Exploiting Protein N-Terminus for Site-Specific Bioconjugation

Abstract

Although a plethora of chemistries have been developed to selectively decorate protein molecules, novel strategies continue to be reported with the final aim of improving selectivity and mildness of the reaction conditions, preserve protein integrity, and fulfill all the increasing requirements of the modern applications of protein conjugates. The targeting of the protein N-terminal alpha-amine group appears a convenient solution to the issue, emerging as a useful and unique reactive site universally present in each protein molecule. Herein, we provide an updated overview of the methodologies developed until today to afford the selective modification of proteins through the targeting of the N-terminal alpha-amine. Chemical and enzymatic strategies enabling the selective labeling of the protein N-terminal alpha-amine group are described.

Keywords: aldehyde protein; azide protein; chemical ligation; chemo-selective reaction; click-chemistry; molecular probe; protein labeling.

Figure 1

Selective protein labeling through the direct targeting of the N-terminal alpha-amine group. (a) Reductive amination with a benzaldehyde probe; (b) imine formation and intramolecular 6-endo-dig cyclization with a 2-ethynylbenzaldehyde (2-EBA) probe; (c) phthalimidation with a N-hydroxy-phthalimide (NHP) probe; (d) aldehyde capture ligation with a selenobenzaldehyde (SBA) ester probe; (e) imine condensation with a 2-pyridinecarbaldehyde (2-PCA) probe.

Figure 2

Selective targeting of N-terminal cysteinyl proteins. (a) Native chemical ligation with a thioester probe; (b) condensation with a 2-cyanobenzothiazole (CBT) probe; (c) thiazolidine ligation with an aldehyde probe; (d) condensation with a 2-formyl phenylboronic acid (2-FPBA) probe.

Figure 3

Site-specific protein labeling through the targeting of N-terminal Ser (or Thr) (a), Trp (b), Pro (c), Gly (d), or Gly-His_n (e).

Figure 4

Two-step N-terminal protein labeling. (a) Transamination reaction with pyridoxal-5′-phosphate (PLP) or with N-methylpyridinium-4-carboxaldehyde benzenesulfonate salt (Rapoport’s salt, RS), leading to a N-terminal ketone protein; (b) oxidation with sodium periodate (NaIO₄) of 1,2 amino alcohol (N-terminal Ser or Thr), affording a N-terminal aldehyde protein. The ketone/aldehyde function installed at the protein N-terminus can be further reacted with an aminooxy-probe via oxime ligation.

Figure 5

Two-step N-terminal protein labeling. (a) N-terminal alpha-amine group can be converted into an azide group by diazotransfer with imidazole-1-sulfonyl azide (I-1-SA); (b) acylation with phenyl ketene (PK) allows for the introduction of a N-terminal alkyne group. After the selective introduction of an alkyne or an azide function at the protein N-terminus, the protein can be further modified through copper-catalyzed [3 + 2] cycloaddition (CuAAC) click chemistry.

Figure 6

Enzyme-catalyzed N-terminal protein labeling. N-terminal protein modification mediated by (a) sortase A (SrtA); (b) subtiligase; (c) N- Myristoyltransferase (NMT); (d) butelase 1; (e) protein trans-splicing (PTS). Aa, amino acid; CoA, coenzyme-A; N-Int, N-terminal domain of a split intein; C-Int, C-terminal domain of a split intein.