Organ- and Cell-Selective Delivery of mRNA In Vivo Using Guanidinylated Serinol Charge-Altering Releasable Transporters.

Selective RNA delivery is required for the broad implementation of RNA clinical applications, including prophylactic and therapeutic vaccinations, immunotherapies for cancer, and genome editing. Current polyanion delivery relies heavily on cationic amines, while cationic guanidinium systems have received limited attention due in part to their strong polyanion association, which impedes intracellular polyanion release. Here, we disclose a general solution to this problem in which cationic guanidinium groups are used to form stable RNA complexes upon formulation but at physiological pH undergo a novel charge-neutralization process, resulting in RNA release. This new delivery system consists of guanidinylated serinol moieties incorporated into a charge-altering releasable transporter (GSer-CARTs). Significantly, systematic variations in structure and formulation resulted in GSer-CARTs that exhibit highly selective mRNA delivery to the lung (∼97%) and spleen (∼98%) without targeting ligands. Illustrative of their breadth and translational potential, GSer-CARTs deliver circRNA, providing the basis for a cancer vaccination strategy, which in a murine model resulted in antigen-specific immune responses and effective suppression of established tumors.

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.

Cocatalytic Activity of the Furfuryl and Oxanorbornane-Substituted Guanidines in the Aldol Reaction Catalyzed by (S)-Proline.

This work investigated the cocatalytic activity of recently prepared guanidinium salts containing an oxanorbornane subunit in an (S)-proline-catalyzed aldol reaction. The activity was interpreted by the diastereoselectivity of the reaction (anti/syn ratio) and for the most interesting polycyclic guanidinium salt, the enantioselectivity of the reaction was determined. The results indicated a negative impact on the oxanorbornane unit if present as the flexible substituent. For most of the tested aldehydes, the best cocatalysts provided enantioselectivities above 90% and above 95% at room temperature and 0 °C, respectively, culminating in >99.5% for 4-chloro- and 2-nitrobenzaldehyde as the substrate. The barriers for forming four possible enantiomers were calculated and the results for two anti-enantiomers are qualitatively consistent with the experiment. Obtained results suggest that the representatives of furfurylguanidinium and rigid polycyclic oxanorbornane-substituted guanidinium salts are good lead structures for developing new cocatalysts by tuning the chemical space around the guanidine moiety.

Anion Binding to Ammonium and Guanidinium Hosts: Implications for the Reverse Hofmeister Effects Induced by Lysine and Arginine Residues.

Anions have a profound effect on the properties of soluble proteins. Such Hofmeister effects have implications in biologics stability, protein aggregation, amyloidogenesis, and crystallization. However, the interplay between the important noncovalent interactions (NCIs) responsible for Hofmeister effects is poorly understood. To contribute to improving this state of affairs, we report on the NCIs between anions and ammonium and guanidinium hosts 1 and 2, and the consequences of these. Specifically, we investigate the properties of cavitands designed to mimic two prime residues for anion-protein NCIs─lysines and arginines─and the solubility consequences of complex formation. Thus, we report NMR and ITC affinity studies, X-ray analysis, MD simulations, and anion-induced critical precipitation concentrations. Our findings emphasize the multitude of NCIs that guanidiniums can form and how this repertoire qualitatively surpasses that of ammoniums. Additionally, our studies demonstrate the ease by which anions can dispense with a fraction of their hydration-shell waters, rearrange those that remain, and form direct NCIs with the hosts. This raises many questions concerning how solvent shell plasticity varies as a function of anion, how the energetics of this impact the different NCIs between anions and ammoniums/guanidiniums, and how this affects the aggregation of solutes at high anion concentrations.

Novel guanidine derivatives targeting leukemia as selective Src/Abl dual inhibitors: Design, synthesis and anti-proliferative activity.

