Exocyclic Amino Research Articles

Mechanistic Insights into Dibasic Iminosugars as pH-Selective Pharmacological Chaperones to Stabilize Human α-Galactosidase.

The use of pharmacological chaperones (PCs) to stabilize specific enzymes and impart a therapeutic benefit is an emerging strategy in drug discovery. However, designing molecules that can bind optimally to their targets at physiological pH remains a major challenge. Our previous study found that dibasic polyhydroxylated pyrrolidine 5 exhibited superior pH-selective inhibitory activity and chaperoning activity for human α-galactosidase A (α-Gal A) compared with its monobasic parent molecule, 4. To further investigate the role of different C-2 moieties on the pH-selectivity and protecting effects of these compounds, we designed and synthesized a library of monobasic and dibasic iminosugars, screened them for α-Gal A-stabilizing activity using thermal shift and heat-induced denaturation assays, and characterized the mechanistic basis for this stabilization using X-ray crystallography and binding assays. We noted that the dibasic iminosugars 5 and 20 protect α-Gal A from denaturation and inactivation at lower concentrations than monobasic or other N-substituted derivatives; a finding attributed to the nitrogen on the C-2 methylene of 5 and 20, which forms the bifurcated salt bridges (BSBs) with two carboxyl residues, E203 and D231. Additionally, the formation of BSBs at pH 7.0 and the electrostatic repulsion between the vicinal ammonium cations of dibasic iminosugars at pH 4.5 are responsible for their pH-selective binding to α-Gal A. Moreover, compounds 5 and 20 demonstrated promising results in improving enzyme replacement therapy and exhibited significant chaperoning effects in Fabry cells. These findings suggest amino-iminosugars 5 and 20 as useful models to demonstrate how an additional exocyclic amino group can improve their pH-selectivity and protecting effects, providing new insights for the design of pH-selective PCs.

A computational study of the structures and base-pairing properties of pyrrolizidine alkaloid-derived DNA adducts

Pyrrolizidine alkaloids (PAs) are found in many plants worldwide, including some in Canada. PAs have been linked to losses in livestock populations and the development of human PA diseases. Four PA-derived dehydrosupinidine (DHP) adducts formed at the exocyclic amino groups of DNA purines have been found in tumor tissue and flagged as potential biomarkers for tumor formation (denoted DHP–G/A–3 and DHP–G/A–4). Four additional adducts (DHP–G/A–1 and DHP–G/A–2) have also been identified, which differ in the stereochemistry at the DHP–nucleobase linker or DHP ring carbon containing the hydroxy group, as well as the length of the DHP–nucleobase linker. Since the impact of these distinct chemical features on adduct mutagenicity is currently unclear, the present work uses density functional theory calculations to uncover the structures and base-pairing properties of these experimentally-observed DHP-derived purine adducts. Adduct Watson–Crick–Franklin base pairs involving the canonical partner are energetically and structurally feasible. However, the G/A–3 and G/A–4 adducts are also highly susceptible to mispairing with G. Indeed, the longer and more flexible DHP–nucleobase linker in these adducts affords interactions between the DHP moiety and the pairing G that are not possible for the G/A–1 and G/A–2 counterparts. Our data thus rationalize the experimental identification of this subset of adducts in liver tumor tissue, and provide key insights to guide future experimental and computational studies that investigate the replication and broader biological outcomes of DHP-derived lesions.

A new series of acylhydrazones derived from metribuzin with modulated herbicidal activity

