113Cd NMR Research Articles

HOST-GUEST INTERACTIONS FACILITATED BY CHALCOGEN BONDING WITHIN SELENADIAZOLE FUNCTIONALISED PORPHYRIN NANOTUBES.

The structural rigidity of tetrakis(4-pyridyl)porphyrin (TPyP) has been utilised to prepare a robust novel porous coordination polymer of composition Cd2(TPyP)(sez)2 (TPyP = 5,10,15,20-tetra(4-pyridyl)porphyrin, sez = 1,2,5-benzoselenadiazole-5-carboxylate). The coordination polymer may be described as a hexagonal porphyrin nanotube (PNT) and has the potential to bind guest molecules through chalcogen bonding. Single crystal X-ray diffraction (SCXRD) data indicate an internal pore diameter ~9 Å which represents ~35% of the crystal volume. Immersion of the PNTs in solvents such as DMSO and CS2 result in the incorporation of these molecules within the nanotubes with chalcogen bonding between host and guest. The crystallographic guest-inclusion investigations are complemented by solid-state 77Se, 13C, 113Cd and 2H NMR studies which provide insights into dynamic behaviour. The porosity of the crystals was further explored using gas adsorption experiments, indicating the reversible uptake of CO2, CH4, H2 and N2. Structure-function relationships are clearly established from complementary crystallographic, NMR and adsorption investigations.

Diphosphonic ionic liquids as anion-exchangers for palladium(II) and platinum(IV): Synthesis, complexation and selective extraction from hydrochloric solutions

The demand for platinum group metals (PGMs) continues to grow. To reduce the negative environmental impact, it is necessary to switch to secondary sources of PGMs and to improve the separation and purification processes of these metals. In this work, new phosphonium-based ionic liquids (diphosphonium salts) were synthesized as extractants for the selective recovery of palladium(II) and platinum(IV) from hydrochloric acid solutions. Substances synthesized contain a doubly-charged cation, which significantly affects the selectivity of extraction compared to singly charged phosphonium extractants. It was found that diphosphonium salts have a high extraction efficiency of metallic ions that form stable doubly-charged chloride anionic complexes. Thus, solutions of synthesized ionic liquids in 3-nitrobenzotrifluoride can selectively extract palladium(II) and platinum(IV) in the presence of all d‐metal ions studied, which are contained in PGMs secondary sources. The extraction efficiency under optimal conditions is 99.6 % and 98.5 % for palladium(II) and platinum(IV), respectively. The extraction mechanism has been investigated by X-ray diffraction, DFT calculations and 31P, 111Cd NMR spectroscopy. Extraction proceeds through the anion-exchange mechanism, however, in the case of the studied compounds, the significant role of bromide as a counterion of ligands was shown. It was found that bromide is likely included in the extracted ion pair, displacing chloride ions from anionic metal complexes during extraction.

Rapid Quantification of Protein Secondary Structure Composition from a Single Unassigned 1D 13C Nuclear Magnetic Resonance Spectrum.

The function of a protein is predicated upon its three-dimensional fold. Representing its complex structure as a series of repeating secondary structural elements is one of the most useful ways by which we study, characterize, and visualize a protein. Consequently, experimental methods that quantify the secondary structure content allow us to connect a protein's structure to its function. Here, we introduce an automated gradient descent-based method we refer to as secondary-structure distribution by NMR that allows for rapid quantification of the protein secondary structure composition of a protein from a single, 1D 13C NMR spectrum without chemical shift assignments. The analysis of nearly 900 proteins with known structure and chemical shifts demonstrates the capabilities of our approach. We show that these results rival alternative techniques such as FT-IR and circular dichroism that are commonly used to estimate secondary structure compositions. The resulting method requires only the primary sequence of the protein and its referenced 13C NMR spectrum. Each residue is modeled in an ensemble of secondary structures with percentage contributions from random coil, α-helix, and β-sheet secondary structures obtained by minimizing the difference between a simulated and experimental 1D 13C NMR spectrum. The capabilities of the method are demonstrated as applied to samples at natural abundance or enriched in 13C, acquired by either solution or solid-state NMR, and even on low magnetic field benchtop NMR spectrometers. This approach allows for rapid characterization of protein secondary structure across traditionally challenging to characterize states including liquid-liquid phase-separated, membrane-bound, or aggregated states.

