Linker Moiety Research Articles

Effect of linker moiety on linear dimeric benzotriazole derivatives as highly stable UV absorber for transparent polyimide film

In this study, we developed a series of highly stable UV absorbers for polymers which require lengthy high temperature processes, such as polyimide (PI). Three types of linear dimeric hydroxyphenyl benzotriazole derivatives were synthesized by linking monomers with different linker moieties (phenyl, disulfide, biphenyl). The arrangement between the monomers was affected by the type of linker moiety introduced in the dimeric hydroxyphenyl benzotriazole derivatives. As a result, the intermolecular interactions changed resulting in changes in thermal stability and solubility. These results revealed that dimeric hydroxyphenyl benzotriazole derivatives had remarkable stability compared to the monomer. Especially, a novel dimeric UV absorber with phenyl linker (PBT) exhibited excellent stability suitable for high temperature processes.

Recent Trends in In Situ Enzyme-Activatable Prodrugs for Targeted Cancer Therapy

Enzyme-activatable anticancer prodrugs are modified medications that are composed of an anticancer drug, cleavable linker, and functional moiety. The purpose of such a prodrug structure is to generate multipurpose functions that traditional drugs cannot perform and to reduce the toxicity of conventional anticancer drugs by the mask of the cleavable linker. Once the cleavable linker is degraded via a specific chemical reaction in the cancer microenvironment, the cytotoxicity of the degraded prodrugs is selectively recovered. Among many factors that cleave the linker, we focus on the overexpressed enzymes in cancer. Because of the selective enzymatic degradation of the cleavable linker and the high local concentration of specific enzymes in cancer, the enzyme-activatable prodrugs could show low toxicity in normal tissues, while showing comparable anticancer effect in tumors. In addition, some prodrugs provide additional features, such as cancer imaging, drug release monitoring, tumor targeting, and enhanced stability, which conventional anticancer drugs cannot possess. In this review, we summarize currently developed enzyme-activatable prodrugs according to their activating enzymes, and categorize them by their additional functions, e.g. targeting, imaging, and delivery. This summary of enzyme-activatable prodrugs may help in the design of anticancer prodrugs, and in the establishment of a personalized cancer treatment strategy.

Structure–Activity Relationship Study on Col-003, a Protein–Protein Interaction Inhibitor between Collagen and Hsp47

This study demonstrates the structure-activity relationship of Col-003, a potent collagen-heat-shock protein 47 (Hsp47) interaction inhibitor. Col-003 analogues were successfully synthesized by Pd(0)-catalyzed cross-coupling reactions of 5-bromosalicylaldehyde derivatives with alkyl-metal species, and the inhibitory activities of the synthetic analogues were evaluated using surface plasmon resonance analysis (BIAcore). We succeeded in discovering two potent inhibitors that showed 85 and 81% inhibition at a concentration of 1.9 µM against the collagen-Hsp47 interaction. This indicates that elongation of an alkyl linker between two aromatic rings could considerably improve inhibitory activity due to the adjustment of a pendant phenyl moiety to an appropriate position, in addition to the hydrophobic interaction with an alkyl linker moiety.

A Combinatorial Library of Lipid Nanoparticles for RNA Delivery to Leukocytes.

Lipid nanoparticles (LNPs) are the most advanced nonviral platforms for small interfering RNA (siRNA) delivery that are clinically approved. These LNPs, based on ionizable lipids, are found in the liver and are now gaining much attention in the field of RNA therapeutics. The previous generation of ionizable lipids varies in linker moieties, which greatly influences in vivo gene silencing efficiency. Here novel ionizable amino lipids based on the linker moieties such as hydrazine, hydroxylamine, and ethanolamine are designed and synthesized. These lipids are formulated into LNPs and screened for their efficiency to deliver siRNAs into leukocytes, which are among the hardest to transfect cell types. Two potent lipids based on their in vitro gene silencing efficiencies are also identified. These lipids are further evaluated for their biodistribution profile, efficient gene silencing, liver toxicity, and potential immune activation in mice. A robust gene silencing is also found in primary lymphocytes when one of these lipids is formulated into LNPs with a pan leukocyte selective targeting agent (β7 integrin). Taken together, these lipids have the potential to open new avenues in delivering RNAs into leukocytes.

