Structures of N-Hydroxy-Type Tetrodotoxin Analogues and Bicyclic Guanidinium Compounds Found in Toxic Newts.

The biosynthesis of tetrodotoxin (TTX, 1), a potent neurotoxin widely distributed in marine and terrestrial metazoans, remains unresolved. A significant issue has been identifying intermediates and shunt products associated with the biosynthetic pathway of TTX. We investigated TTX biosynthesis by screening and identifying new TTX-related compounds from Cynops ensicauda popei and Taricha granulosa. Mass spectrometry (MS)-guided screening identified two new N-hydroxy TTX analogues in newts: 1-hydroxy-8-epiTTX (2) and 1-hydroxy-8-epi-5,11-dideoxyTTX (3, previously reported as 1-hydroxy-5,11-dideoxyTTX). We prepared a new analogue, 8-epi-5,11-dideoxyTTX (4), from 3 via N-OH reduction and confirmed the presence of 4 in T. granulosa using hydrophilic interaction liquid chromatography (HILIC)-LCMS. The presence of 8-epi-type TTX analogues in both Cynops and Taricha supports a branched biosynthetic pathway of terrestrial TTX, which produces 6- and 8-epimers. In addition, new bicyclic guanidinium compounds Tgr-238 (5) and Tgr-240 (6) were identified as putative shunt products of our proposed TTX biosynthesis pathway. A structural analysis of Cep-228A (7), another bicyclic compound, was performed using NMR. Based on the structures of 5-7 and their analogues, we propose a model of the shunt and metabolic pathways of the terrestrial TTX biosynthesis.

Virus and chlorine adsorption onto guanidine modified cellulose nanofibers using covalent and hydrogen bonding.

Unsafe drinking water leads to millions of human deaths each year, while contaminated wastewater discharges are a significant threat to aquatic life. To relieve the burden of unsafe water, we are in search of an inexpensive material that can adsorb pathogenic viruses from drinking water and adsorb toxic residual chlorine from wastewater. To impart virus and chlorine removal abilities to cellulosic materials, we modified the primary hydroxyl group with a positively charged guanidine group, to yield guanidine modified cellulose derivatives. Microcrystalline cellulose (MC) bearing covalently bonded guanidine hydrochloride (MC-GC) and hydrogen-bonded guanidine hydrochloride (MC-GH) were synthesized, and electrospun into nanofibers after blending with the non-ionogenic polyvinyl alcohol (PVA), to produce large pore sized, high surface area membranes. The MC-GC/PVA and MC-GH/PVA nanofibers were stabilized against water dissolution by crosslinking with glutaraldehyde vapor. The water-stable MC-GC/PVA mats were able to remove more than 4 logs of non-enveloped porcine parvovirus (PPV) and enveloped Sindbis virus and reached 58% of chlorine removal. The MC-GC/PVA nanofibers demonstrated better performance for pathogen removal and dechlorination than MC-GH/PVA nanofibers. This first study of MC-GC/PVA electrospun mats for virus removal shows they are highly effective and merit additional research for virus removal.

Diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotide: Isolation and properties.

Recently, a new type of nucleic acid analogues with modified phosphate group, namely, phosphoryl guanidine oligonucleotides, has been described. In the present work, we assess the difference between diastereomers of a mono-substituted phosphoryl guanidine oligonucleotide and analyze their resistance to nuclease digestion. Individual diastereomers ('fast' and 'slow') of a trideoxynucleotide d (TpCp*A) were isolated by reverse-phase HPLC. Snake venom phosphodiesterase digestion showed that the native trideoxynucleotide was fully degraded after 30 min, whereas both 'fast' and 'slow' diastereomers of d (TpCp*A) were not completely digested even after 7 days. UV and CD spectra revealed similarities in the structure of the diastereomers. Structural analysis by 1D and 2D NMR spectroscopy also uncovered significant similarity in the properties of Rp and Sp diastereomers. Structural analysis of nuclear Overhauser effect spectroscopy (NOESY) data and restrained molecular dynamics methods showed very flexible single-stranded oligonucleotide structures. Detailed computational analysis of restraint penalty energies via restrained molecular dynamics simulations with the 2D NMR interproton distance data allowed us to conclude that most likely, the 'fast' isomer is the Sp diastereomer, and the 'slow' isomer is the Rp diastereomer.

