Structural and Functional Insights into Host Death Domains Inactivation by the Bacterial Arginine GlcNAcyltransferase Effector.

Enteropathogenic E. coli NleB and related type III effectors catalyze arginine GlcNAcylation of death domain (DD) proteins to block host defense, but the underlying mechanism is unknown. Here we solve crystal structures of NleB alone and in complex with FADD-DD, UDP, and Mn2+ as well as NleB-GlcNAcylated DDs of TRADD and RIPK1. NleB adopts a GT-A fold with a unique helix-pair insertion to hold FADD-DD; the interface contacts explain the selectivity of NleB for certain DDs. The acceptor arginine is fixed into a cleft, in which Glu253 serves as a base to activate the guanidinium. Analyses of the enzyme-substrate complex and the product structures reveal an inverting sugar-transfer reaction and a detailed catalytic mechanism. These structural insights are validated by mutagenesis analyses of NleB-mediated GlcNAcylation in vitro and its function in mouse infection. Our study builds a structural framework for understanding of NleB-catalyzed arginine GlcNAcylation of host death domain.

Guanidine Hydrochloride-Induced Hepatitis B Virus Capsid Disassembly Hysteresis.

Hepatitis B virus (HBV) displays remarkable self-assembly capabilities that interest the scientific community and biotechnological industries as HBV is leading to an annual mortality of up to 1 million people worldwide (especially in Africa and Southeast Asia). When the ionic strength is increased, hepatitis B virus-like particles (VLPs) can assemble from dimers of the first 149 residues of the HBV capsid protein core assembly domain (Cp149). Using solution small-angle X-ray scattering, we investigated the disassembly of the VLPs by titrating guanidine hydrochloride (GuHCl). Measurements were performed with and without 1 M NaCl, added either before or after titrating GuHCl. Fitting the scattering curves to a linear combination of atomic models of Cp149 dimer (the subunit) and T = 3 and T = 4 icosahedral capsids revealed the mass fraction of the dimer in each structure in all the titration points. Based on the mass fractions, the variation in the dimer-dimer association standard free energy was calculated as a function of added GuHCl, showing a linear relation between the interaction strength and GuHCl concentration. Using the data, we estimated the energy barriers for assembly and disassembly and the critical nucleus size for all of the assembly reactions. Extrapolating the standard free energy to [GuHCl] = 0 showed an evident hysteresis in the assembly process, manifested by differences in the dimer-dimer association standard free energy obtained for the disassembly reactions compared with the equivalent assembly reactions. Similar hysteresis was observed in the energy barriers for assembly and disassembly and the critical nucleus size. The results suggest that above 1.5 M, GuHCl disassembled the capsids by attaching to the protein and adding steric repulsion, thereby weakening the hydrophobic attraction.

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.

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.

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.

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.

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).

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.

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.

Impact of guanidine-containing backbone linkages on stereopure antisense oligonucleotides in the CNS.

Attaining sufficient tissue exposure at the site of action to achieve the desired pharmacodynamic effect on a target is an important determinant for any drug discovery program, and this can be particularly challenging for oligonucleotides in deep tissues of the CNS. Herein, we report the synthesis and impact of stereopure phosphoryl guanidine-containing backbone linkages (PN linkages) to oligonucleotides acting through an RNase H-mediated mechanism, using Malat1 and C9orf72 as benchmarks. We found that the incorporation of various types of PN linkages to a stereopure oligonucleotide backbone can increase potency of silencing in cultured neurons under free-uptake conditions 10-fold compared with similarly modified stereopure phosphorothioate (PS) and phosphodiester (PO)-based molecules. One of these backbone types, called PN-1, also yielded profound silencing benefits throughout the mouse brain and spinal cord at low doses, improving both the potency and durability of response, especially in difficult to reach brain tissues. Given these benefits in preclinical models, the incorporation of PN linkages into stereopure oligonucleotides with chimeric backbone modifications has the potential to render regions of the brain beyond the spinal cord more accessible to oligonucleotides and, consequently, may also expand the scope of neurological indications amenable to oligonucleotide therapeutics.

In this study, the authors explore the impact of nitrogen-containing (PN) backbones on oligonucleotides that promote RNase H-mediated degradation of a transcript in the central nervous system (CNS). Using Malat1, a ubiquitously expressed non-coding RNA that is predominately localized in the nucleus, and C9orf72, a challenging RNA target requiring a more nuanced targeting strategy, as benchmarks, they show that chimeric oligonucleotides containing stereopure PS and one of the more promising PN backbones (PN-1) have more potent and durable activity throughout the CNS compared with more traditional PS-modified molecules in mouse models. They demonstrate that potency and durability benefits in vivo derive at least in part from increased tissue exposure, especially in more difficult to reach regions of the brain. Ultimately, these benefits enabled the authors to demonstrate pharmacodynamic effects on Malat1 and C9orf72 RNAs in multiple brain regions with relatively low doses.

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.

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.

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.

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.

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.

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.

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.

Guanidine-II aptamer conformations and ligand binding modes through the lens of molecular simulation.

Regulation of gene expression via riboswitches is a widespread mechanism in bacteria. Here, we investigate ligand binding of a member of the guanidine sensing riboswitch family, the guanidine-II riboswitch (Gd-II). It consists of two stem-loops forming a dimer upon ligand binding. Using extensive molecular dynamics simulations we have identified conformational states corresponding to ligand-bound and unbound states in a monomeric stem-loop of Gd-II and studied the selectivity of this binding. To characterize these states and ligand-dependent conformational changes we applied a combination of dimensionality reduction, clustering, and feature selection methods. In absence of a ligand, the shape of the binding pocket alternates between the conformation observed in presence of guanidinium and a collapsed conformation, which is associated with a deformation of the dimerization interface. Furthermore, the structural features responsible for the ability to discriminate against closely related analogs of guanidine are resolved. Based on these insights, we propose a mechanism that couples ligand binding to aptamer dimerization in the Gd-II system, demonstrating the value of computational methods in the field of nucleic acids research.

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.