Author: Mark Butler

More environmentally friendly alkaloid extraction methods

Background: I started my natural products journey looking at marine sponge chemistry. The sponges were stored in individual containers in an ethanol (EtOH) solution in a freezer or frozen sponge material was extracted freshly with EtOH. Nearly always there was a limited supply of material and care was taken to keep the temperature as low as possible as compounds were often unstable. After several compounds I was working on underwent relatively rapid degradation, subsequently isolated compounds were stored at -70 °C in vials or flasks wrapped in aluminium foil under argon as an oil/solid or in solution! Moving forward several years, I also started working on plants and became very interested in alkaloids. I couldn’t believe what I read – extract ground plant material at room temperature with chloroform (CHCl3), add 1M hydrochloric acid (HCl) and remove the organic layer. Then basify the aqueous layer using 1M NH4OH (on ice), partition with CHCl3 and remove the CHCl3 using rotary evaporation to give enriched alkaloids. Definitely more cavalier compared to my previous experiences with marine natural products.

The study: This recent paper in the Journal of Natural Products caught my attention: “Toward a More Sustainable Sample Preparation in Phytochemistry: Case Studies in Four Subclasses of Alkaloids”. This article details a study that evaluated sustainable extraction and purification methods for alkaloids. As described in the first paragraph, alkaloid enrichment usually uses potentially hazardous chemicals (inhalation, skin contact, environmental if improperly disposed of) such as HCl and dichloromethane (CH2Cl2)/CHCl3. Also, these chlorinated solvents can sometimes react with alkaloids.

This study evaluated the extraction of four alkaloids (harmine, boldine, vincamine and mescaline) using less toxic alternatives. It was found that citric acid, which is used as a preservative in food and pharmaceutical formulations, could replace HCl without loss of extraction efficiency. Also, ethyl acetate (EtOAc) could replace CH2Cl2 in three out of four cases without harming extraction efficiency. Alternative solvents such as tert-amyl methyl ether (TAME), n-butyl acetate (BuOAc) and anisole also showed potential.

Take home message: Consider using more environmentally friendly acids and solvents if performing alkaloid enrichment studies. This is especially relevant for processes undertaken multiple times like re-isolation or generation of extract libraries.

Tetracycline has left its (fluorescent) mark on the world

Evidence of tetracycline containing fermentations in northern Africa around 1,500 years ago.

This is some older research published in 2010 (available here) that I recently read, which I wanted to share.

Some bones excavated from northern Africa (modern day Egypt and Sudan) burials have bright yellow-green fluorescent bands (λ 490 nm), which was similar to those observed in people treated with first generation tetracycline antibiotics. When first found, fluorescent bands were conjectured to have formed post death or through infection. More on the history of tetracycline (ancient uses and its rediscovery) can be found in this lecture text (available here).

Bones from a four-year-old Nubian child buried around 1,500 years ago were demineralised with hydrofluoric acid (HF) to release the fluorescent compound(s) (available here). HPLC-MS analysis showed two closely eluting peaks with a m/z of 427.1 Da. Authentic standards were derived from HF treatment of tetracycline, oxytetracycline and chlortetracycline, which resulted in aromatisation of the B ring and racemisation of the A ring N-dimethyl group at C6 (see Figure 1). These diastereomers could be separated by HPLC-MS and it was shown that the bone-derived fluorescent compounds had the same retention times and MS as the acid treated products from tetracycline. This study provided unequivocal evidence that the Nubians had access to a tetracycline producing Actinomycetes, which they presumably used for medical purposes (treatment and/or prophylactic). Tetracycline antibiotics were not used in modern medicine until their re-discovery in the 1950s. Since then, several semi-synthetic and synthetic tetracycline drugs have been approved and are used to treat a variety of infections.

Figure 1. Bone was treated with HF, which liberated anhydrotetracycline diastereomers that were analysed by LC-MS and an authentic standard. The bone picture used was sourced from “Ancient brew masters tapped drug secrets”, Emory University 2010 (available here).

