Researcher(s)
- Benjamin Brenner, Biochemistry, University of Delaware
Faculty Mentor(s)
- Joseph Fox, Chemistry and Biochemistry, University of Delaware
Abstract
Tetrazine trans-Cyclooctene (Tz-TCO) ligation was developed in 2008 by the Fox Group and remains the fastest known reaction in bioorthogonal chemistry with a rate constant of 104-106 M-1s-1. During this reaction, electron-rich tetrazine undergoes a 3+2 cycloaddition (Inverse Electron Demand Diels Alder) with TCO, an electron-poor dienophile. This intermediate is followed by a retro Diels Alder reaction, releasing inert nitrogen gas. Many derivatives of TCO are made from (E)-cyclooct-4-enone (Keto TCO), which is synthetically accessible from 5-hydroxy-1-cyclooctene using Swern oxidation and a flow photoisomerization apparatus. This work explores the further functionalization of keto TCO using alpha alkylation. Alpha alkylation of carbonyls proceeds as follows: A strong base is used to deprotonate the alpha carbon, forming an enolate. The enolate proceeds to react with a brominated substituent by SN2. Due to the conformation assumed TCO, this alkylation is stereospecific. Previous experimentation with alpha alkylation using Lithium bis(trimethylsilyl)amide at -78॰C showed incomplete enolate formation. At 0॰C with inverse addition, kinetic enolate formation proceeds, but some thermodynamic enolate still forms as a byproduct. Further addition of a variety of bromyl alkylating agents above 1 equivalent allows for dialkylation products. Alkylithium nucleophilic attack products are also observed to form when allowed to warm to room temperature. An updated method limits byproducts to kinetic and thermodynamic mono alkylated products at a 5:1 ratio. These products (unlike mono and dialkylated TCO) are separable by traditional flash column chromatography in place of HPLC.