This paper reports the preparation and herbicidal evaluation of a small library of acylhydrazones based on the synthetic herbicide metribuzin. The hydrazone linkage easily obtained by reaction of metribuzin with aliphatic and aromatic aldehydes, masks efficiently the exocyclic amino group, thereby altering significantly H-bonding with the receptor and increasing the lipophilicity relative to the parent herbicide. The structures of all compounds, including key stereochemical issues on conformation and E/Z configuration around the CN bond were thoroughly elucidated by spectroscopic methods, and unambiguously corroborated by X-ray diffraction analysis. The herbicidal assays using an aliphatic and an aromatic acylhydrazone were performed on tomato and rapeseed plants grown in greenhouse. Our results demonstrate, regardless of rate application, that such acylhydrazone formulations do not alter the selectivity of metribuzin. Moreover, the herbicide activity was even higher in the alkyl derivative than that achieved by commercial metribuzin, thus suggesting that this substance can be applied with no need of combination with chemical coadjuvants, unlike most formulations of commercially available herbicides. Therefore, the study shows the promising effect of chemical derivatization of a common herbicide as metribuzin, to improve the herbicide activity without compromising selectivity, and allowing the farmers its use in crop protection safely and effectively.

N2 modified cap analogues as translation inhibitors and substrates for preparation of therapeutic mRNA.

In recent years many scientists have begun to focus on the mRNA molecule's emeregence as a new type of drug. Its fast-moving and successful career as a vaccine technology cannot be underestimated. mRNA provides new opportunities and allows for the rapid preparation of effective drugs at low cost. These extensive possibilities stem from a number of factors, but the small cap structure located at the 5' end of the mRNA is one contributing factor. Cap protects mRNA and ensures efficient recruitment to the biosynthesis machinery. Furthermore, it allows for the easy introduction of various modifications that influence the activity of the entire mRNA. Among the many different cap analogues that have been reported, those modified at the N2 position of guanosine have been systematically developed. N2-modified caps in the form of nucleoside monophosphates or dinucleotides show favorable biological properties, as well as a high capacity to inhibit the translation process in the cell-free RRL system. Modified N2 dinucleotides are efficiently incorporated into the structure of the mRNA transcript, and in specific circumstances with the correct orientation, making them an interesting alternative for ARCA-type analogues. Moreover, mRNA transcripts containing cap structures modified within the exocyclic amino group show very high translational activity. Therefore, analogues modified at the N2 position may have future applications as therapeutics against various manifestations of cancer and as desirable tools in RNA engineering.

N,O-, N,N-, N,S-, and N,N,S-Heterocycles with an Exocyclic Amino Group in the Synthesis of 1,5,3,7-Diazadiphosphacyclooctanes

N,O-, N,N-, N,S-, and N,N,S-Heterocycles with an Exocyclic Amino Group in the Synthesis of 1,5,3,7-Diazadiphosphacyclooctanes

Vertical Excitation Energies and Lifetimes of the Two Lowest Singlet Excited States of Cytosine, 5-Aza-cytosine, and the Triazine Family: Quantum Mechanics-Molecular Mechanics Studies.

A swarm of semi-classical quantum mechanics/molecular mechanics molecular-dynamics simulations where OM2/MNDO is combined with the Gromacs program for consideration of explicit water is performed, solving the time-dependent Schrödinger equation in each step of the trajectories together with the Tully's fewest switches algorithm. Within this stochastic treatment, time dependent probabilities of the three lowest electronic states are determined. The fact that nucleobases are quickly deactivated is confirmed in the cytosine case where our best lifetime estimation is τ1=0.82 ps for the model with 100 water molecules with the SPCE force field and a time step of 0.1 fs. Lifetimes of the remaining molecules are visibly longer: 5-azacytosine, 2,4-diamino-1,3,5-triazine (DT), and 2,4,6-triamino-1,3,5-triazine (TT) molecules have an S1 → S0 de-excitation time of slightly above 10 ps. The lifetimes of the triazine family increases with the increasing number of exocyclic amino groups, that is, s-triazine < 2-amino-1,3,5-triazine < DT < TT. This can be explained by a higher mobility of the carbon-bonded hydrogen atoms in comparison with heavier amino groups since their movement is slowed down due to a substantially higher mass than hydrogen atoms, which can easier reach the out-of-plane positions required in the conical intersection structures. Moreover, bulkier NH2 ligands suffer due to greater friction caused by the surrounding water environment. These mechanical aspects caused a change in the explored lifetime dependences in comparison with our previous gas-phase study.

