Site-specific labeling of proteins and peptides with trans-cyclooctene containing handles capable of tetrazine ligation

Abstract

There is a growing library of functionalized non-natural substrates for the enzyme protein farnesyltransferase (PFTase). PFTase covalently attaches these functionalized non-natural substrates to proteins ending in the sequence CAAX, where C is a cysteine that becomes alkylated, A represents an aliphatic amino acid, and X is Ser, Met, Ala, or Gln. Reported substrates include a variety of functionalities that allow modified proteins to undergo subsequent bioconjugation reactions. To date the most common strategy used in this approach has been copper catalyzed azide-alkyne cycloaddition (CuAAC). While being fast and bioorthogonal CuAAC has limited use in live cell experiments due to copper's toxicity.(1) Here, we report the synthesis of trans-cyclooctene geranyl diphosphate. This substrate can be synthesized from geraniol in six steps and be enzymatically transferred to peptides and proteins that end in a CAAX sequence. Proteins and peptides site-specially modified with trans-cyclooctene geranyl diphosphate were subsequently targeted for further modification via tetrazine ligation. As tetrazine ligation is bioorthogonal, fast, and is contingent on ring strain rather than the addition of a copper catalyst, this labeling strategy should prove useful for labeling proteins where the presence of copper may hinder solubility or biological reactivity.

Keywords: bioorthogonal; inverse-electron-demand Diels-Alder; protein farnesyltransferase; protein prenylation; tetrazine ligation; trans-cyclooctene.

Figure 1

Farnesyl diphosphate (1), trans-cyclooctene geranyl diphosphate (2), dipyridyl-tetrazine (3), and benzylamino-tetrazine (4).

Figure 2

A Fluorescence-based enzyme assay was used to calculate the rate for enzymatic modification using different concentrations of 2. The plot of rate versus substrate concentration showed that compound 2 was a substrate for PFTase with a K_M,app of 0.76 μM. Each measurement was performed in triplicate and the average plotted with the SD indicated by the error bars.

Figure 3

(A) ESI-MS of eGFP-CVIA (12). (B) Deconvoluted mass spectrum of eGFP-CVIA. (C) Deconvoluted mass spectra of eGFP-CVIA that was subjected to enzymatic modification using trans-cyclooctene geranyl diphosphate (2) and PFTase. (D) Deconvoluted mass spectra of trans-cyclooctene labeled eGFP-CVIA that underwent tetrazine ligation with benzylamino-tetrazine (4).

Scheme 1

Synthesis of trans-cyclooctene geranyl diphosphate (2).

Scheme 2

Enzymatic modification of a CAAX-box containing peptide with trans-cyclooctene geranyl diphosphate (2) and subsequent tetrazine ligation with dipyridyl-tetrazine (3).

Scheme 3

Enzymatic modification of eGFP-CVIA with trans-cyclooctene geranyl diphosphate (2) and subsequent tetrazine ligation with benzylamino-tetrazine (4).