Microorganism Coculture-independent Synthesis of

Berkeleypenostatin A

Kun Wei

Dulwich College Beijing, Beijing, 101300, China

Keywords: Retrosynthesis, Berkeleypenostatin, Anti-Cancer, Microorganism Coculture-independent.

Abstract: In 2021, berkeleypenostatins A-G have been biosynthesized by the coculture fermentation of microbes -

Penicillium fuscum and P. camembertii/clavigerum - isolated from the Berkeley Pit Lake (Stierle, Stierle,

Decato, Alverson, Apedaile, 2021). Tested by the NCI Developmental Therapeutics Program,

berkeleypenostatin A effectively inhibited the migration of human pancreatic carcinoma cells (HPAF-II)

(Stierle, Stierle, Decato, Alverson, Apedaile, 2021). However, despire the potent anti-tumor activity

demonstrated by berkeleypenostatin A, its production from terrestrial extremophilic fungi presents challenges

such as low-cell growth and high shear sensitivity (Ludlow, Clark 1991). Considering the potential of

berkeleypenostatin A in pancreatic cancer treatment, this report proposes a laboratory synthesis of

berkeleypenostatin A as an alternative to fungal coculture. The report analyzes berkeleypenostatin A’s

common atoms, deduces its retrosynthesis disconnections, and plans an effective synthesis route. To

maximize the convergency of the synthesis, it begins with the reaction between a strong nucleophile and

electrophile, proceeds to the McMurry coupling and the Diels-Alder reaction, and ends with the addition of

glucose. Such a universal and simple synthesis introduces a series of rapid steps in producing

berkeleypenostatin A, a potential anti-cancer material, which offers an innovative insight to the future

treatment of pancreatic cancer.

1 INTRODUCTION

With a 5-year survival rate of 10%, pancreatic ductal

adenocarcinoma (PDAC) is leading the cancer-

related deaths worldwide (Osuna de la Peña, D.,

Trabulo, S.M.D., Collin, E. et al. 2021). Most patients

have advanced or metastatic disease at diagnosis

(Park, W., Chawla, A., & O’Reilly, E. M. 2021).

Existing treatments, including gemcitabine and nab-

paclitaxel, have limited efficacies due to various

complex factors affecting PDAC, such as

desmoplasia and hypervascularization (Osuna de la

Peña, D., Trabulo, S.M.D., Collin, E. et al. 2021).

Although chemotherapy with gemcitabine is the

standard therapy for advanced or metastatic disease,

its efficacy is highly limited by undesirable qualities

of rapid plasma degradation, toxicity, and drug

resistance (Tada 2011, Correia, Xavier, Duarte,

Ferreira, Moreira, Vasconcelos, Vale 2020).

Meanwhile, nab-paclitaxel demonstrates multiple

adverse side-effects, including alopecia, neutropenia

and nausea (Vishnu, Roy 2011). Therefore, there is

an urgent demand for developing effective

therapeutics for pancreatic cancer.

As a recently isolated compound,

berkeleypenostatin A displays the anti-tumor

qualities for a potentially effective therapeutic. The

structure and configuration of berkeleypenostatins A-

G have been deduced from spectral data and single-

crystal X-ray crystallography (Stierle, Stierle,

Decato, Alverson, Apedaile, 2021). After being

produced in coculture, berkeleypenostatins were

tested for anti-cancer activity. Among these

molecules, berkeleypenostatin A was identified as a

moderate inhibitor (50−100 μM) of MMP-3, an

enzyme that promotes metastasis in pancreatic tumor

cells (Suhaimi, Chan, Rosli 2020, Yang et al 2020).

Berkeleypenostatin A also induces reduced cell

migration of human pancreatic carcinoma cells

(HPAF-II) by 30% at a concentration of 1.25 μM over

a 24 h period (Stierle, Stierle, Decato, Alverson,

Apedaile, 2021). The results shed light on the

research for pancreatic cancer, which has been a

challenging issue with patients exhibiting a low

survival rate within five years after diagnosis and

Wei, K.

Microorganism Coculture-independent Synthesis of Berkeleypenostatin A.

DOI: 10.5220/0011296500003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 797-802

ISBN: 978-989-758-595-1

797

diagnosed in the advanced stages (Rawla, Sunkara,

Gaduputi 2019, American Cancer Society. 2021).

Although the current results indicate its potential in

cancer treatment, further biological assays are

essential to understanding more about the biological

activity of berkeleypenostatin A. Such inquiry

introduces the need to prepare berkeleypenostatin A

for analysis and evaluation.

However, the existing synthesis of

berkeleypenostatin A comes with various drawbacks

introduced by the complexity and time-consuming

nature of fungal coculture. Berkeleypenostatins are

examples of secondary metabolites grown in axenic

culture from microorganisms in the Berkeley Pit,

acidic metal-rich waste lake (Giddings, Newman

2015). To combat the inconveniences of producing

berkeleypenostatin A from microorganism coculture,

this report proposes a coculture-independent method

to synthesize berkeleypenostatin A with simple and

fast steps. It is expected to prepare berkeleypenostatin

A using the devised synthesis outlines below.

Figure 1. Structure of Berkeleypenostatin A

2 METHOD

As shown in Figure 1, the core of berkeleypenostatin

A has 11 stereocenters, which results in 2

potential

stereoisomers. Therefore, the final products of the

synthesis may include stereoisomers of this target

molecule. To maximize the simplification and

convergency of each retrosynthetic step, the strategy

is to disconnect bonds linking two or more common

atoms of the polycyclic core.