Effects of Local Sequence, Reaction Conditions, and Various Additives on the Formation and Stability of Interstrand Cross-Links Derived from the Reaction of an Abasic Site with an Adenine Residue in Duplex DNA.

The experiments described here examined the effects of reaction conditions, various additives, and local sequence on the formation and stability interstrand cross-links (ICLs) derived from the reaction of an apurinic/apyrimidinic (AP) site with the exocyclic amino group of an adenine residue on the opposing strand in duplex DNA. Cross-link formation was observed in a range of different buffers, with faster formation rates observed at pH 5. Inclusion of the base excision repair enzyme alkyladenine DNA glycosylase (hAAG) which binds tightly to AP-containing duplexes decreased, but did not completely prevent, formation of the dA-AP ICL. Formation of the dA-AP ICL was not altered by the presence of the biological metal ion Mg2+ or the biological thiol, glutathione. Several organocatalysts of imine formation did not enhance the rate of dA-AP ICL formation. Duplex length did not have a large effect on dA-AP yield, so long as the melting temperature of the duplex was not significantly below the reaction temperature (the duplex must remain hybridized for efficient ICL formation). Formation of the dA-AP ICL was examined in over 40 different sequences that varied the neighboring and opposing bases at the cross-linking site. The results indicate that ICL formation can occur in a wide variety of sequence contexts under physiological conditions. Formation of the dA-AP ICL was strongly inhibited by the aldehyde-trapping agents methoxyamine and hydralazine, by NaBH3CN, by the intercalator ethidium bromide, and by the minor groove-binding agent netropsin. ICL formation was inhibited to some extent in bicarbonate and Tris buffers. The dA-AP ICL showed substantial inherent stability under a variety of conditions and was not a substrate for AP-processing enzymes APE1 or Endo IV. Finally, we characterized cross-link formation in a small (11 bp) stem-loop (hairpin) structure and in DNA-RNA hybrid duplexes.

Synthesis of 7-Arylpurines from Substituted Pyrimidines

AbstractA simple three-step approach for the synthesis of substituted N7-arylpurines with an overall yield of the whole sequence from 40% to 71% is described. N7-Arylpurines were constructed by de novo synthesis from commercially available substituted 4-chloropyrimidine-5-amines. Different substituents at purine C2 and C6 were obtained by changing the corresponding substituents of the starting pyrimidine. Further, heteroaromatic, electron-deficient, and electron-rich aromatic groups were attached to the exocyclic amino group by iodane reagents under copper catalysis. This moiety is prepared to become purine N7 position after the ring closure. Finally, purine C8 substitution was varied during the last step of the developed sequence by employing different reagents for the purine ring closing reactions or post functionalization.

Hepatic RNA adduction derived from metabolic activation of retrorsine in vitro and in vivo

Pyrrolizidine alkaloids (PAs) are among the most significant hepatotoxins widely distributed in plant species. Incidence of liver injuries caused by PAs has been reported worldwide, and the reactive metabolites of PAs are known to play a critical role in causing the hepatotoxicity. To better understand the toxicity-induction mechanisms, we explored the interactions of PA metabolites with cellular RNA molecules, and examined their effects on the biochemical and metabolic properties of hepatic RNAs. After exposure to retrorsine, adduction on adenosine and guanosine were detected in mouse liver microsomal incubations, cultured mouse primary hepatocytes, and mouse liver tissues. NMR analysis showed that the exocyclic amino group participated in the adduction. We found drastically altered properties and metabolism of the adducted RNA such as reverse-transcriptability, translatability, and RNase-susceptibility. In addition, endogenous modification of N6-methyladenosine (m6A) was remarkably reduced.

