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[Music]

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[Applause]

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in the my name is Trent Stubbs I'm a

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rising senior undergraduate student at

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Furman University and I've been working

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into the direction of dr. Greg

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Springsteen for the past two summers at

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a part of NASA and NSF funded Center for

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chemical evolution so I would like to

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start today by discussing the classic

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miller-urey experiment done in the 1950s

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it's really launched the field of

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prebiotic chemistry of course by

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demonstrating being able to transform

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these small organic materials into

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larger biologically relevant species

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like amino acids to other some more

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related experiments being formaldehyde

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condensation producing saccharides or

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HCN polymerization and hydrolysis

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generating a lot of precursors to

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nucleobases but as you can tell from the

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two HPLC chromatograms

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there was no thermodynamic end product

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to these processes and it's hard to

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imagine that biology could then take

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this system which is somewhat of an

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organic tar-like mess and be able to

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evolve from it and as some sort of

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established foundation so when we were

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designing our experiments to investigate

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the origins of metabolism we wanted

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something a little bit more robust and

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so we wanted to study something that

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exists in every living system ever and

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that is the circuit citric acid cycle

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these transformations now have many

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roles in modern biology three of which

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are to capture redox potential

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biosynthesize other relevant molecules

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and then fixate carbon in the form of

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the reductive citric acid cycle

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now these specific transformations to

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turn pyruvate an acetyl co a into these

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larger building block intermediates

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require eleven different enzymes and

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multiple Co factors including nad plus F

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ad and GDP but but of course we were

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just looking more for a some sort of

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remnant of these robusta geochemical

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pathways that existed within and many

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researchers before have tried to

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recapitulate these exact transformations

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that occur within the citric acid cycle

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and only two of which really proceeded

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in high yield that's the oxidative

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decarboxylation and the beta

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decarboxylation and and it makes very

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good sense for why these transformations

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don't occur the carboxylate is just

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simply not reactive enough

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the carbonyl carbon is not electrophilic

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enough for attack and then the carbon

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alpha 2 the carboxylate has a pKa of

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about 27 so it's just not something that

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is nucleophilic enough to be able to

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establish an appreciable equilibrium

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between the carboxylate and the either

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email or enolate to attack out of so we

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had an idea and now is to replace this

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carboxylate functional group with

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something more reactive and that more

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reactive group turns out to be something

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called an alpha keto acid and it's shown

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below on the bottom half of the screen

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and these alpha keto acids are much more

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electrophilic at that carbonyl carbon

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and are susceptible to attack and

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they're much more nucleophilic that

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alpha carbon next to the keto acid has a

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pKa of about 12 so now in an aqueous

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environment we can begin to

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deprotonating and start form the enol or

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enolate to attack out of as a good

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nucleophile and somewhat remarkably this

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alpha keto acid can then be transformed

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right back into the canonical carboxylic

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acid in near quantitative yield just

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through the oxidative decarboxylation so

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our idea was to take the citric acid

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cycle something robust in every

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biological system that exists and

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replaced one of those carboxylate

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functional groups with this more

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reactive alpha keto acid functional

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group and that's shown on the second

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line so for example Malley becomes now

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mal loyal for mate or fumarate becomes

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few more oil for me and you may even see

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that some keto acids exist now already

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in the citric acid cycle like alpha

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ketoglutarate and oxaloacetate and

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here's just an example of one reference

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in the literature right now utilizing

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the power of these alpha keto acids this

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was done by kim @l in the 70s where they

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were able to show the generation of

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citrate through a self condensation of

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oxaloacetate followed by beta

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decarboxylation and then oxidative

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decarboxylation to give reasonable

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yields in the aqueous environment at a

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pH of 5 in pH of 7 we wanted to test our

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prebiotic pathway and so we did that by

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reacting 200 millimolar pyruvate with

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three equivalents of glyoxylate and a pH

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7 buffer and that gave us these first

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these two NMR's shown and it was

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somewhat remarkable and that not only

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did we see the first aldol addition

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product that mal oil form eight species

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but we also saw the condensation product

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and then reduction product and then

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subsequent aldol addition and

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condensation product as well and this

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was really exciting for us and that's

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what the both NMR's are showing us a

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being starting from pyruvate and then be

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starting from alpha ketoglutarate where

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nearly everything we produced in

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solution was one of these citric acid

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cycle equivalence right just with the

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alpha keto acid functional group and

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then from there we wanted to transform

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this keto acid group back into the

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carboxylate functional group and we did

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that just through the oxidative

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decarboxylation ECAR box elation

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mechanism with five equivalents of

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hydrogen peroxide just at room

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temperature and in mere 15 minutes it

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gave us this beautiful spectra below

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where now everything in solution is now

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these citric acid cycle intermediates

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and this all stemmed from a single

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reaction pot a single system of just

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pyruvate and glyoxylate the smallest

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alpha keto acid and the second smallest

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alpha keto acid another way which we

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were able to visualize this reaction

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progression was by HPLC so two of the

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molecules in particular having a double

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bond really have a nice UV trace one

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being the few more oil formate labeled B

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and the other being a Cana torial

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formate labeled C so as we see that

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concentration of fumarole formate

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increase as we begin to go through the

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cycle we then see it reached its maximum

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and then start to fall as the

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concentration of the transit connot

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eight begins to increase so this was a