Identification by HSQC and quantification by qHNMR innovate pharmaceutical amino acid analysis

This study introduces a new NMR-based methodology for identification (ID) and quantification (purity, strength) assays of widely used amino acids. A detailed analysis of four amino acids and their available salts was performed with both a high-field (600 MHz) and a benchtop (60 MHz) NMR instrument. To assess sensitivity constraints, samples for 1H NMR analysis were initially prepared using only 10 mg of analyte and 1 mg of maleic acid (MA) as an internal calibrant (IC) and secondary chemical shift reference. The characteristic dispersion of the peak patterns indicating the presence or absence of a counterion (mostly chloride) was conserved at both high and low-field strength instruments, showing that the underlying NMR spectroscopic parameters, i.e., chemical shifts and coupling constants, are independent of the magnetic field strength. However, as the verbal descriptions of 1H NMR spectra are challenging in the context of reference materials and pharmaceutical monographs, an alternative method for the identification (ID) of amino acids is proposed that uses 13C NMR patterns from multiplicity-edited HSQC (ed-HSQC), which are both compound-specific and straightforward to document. For ed-HSQC measurements, the sample amount was increased to 30 mg of the analyte and several acquisition parameters were tested, including t1 increments used in the pulse program, number of scans, and repetition time. Excellent congruence with deviations <0.1 ppm was achieved for the 13C chemical shifts from 1D 13C NMR spectra (150 MHz) vs. those extracted from ed-HSQC (15 MHz traces). Finally, all samples of amino acid candidate reference materials were quantified by 1H qNMR (abs-qHNMR) at both 600 and 60 MHz. At high field, both IC and relative quantitations were performed, however, with the low-field instrument, only the IC method was used. The results showed that the analyzed reference material candidates were generally highly pure compounds. To achieve adequately low levels of uncertainty for such high-purity materials, the sample amounts were increased to 100 mg of analytes and 10 mg of the IC and replicates were analyzed for selected amino acids.

Development of a Simple Cost Effective Oxygenation System for In Vivo Solution State NMR in 10 mm NMR Tubes.

In vivo NMR is evolving into an important tool to understand biological processes and environmental responses. Current approaches use flow systems to sustain the organisms with oxygenated water and food (e.g., algae) inside the NMR. However, such systems have the potential to leak and clog (potentially damaging costly hardware), require large volumes of media, and multiple expensive HPLC pumps. The proposed "oxygenation system", uses a simple "double slit" adapter and a single air/oxygen flow line into the NMR. The design is especially suited to larger diameter probes given that standard flow systems would require higher flow rates thus amplifying the potential and impact of leaks/clogs. Traditionally, in vivo NMR of small organisms (e.g., Daphnia) have required 2D NMR in combination with 13C enrichment to overcome susceptibility distortions and provide information rich metabolic profiles. Here Daphnia magna, Eisenia fetida and Artemia franciscana are used to demonstrate the potential of the oxygenation system. Survivability tests and 1H time-resolved monitoring were first performed on D. magna, while E. fetida contained enough biomass to permit 1H-13C HSQC, 13C-1H HETCOR and 31P NMR without isotopic enrichment. Finally, STOCSY of 1D 13C NMR was used to follow the growth of A. franciscana (without 13C enrichment) for 48 h after birth, which helps visualize trends across a series of 1D in vivo data. In summary, application of the oxygenation system toward larger diameter probes allows the collection of NMR data without enrichment, offering a promising solution to better understand processes in vivo.

A Chiral [2+3] Covalent Organic Cage Based on 1,1'-Bi-2-naphthol (BINOL) Units.