Regioselectivity in the reaction of 5-amino-3-anilino-1H-pyrazole-4-carbonitrile with cinnamonitriles and enaminones: Synthesis of functionally substituted pyrazolo[1,5-a]pyrimidine derivatives

Abstract The development of efficient methods for the synthesis of polyfunctional N-heterocycles is an important area of research in organic and medicinal chemistry. Pyrazolo[1,5-a]pyrimidine derivatives are purine analogous of biomedical importance and have been extremely studied for their broad spectrum of biological activities. Recently, they have attracted great interest in materials science owing to their photophysical properties. 3(5)-Aminopyrazoles are extensively utilized in the synthesis of condensed heterocyclic systems, particularly pyrazolo[1,5-a]pyrimidines via the reaction with 1,3-biselectrophilic reagents. However, the information available in the literature provides little in the way of reasoning their cyclization, particularly the initial attack either by the exocyclic amino group or endocyclic nitrogen. Unfortunately, the relative nucleophilicity of exo- and endocyclic nitrogen atoms in 1-unsubstituted 3(5)-aminopyrazoles is not clear and contradicting. It has been found that other factors can modulate the regioselectivity rather than basicity or steric hindrance for both active sites. The reported studies in the structure–activity relationship revealed that pyrazolo[1,5-a]pyrimidines having a substitution at fifth, sixth, and seventh positions possess potent biological activities, especially those with an amino group at the seventh position. We here developed a regioselective, high yield synthesis of 7-amino-5-arylpyrazolo[1,5-a]pyrimidine-3,6-dicarbonitriles by the reaction of N-(5-amino-4-cyano-1H-pyrazole-3-yl)-benzamide with various cinnamonitriles and enaminones in pyridine at 120°C under controlled microwave heating conditions. All structures of newly synthesized compounds were established by analytical and spectral data as well as single-crystal diffraction and rationalized for their formation.

Optimization of 2-Amino-4,6-diarylpyrimidine-5-carbonitriles as Potent and Selective A1 Antagonists.

We herein document a large collection of 108 2-amino-4,6-disubstituted-pyrimidine derivatives as potent, structurally simple, and highly selective A1AR ligands. The most attractive ligands were confirmed as antagonists of the canonical cyclic adenosine monophosphate pathway, and some pharmacokinetic parameters were preliminarilly evaluated. The library, built through a reliable and efficient three-component reaction, comprehensively explored the chemical space allowing the identification of the most prominent features of the structure–activity and structure–selectivity relationships around this scaffold. These included the influence on the selectivity profile of the aromatic residues at positions R4 and R6 of the pyrimidine core but most importantly the prominent role to the unprecedented A1AR selectivity profile exerted by the methyl group introduced at the exocyclic amino group. The structure–activity relationship trends on both A1 and A2AARs were conveniently interpreted with rigorous free energy perturbation simulations, which started from the receptor-driven docking model that guided the design of these series.

Ambident reactivity of 5-aminopyrazoles towards donor-acceptor cyclopropanes.

Lewis acid-catalysed reactions of donor-acceptor cyclopropanes with 1,3-disubstituted 5-aminopyrazoles were investigated. Under catalysis with gallium(III) chloride, products of the three-membered ring opening via a nucleophilic attack of the exocyclic amino group were obtained in a chemoselective manner. Oppositely, in the presence of scandium(III) triflate, products of either N-alkylation or C(4)-alkylation, or a mixture of both were formed. The products of the C(4) alkylation were transformed in one step into tetrahydropyrazolo[3,4-b]azepines that are attractive for medicinal chemistry and pharmacology.

Purine adducts as a presumable missing link for aristolochic acid nephropathy-related cellular energy crisis, potential anti-fibrotic prevention and treatment.

Aristolochic acid nephropathy is a progressive exposome-induced disease characterized by tubular atrophy and fibrosis culminating in end-stage renal disease and malignancies. The molecular mechanisms of the energy crisis as a putative cause of fibrosis have not yet been elucidated. In light of the fact that aristolochic acid forms DNA and RNA adducts by covalent binding of aristolochic acid metabolites to exocyclic amino groups of (deoxy)adenosine and (deoxy)guanosine, we hypothesize here that similar aristolochic acid adducts may exist with other purine-containing molecules. We also provide new insights into the aristolochic acid-induced energy crisis and presumably a link between already known mechanisms. In addition, an overview of potential targets in fibrosis treatment is provided, which is followed by recommendations on possible preventive measures that could be taken to at least postpone or partially alleviate aristolochic acid nephropathy.

