Brevijanazines: structure and biosynthesis of new fungal-derived para-nitrobenzamide piperazines

I wanted to tell you about some work I was involved with that was recently published in Chemical Communications. I was able to solve the structures of two new para-nitrobenzoic acid containing piperazines, which were named brevijanazine A and B, isolated from the fungus Aspergillus brevijanus (NRRL 1935).

The NMR data of the natural products were broad and had multiple sets of resonances due to slow conformational changes of the amide bond(s). The NMR data of the p-nitrobenzoic acid moieties, which are relatively rare in nature, were consistent with those reported for other natural products such as waspergillamide A. This is where the rest of the team (from Microbial Screening Technologies, Macquarie University, The University of Western Australia and Sun Yat-sen University) went into action and the structure was confirmed though X-ray analysis and total synthesis. Probably one of the most interesting aspects was the heterologous biosynthesis, precursor feeding and in vitro microsomal assays that showed that a cytochrome P450 oxygenase converts p-aminobenzoic acid to p-nitrobenzoic acid. It wasn’t too long ago that manipulating the genetics of fungi seemed like a distant dream.

Falcitidins revisited – a new study identifies over 30 analogues using molecular networking

In 2013, my team from MerLion Pharmaceuticals in Singapore, in collaboration with Martin Lear (then at NUS), published the structure of a new acyl tetrapeptide, which was an inhibitor of an antimalarial cysteine protease drug target named falcipain-2, in The Journal of Antibiotics. We named this compound falcitidin. In our work, a small amount of a relatively pure active fraction was secured using bioassay-guided isolation after several fermentation attempts and media changes. The planar structure was elucidated by NMR and MS/MS analysis but attempts to isolate further material for biological testing were hampered by an inconsistent production and a low yield (< 0.1 mg/L). As a consequence, we decided to use an alternative approach to fermentation. First, the absolute configuration was determined by Marfey’s analysis and then the structure was confirmed using total synthesis to be isovaleric acid-D-His-L-Ile-L-Val-L-Pro-NH2. We also explored some preliminary SAR that was published in Tetrahedron Letters the next year.

This is where the story was left until a paper in ACS Chemical Biology was recently published. This team led by Armin Bauer and Till Schäberle used molecular networking to identify over 30 naturally occurring falcitidin analogues from 25 different strains of the bacteria Chitinophaga sp. The team also investigated their biosynthesis. An example of a more potent analogue was pentacitidin A, which is shown above. Interestingly, synthetic falcitidin was not active when tested at 50 uM in their assays and they hypothesise that this could be due to different assay systems. Please read their in-depth study for further details.

My take home points:

  • Bioassay-guided isolation can be difficult on occasions but is important as it can unveil new pharmacophores. Don’t give up easily if the biological signal is clear, but always use dereplication to identify known actives and to group ‘like extracts’.
  • Molecular networking is a very powerful tool for identifying analogues as clearly demonstrated in this ACS Chem. Biol. paper.
  • Analogue isolation and synthesis are important that led to a deeper understanding of the biological activity and potential utility of these compounds.

Pictures around CRI and ASU from 1994

In the 3rd part of my Blog about my time doing a postdoc with Prof Bob Pettit from 1993-4, I wanted to share some pictures that I have scanned from that that time. The first is the view along Palm Walk from outside the entrance to what was then the Cancer Research Institute (CRI). Here’s what it looks like today – link. The second picture captures the ASU bridge on E. University Drive and the last is the bike rack where I parked my primary mode of transport. During summer it was always advisable to get there before 8 am as it became very hot, very quickly. This could be a struggle on a Friday morning after heading out with everyone the night before.

Palm Walk, outside the Bateman Physical Sciences Center, looking towards E. University Drive, Tempe, Arizona
Arizona State University Bridge on E. University Drive, Tempe, Arizona
Bike racks behind CRI (sorry for the poor image quality). Not too exciting but may be of interest to some.

Pettit Lab at the Cancer Research Institute, Arizona State University (ca. 1994)

Following up on my post commenting on Prof Bob Pettit’s life and accomplishments, I have scanned a few photos from 1994 that I took in a Cancer Research Institute (CRI) lab.

Definitely the largest Sephadex LH-20 columns that I ever ran, CRI circa 1994.

A few things stand out from the lab: (1) the absolute focus on bioassay-guided isolation and (2) Kupchan-type partitions followed by Sephadex LH-20 chromatography using a variety of solvent systems. For example, see the isolation of cephalostatins 10 and 11 from a 1994 JNP paper: solvent partitioning, followed by Sephadex LH-20 columns eluting with (1) MeOH, (2) CH2Cl2-MeOH [3:2], (3) hexane-toluene-MeOH [3:1:1], (4) hexane-iPrOH-MeOH [8:1:1], and (5) hexane-CH2Cl2-MeOH [5:1:1], with the last step being reversed-phase HPLC.

Having this type of steely focus on bioassay-guided isolation, really helped to lay the groundwork for my subsequent teams and I to be able to successfully biologically purify active compounds that account for the extract’s activity – even if it was not straightforward. I also remember a list of crystallisation solvents that Bob used to carry around with him. One started at the top and worked your way through the list until crystals were obtained that were suitable for structure confirmation using X-ray crystallography.

Out of focus picture of the working lab bench at the CRI, circa 1994.

In memory of George Robert “Bob” Pettit II (8 June 1929 – 21 September 2021)

I wanted to write a short note to commemorate my postdoc supervisor, Prof Bob Pettit, who passed away in September last year. He was a colossus of natural product chemistry and cancer research who made seminal discoveries such as the bryostatins, dolastatins, combretastatins, cephalostatins and spongistatins. I worked for nearly two years from 1993 at the Cancer Research Institute at Arizona State University. Phoenix was a great place to live, and I had a very enjoyable times working with many colleagues in the lab and exploring Phoenix, Arizona and beyond.

The last (and only) picture I have of Bob and I was taken on the New York Harbour cruise at the 43rd ASP Annual Meeting held from 27 –31 July 2002 in New Brunswick, New Jersey, as well as a picture of him delivering his lecture at this conference.

Mark Butler and Bob Pettit, ASP Conference 2002

I had been in Singapore for a few by then and was starting to work on antibacterials. I asked him if he was interested in also looking at antibiotics. I still remember him saying to me, “Mark, I will look at antibiotics after I have solved cancer”. RIP.

Bob Pettit lecture in 2002 at the 43rd ASP Annual Meeting, New Jersey.

Antibacterial pipeline discussion available on YouTube…

A week or so ago, Prabha Fernandes (GARDP and National Biodefense Science Board), Peter Beyer (WHO) and I were involved in a discussion with Cesar Arias (AAC Chief Editor and Houston Methodist Hospital) about the state of the antibacterial pipeline as a companion to our recent Antimicrobial Agents and Chemotherapy review. The podcast link is here and the YouTube video is below.

The discussion first talks about the WHO’s current roles in antimicrobial resistance (AMR). Next, the state of the antibacterial pipeline was discussed and finishes with some closing remarks around the future of antibacterial research.