A new series of benzene-sulfonamide derivatives 3a-i was designed and synthesized via the reaction of N-(pyrimidin-2-yl)cyanamides 1a-i with sulfamethazine sodium salt 2 as dual Src/Abl inhibitors. Spectral data IR, 1H-, 13C- NMR and elemental analyses were used to confirm the structures of all the newly synthesized compounds 3a-i and 4a-i. Crucially, we screened all the synthesized compounds 3a-i against NCI 60 cancer cell lines. Among all, compound 3b was the most potent, with IC50 of 0.018 µM for normoxia, and 0.001 µM for hypoxia, compared to staurosporine against HL-60 leukemia cell line. To verify the selectivity of this derivative, it was assessed against a panel of tyrosine kinase EGFR, VEGFR-2, B-raf, ERK, CK1, p38-MAPK, Src and Abl enzymes. Results revealed that compound 3b can effectively and selectively inhibit Src/Abl with IC500.25 µM and Abl inhibitory activity with IC500.08 µM, respectively, and was found to be more potent on these enzymes than other kinases that showed the following results: EGFR IC500.31 µM, VEGFR-2 IC500.68 µM, B-raf IC500.33 µM, ERK IC501.41 µM, CK1 IC500.29 µM and p38-MAPK IC500.38 µM. Moreover, cell cycle analysis and apoptosis performed to compound 3b against HL-60 suggesting its antiproliferative activity through Src/Abl inhibition. Finally, molecular docking studies and physicochemical properties prediction for compounds 3b, 3c, and 3 h were carried out to investigate their biological activities and clarify their bioavailability.

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).

Liver glycogen fragility in the presence of hydrogen-bond breakers.

Glycogen, a complex branched glucose polymer, is responsible for sugar storage in blood glucose homeostasis. It comprises small ß particles bound together into composite α particles. In diabetic livers, α particles are fragile, breaking apart into smaller particles in dimethyl sulfoxide, DMSO; they are however stable in glycogen from healthy animals. We postulate that the bond between ß particles in α particles involves hydrogen bonding. Liver-glycogen fragility in normal and db/db mice (an animal model for diabetes) is compared using various hydrogen-bond breakers (DMSO, guanidine and urea) at different temperatures. The results showed different degrees of α-particle disruption. Disrupted glycogen showed changes in the mid-infra-red spectrum that are related to hydrogen bonds. While glycogen α-particles are only fragile under harsh, non-physiological conditions, these results nevertheless imply that the bonding between ß particles in α particles is different in diabetic livers compared to healthy, and is probably associated with hydrogen bonding.

Scaffold-Oriented Asymmetric Catalysis: Conformational Modulation of Transition State Multivalency during a Catalyst-Controlled Assembly of a Pharmaceutically Relevant Atropisomer.

A new class of superbasic, bifunctional peptidyl guanidine catalysts is presented, which enables the organocatalytic, atroposelective synthesis of axially chiral quinazolinediones. Computational modeling unveiled the conformational modulation of the catalyst by a novel phenyl urea N-cap, that preorganizes the structure into the active, folded state. A previously unanticipated noncovalent interaction involving a difluoroacetamide acting as a hybrid mono- or bidentate hydrogen bond donor emerged as a decisive control element inducing atroposelectivity. These discoveries spurred from a scaffold-oriented project inspired from a fascinating investigational BTK inhibitor featuring two stable chiral axes and relies on a mechanistic framework that was foreign to the extant lexicon of asymmetric catalysis.

Human apo-metallothionein 1a is not a random coil: Evidence from guanidinium chloride, high temperature, and acidic pH unfolding studies.

The structures of apo-metallothioneins (apo-MTs) have been relatively elusive due to their fluxional, disordered state which has been difficult to characterize. However, intrinsically disordered protein (IDP) structures are rather diverse, which raises questions about where the structure of apo-MTs fit into the protein structural spectrum. In this paper, the unfolding transitions of apo-MT1a are discussed with respect to the effect of the chemical denaturant GdmCl, temperature conditions, and pH environment. Cysteine modification in combination with electrospray ionization mass spectrometry was used to probe the unfolding transition of apo-MT1a in terms of cysteine exposure. Circular dichroism spectroscopy was also used to monitor the change in secondary structure as a function of GdmCl concentration. For both of these techniques, cooperative unfolding was observed, suggesting that apo-MT1a is not a random coil. More GdmCl was required to unfold the protein backbone than to expose the cysteines, indicating that cysteine exposure is likely an early step in the unfolding of apo-MT1a. MD simulations complement the experimental results, suggesting that apo-MT1a adopts a more compact structure than expected for a random coil. Overall, these results provide further insight into the intrinsically disordered structure of apo-MT.

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.