Correction to: Synthesis and characterization of processable aromatic poly(ether ether ketone amide)s modified by phenoxy and 1,3 ketone moiety linkages

Correction to: Synthesis and characterization of processable aromatic poly(ether ether ketone amide)s modified by phenoxy and 1,3 ketone moiety linkages

A Non-Genotoxic Anti-CD117 Antibody Drug Conjugate (ADC) Designed for Patient Conditioning Prior to Stem Cell Transplant and HSC-Based Gene Therapy Has a Broad Therapeutic Window across Species

Current approaches to conditioning prior to transplant and gene therapy employ non-specific genotoxic agents that are associated with severe acute and long-term toxicities. Consequently, many transplant eligible patients either refuse transplant or receive reduced intensity regimens, which still have adverse toxicities with increased risk of relapse and graft failure. Importantly, patients with non-malignant disease may choose to forgo a potentially curative transplant due to risks of conditioning-associated toxicities, potentially limiting access of these patients to transplant and emerging HSC-based gene therapy. Thus, safe and effective targeted conditioning agents would have broad application to transplant in both malignant and non-malignant settings as well as gene therapy. We developed a CD117 antibody conjugated to amanitin (anti-CD117-AM) to specifically deplete hematopoietic stem and progenitor cells (HSPCs) as a novel approach to patient preparation for allogeneic and autologous transplant. ADCs have significant single agent anti-tumor activity in the clinic, however, achieving suitable tolerability at efficacious doses has presented a clinical challenge. We engineered the properties of the anti-CD117-AM to provide a broad therapeutic window across pre-clinical models. Through modification of the linker moiety, the maximum tolerated dose (MTD) of the ADC in C57Bl6 mice was improved 17-fold compared to previous versions. The engineered anti-CD117-AM maintained potent cytotoxicity on primary human CD34+ cells and a CD117+ AML cell line ( 95% of human HSPCs (Fig. 1A). Single doses of the anti-CD117-AM demonstrated robust tumor control in xenograft models of AML, despite its purposefully shortened half-life as a targeted agent for transplant indications (Fig. 1B). The tolerability of the engineered anti-CD117-AM was assessed in cynomolgus NHPs. The ADC was well-tolerated with an MTD at ≥ 2 mg/kg (Fig. 1C). A single dose of the ADC was able to drive marked depletion of HSPCs and downstream progeny. The pharmacokinetics of the ADC exhibited linear clearance supporting tolerability well above target saturation. In summary, optimization of the chemical linker between antibody and payload yielded a well-tolerated ADC with wide safety margins that could eliminate HSPCs and confer survival benefit in AML xenograft models. ADC-mediated conditioning through selective depletion of CD117+ cells may provide an improved approach to patient preparation for transplant in malignant and non-malignant disease including gene therapy, broadening patient access to potentially curative treatments.

Synthesis and evaluation of novel radioiodinated PSMA targeting ligands for potential radiotherapy of prostate cancer

Radioligand therapy (RLT) using prostate-specific membrane antigen (PSMA) targeting ligands is an attractive option for the treatment of Prostate cancer (PCa) and its metastases. We report herein a series of radioiodinated glutamate-urea-lysine-phenylalanine derivatives as new PSMA ligands in which l-tyrosine and l-glutamic acid moieties were added to increase hydrophilicity concomitant with improvement of in vivo targeting properties. Compounds 8, 15, 19a/19b and 23a/23b were synthesized and radiolabeled with 125I by iododestannylation. All iodinated compounds displayed high binding affinities toward PSMA (IC50 = 1–13 nM). In vitro cell uptake studies demonstrated that compounds containing an l-tyrosine linker moiety (8, 15 and 19a/19b) showed higher internalization than MIP-1095 and 23a/23b, both without the l-tyrosine linker moiety. Biodistribution studies in mice bearing PC3-PIP and PC3 xenografts showed that [125I]8 and [125I]15 with higher lipophilicity exhibited higher nonspecific accumulations in the liver and intestinal tract, whereas [125I]19a/19b and [125I]23a/23b containing additional glutamic acid moieties showed higher accumulations in the kidney and implanted PC3-PIP (PSMA+) tumors. [125I]23b displayed a promising biodistribution profile with favorable tumor retention, fast clearance from the kidney, and 2–3-fold lower uptake in the liver and blood than that observed for [125I]MIP-1095. [125/131I]23b may serve as an optimal PSMA ligand for radiotherapy treatment of prostate cancer over-expressing PSMA.