A [2+3] chiral covalent organic cage is produced through a dynamic covalent chemistry approach by mixing two readily available building units, viz. an enantiopure 3,3'-diformyl 2,2'-BINOL compound (A) with a triamino spacer (B). The two enantiomeric (R,R,R) and (S,S,S) forms of the cage C are formed nearly quantitatively thanks to the reversibility of the imine linkage. The X-ray diffraction analysis of cage (S,S,S)-C highlights that the six OH functions of the BINOL fragments are positioned inside the cage cavity. Upon reduction of the imine bonds of cage C, the amine cage D is obtained. The ability of the cage D to host the 1-phenylethylammonium cation (EH+) as a guest is evaluated through UV, CD and DOSY NMR studies. A higher binding constant for (R)-EH+ cation (Ka=1.7 106±10 % M-1) related to (S)-EH+ (Ka=0.9 106±10 % M-1) is determined in the presence of the (R,R,R)-D cage. This enantiopreference is in close agreement with molecular dynamics simulation.

Efficient Access to Elusive 1D 13C NMR Spectra through Highly Resolved 1H, 13C-Long-Range Correlation Spectroscopy.

A method for obtaining 1D 13C NMR spectra from natural products or metabolites using proton detection is described. The approach delivers singlets for every 13C signal without conducting any broadband 1H decoupling (CPD) and is based on calculating 13C projections from constant-time HMBC and conventional HSQC experiments, recorded at high digital resolution and processed to pure phases. Paramount to the proposed method is the implication of nonuniform sampling and echo processing. The echo processing produces phase-sensitive 2D CT-HMBC spectra with narrow 13C signal line shapes. Two simple HMBC pulse sequences are utilized with the suppression of homo- and heteronuclear couplings. Due to the removal of the 1H multiplet structure in F1 (no tilt at higher digital resolution), 13C singlets arise. An overall increase in 13C signal-to-noise (SINO) for all types of carbon multiplicities is observed, making the proposed technique superior compared to direct 13C excitation. For otherwise difficult-to-measure quaternary carbon atoms, a SINO enhancement of up to 6 and 12 depending on F1 resolution (3 and 6 Hz/point) is reported. Echo/anti-Echo signal detection cleans up the spectrum. Nonuniform sampling (NUS) lays the groundwork to significantly reduce the total acquisition time. Final 1D 13C projections are obtained by combining the 13C projection from CT HMBC and conventional HSQC. This orthogonal concept of combining the 13C projections from different spectra inherently minimizes the risk of missing 13C cross-peaks by inappropriate setting of long-range nJHC coupling delays and the shortcoming of T2 relaxations. The advantages and some limitations of the concept are discussed.

The Scope of the Applicability of Non-relativistic DFT Calculations of NMR Chemical Shifts in Pyridine-Metal Complexes for Applied Applications.

Heavy metals are toxic, but it is impossible to stop using them. Considering the variety of molecular systems in which they can be present, the multicomponent nature and disorder of the structure of such systems, one of the most effective methods for studying them is NMR spectroscopy. This determines the need to calculate NMR chemical shifts for expected model systems. For elements beyond the third row of the periodic table, corrections for relativistic effects are necessary when calculating NMR parameters. Such corrections may be necessary even for light atoms due to the shielding effect of a neighboring heavy atom. This work examines the extent to which non-relativistic DFT calculations are able to reproduce experimental 15N and 113Cd NMR chemical shift tensors in pyridine-metal coordination complexes. It is shown that while for the calculation of 15N NMR chemical shift tensors there is no real need to consider relativistic corrections, for 113Cd, on the contrary, none of the tested calculation methods could reproduce the experimentally obtained tensor to any extent correctly.

Reactions of Cadmium(II) Halides and Di-2-Pyridyl Ketone Oxime: One-Dimensional Coordination Polymers.

