Noncanonical amino acids as doubly bio-orthogonal handles for one-pot preparation of protein multiconjugates

Abstract

Genetic encoding of noncanonical amino acid (ncAA) for site-specific protein modification has been widely applied for many biological and therapeutic applications. To efficiently prepare homogeneous protein multiconjugates, we design two encodable noncanonical amino acids (ncAAs), 4-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (pTAF) and 3-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (mTAF), containing mutually orthogonal and bioorthogonal azide and tetrazine reaction handles. Recombinant proteins and antibody fragments containing the TAFs can easily be functionalized in one-pot reactions with combinations of commercially available fluorophores, radioisotopes, PEGs, and drugs in a plug-and-play manner to afford protein dual conjugates to assess combinations of tumor diagnosis, image-guided surgery, and targeted therapy in mouse models. Furthermore, we demonstrate that simultaneously incorporating mTAF and a ketone-containing ncAA into one protein via two non-sense codons allows preparation of a site-specific protein triconjugate. Our results demonstrate that TAFs are doubly bio-orthogonal handles for efficient and scalable preparation of homogeneous protein multiconjugates.

Non-canonical capsid engineering highlights new possibilities for AAV vectorology

“…This is in contrast to the majority of previous studies, which have employed ncAAs as “molecular handles” to facilitate the development of antibody-drug conjugates through click chemistry reactions, utilizing biorthogonal functional group such as azides. 4 , 5 , 6

Figure 1 Schematic describing applications of AAV engineering with ncAAs, including that described by Chang et al., versus historical engineering strategies focused on bioconjugation

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The Application of Bio-orthogonality for In Vivo Animal Imaging

“…In addition, the tissue penetrance of NIRF is considerably high. Bio-orthogonal chemistry has been used for NIRF imaging via several strategies: − (1) incorporation of noncanonical amino acids (ncAAs) with alkyne or azide tags that can be conjugated with a fluorophore via bio-orthogonal reactions; (2) pretargeting that uses a molecule with a clickable tag to bind to the target of interest; (3) incorporation of clickable tags into glycans and lipids. The ncAA strategy has been widely used for cell imaging, and the pretargeting and glycan/lipid tagging strategies have been used for in vivo animal imaging.…”

“…Remarkably, Wang et al recently demonstrated that it was feasible to have two orthogonal tags in one ncAA. In this study, they demonstrated that 4-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (pTAF) and 3-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (mTAF) could be incorporated into recombinant proteins and antibody fragments . This method could enable combinations of commercially available fluorophores, radioisotopes, poly­(ethylene glycol) (PEG), and drugs in a plug-and-play manner in one pot.…”

Bioorthogonal Chemistry in Translational Research: Advances and Opportunities

“…[46] (b) Schematic of dual labeling of HER2-(scFv) antibody with FITC-tagged TCO and CY5-tagged DBCO via IEDDA and SPAAC reactions, respectively. [47] (c) Functionalization of Trastuzumab-Im10-TAMRA conjugate and bioorthogonal cleavage induced by cyclooctyne B. [48] Recently, Yang and Liu showed double labeling of antibodies by incorporating clickable azide and Tz handles into multiple non-canonical amino acids to carry out azide-cyclooctyne cycloaddition.…”

“…Yang and Liu introduced another design strategy for the preparation of homogeneous protein multiconjugates using two genetically encodable ncAAs: 4-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (pTAF) and 3-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (mTAF). [47] These azide and tetrazine containing ncAAs participated in IEDDA as well as SPAAC reactions in a single reaction vessel without interfering with each other (Figure 6b). They demonstrated that recombinant proteins and antibody (Her2) fragments containing the TAFs could be easily functionalized in one-pot reactions with commercially available fluorophores, radioisotopes, PEGs, and drugs, enabling the evaluation of tumor diagnosis, imageguided surgery, and targeted therapy in mouse models.…”