Integrating Flavin Cofactor into Peptide Amphiphiles for Electron Transport

Flavin is a redox cofactor found in many biological systems. Its ability to support electron transfer makes them a valuable tool to study this process in nature. In this project, we are synthesizing an Fmoc-protected flavin amino acid for use in peptide-based model systems that mimic redox-active proteins. This work is part of a broader effort to design an artificial system that isolates the biological properties underlying natural protein functions while being free from the complexity of evolutionarily affected systems. These simplified systems will ultimately help us understand redox behavior in proteins and contribute to the development of next generation bioelectronic materials.

Slide 1

Hello everyone. My name is Nathan Hernandez, and I’ll be presenting the integration of Flavin cofactor into peptide amphiphiles for electron transport.

Slide 2

So our original research question was whether or not we could embed the flavin cofactor into peptide amphiphiles to create a synthetic material that supports directional electron transport.

Slide 3

My research question stems from the fact that life relies on electron transport and is seen in processes like cellular respiration and photosynthesis. Throughout these processes, there are proteins that guide electron transport in a very specific manner. They rely on well positioned co-factors like hemes, Flavins, and Quinones. 
But the issue with these systems is that they’re super complex and sensitive to environments, and it makes it difficult to isolate and reengineer in controlled environments.

Slide 4

I decided to choose flavin because it’s a cofactor that’s already found in the body and is able to support 2 electron and one electron transfer. It’s an extremely versatile co-factor with predictable redox potential. It’s chemically robust enough to survive acids and solid-state peptide synthetic conditions. And there’s also an existing protocol for synthesizing flavor modified amino acids, which can then later be introduced into peptides.

Slide 5

A peptide amphiphile is a short peptide sequence with a hydrophobic tail. These sequences under basic aqueous conditions form fibers. The really cool part about peptide amphiphiles is that we’re able to modularly design them and change the charges, hydrophobicity, and change the sequencing. 
Which means we’re able to place our co factors in specific positions along the peptide. And due to their ability to form fibers, we’re able to control highly ordered structures which allow us to create a potential for directional electron movement.

Slide 6

To synthesize the flavor modified amino acid, we follow synthetic steps set by carrel et al. in 1998. This includes the oxidation of the precursor with potassium, persulfate in an acid to introduce the nitroso intermediate, which is an orange crystal. Highly activated for nucleophilic aromatic substitution. 
And the ipso substitution with Boc protected lysine and pyridine for 72 hours. Which yields a deep red product and forms the key carbon nitrogen bond linking flavor, precursor to the amino acid.

Slide 7

The next step is a reduction done by a hydrogenation catalyzed by palladium on carbon to convert the nitro group into an aniline group. Alloxan monohydrate and boric acid are added to cyclolize the isoalloxazine core. This step is crucial and must be done in the dark to avoid photodegradation. This is followed by Fmoc protection. Through the addition of Fmoc OSU, which installs a protecting group that is suitable for solid-state peptide synthesis. The final product is a bright yellow powder, which is the flavin modified amino acid.

Slide 8

After the amino acid is synthesized, we incorporate it into a peptide using our purepep chorus automated peptide synthesizer. Since the flavor monomer is bulky, we use a double coupling cycle to ensure that it is fully incorporated into our peptide. The peptide is then cleaved off the resin and precipitated with cold ether to yield the product.

Slide 9

The crude peptide is then purified through high performance liquid chromatography, and its mass is verified through mass spectrometry.

Slide 10

Our current results are that we’ve been able to synthesize the flavin modified amino acid and verified structure through NMR. We’ve also been able to synthesize crude peptide in high yield. However, we have been unable to fully purify this through high performance liquid chromatography.

Slide 11

Once our peptide is pure, we plan to analyze its structure through atomic force microscopy. This will confirm nano fiber formation, length, height, and bundling of those nanofibers. We will also use conductive atomic force microscopy to analyze the conductive properties of these fibers.

Slide 12

We will use redox Titrations with oxidizing and producing agents to analyze the electrochemical properties of our nano fibers. This will be done through step-wise edition of oxidants and reductants to cycle the flavin between oxidized and reduced states. We will monitor these changes through UV-ViS, spectroscopy, to track the characteristic flavin bands.

Slide 13

This work creates a simplified platform to study electron transport while avoiding the complexity of full proteins. This has potential applications and bioelectronic interfaces, implantable or wearable sensors in the next generation of circuitry.