The coordination chemistry of 2-pyridyl ketoximes continues to attract the interest of many inorganic chemistry groups around the world for a variety of reasons. Cadmium(II) complexes of such ligands have provided models of solvent extraction of this toxic metal ion from aqueous environments using 2-pyridyl ketoxime extractants. Di-2-pyridyl ketone oxime (dpkoxH) is a unique member of this family of ligands because its substituent on the oxime carbon bears another potential donor site, i.e., a second 2-pyridyl group. The goal of this study was to investigate the reactions of cadmium(II) halides and dpkoxH in order to assess the structural role (if any) of the halogeno ligand and compare the products with their zinc(II) analogs. The synthetic studies provided access to complexes {[CdCl2(dpkoxH)∙2H2O]}n (1∙2H2O), {[CdBr2(dpkoxH)]}n (2) and {[CdI2(dpkoxH)]}n (3) in 50-60% yields. The structures of the complexes were determined by single-crystal X-ray crystallography. The compounds consist of structurally similar 1D zigzag chains, but only 2 and 3 are strictly isomorphous. Neighboring CdII atoms are alternately doubly bridged by halogeno and dpkoxH ligands, the latter adopting the η1:η1:η1:μ (or 2.0111 using Harris notation) coordination mode. A terminal halogeno group completes distorted octahedral coordination at each metal ion, and the coordination sphere of the CdII atoms is {CdII(η1 - X)(μ - X)2(Npyridyl)2(Noxime)} (X = Cl, Br, I). The trans-donor-atom pairs in 1∙2H2O are Clterminal/Noxime and two Clbridging/Npyridyl; on the contrary, these donor-atom pairs are Xterminal/Npyridyl, Xbridging/Noxime, and Xbridging/Npyridyl (X = Br, I). There are intrachain H-bonding interactions in the structures. The packing of the chains in 1∙2H2O is achieved via π-π stacking interactions, while the 3D architecture of the isomorphous 2 and 3 is built via C-H∙∙∙Cg (Cg is the centroid of one pyridyl ring) and π-π overlaps. The molecular structures of 1∙2H2O and 2 are different compared with their [ZnX2(dpkoxH)] (X = Cl, Br) analogs. The polymeric compounds were characterized by IR and Raman spectroscopies in the solid state, and the data were interpreted in terms of the known molecular structures. The solid-state structures of the complexes are not retained in DMSO, as proven via NMR (1H, 13C, and 113Cd NMR) spectroscopy and molar conductivity data. The complexes completely release the coordinated dpkoxH molecule, and the dominant species in solution seem to be [Cd(DMSO)6]2+ in the case of the chloro and bromo complexes and [CdI2(DMSO)4].

Quantitative analysis of the β-1 structure in lignin by administration of [ring-1-13C]coniferin

Abstract Lignin dimeric units are characterized by various inter-unit linkage types such as β-O-4, β-5, β-β, and β-1. Spirodienones are the native form of the β-1 structures, but the content in lignin has not been clarified. In this study, the ring-1-13C labeled coniferin was synthesized and administered to Ginkgo biloba shoots, obtaining ring-1 selectively labeled xylem samples. Enzymatically saccharified lignin (EL) samples were prepared from the xylem sample (400–600 µm distant region from the cambial zone), and solution-state quantitative 13C NMR and solid-state CP/MAS NMR measurements were conducted. Acetylated EL (ELAc) was also prepared from the xylem sample (600–800 µm distant region from the cambial zone), and solution-state quantitative 13C NMR and 1H–13C 2D NMR measurements were conducted. Difference spectra obtained by subtracting the unlabeled spectra from the ring-1 labeled spectra showed that the ring-1 was responsible for broad signals at 134 ppm and signals of the spirodienone structure at 56.11 ppm (in solid-state), 54.70 ppm (EL in solution-state), and 54.72 ppm (ELAc in solution-state). The ratio of spirodienone structure was evaluated as 0.68 % (EL) and 0.72 % (ELAc) by the solution-state quantitative 13C difference spectra, and 2.3 % (ELAc) by HSQC volume ratio of Cα′-H to G2-H.

NMR analysis of structural geometry and molecular dynamics in perovskite-type N(CH3)4CdBr3 crystal near high-temperature phase transition.