Some thoughts: It is fascinating that tetracycline fermentation broths were used at least 1,500 years ago. We don’t know what happened, but I hope that some of these people weren’t some of the first to be infected with drug-resistant bacteria. Although some traditional fermentation knowledge has survived (e.g. alcohol production and Chinese red yeast rice), I wonder if other crude antibiotics were used by the Nubians or other peoples, but no traces were left (or have been discovered yet). It was hypothesised that tetracycline-containing broths were drunk. However, the tetracycline production levels must have been quite high to leave multiple fluorescent bone staining bands. Perhaps concentration steps could have used such as cloth filtration and/or evaporation. Actinomycetes can also grow on solid media and there is a possibility that a grain could have been used in an analogous way to red yeast rice.

Taurine – three short stories

Story 1: Marine sponges. The first time I encountered taurine was during my PhD where we routinely isolated the zwitterionic taurine and trigonelline from marine sponges. A paper from around that time found that taurine was often the most abundant amino acid in marine sponges, along with hypotaurine and glycine. Interestingly recent work has reported that taurine plays a role in host-symbiont interactions in Ianthella basta – a sponge that I worked on during my PhD and Postdoc (here and here).

Story 2: Antibacterial. In November 2023, the FDA approved the use of a taurolidine and heparin (DefenCath®) as a “catheter lock solution to reduce catheter-related bloodstream infections in adult patients with kidney failure who are receiving chronic hemodialysis through a central venous catheter.” A taurolidine containing catheter lock solution TauroSept® has been available in Europe since 2006. The use of catheter lock solutions was reviewed in 2022 for those interested. Taurolidine has weak broad spectrum antibacterial activity against both G+ve and G-ve bacteria. In aqueous solution, taurolidine is equilibrium with several species, which have antibacterial activity: the methylol groups can react with peptidoglycan, endotoxins and exotoxins, while taurine and taurinamide also have weak antibacterial activity.

Story 3: Energy drinks. In addition to caffeine, energy drinks such as Red Bull contain taurine, several B vitamins and sugars/sugar substitutes. In Australia, there is 400 mg/100mL of taurine and 32 mg/100 mL of caffeine, but these levels can vary by country. Red Bull GmbH is one of the most interesting companies in the world, which was recently explored in Founders podcast #333 (highly recommended).

Zosurabalpin — a new class of Acinetobacter targeting antibiotic

The discovery, antibacterial profile, and mode of action (MoA) of zosurabalpin, which belongs to a new antibacterial class called ‘tethered macrocyclic peptides’ (MCPs), was recently disclosed in two Nature papers (here and here, as well as a commentary). Zosurabalpin is currently being evaluated in a phase 1 trial by Roche for the treatment of carbapenem-resistant Acinetobacter baumannii (CRAB) infections. A. baumannii is classed a Priority Pathogen by the WHO and these infections, while being relatively rare at this moment in time, have high mortality and morbidity. These two papers are very detailed and this blog highlights only some of the work undertaken!

Hit Discovery: Whole-cell phenotypic screening of a 44,985 member MCP library identified a cluster of active MCPs. One of these hits, RO7036668, displayed selective activity against A. baumannii, but no cytotoxicity (promising).

Hit to lead to clinical candidate: Iterations of medicinal chemistry and testing led to the identification of MCPs such as RO7075573 with more potent in vitro and in vivo mouse model activity against A. baumannii; however, it was observed during pre-clinical toxicology studies that RO7075573 caused aggregation of low-density lipoprotein/high-density lipoprotein vesicles in rat plasma through an unknown mechanism. Using an in-house developed rat plasma precipitation assay, the team showed that zwitterionic (definition) tethered MCPs had significantly reduced plasma precipitation and zosurabalpin was ultimately selected for development. It had been previously shown that zwitterionic compounds can have enhanced Gram-negative bacterial membrane transport — see O’Shea & Moser and Muñoz & Hergenrother for discussions on bacterial entry.

Mode of action (MoA): Sequencing of resistant mutants suggested that the zosurabalpin’s target was likely to be the protein complex LptB2FGC, which is an ATP-binding cassette used by Gram-negative bacteria to move lipopolysaccharide (LPS) from their inner membrane to the outer layer of the outer membrane. It was shown that MCPs helped to trap LPS in the protein complex by several orthogonal methods including Cryo-EM.