Figure 2. Retrosynthesis Strategy of Berkeleypenostatin A.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

798

After evaluating several retrosynthesis methods,

the final version, with the highest expected

efficiency, is presented in Figure 2. The

retrosynthesis is proposed to begin with a hydrolysis

reaction under acidic conditions, separating the R

group, β-D-glucose, from the rest of

berkeleypenostatin A 1. Berkeleypenostatin B 2 is

expected to undergo a Diels-Alder reaction to

disassemble the fused rings resulting in a bridged

bicyclic ring system composed of a pyrone and a 10-

carbon ring. Followed by a McMurry coupling, the

alkene group of molecule 3 will split into two chains

ending with an aldehyde group. When the 1,4

dioxygenated system of an ether group and an

aldehyde group cleave, molecules 5 and 6 will be

produced. These synthesized molecules will react as

the starting materials of berkeleypenostatin A.

Figure 3. Preparation of Molecule 5 as an Improved Nucleophile.

Figure 4. Preparation of Molecule 6 as an Improved Electrophile.

Before the formal synthesis, molecules 5 and 6 are

prepared and tuned using the procedure presented in

Figures 3 and 4. The hydroxyl groups on molecule 5

are reactive, which may later interfere with essential

Microorganism Coculture-independent Synthesis of Berkeleypenostatin A

799

steps in the synthesis. Therefore, the hydroxyl groups

need to be masked with protective agents with

varying strengths. With lithium diisopropylamide

(LDA), molecule 9 forms a carbanion, which will

react with malonaldehyde under acidic conditions

with the existence of water to form molecule 11.

Under these conditions, the weak protective agent on

molecule 11, the silyl ether, will eventually be

converted to a hydroxyl group, forming molecule 5.

Under alkaline conditions, the hydroxyl group on

molecule 5 will be ionized to form an oxygen anion,

resulting in a stronger nucleophile.

Molecule 6 cannot be found as a commercial

reagent, so it is synthesized using simpler, more

accessible molecules. The starting materials,

acetaldehyde 13 and 2,3-butanedione 14, will join

together to form 2,5-hexanedione 15 under alkaline

conditions. 2,5-Hexanedione 15 will undergo a

functional group interconversion by eliminating

water, which transforms the hydroxyl group to an

alkene group. Next, a bromide ion will be added to 3-

Hexene-2,5-dione 16 using bromine liquid under

strong alkaline conditions. In the Wittig reaction, the

phosphorus of triphenylphosphine (Ph



P) attacks the

carbon next to the bromide group in molecule 17,

forming a ylide that reacts with heptaldehyde 18,

generating molecule 19. The ketone group on

molecule 19 is then converted to a hydroxyl group,

forming molecule 6. To improve the electrophilic

properties of molecule 6, the hydroxyl group is

converted to a bromide group. The enhanced

nucleophilic and electrophilic properties of molecules

20 and 12, respectively, prepare for their reaction.

Figure 5. Synthesis Strategy of Berkeleypenostatin A.

The steps to synthesizing berkeleypenostatin A

are shown in Figure 5. Molecules 12 and 20 will be

mixed under alkaline conditions, forming molecule 4.

With titanium chloride acting as a reducing agent, the

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800

two aldehyde groups at the ends of molecule 4 will

connect to form an alkene group, undergoing the

McMurry reaction. The bicyclic bridged system of

pyrone 3 will fuse to form three fused rings under

heat, carrying out the Diels-Alder reaction. Finally,

by eliminating water via heating, β-D-glucose may be

added to berkeleypenostatin B 2 in a condensation

reaction, synthesizing berkeleypenostatin A.

3 RESULTS & DISCUSSION

By following the synthesis proposal,

berkeleypenostatin A is expected to be produced with

a few challenges. In the Wittig reaction, the aldehyde

group on the heptaldehyde may be affected given that

it is more reactive than the ketone group on molecule

17, producing an undesired outcome as the aldehyde

group converts into a hydroxyl group. If such

possibility is verified by experiment that it hugely

impacts the result, an alternative step should be

devised.

A notable limitation of the synthesis is the

production of undesired stereochemical outcomes.

For instance, when the malonaldehyde is added to

molecule 10, the product consists of one stereocenter,

forming two enantiomers of molecule 11 in a 1:1

ratio. Therefore, it is expected that

berkeleypenostatin A will be mixed with some

unexpected stereoisomers in the final products.

To boost the yield of berkeleypenostatin A, the

future direction of the synthesis is to develop an

enantioselective strategy of adding the

malonaldehyde to molecule 10, which may involve

the use of a catalyst.

4 CONCLUSION

In conclusion, this report suggests a synthesis

strategy of berkeleypenostatin A with significantly

maximized convergency. The logistics of the route

may be improved by experiments and testing the

optimal temperatures at different stages of the

synthesis, including the necessary heating during the

Diels-Alder reaction and the condensation reaction.

The yield of the synthesis may be enhanced by

stereoselective steps. Nevertheless, the report devised

a strategic laboratory method to synthesize

berkeleypenostatin A by reacting a nucleophile with

an electrophile in a minimal number of steps. Overall,

this microbe coculture-independent synthesis route of

berkeleypenostatin A has high prospects, offering a

new way of producing this anti-tumor reagent without

limitations of fungal coculture. Such a facile and

universal synthesis of berkeleypenostatin A would

make the material more readily available for

biological assays in evaluating its efficacy for

pancreatic cancer treatment.

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