The NMR chemical shifts, linewidths, spin-lattice relaxation times in the rotating system T1ρ, and spin-lattice relaxation times in the laboratory system T1 were evaluated for the perovskite-type N(CH3)4CdBr3 crystal, aiming to understand the changes in the structural geometry and molecular dynamics from phase I to phase II. From the temperature-dependence of the 1H, 13C, 14N, and 113Cd NMR chemical shifts, the structural geometry underwent a continuous change, without anomalous changes around (TC = 390 K). However, the linewidths in phase I were narrower than those in phase II, indicating that the motional averaging effects were caused by the rapid rotation of the N(CH3)4 group. Sudden changes in T1 and T1ρ were observed near TC, for which the activation energy Ea in phase I was approximately 12 times larger than that in phase II; the small Ea values in phase II indicate a large degree of freedom for the methyl group and CdBr6 octahedra, whereas the large Ea in phase I was primarily attributed to the overall N(CH3)4 and the 113Cd in the CdBr6 groups. Consequently, the phase transition mechanisms of N(CH3)4CdBr3 are related to reorientation of the N(CH3)4 group and the arrangement of the CdBr6 groups.

Surface Sites and Ligation in Amine‐capped CdSe Nanocrystals**

AbstractConverting colloidal nanocrystals (NCs) into devices for various applications is facilitated by designing and controlling their surface properties. One key strategy for tailoring surface properties is thus to choose tailored surface ligands. In that context, amines have been universally used, with the goal to improve NCs synthesis, processing and performances. However, understanding the nature of surface sites in amine‐capped NCs remains challenging, due to the complex surface compositions as well as surface ligands dynamic. Here, we investigate both surface sites and amine ligation in CdSe NCs by combining advanced NMR spectroscopy and computational modelling. Notably, dynamic nuclear polarization (DNP) enhanced 113Cd and 77Se 1D NMR helps to identify both bulk and surface sites of NCs, while 113Cd 2D NMR spectroscopy enables to resolve amines terminated sites on both Se‐rich and nonpolar surfaces. In addition to directly bonding to surface sites, amines are shown to also interact through hydrogen‐bonding with absorbed water as revealed by 15N NMR, augmented with computations. The characterization methodology developed for this work provides unique molecular‐level insight into the surface sites of a range of amine‐capped CdSe NCs, and paves the way to identify structure‐function relationships and rational approaches towards colloidal NCs with tailored properties.

Surface Sites and Ligation in Amine-capped CdSe Nanocrystals.

Converting colloidal nanocrystals (NCs) into devices for various applications is facilitated by designing and controlling their surface properties. One key strategy for tailoring surface properties is thus to choose tailored surface ligands. In that context, amines have been universally used, with the goal to improve NCs synthesis, processing and performances. However, understanding the nature of surface sites in amine-capped NCs remains challenging, due to the complex surface compositions as well as surface ligands dynamic. Here, we investigate both surface sites and amine ligation in CdSe NCs by combining advanced NMR spectroscopy and computational modelling. Notably, dynamic nuclear polarization (DNP) enhanced 113 Cd and 77 Se 1D NMR helps to identify both bulk and surface sites of NCs, while 113 Cd 2D NMR spectroscopy enables to resolve amines terminated sites on both Se-rich and nonpolar surfaces. In addition to directly bonding to surface sites, amines are shown to also interact through hydrogen-bonding with absorbed water as revealed by 15 N NMR, augmented with computations. The characterization methodology developed for this work provides unique molecular-level insight into the surface sites of a range of amine-capped CdSe NCs, and paves the way to identify structure-function relationships and rational approaches towards colloidal NCs with tailored properties.

Seventeen undescribed iridoid derivatives with anti-inflammatory effects from Hedyotis diffusa and their structure-activity relationships

Seventeen undescribed iridoid derivatives (1–17) and four known compounds (18–21) were isolated from the whole plant of Hedyotis diffusa Willd. Their structures were elucidated based on unambiguous spectroscopic data (UV, IR, HRESIMS, CD, and 1D and 2D NMR). It is noteworthy that compounds 1–8, which possess unique long-chain aliphatic acid moiety, were reported for the first time among the iridoid natural products. All compounds were evaluated for their anti-inflammatory activities in lipopolysaccharide-induced RAW 264.7 cells. Compounds 2, 4, and 6 showed significant suppression effects on nitric oxide production, with IC50 values of 5.69, 6.16, and 6.84 μM, respectively. The structure-activity relationships of these compounds indicated that long-chain aliphatic moieties at C-10 might be the key group for their anti-inflammatory activities. The therapeutic properties of these iridoid derivatives could give an insight into utilizing H. diffusa as a natural source of anti-inflammatory agents.