Did you know that A. baumannii can survive without LPS in its outer membrane? Amazingly it can! It was shown that MCPs failed to inhibit the growth of LPS-deficient A. baumannii, which supported the proposed MoA. The LPS-deficient phenotype can be produced in response to polymyxin treatment, but there is a significant fitness cost, as well as increased susceptibility to other antibiotics and disinfectants.

Looking forward: Zosurabalpin has only just started its clinical development (safety and dosing). Although the total number of A. baumannii infections is (mercifully) relatively low, perhaps a multi-site, multi-country clinical strategy could be used in phase 3, similar to the trial successfully completed for the β-lactam and β-lactamase inhibitor combination sulbactam-durlobactam. Although it could be theoretically possible to undertake an Acinetobacter specific trial using rapid diagnostics, the frequency of polymicrobial infections in clinical settings could lead to the need for the development of combination therapies with other antibiotics with activity against other pathogens such as Pseudomonas, Klebsiella and E. coli, may be even MRSA. It will be interesting to what directions are taken.

Concluding remarks: The discovery and development of zosurabalpin and the unravelling of its clinically unexploited MoA is very impressive. Importantly, the team also included how they overcame development hurdles, which is very valuable information for antibacterial drug discoverers. Recent successful clinical trials have shown that it is possible to run multi-site, multi-country trials for these deadly Gram-negative infections and the approval of a novel class of antibiotics to treat CRAB infections will be welcomed by clinicians. Now the world needs to make sure that adequate funding is available for these types of phase 3 trials, as well as ensure that incentives such as the Pasteur Act or similar pull incentives are introduced as soon as possible. Otherwise, novel antibiotics with life saving potential may not make it to patients. Remember, paraphrasing Mike Tyson “Everyone has a plan until they get punched in the mouth a drug-resistant Gram-negative infection.”

Talcarpones — interesting new antimicrobials from Talaromyces johnpittii

I was involved in some recent work published in the Journal of Antibiotics (co-authors from Microbial Screening Technologies, Macquarie University, and Queensland Plant Pathology Herbarium) that describes the isolation, structure elucidation and antimicrobial activity of the talcarpones, which were isolated along with a known naphthazarin, aureoquinone. The paper is available here.

Interesting points:

  • These compounds were isolated from Talaromyces johnpittii sp. nov., which was named after John Ingram Pitt (1937–2022), an Australian food microbiologist and mycologist.
  • The structures and relative stereochemistry of talcarpones A and B were secured by analysis of MS and NMR data. The absolute configuration of the talcarpones was tentatively assigned as 1 S,10 S,1′S,10′S using ECD calculations.
  • The talcarpones and aureoquinone are homologues of hybocarpone and boryquinone, respectively. This is the first report of a homologue of hybocarpone, which had been previously reported from several fungal lichen symbionts. Although the relative configuration of hybocarpone was previously confirmed by X-ray crystallography and total syntheses, the absolute configuration was not assigned.
  • Talcarpone A had MICs of 1.6 µg/mL against Candida albicans and Saccharomyces cerevisiae, and 12.5 µg/mL against Bacillus subtilis and Staphylococcus aureus.
  • Talcarpone B was shown to convert to talcarpone A in aqueous acetonitrile, which then partially converted to aureoquinone by an unknown mechanism.
  • The NMR data of aureoquinone showed that it contained a plane of symmetry. We hypothesised that facile intramolecular proton transfer mediated by proton tunnelling leads to very rapid tautomerisation and averaging of the quinone/quinol carbon resonances in aureoquinone, as has been reported for other symmetrical naphthazarins. Interesting, proton tunnelling is eliminated in asymmetrical environments such as hydroxydroserone.

2023 ASM Top-Cited

The culmination of my time consulting for the WHO was the publication of “Analysis of the Clinical Pipeline of Treatments for Drug-Resistant Bacterial Infections: Despite Progress, More Action Is Needed”, which was just announced as one of the Antimicrobial Agents and Chemotherapy (AAC) 2023 Top Cited Collections. Open access here.