Synthesis and characterization of processable aromatic poly(ether ether ketone amide)s modified by phenoxy and 1,3 ketone moiety linkages

New series of aromatic poly(ether ether ketone amide)s were synthesized by low-temperature solution polymerization of novel aromatic diamine, namely 1,3-bis-4′-(4″-aminophenoxy benzoyl)benzene (XIV), and aromatic diacid chlorides, viz. isophthaloyl chloride (IPC) and terephthaloyl chloride (TPC). Co-poly(ether ether ketone amide)s were also synthesized by employing various mole proportions of IPC and TPC with diamine (XIV). These poly(ether ether ketone amide)s were characterized by FTIR, solubility, inherent viscosity, TGA, DSC, and XRD. Inherent viscosities of these poly(ether ether ketone amide)s were in the range of 0.41–0.52 dL/g in DMAc, indicating the formation of moderate to high molecular weight of polymers. Poly(ether ether ketone amide)s showed good solubility in polar aprotic solvents such as N,N-dimethyl acetamide (DMAc), N-methyl 2-pyrrolidone, N,N-dimethylformamide, and dimethyl sulfoxide. These poly(ether ether ketone amide)s had glass transition temperatures, as determined by DSC, in the range of 252–302 °C. These polymers showed similar decomposition patterns and had no weight loss below 335 °C, and temperatures for 10% weight loss (T10) were in the range of 397–406 °C, indicating that these polymers showed good thermal stability.

Essential elements regulating HDAC8 inhibition: a classification based structural analysis and enzyme-inhibitor interaction study of hydroxamate based HDAC8 inhibitors

Histone deacetylase 8 (HDAC8) expressions are correlated with a variety of cancer and tumor conditions. For the pathophysiological contributions of HDAC8, it is classified as an important target for cancer research. The hydroxamate derivatives are identified as more efficient HDAC8 inhibitors. However, strong chelating properties of hydroxamate group with the catalytic zinc ion of HDAC8 resulted in some demerits. Hence, in this current study, classification based chemoinformatic approaches including Bayesian modeling and recursive partitioning studies were conducted on a large and diverse set of 607 hydroxamates having less, very poor to high HDAC8 inhibitory properties. The main motto of this study is to identify and analyze the pivotal structural features of the cap and linker moieties required to obtain better HDAC8 inhibition. Moreover, a scrutiny of the HDAC8 crystal structure bound inhibitors was performed to correlate enzyme-inhibitor interactions with important molecular features resulted from these two classification-based models. The approach may be used to design novel HDAC8 inhibitors.Communicated by Ramaswamy Sarma

Twisted ladder-like donor-acceptor polymers as electrode materials for flexible electrochromic supercapacitors

Three ladder-like donor-acceptor (D-A) polymers PI-IDT, PI-BT, PI-DPP containing twisted spirobifluorene unit were synthesized and applied as electrochromic supercapacitor electrodes. By using density functional theory (DFT), we found that the polymer exhibited a distinct HOMO/LUMO wave function distribution when the linker moiety was electron-donors and electron-acceptors, respectively. Due to the introduction of the twisted unit, PI-IDT based on electron donor indacenodithiophene (IDT) unit as the linker moiety expressed a completely separated HOMO/LUMO wave function，and possessed independent p/n-dopable redox properties. With the separation of p-doped site and n-doped site, PI-IDT exhibited excellent performances in p-doping, n-doping and full cyclic scan including p-doping and n-doping processes. The area specific capacitance could reach 6.2 mF cm−2 during p-doping process, and 2.4 mF cm−2 during n-doping process. While the other two polymers PI-BT, PI-DPP based on the benzodithiazole (BT) and diketopyrrolopyrrole (DPP) as the linking unit, respectively, were characterized as that the p-doped sites and n-doped sites were located on one moiety, showing limited capacitance and poor stability. Besides, the selection of different linking units also directly affected the electrode-electrolyte interface contact, film-forming properties, electrochromic and electrochemical properties of the polymer. In addition, PI-IDT had a rich color change (orange, green, blue) and excellent coloring efficiency (155 cm2 C−1). The asymmetric supercapacitor PI-IDT//W0.71Mo0.21O3-x with PI-IDT as anode material showed discoloration from orange to blue, and delivered an energy density of 3.89 W h kg −1.