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beautiful pathway to really show that we

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were following this this cyclic system

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like that of the reductive reductive

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citric acid cycle and we believe that

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that transformation from be the fumarole

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format to the alpha ketoglutarate is the

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rate limiting step and it is presumed

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that that might proceed through a Kaunas

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ro type reduction of a hydride through

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the hydrate of glyoxylate additionally

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this helped us visualize that we were

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also producing some sis Akana Tate which

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is the biologically relevant ice

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of ikana tate and so this is the pathway

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in a nutshell the outer cycle being the

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modern reductive citric acid cycle and

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the inner cycle being our keto acid

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equivalent pathway starting from

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pyruvate and glyoxylate proceeding

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through the exact inner the exact

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equivalent of the intermediates in the

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citric acid cycle in a single reaction

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pot a single reaction system at a pH of

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seven and 50 degrees C and in only 21

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hours now you may see the one gap in

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between the Econo toil format in central

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formate that's one area in which we

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wanted to investigate in the future and

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we have now an idea for being able to

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transform that molecule the medial econo

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toll format we call it into the central

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forming if medial kana toriel form eight

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just hydrates in water it would be a

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conjugate addition and it would add not

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at the tertiary carbon which is give us

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the Satori form eight so we need to

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somewhat rearrange this keto acid

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functional group and we found that while

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taking an NMR of this species we would

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exchange protons with a deuterium in d2o

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at the beta carbon which would suggest

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we somehow activated that beta carbon

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and when you look at the structures and

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particularly the resonance structures of

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that species it makes sense why that

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carbon was activated it's now dumping

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into the carboxylate adjacent to it and

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then through the double bond into

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another carboxylate as well so in theory

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now we can add a second equivalent of

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glyoxylate ad at the beta position and

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then hydrate in retro out all that first

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glyoxylate addition giving us now

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terminal Akana toil for mate same

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molecule just having that car box re the

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keto acid a functional group in a

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different location this would then

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hydrate in the tertiary position we

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believe which would retro a little

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giving us two molecules of pyruvate and

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one molecule of carbon dioxide so if we

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can achieve this transformation now in

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the future that would have taken us from

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one pyruvate starting the cycle to two

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pyruvates and in excess of glyoxylate

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something else we wanted to demonstrate

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was how far can we take these alpha keto

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acids how

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can we utilize their reactiveness and

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and we wanted to generate some amino

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acids much like biology does with them

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now and so we did that through a

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transamination with the simplest amino

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acid glycine

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and a potassium aluminium salt at 80

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degrees C and a pH of 5 for about five

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hours and NMR a showing the generation

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of glutamate from alpha ketoglutarate

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and then B showing the generation of

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alanine from pyruvate in the presence of

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the glyoxylate and this potassium

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aluminium salt and then now somewhat

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interestingly is as well the glycine

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species then is also it's undergoing

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that transamination so it is producing

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glyoxylate which is the feed stock for

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our prebiotic cycle as shown on the

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previous slide and so just one more time

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just to kind of wrap up everything that

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was done on this project the inner cycle

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being our alpha keto acid equivalent in

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the outer cycle being the canonical

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citric acid cycle and a couple key

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points I want to emphasize or we're not

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suggesting that it was hydrogen peroxide

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or a potassium aluminium salt which

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under did these exact transformations

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but what we're trying to show is that

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going from these keto acids to the

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canonical carboxylates or two amino

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acids would have been really easy and

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really efficient and even advantageous

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for an evolving enzymatic system to to

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to use once it requires regulation

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because once we get the car nautical

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citric acid cycle intermediates as I

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showed on I think slide 2 they're dead

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ends and we can't undergo any further

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transformations unless we have that

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enzyme so we can imagine this as a

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possible prebiotic we plausible system

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for now in evolving enzyme to really get

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ahold of and grasp and be and to

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transform and evolve so with that I

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would just like to thank everyone who

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helped support this project Furman

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University and the Furman advantage for

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funding my summer research experience

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NASA and the NSF for funding the Center

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for chemical evolution the Scripps

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Research Institute specifically the lab

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of dr. Rama Krishna Murthy for all their

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help

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operation on this product and lastly my

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lab members ELISA clay who's now at

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Stanford Rachel Cooke who will be giving

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a poster presentation tonight at seven

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and my p i-- and and mentor dr. Greg

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Springsteen so with that thank you so

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much for your attention I'd be happy to

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take any questions so we have time for

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yes right so we do see some oxalate in

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the NMR and particularly we believe

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there are a couple of competing pathways

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which might produce this the alpha

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ketoglutarate another one being through

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a second edition of glyoxylate but

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that's something we're still

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investigating and wanting to look down

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the road to really confirm that

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particularly with some isotopic labeling

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studies though I think it's a great

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question I think that's one of the the

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strongest points we have for this

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prebiotic cycle is that it runs by

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itself and therefore for some sort of

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living system there would be no

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regulation and there would be no control

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over what's occurring it can just go in

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water by itself pretty remarkable so

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then once the enzyme catalysis were

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available it could really kind of

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capture it and get a hold of what was

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going on that's exactly right

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so right now that that's just an

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oxidative decarboxylation mechanism and

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the currently the the cofactor to do

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that is TPP but we can do that with

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hydrogen peroxide which is just the

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byproduct of the photo oxidation of