Supermetalation of Cd-MT3 beyond the two-domain model

The flexibility of mammalian metallothioneins (MTs) has contributed to the difficulty in obtaining structural information for this family of metalloproteins that bind divalent metals with its twenty cysteines. While the two-domain structure for Cd7MT is well-established as a Cd4S11 and Cd3S9, a third structure has been reported when 8 Cd(II) ions bind to MT1. Isoform 3 of the MT family, MT3, has been of interest to the research community since its isolation as a growth inhibitory factor isolated in brain tissue, and has since been noted as a prominent participant in the mediation of neurodegenerative diseases and regular brain development. The differences between MT3 and the other isoforms of MT include an additional hexapeptide insertion of acidic residues in the α domain as well as the introduction of two prolines in the β domain. It is unclear whether these changes impact the metalation properties of MT3. We report the formation of a Cd8MT3 species is characterized by electrospray ionization mass spectrometry and UV–visible absorption spectroscopy. We report that the spectroscopic properties of this supermetalated Cd8MT3 are similar to those of the supermetalated Cd8MT1, with a clear indication of changes in structure from “fully-metalated” Cd7MT3 to supermetalated Cd8MT3 from circular dichroism spectra and both 1D 113Cd and 2D 1H-113Cd HSQC NMR spectra. We conclude that the metalation properties are not impacted significantly due to the amino acid changes in MT3, and that the cysteinyl thiols are the key players in determining the capacity of metal-binding and the structure of metal-thiolate clusters.

Metabolites analysis and new bioactive pigments from a Monascus purpureus strain with low citrinin

A Monascus purpureus strain with high mycelial biomass and no detected citrinin in liquid fermentation was selected from 20 strains. HPLC-DAD-HRMS analysis revealed that the EtOAc extract of the fermented mash contained 2 unknown compounds and 11 known metabolites of Monascus fungi. Isolations with preparative and semi-preparative HPLC resulted in four brownish red pigments. The two unknown pigments were identified with HRMS, CD, ECD, 1D and 2D NMR, and designated as monascuskaolin A (3) and B (8). The two known compounds were verified with 13C NMR as monascusazaphilone C and monasfluol B. Result of bioactivity tests showed that the two new pigments were able to significantly reduce the transcription levels of IL-1β, TNF-α, and TGF-β, and significantly promote the proliferation of 293 T cells, suggesting that they have anti-inflammatory function and may be good for human kidney. Metabolites studies suggested that M. purpureus RCEF7056 is a safe strain with functional food utilization potential.

Synthesis, X-ray Diffraction, NMR and Thermolysis Studies of Cadmium Tin Sulfido Complexes

Three molecular sulfido complexes containing a Cd-S-Sn structure motive, [{(tmeda)Cd}3(SnPh)2S6] (1), [{(tmeda)Cd}2(SnPh2)2S4] (2) and [{(iPr3P)Cd}3Cd4(SnPh2)6S13] (3), have been synthesized and characterized by single-crystal X-ray diffraction. So far, 2 is the first cadmium tin sulfido complex soluble in common organic solvents. Analysis by 113Cd and 119Sn NMR spectroscopy shows a slow rearrangement in solution, while spin coupling between the nuclei can be used to identify the species in solution. Thermolysis of 2 results in CdS and SnS as solid thermolysis products.