Neuraminidase-triggered activation of prodrug-type substrate of 4-nitroaniline

Artificial substrates for probing neuraminidase activity are powerful tools for studying the physiological and pathological roles of neuraminidases. Most of the substrates are α-O-linked sialosides involving hydroxyl-containing reporters for visualization, and neuraminidase-catalyzed cleavage of the sialic acid residues directly activates the reporters. However, the use of amine-containing reporters has been avoided because α-N-linked sialosides are marginal substrates for neuraminidases. To expand the applicability of reporters to amine-containing compounds, we have focused on prodrug design. Herein we describe the synthesis and enzymatic study of a model substrate involving 4-nitroaniline as an amine-containing chromogenic reporter. The substrate can respond to neuraminidase from Clostridium perfringens. Neuraminidase-mediated hydrolysis of the sialic acid moiety of the substrate initiates self-immolative elimination of the linker moiety, leading the liberation of yellow-colored reporter 4-nitroaniline. The elimination process involves generation of quinone methide intermediate, which causes to neutralize neuraminidase. The substrate, thus, works as not only a chromogenic substrate but also a suicide inactivator.

Tuning the Photonic Behavior of Symmetrical bis-BODIPY Architectures: The Key Role of the Spacer Moiety.

Herein we describe the synthesis, computationally assisted spectroscopy, and lasing properties of a new library of symmetric bridged bis-BODIPYs that differ in the nature of the spacer. Access to a series of BODIPY dimers is straightforward through synthetic modifications of the pending ortho-hydroxymethyl group of readily available C-8 (meso) ortho-hydroxymethyl phenyl BODIPYs. In this way, we have carried out the first systematic study of the photonic behavior of symmetric bridged bis-BODIPYs, which is effectively modulated by the length and/or stereoelectronic properties of the spacer unit. The designed bis-BODIPYs display bright fluorescence and laser emission in non-polar media. The fluorescence response is governed by the induction of a non-emissive intramolecular charge transfer (ICT) process, which is significantly enhanced in polar media. The effectiveness of the fluorescence quenching and also the prevailing charge transfer mechanism (from the spacer itself or between the BODIPY units) rely directly on the electron-releasing ability of the spacer. Moreover, the linker moiety can also promote intramolecular excitonic interactions, leading to excimer-like emission characterized by new spectral bands and the lengthening of lifetimes. The substantial influence of the bridging moiety on the emission behavior of these BODIPY dyads and their solvent-sensitivity highlight the intricate molecular dynamics upon excitation in multichromophoric systems. In this regard, the present work represents a breakthrough in the complex relationship between the molecular structure of the chromophores and their photophysical signatures, thus providing key guidelines for rationalizing the design of tailored bis-BODIPYs with potential advanced applications.

Biophysical Methods for Characterization of Antibody-Drug Conjugates.

Antibody-drug conjugates (ADC) are made up of three components: (1) a mAb specific to cells of choice, (2) a small molecule with desired end goal, and (3) a linker to covalently link drug molecule to the antibody. Bringing together the mAb, drug molecule, and the linker results in the formation of an immunoconjugate designed to selectively deliver the drug molecule to a cell of interest. Synergic effects of the mAb and drug molecule lead to destroying the target tumor cells while leaving the normal cells unharmed. However, the development of ADCs is associated with challenges due to the heterogeneity of the ADC molecules created from the conjugation process. Addition of the linker and drug moieties during processing as well as the hydrophobicity of the drug itself can lead to structural changes that may affect the stability and functional profile of the conjugated molecule. Furthermore, linkers site of attachment plays a major role in determining the conformational and colloidal properties of the ADCs. In this chapter, several characterization methods are introduced to determine the biophysical characteristics of the ADC. Protocols, data analysis as well as notes for circular dichroism, intrinsic fluorescence, ANS fluorescence, differential scanning calorimetry, and dynamic scanning fluorimetry are outlined in detail.

Synthesis of Monovalent N-Acetylgalactosamine Phosphoramidite for Liver-Targeting Oligonucleotides.

