cytotoxic activity in human prostate cancer cells PC3, PC3-DR, and 22Rv1 at low micromolar concentrations and inhibited colony formation and survival of the cancer cells. Batzelladines O (1) and P (2) induced apoptosis, which was detected by Western blotting as caspase-3 and PARP cleavage. Additionally, induction of pro-survival autophagy indicated as upregulation of LC3B-II and suppression of mTOR was observed in the treated cells. In line with this, the combination with autophagy inhibitor 3-methyladenine synergistically increased the cytotoxic activity of batzelladines O (1) and P (2). Both compounds were equally active in docetaxel-sensitive and docetaxel-resistant prostate cancer cells, despite exhibiting a slight p-glycoprotein substrate-like activity. In combination with docetaxel, an additive effect was observed. In conclusion, the isolated new guanidine alkaloids are promising drug candidates for the treatment of taxane-resistant prostate cancer.

Guanidinium-amino acid hydrogen-bonding interactions in protein crystal structures: implications for guanidinium-induced protein denaturation.

In the present work, 86 available high resolution X-ray structures of proteins that contain one or more guanidinium ions (Gdm+) are analyzed for the distribution and nature of noncovalent interactions between Gdm+ and amino-acid residues. A total of 1044 hydrogen-bonding interactions were identified, of which 1039 are N-H⋯O, and five are N-H⋯N. Acidic amino acids are more likely to interact with Gdm+ (46% of interactions, 26% Asp and 20% Glu), followed by Pro (19% of interactions). DFT calculations on the identified Gdm+-amino acid hydrogen-bonded pairs reveal that although Gdm+ interacts primarily with the backbone amides of nonpolar amino acids, Gdm+ does interact with the sidechains of polar and acidic amino acids. We classified the optimized Gdm+-amino acid pairs into parallel [p], bifurcated [b], single hydrogen bonded [s] and triple hydrogen bonded [t] types. The [p] and [t] type pairs possess higher average interaction strength that is stronger than that of [b] and [s] type pairs. Negatively charged aspartate and glutamate residues interact with Gdm+ ion exceptionally tightly (-76 kcal mol-1) in [p] type complexes. This work provides statistical and energetics insights to better describe the observed destabilization or denaturation process of proteins by guanidinium salts.

Insights into the binding of arginine to adenosine phosphate from mimetic complexes.

The amino acid arginine plays a key role in the interaction of proteins with adenosine phosphates, as its protonated guanidinium side group is capable of building multipodal H-bonding interactions with the oxygen atoms of the phosphate, phosphoester and ribose moieties and with the nitrogen atoms of adenine. Protein interactions often take place in competition with other ionic species, typically metal cations, which are prone to build concerted coordination arrangements with the same centers of negative charge as guanidinium. We report on a vibrational spectroscopy and computational investigation of a positively charged ternary complex formed by adenosine monophosphate (AMP) with methyl guanidinium and Na+. Following a bottom-up approach, an analogous complex with ribose phosphate is characterized as well, which serves to assess the individual role of the phosphate, sugar and adenine moieties in the binding process and to compare, within a single complex, the interactions associated with diffuse versus localized charge distributions of guanidinium and the alkali cation, respectively. The results indicate that Na+ is preferentially hosted in a semi-rigid pocket formed by the phosphoester-adenosine backbone of AMP and displaces guanidinium to a peripheral binding to the phosphate anionic end group. This suggests that the control of the salt concentration may constitute an effective route to modulate protein-AMP complexation.