Crambescidin 800, Isolated from the Marine Sponge Monanchora viridis, Induces Cell Cycle Arrest and Apoptosis in Triple-Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is currently the only group of breast cancers without an effective targeted therapy. Marine sponges have historically been a source of compounds with anticancer activity. In this study, we screened extracts from twenty marine sponges collected off the coast of Western Australia for cytotoxic activity against TNBC cells. One very active extract derived from the sponge Monanchora viridis was selected for bioactivity-guided fractionation. Through multiple steps of purification, we isolated a potent cytotoxic compound, which was identified as crambescidin 800 (C800). We found that C800 exhibited cytotoxic potency in a panel of breast cancer cells, of which TNBC and luminal cancer cell models were the most sensitive. In addition, C800 induced cell cycle arrest at the G2/M phase, resulting in a decline in the expression of cyclin D1, CDK4, and CDK6 in TNBC cells. This effect was associated with the inhibition of phosphorylation of Akt, NF-κB, and MAPK pathways, resulting in apoptosis in TNBC cells.

Argininosecologanin, a secoiridoid-derived guanidine alkaloid from the roots of Lonicera insularis.

A new secoiridoid-derived guanidine alkaloid, argininosecologanin (1), along with 12 known iridoids and secoiridoids (2-13), was isolated from the roots of Lonicera insularis. The structures of the isolated compounds were established by the spectroscopic analysis and comparison of their spectral data with previously reported data. Compound 1 was assigned as the first secoiridoid-derived guanidine alkaloid isolated as a natural product. A plausible biogenetic pathway for 1 is suggested based on its structural similarity to (E)-aldosecologanin (4).

A new approach for trace analysis of guanidine compounds in surface water with resorcinarene-based ion chromatography columns.

Trace levels of pharmaceuticals have been detected in surface water and may pose a health risk to humans and other organisms. New chromatographic materials will help identify and quantify these contaminants. We introduce a new ion chromatographic (IC) material designed to separate cationic pharmaceuticals and report its ability to separate a group of guanidine compounds. Guanidine moieties are strongly basic and protonated under acid conditions, and therefore can potentially be separated on the newly designed stationary phase and detected by ion exchange chromatography. The new column packing material is based on glutamic acids bonded to resorcinarene moieties that in turn are bound to divinylbenzene macroporous resin. Detection limits in the range of 5-30 µg L(-1) were achieved using integrated pulsed amperometric detection (IPAD) for guanidine (G), methylguanidine (MG), 1,1-dimethylbiguanide (DMG), agmatine (AGM), guanidinobenzoic acid (GBA) and cimetidine (CIM). Suppressed conductivity (CD) and UV-vis detection resulted in limits of detection similar to IPAD, in the range of 2-66 µg L(-1), but were not able to detect all of the analytes. Three water sources, river, lake, and marsh, were analyzed and despite matrix effects, sensitivity for guanidine compounds was in the 100 µg L(-1) range and apparent recoveries were 80-96%. The peak area precision was 0.01-2.89% for IPAD, CD and UV-vis detection.

Antifouling compounds from the sub-arctic ascidian Synoicum pulmonaria: synoxazolidinones A and C, pulmonarins A and B, and synthetic analogues.

The current study describes the antifouling properties of four members belonging to the recently discovered synoxazolidinone and pulmonarin families, isolated from the sub-Arctic sessile ascidian Synoicum pulmonaria collected off the Norwegian coast. Four simplified synthetic analogues were also prepared and included in the study. Several of the studied compounds displayed MIC values in the micro-nanomolar range against 16 relevant marine species involved in both the micro- and macrofouling process. Settlement studies on Balanus improvisus cyprids indicated a deterrent effect and a low toxicity for selected compounds. The two synoxazolidinones displayed broad activity and are shown to be among the most active natural antifouling bromotyrosine derivatives described. Synoxazolidinone C displayed selected antifouling properties comparable to the commercial antifouling product Sea-Nine-211. The pulmonarins prevented the growth of several bacterial strains at nanomolar concentrations but displayed a lower activity toward microalgae and no effect on barnacles. The linear and cyclic synthetic peptidic mimics also displayed potent antifouling activities mainly directed against bacterial adhesion and growth.

Monanchomycalin C, a new pentacyclic guanidine alkaloid from the far-eastern marine sponge Monanchora pulchra.

A new pentacyclic guanidine alkaloid, monanchomycalin C (1), along with the earlier known ptilomycalin A (2), were isolated from the Far-Eastern marine sponge Monanchora pulchra. The structure of 1 was elucidated using 1D and 2D NMR spectroscopic and ma ss spectrometric da ta. Compounds 1 and 2 exhibited cytotoxic activities against human breast cancer MDA-MB-231 cells with IC50 values of 8.2 microM and 4.3 pM, respectively.