Ligand-targeted drug delivery (LTDD) has emerged as an attractive option in the field of oligonucleotide drugs following the great success of N-acetylgalactosamine (GalNAc)-conjugated siRNA and antisense oligonucleotides. GalNAc is a well-known ligand of the asialoglycoprotein receptor (ASGPR), and is classified as a C-type lectin associated with the metabolism of desialylated glycoproteins. This article describes the synthesis of a non-nucleosidic monovalent GalNAc phosphoramidite-a useful reagent for facilitating the conjugation of GalNAc epitopes into oligonucleotides using DNA synthesizers-together with some important caveats. The monomeric GalNAc consists of three parts: (1) a GalNAc moiety, (2) a linker moiety, and (3) a trans-4-hydroxyprolinol (tHP) branch point. The GalNAc moiety and the tHP moiety are coupled via a condensation reaction to prepare the monovalent GalNAc phosphoramidite. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of N-acetylgalactosamine ligand Basic Protocol 2: Preparation of trans-4-hydroxyprolinol building block Basic Protocol 3: Preparation of GalNAc phosphoramidite.

Effect of the bridge structure on the binding mode of the binuclear ruthenium complex to native DNA

Ruthenium(II) polypyridyl dimer containing large planar aromatic bridging ligands [μ-(tpphz)(phen)4Ru2]4+ (phen = 1,10-phenanthroline, tpphz = tetrapyrido[3,2-a:2′,3′-c:3″, 2″-h:2‴, 3‴-j]phenazine, bis-Ru-tpphz) were synthesized and characterized by elemental analysis, and 1H nuclear magnetic spectroscopy. The binding mode of the synthesized bis-Ru-tpphz to DNA was compared with those of the bis-Ru-bip, and bis-Ru-tatpp complexes. The structures of the connecting bridge of bis-Ru complexes were different. The absorption and reduced linear dichroism spectrum showed that the planar linker moiety of the bis-Ru-tatpp complex is inserted between the DNA base-pairs despite the steric hindrance originating from its large ruthenium ligand part, whereas the bis-Ru-bip complex has flexible linker binds at the groove of the DNA. The binding mode of the bis-Ru-tpphz complex, which has fused shorter bridge ligands than the bis-Ru-tatpp complex, was in contrast with the other two complexes. The bis-Ru-tpphz complex binds either at the groove of the DNA or to the negatively charged phosphate group; intercalative binding was prevented by either short bridge structure or steric hindrances from the phenanthroline ligand. In conclusion, the structure of the bridge of the bis-Ru(II) complex plays an important role in determining the binding mode of these compounds to DNA. To have the mode of insertion, the bridge ligand structure must be fused and have a sufficiently long distance.

Structure based design of macrocyclic factor XIa inhibitors: Discovery of cyclic P1 linker moieties with improved oral bioavailability.

This manuscript describes the discovery of a series of macrocyclic inhibitors of FXIa with oral bioavailability. Assisted by structure based drug design and ligand bound X-ray crystal structures, the group linking the P1 moiety to the macrocyclic core was modified with the goal of reducing H-bond donors to improve pharmacokinetic performance versus 9. This effort resulted in the discovery of several cyclic P1 linkers, exemplified by 10, that are constrained mimics of the bioactive conformation displayed by the acrylamide linker of 9. These cyclic P1 linkers demonstrated enhanced bioavailability and improved potency.

Design, synthesis, biological evaluation and molecular docking study of arylcarboxamido piperidine and piperazine-based hydroxamates as potential HDAC8 inhibitors with promising anticancer activity

HDAC8 has been established as one of the vital targets as far as the cancer is concerned. Different compounds having potential HDAC inhibitory activity have been approved by USFDA. However, none of these compounds are selective towards specific HDAC isoform. In this current study, some new hydroxamate derivatives with alkylpiperidine and alkylpiperazine linker moieties have been designed, synthesized and biologically evaluated. All these compounds are effective HDAC8 inhibitors comprising more or less similar cytotoxic potential against different cancer cell lines. It is observed that the piperazine scaffold containing compound is more active than the compound with piperidine scaffold for exerting HDAC8 inhibitory activity. Moreover, the 4-quinolyl cap group is better than the biphenyl group which is better than the benzyl group for producing higher HDAC8 inhibition as well as cytotoxicity. These compounds displayed selective HDAC8 inhibition over HDAC3. Moreover, these compounds showed an increased caspase3/7 activity suggesting their anticancer potential through modulation of apoptotic pathways. Molecular docking study with three potent compounds was performed with both HDAC3 and HDAC8 enzymes to understand the selectivity profile of these compounds. Compound containing 4-quinolyl cap group with alkyl piperazinyl urea linker moiety has been emerged out as the lead molecule that may be further modified to design more effective and selective HDAC8 inhibitors in future.