Investigating the physicochemical properties, structural attributes, and molecular dynamics of organic–inorganic hybrid [NH3(CH2)2NH3]2CdBr4·2Br crystals

An in-depth understanding of the physicochemical properties of the organic–inorganic hybrid [NH3(CH2)2NH3]2CdBr6 whose structure corresponds to the formulation [NH3(CH2)2NH3]2CdBr4· 2Br is essential for its application in batteries, supercapacitors, and fuel cells. Therefore, this study aimed to determine the crystal structure, phase transition, structural geometry, and molecular dynamics of these complexes. Considering its importance, a single crystal of [NH3(CH2)2NH3]2CdBr6 was grown; the crystal structure was found to be monoclinic. The phase transition temperatures were determined to be 443, 487, 517, and 529 K, and the crystal was thermally stable up to 580 K. Furthermore, the 1H, 13C, 14N, and 113Cd NMR chemical shifts caused by the local field surrounding the resonating nucleus of the cation and anion varied with increasing temperature, along with the surrounding environments of their atoms. In addition, 1H spin–lattice relaxation time T1ρ and 13C T1ρ, which represent the energy transfer around the 1H and 13C atoms of the cation, respectively, varied significantly with temperature. Consequently, changes in the coordination geometry of Br around Cd in the CdBr6 anion and the coordination environment around N (in the cation) were associated with changes in the N–H···Br hydrogen bond. The structural geometry revealed critical information regarding their basic mechanism of organic–inorganic hybrid compounds.

Structural Characterization, Cytotoxicity, and the Antifungal Mechanism of a Novel Peptide Extracted from Garlic (Allium sativa L.)

Garlic (Allium sativa L.) is a traditional plant with antimicrobial activity. This study aimed to discover new antifungal peptides from garlic, identify their structure, and explore the antimicrobial mechanism. Peptides were separated by chromatography and identified by MALDI-TOF analysis. Structure and conformation were characterized by CD spectrum and NMR analysis. Mechanism studies were conducted by SEM, membrane depolarization, and transcriptomic analysis. The cytotoxicity to mammalian cells as well as drug resistance development ability were also evaluated. A novel antifungal peptide named NpRS with nine amino acids (RSLNLLMFR) was obtained. It was a kind of cationic peptide with a α-helix as the dominant conformation. NOESY correlation revealed a cyclization in the molecule. The peptide significantly inhibited the growth of Candida albicans. The mechanism study indicated that membrane destruction and the interference of ribosome-related pathways might be the main mechanisms of antifungal effects. In addition, the resistance gene CDR1 for azole was down-regulated and the drug resistance was hardly developed in 21 days by the serial passage study. The present study identified a novel antifungal garlic peptide with low toxicity and provided new mechanism information for the peptide at the gene expression level to counter drug resistance.

Structurally diverse alkaloids with nine frameworks from Zephyranthes candida and their acetylcholinesterase inhibitory and anti-inflammatory activities

Twenty-six structurally diverse Amaryllidaceae alkaloids, including ten undescribed compounds named zephyranines A–I and 6-O-ethylnerinine, two undescribed natural products zephyranthine-6-one and 3-O-deacetyl-sternbergine, were isolated from whole plants of Zephyranthes candida. Their structures were determined by HRESIMS, 1D and 2D NMR, CD data analysis, NMR and ECD calculations, and single-crystal X-ray diffraction analysis. All structures were classified into nine framework types: 10b,11-seco-crinine, graciline, crinine, homolycorine, trisphaeridine, lycorine, galasine, tazettine, and belladine. Zephyranine A represents the first naturally occurring 10b,11-seco-crinine type alkaloid, and zephyranine B is the sixth graciline type alkaloid. 6-O-ethylnerinine is an artifact from the extraction and isolation. All isolates were evaluated for their acetylcholinesterase (AChE) inhibitory and anti-inflammatory activities. Zephyranines A, G, and H exhibited moderate AChE inhibitory activities, with IC50 values of 8.2, 39.0, and 10.8 μM, respectively. Zephyranine B, haemanthamine, haemanthidine, 11-hydroxyvittatine, and 8-demethoxy-10-O-methylhostasine exhibited potent anti-inflammatory activity on the LPS-induced NO production in RAW264.7 mouse macrophages with IC50 values of 21.3, 4.6, 12.2, 5.6, and 17.4 μM, respectively. Structure-activity-relationship analysis and docking studies indicated that interactions with the key Trp286 and Tyr337 residues are required for potent AChE inhibitors.