Neprirodne aldolne adicije fosfoenolpiruvata na alifatske i aromatske aldehide katalizirane PEP-ovisnim sintazama

master's thesis

• Title (english): Promiscuous aldol additions of phosphoenolpyruvate to aliphatic and aromatic aldehydes catalysed by PEP-dependent synthases
• Author: Karlo Borović
• Mentor: Zvjezdana Findrik Blažević (mentor)
• Committee member: Zvjezdana Findrik Blažević (predsjednik povjerenstva)
• Committee member: Martina Sudar (član povjerenstva)
• Committee member: Marija Vuković Domanovac (član povjerenstva)
• Granter: University of Zagreb; Faculty of Chemical Engineering and Technology; Zagreb
• Defense date and country: 2024-09-04, Croatia
• Scientific / art field, discipline and subdiscipline: TECHNICAL SCIENCES; Chemical Engineering

Abstract

Stvaranje C-C veze ključna je u sintetskoj organskoj kemiji jer povezuje manje podjedinice u složenije molekule. Klasična sinteza zahtijeva sofisticirane asimetrične metode i zaštitu funkcionalnih skupina. Biokatalitička kondenzacija postaje sve zanimljivija jer omogućava dobivanje sterički čistih molekula bez potrebe za zaštitom funkcionalnih skupina. Ovaj rad istražuje neprirodne aldolne adicije fosfoenolpiruvata (PEP) na alifatske i aromatske aldehide, katalizirane PEP-ovisnim aldolazama, fokusirajući se na enzime DHAPH i KDOH iz metagenomske zbirke. Cilj rada bio je optimizirati reakcijske uvjete i ispitati konverziju i enantioselektivnost ovih enzima kako bi se utvrdila njihova primjenjivost u formiranju C-C veze. U eksperimentalnom dijelu, istraženo je 38 enzima iz metagenomske zbirke s različitim aldehidima. Najbolji rezultati postignuti su s enzimom KDOH i aldehidom fenilacetaldehidom, gdje su konverzije dosegle do 92,7 %. Posebno su se istaknuli karbamatni spojevi, s konverzijama koje su prelazile 56,6 %. Optimizacija udjela dimetilformamida (DMF) pokazala je da smanjenje DMF-a povećava enzimske aktivnosti, ali je njegovo potpuno uklanjanje nepoželjno zbog netopivosti aldehida u vodi. DMF udio od 5 % naspram 20 % doveo je do značajnog povećanja konverzije, čak do 44,9 %. Daljnja ispitivanja pročišćenih enzima pokazala su niže stope konverzije u usporedbi s početnim probirom, a neki od razloga mogu biti gubitk stabilizirajućih faktora ili parcijalne denaturacije enzima tijekom pročišćavanja. Također, enantioselektivnost se nije mogla jasno odrediti zbog niske aktivnosti enzima i formiranja nusprodukata umjesto ciljanih produkata reakcije. DHAPH i KDOH enzimi imaju potencijal za daljnju primjenu u formiranju C-C veze, ali su potrebna dodatna istraživanja i optimizacija reakcijskih uvjeta kako bi se postigli zadovoljavajući rezultati.

Abstract (english)

The formation of C-C bonds is crucial in synthetic organic chemistry as it connects smaller subunits into more complex molecules. Sophisticated asymmetric methods and protection of functional groups are required in classical synthesis. Biocatalytic condensation is becoming increasingly interesting as it allows the production of sterically pure molecules without the need for functional group protection. Unnatural aldol additions of phosphoenolpyruvate (PEP) to aliphatic and aromatic aldehydes, catalyzed by PEP-dependent aldolases, are investigated in this study, focusing on DHAPH and KDOH enzymes from a metagenomic library. The aim was to optimize reaction conditions and examine the conversion and enantioselectivity of these enzymes to determine their applicability in C-C bond formation.
In the experimental part, 38 enzymes from the metagenomic library were tested with different aldehydes. The best results were achieved with the KDOH enzyme and phenylacetaldehyde, with conversions reaching up to 92,7 %. Carbamate compounds were particularly notable, with conversions exceeding 56,6 %. Optimization of dimethylformamide (DMF) content showed that reducing DMF increases enzyme activities, but its complete removal is undesirable due to the insolubility of aldehydes in water. A DMF content of 5 % compared to 20% led to a significant increase in conversion, up to 44,9 %. Further testing of purified enzymes showed lower conversion rates compared to initial screening, possibly due to the loss of stabilizing factors or partial denaturation of enzymes during purification. Additionally, enantioselectivity could not be clearly determined due to low enzyme activity and the formation of by-products instead of target reaction products. DHAPH and KDOH enzymes have potential for further application in C-C bond formation, but additional research and optimization of reaction conditions are needed to achieve satisfactory results.

• Keywords: C-C veza; aldolne adicije; PEP-ovisne aldolaze; PEP; DHAPH; KDOH
• Keywords (english): C-C bond; aldol addition; PEP-dependant synthases; PEP; DHAPH; KDOH
• Language: croatian
• URN:NBN: urn:nbn:hr:149:714458
• Study programme: Title: Applied Chemistry - Graduate study; specializations in: Environmental chemistry and green technologies, Advanced materials and technologies, Applied organic chemistry; Course: Applied organic chemistry; Study programme type: university; Study level: graduate; Academic / professional title: sveučilišni/a magistar/magistra primijenjene kemije (sveučilišni/a magistar/magistra primijenjene kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2026-09-01
• Created on: 2024-09-12 09:12:17