Sulfonamide-Based Inhibitors of Biotin Protein Ligase as New Antibiotic Leads.

Here, we report the design, synthesis, and evaluation of a series of inhibitors of Staphylococcus aureus BPL (SaBPL), where the central acyl phosphate of the natural intermediate biotinyl-5'-AMP (1) is replaced by a sulfonamide isostere. Acylsulfamide (6) and amino sulfonylurea (7) showed potent in vitro inhibitory activity (Ki = 0.007 ± 0.003 and 0.065 ± 0.03 μM, respectively) and antibacterial activity against S. aureus ATCC49775 with minimum inhibitory concentrations of 0.25 and 4 μg/mL, respectively. Additionally, the bimolecular interactions between the BPL and inhibitors 6 and 7 were defined by X-ray crystallography and molecular dynamics simulations. The high acidity of the sulfonamide linkers of 6 and 7 likely contributes to the enhanced in vitro inhibitory activities by promoting interaction with SaBPL Lys187. Analogues with alkylsulfamide (8), β-ketosulfonamide (9), and β-hydroxysulfonamide (10) isosteres were devoid of significant activity. Binding free energy estimation using computational methods suggests deprotonated 6 and 7 to be the best binders, which is consistent with enzyme assay results. Compound 6 was unstable in whole blood, leading to poor pharmacokinetics. Importantly, 7 has a vastly improved pharmacokinetic profile compared to that of 6 presumably due to the enhanced metabolic stability of the sulfonamide linker moiety.

Hydrazide Derivatives of Luminol for Chemiluminescence-Labelling of Macromolecules.

A series of chemiluminescent compounds containing a hydrazide group as a nucleophilic functional group has been synthesized. The syntheses were started from chemiluminescent luminol and isoluminol. The linker moiety was easily introduced onto non-nucleophilic exocyclic amino groups of luminol and isoluminol by gentle heating with cyclic acid anhydrides such as glutaric anhydride. The resulting carboxy group was converted to hydrazide by a simple condensation reaction using carbodiimide. Although majority of the synthesized compounds did not emit strong light, a sufficient chemiluminescence intensity was obtained from luminol-amido-C2-hydrazide (L2H) comprising of luminol scaffold with a dimethylene linker. The ability of L2H to form a covalent bond with a macromolecule was further investigated by incubation with oxidized horseradish peroxidase. The analysis on matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) MS revealed that the coupling efficiency of L2H was similar to that of commercially available labelling reagent having a hydrazide group. These results suggested that L2H, the luminol hydrazide containing a dimethylene linker, could be useful for the labelling of macromolecules in the sensitive bioassay such as chemiluminescence immunoassay.

Structural and functional analysis of an l‐serine O‐phosphate decarboxylase involved in norcobamide biosynthesis

Structural diversity of natural cobamides (Cbas, B12 vitamers) is limited to the nucleotide loop. The loop is connected to the cobalt-containing corrin ring via an (R)-1-aminopropan-2-ol O-2-phosphate (AP-P) linker moiety. AP-P is produced by the l-threonine O-3-phosphate (l-Thr-P) decarboxylase CobD. Here, the CobD homolog SMUL_1544 of the organohalide-respiring epsilonproteobacterium Sulfurospirillummultivorans was characterized as a decarboxylase that produces ethanolamine O-phosphate (EA-P) from l-serine O-phosphate (l-Ser-P). EA-P is assumed to serve as precursor of the linker moiety of norcobamides that function as cofactors in the respiratory reductive dehalogenase. SMUL_1544 (SmCobD) is a pyridoxal-5'-phosphate (PLP)-containing enzyme. The structural analysis of the SmCobD apoprotein combined with the characterization of truncated mutant proteins uncovered a role of the SmCobD N-terminus in efficient l-Ser-P conversion.