Trace analysis of biocidal oligoguanidines in environmental water samples.

This paper demonstrates the determination of residues of biocidal oligoguanidines manufactured by polycondensation of guanidine hydrochloride and 2,2'-(ethylenedioxy)bis(ethylamine) in environmental water samples. The analytes were preconcentrated from samples adjusted to pH 4 by solid-phase extraction using a mixed-mode sorbent with weak cation exchange functionalities. Elution from the sorbent was achieved by 2M hydrochloric acid in methanol. After evaporation and reconstitution in water, the extract was analyzed by reversed-phase high performance liquid chromatography combined with quadrupole/time-of-flight or triple quadrupole mass spectrometry employing electrospray ionization. A preconcentration factor of 10,000 could be achieved and detection limits were in the sub-µgL(-1) range. This method looks promising to monitor the fate of these biocides released into the aquatic environment during different applications.

Rapid detection of economic adulterants in fresh milk by liquid chromatography-tandem mass spectrometry.

A method to aid in the detection of the economically driven adulteration of fresh milk with a range of small, nitrogen containing compounds, including melamine, ammeline, ammelide, cyanuric acid, allantoin, thiourea, urea, biuret, triuret, semicarbazide, aminotriazine, 3- and 4-aminotriazole, cyanamide, dicyandiamide, guanidine, choline, hydroxyproline, nitrate, and a range of amino acids, has been developed. (15)N2-Urea is used as an internal standard. The adulteration of milk with exogenous urea has previously been difficult to detect because of the variation in the naturally occurring levels of urea in milk. However, by monitoring the contaminants biuret and triuret, which comprise up to 1% of synthetic urea, the adulteration of milk with urea-based fertilizer can be detected. We estimate that to be economically viable, adulteration of the order of 90-4000ppm of the above adulterants would need to be added to fresh milk. For most of the compounds, an arbitrary detection threshold of 2ppm is therefore more than sufficient. For biuret, a lower detection threshold, better than 0.5ppm, is desirable and the sensitivity for biuret and triuret can be improved by the post-column addition of lithium to create lithium adducts under electrospray ionisation. Sample handling involves a two-step solvent precipitation method that is deployed in a 96-well plate format, and the hydrophilic interaction liquid chromatography uses a rapid gradient (1.2min). Three separate injections, to detect the positively charged compounds, the negatively charged compounds and amino acids and finally the lithium adducts, are used. This rapid and qualitative survey method may be deployed as a second tier screening method to quickly reduce sample numbers indicated as irregular by an FTIR based screening system, and to direct analysis to appropriate quantification methods.

Method for protein subfractionation of cardiovascular tissues before DIGE analysis.

Difference gel electrophoresis (DIGE) (Electrophoresis 18, 2071-2077, 1997, 1) is widely used in cardiovascular research. However, the dynamic range limitations stemming from contaminating plasma proteins and highly abundant extracellular matrix components can make cardiovascular tissues difficult to analyze. Here we describe a novel methodology for biochemical subfractionation of cardiovascular tissues before DIGE analysis.

A guanidine derivative from seeds of Plantago asiatica.

A new guanidine derivative named plantago-guanidinic acid was isolated from the seeds of Plantago asiatica. The structure was elucidated by two-dimensional (2D) nuclear magnetic resonance (NMR) spectral and other spectral methods.

Synthesis of N-succinimidyl 4-guanidinomethyl-3-[*I]iodobenzoate: a radio-iodination agent for labeling internalizing proteins and peptides.

This protocol describes a detailed procedure for the synthesis of N-succinimidyl 4-guanidinomethyl-3-[*I]iodobenzoate ([*I]SGMIB), an agent useful in the radio-iodination of proteins, including monoclonal Abs, and peptides that undergo internalization after receptor or antigen binding. In this procedure, the tin precursor N-succinimidyl 4-[N1,N2-bis(tert-butyloxycarbonyl)guanidinomethyl]-3-(trimethylstannyl)benzoate (Boc-SGMTB, 3) was first radio-iodinated to [*I]Boc-SGMIB, a derivative of [*I]SGMIB with the guanidine function protected with Boc groups. Treatment of [*I]Boc-SGMIB with trifluoroacetic acid delivered the final product. The total time for the synthesis and purification of [*I]Boc-SGMIB and its subsequent de-protection is approximately 140 min.

Use of protein folding reagents.

The reagents and methods for purification of the denaturants guanidine hydrochloride (guanidine-HCl) and urea are described. Sulfhydryl reagents (reducing agents) and "oxido-shuffling" (or oxidative regeneration) systems are also discussed.