Safety improvements for the synthesis of menaquinone derivatives

2024 Fall ACS National

Raising Safety in Research

August 21^st, 2024

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Skyler A. Markham

Special Thanks

ACS Division of Chemnical Health and Safety

Debbie Crans

LST Team

This wouldn’t have been possible without all of you. Thank you!!

The Electron Transport Chain

Oxidative phosphorylation: Figure 1", by OpenStax College

Coenzyme Q: How much do we learn about in freshman biology?

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We know it is there, but only learn it shuttles electrons.

“Coenzyme Q”, aka Quinones

Menaquinones (MKs)

Demethylmenaquinones (DMKs)

• Major electron carriers in bacteria
• Participate in anaerobic respiration

Ubiquinones (UQs)

• Participate in anaerobic respiration
• Precursors for biosynthesis of MKs
• Methylated by menG, a methyltransferase enzyme

• Major electron carrier for E. coli aerobic respiration
• Major electron carrier in eukaryotes

Why focus on Coenzyme Q?

• Tuberculosis causes ~1.5 million deaths annually.
• Function of these lipoquinones is well understood, but location and conformation of these molecules is debated.
• We are currently synthesizing MK derivatives to determine both structure and electrochemical properties of these complexes.

Ulrich, E. L.; Girvin, M. E.; Cramer, W. A.; Markley, J. L., Biochemistry 1985, 24, 2501–2508.

Koehn, J. T.; et al. J. Org. Chem. 2018b, 83, 275-288.

Koehn, J. T., et al. ACS Omega 2018, 3, 14889-14901

Why is Structure important?

Braasch-Turi, M.; Koehn, J. T. et. al. Frontiers 2022, 10, 827530.

UQ-10

Lipid membrane

Where does it sit, and how?

Advancements made by the Crans group

Structural Determination in various solvents shows folding conformation

Koehn, J. T.; et al. J. Org. Chem. 2018b, 83, 275-288.

Koehn, J. T., et al. ACS Omega 2018, 3, 14889-14901 c

Braasch-Turi, M.; Crans, D. C. Synthesis of Naphthoquinone Derivatives: Menaquinones, Lipoquinones and Other Vitamin K Derivatives. Molecules 2020, 25 (19), 4477.

Synthetic Interest of MK derivatives

• Quinones have 1,4-dione systems, which have opposing electronics for both 1,2 and 1,4 additions, making it nontrivial
• Syntheses of these molecules really hasn’t changed in decades
• Purification takes a lot of time and work for low yields

Azerad, R.; Cyrot, M. O. Bull. Soc. Chim. Fr. 1965, 12, 3740−3745.

Koehn, J. T.; et al. J. Org. Chem. 2018b, 83, 275-288.

Koehn, J. T., et al. ACS Omega 2018, 3, 14889-14901

Suhara, Y.; Wada, A.; Tachibana, Y.; Watanabe, M.; Nakamura, K.; Nakagawa, K.; Okano, T. Bioorg. Med. Chem. 2010, 18, 3116−3124.

Importance to minimize Safety

Safety in organic labs is typically more involved

i.e. increased number of hazardous chemicals and techniques to focus on

How do we as students running the reactions limit safety?

1. New synthetic design
2. Vigilance of techniques/Decreasing

# of strenuous techniques

The Innate Safety Problem with Organic Synthesis

Common Safety hazards associated with organic synthesis:

• Flammable
• Volatile
• Carcinogens
• Pyrophoric
• Acute Toxicity
• Aspiration Hazard
• Explosive
• Corrosion
• Pressurized Gas
• Organic Peroxide Formers

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Found just from standing at my fume hood!!!

This is just the chemical hazards!

Combined with several techniques needed:

Standard equipment needed for our reactions:

• Schlenk Manifold
• High vacuum
• Reflux apparatus
• Specific Setups to manage hazardous

chemicals

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Needless to say, there is

A LOT to manage

Previous Synthesis and Safety Risks

Major safety hazards:

• EtOAc is flammable and volatile
• Oral toxicity
• Eye irritation/damage

Major safety hazards:

• EtOAc and Dioxane is flammable and volatile
• Dioxane is a peroxide former
• BF₃ Etherate is it’s own beast
• Flammable, strong reactions with water, Strong HF Releaser

What can we control?

• What Catalyst we use
• Look for solvents with less hazards
• Our surrounding – A clean lab is a happy Lab

Video: What it looks like

Synthetic design considering Safety

Our goal: Synthesize Menaquinone derivatives using less harsh conditions

OR more manageable conditions

Malkov, A. V.; Baxendale, I. R.; Dvořák, D.; Mansfield, D. J.; Kočovský, P. Molybdenum(II)- and Tungsten(II)-Catalyzed Allylic Substitution. J. Org. Chem. 1999, 64 (8), 2737–2750.

New Safety Risks

Other known risk:�Carbonyl ligands can go through skin and carbonylate hemoglobin.

Proper PPE and CLEAN environment at ALL COSTS

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Br₂ also has several hazards associated with, but we have lots of experience with Br₂

Molybdenum hexacarbonyl SDS. Accessed 8/2/2024 https://www.sigmaaldrich.com/US/en/sds/ALDRICH/577766?sdslanguage=EN&srsltid=AfmBOop_i36LCDJJN1feqTe7xDp_EeydNXSk6EKo_T7a4LGPAqJsS18V

Safety Comparison of the 2 Reactions

• Solvents will likely still be volatile/flammable
• No use of Peroxide Formers
• The use of silica gel for columns
• Theoretically synthesize a purer product, less silica use
• Will likely have to continue refluxing the reaction
• Standard workplace risk
• BF₃ fumes constantly, multiple waves of safety issues
• Molybdenum catalyst is not a fine solid, hard to inhale

So, how does the reaction perform?

Pure MK-2

Mo Catalyst Reaction

Safety Implications on reaction setup:

MUCH safer than previous:

• Anhydrous technique necessary
• But not intense Schlenk technique
• Catalyst reaction runs at -78^oC
• Simple but mindful setup
• Same reflux-type setup
• No increase in hazards

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No more:

• Peroxides formers or pyrophoric chemicals

Comparison of compound purity

BF₃ Reaction

Mo Cat Reaction

Planned route to improve synthetic yields:

Limiting factor to yields:

• Menadione formation
• Opposing electronics
• Low solubility of menadiol in several organic solvents
• How to solve this issue?

Other pros: Solubility of silyl alcohol has significantly higher solubility in an array of solvents

Safety Summary of the new reaction:

What did we improve?

• Chemicals are much safer to work with in comparison!!
• Huge success to get away from BF₃ etherate
• Solvent choice is much safer
• Overall, reaction procedure is much simpler than previous
• Therefore, we limit personal safety risks involved

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What can we further improve?

• Solvent choice still not great
• New silyl alcohol should help improve
• Ultimately, further development of a new non-carbonyl based catalyst would be ideal

Conclusions:

Safety Impacts:

• Overall, catalyst used is safer with proper PPE
• Solvents used aren’t major peroxide formers

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Research Impacts:

• A new method for synthesizing MK-2 has been developed
• Purity of the compound is same quality
• Developments can be made to further improve reaction

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Acknowledgements:

Funding:

National Science Foundation

Crans Group Members:

Dr. Debbie Crans

Andrew Bates

Kameron Klugh

Connor Dolan

Drew Schiink

Chris Chang

Sho Nakayama

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Crick Lab:

Dean Crick

Andrea Russell

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Analytical Resource Core (ARC) at CSU

Fearless Leader

Dean Crick

In memory of our lab member:

John Manganaro

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Questions?