Water structure around a left-handed Z-DNA fragment analyzed by cryo neutron crystallography.

Even in high-quality X-ray crystal structures of oligonucleotides determined at a resolution of 1 Å or higher, the orientations of first-shell water molecules remain unclear. We used cryo neutron crystallography to gain insight into the H-bonding patterns of water molecules around the left-handed Z-DNA duplex [d(CGCGCG)]2. The neutron density visualized at 1.5 Å resolution for the first time allows us to pinpoint the orientations of most of the water molecules directly contacting the DNA and of many second-shell waters. In particular, H-bond acceptor and donor patterns for water participating in prominent hydration motifs inside the minor groove, on the convex surface or bridging nucleobase and phosphate oxygen atoms are finally revealed. Several water molecules display entirely unexpected orientations. For example, a water molecule located at H-bonding distance from O6 keto oxygen atoms of two adjacent guanines directs both its deuterium atoms away from the keto groups. Exocyclic amino groups of guanine (N2) and cytosine (N4) unexpectedly stabilize waters H-bonded to O2 keto oxygens from adjacent cytosines and O6 keto oxygens from adjacent guanines, respectively. Our structure offers the most detailed view to date of DNA solvation in the solid-state undistorted by metal ions or polyamines.

Interstrand Cross-Link Formation Involving Reaction of a Mispaired Cytosine Residue with an Abasic Site in Duplex DNA.

The formation of interstrand cross-links in duplex DNA is important in biology, medicine, and biotechnology. Interstrand cross-links arising from the reaction of the aldehyde residue of an abasic (apurinic or AP) site with the exocyclic amino groups of guanine or adenine residues on the opposing strand of duplex DNA have previously been characterized. The canonical nucleobase cytosine has an exocyclic amino group but its ability to form interstrand cross-links by reaction with an AP site has not been characterized before now. Here it is shown that substantial yields of interstrand cross-links are generated in sequences having a mispaired cytosine residue located one nucleotide to the 3'-side of the AP site on the opposing strand (e.g., 5'XA/5'CA, where X = AP). Formation of the dC-AP cross-link is pH-dependent, with significantly higher yields at pH 5 than pH 7. Once formed, the dC-AP cross-link is quite stable, showing less than 5% dissociation over the course of 96 h at pH 7 and 37 °C. No significant yields of cross-link are observed when the cytosine residue is paired with its Watson-Crick partner guanine. It was also shown that a single AP site can engage with multiple nucleobase cross-linking partners in some sequences. Specifically, the dG-AP and dC-AP cross-links coexist in dynamic equilibrium in the sequence 5'CXA/5'CAG (X = AP). In this sequence, the dC-AP cross-link dominates. However, in the presence of NaBH3CN, irreversible reduction of small amounts of the dG-AP cross-link present in the mixture shifts the equilibria away from the dC-AP cross-link toward good yields of the dG-APred cross-link.

XRD, NMR, FT-IR and DFT structural characterization of a novel organic-inorganic hybrid perovskite-type hexabromotellurate material

The synthesis of a new hexabromotellurate of triazole ring-containing amino acid is described. XRD, NMR and DFT analysis of the structure of protonated [(5-amino-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetic acid testifies the protonation of the first nitrogen atom of the triazole ring. Hirshfeld surface analysis identifies numerous intermolecular interactions in the newly synthesized organic-inorganic hybrid hexabromotellurate. The DFT based comprehensive analysis of the structures of possible conformers also testifies the higher thermodynamic stability of the 1-protonated triazole cation, whereas the starting [(5-amino-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetic acid exists in solution in neutral form with the intermolecular hydrogen bond between the carboxylic acid OH group and the second nitrogen of the triazole ring. The experimentally observed NMR upfield shift of the signal of the exocyclic amino group was studied via GIAO computations and was explained through the analysis of the HOMOs of the neutral and protonated forms of the triazole compound.

Interaction of GelRed™ with single-stranded DNA oligonucleotides: Preferential binding to thymine-rich sequences

GelRed (GR) is a widely used nucleic acid-binding dye. It interacts with double-stranded DNA (dsDNA) via intercalation between the base-pairs, which enhances the dye's fluorescence. Binding of GR to single-stranded DNA (ssDNA) is much less efficient, yet the dye is still used for ssDNA staining in such applications as gel electrophoresis. The nature of GR interactions with ssDNA is not well understood. Here, preferential binding of GR to thymine-rich sequences was demonstrated, with ~20-fold increase in the dye's fluorescence in the presence of equimolar T20 concentration. Based on pH and ionic strength dependences, electrostatic interactions with oligonucleotide backbone and hydrogen bonding between thymine and exocyclic amino groups of GR were proposed as likely stabilizing forces in GR-oligothymidylate binding. Lack of the C5-methyl group in dU20 decreased GR affinity to the oligonucleotide ~2-fold. Molecular dynamic simulations were used to propose a possible structure of the GR-oligothymidilate complex.

NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination.

Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of ∼50% were detected in defined amplicons, and this threshold can be lowered to ∼10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.

Dynamic Structure and Stability of DNA Duplexes Bearing a Dinuclear Hg(II)-Mediated Base Pair.

Quantum mechanical (QM) and hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations of a recently reported dinuclear mercury(II)-mediated base pair were performed aiming to analyse its intramolecular bonding pattern, its stability, and to obtain clues on the mechanism of the incorporation of mercury(II) into the DNA. The dynamic distance constraint was employed to find initial structures, control the dissociation process in an unbiased fashion and to determine the free energy required. A strong influence of the exocyclic carbonyl or amino groups of neighbouring base pairs on both the bonding pattern and the mechanism of incorporation was observed. During the dissociation simulation, an amino group of an adenine moiety of the adjacent base pair acts as a turnstile to rotate the mercury(II) ion out of the DNA core region. The calculations provide an important insight into the mechanism of formation of this dinuclear metal-mediated base pair and indicate that the exact location of a transition metal ion in a metal-mediated base pair may be more ambiguous than derived from simple model building.

Synthesis of 1-aroyl-3-methylsulfanyl-5-amino-1,2,4-triazoles and their analysis by spectroscopy, X-ray crystallography and theoretical calculations

N-Aroyl-1,2,4-triazoles TAM and TACl were regioselectively synthesized in excellent yields through an efficient N-acylation reaction of 3-amino-5-methylsulfanyl-1H-1,2,4-triazole with aroyl chlorides. Structures of N-aroyl-1,2,4-triazoles were studied by single-crystal X-ray diffraction, observing that their crystal structures are characterized by the formation of dimers through N‒H‧‧‧N bonds. The supramolecular assembly depends on the sort of connections between dimers, which notably change with the para-substituent on the aroyl group. The directly calculated ionization potential (IP), electron affinity (EA), electronegativity (χ), electrophilicity index (ω), hardness (η), and chemical potential (μ) are correlated with the HOMO and LUMO orbital energies. Moreover, molecular electrostatic potential maps of both molecules have been calculated showing a negative region at N2 atom of the 1,2,4-triazole ring instead of exocyclic amino group. The vibrational spectral analysis was carried out using infrared spectroscopy in the range 4000–400 cm−1 for N-aroyl-1,2,4-triazoles TAM and TACl. The experimental spectra were recorded in the solid state. The fundamental vibrational frequencies and intensity of vibrational bands were evaluated using density functional theory (DFT) with the standard B3LYP/6–31G(d,p) method and basis set yielding fairly good agreement between observed and calculated frequencies. Simulation of infrared spectra utilizing the results of these calculations led to an excellent overall agreement with the observed spectral patterns.