Chemistry Reference
In-Depth Information
O
H
N
Intein
NH
2
Target protein
S
-
O
Peptide
SH
O
NS acyl shift
O
O
O
N
NH
2
PhSH
transthio-
esterication
S
S
Intein
Peptide
NCL
Target protein
Target protein
Target protein
O
O
SH
scheme 2.23
Synthesis of protein-peptide conjugate via expressed protein ligation.
O
O
NH
2
S
Intein
Protease substrate
r.t., 2 h
NHNH
Qdot
Luciferase
Protease substrate
Luciferase
O
Qdot
NHNH
2
scheme 2.24
Construction of quantum dot-protein conjugates via a modified intein-mediated ligation.
nanosensor to detect matrix metalloproteinase (MMP) activity [258]. The attachment of luciferase and protease substrate
fusion protein to Qds was achieved through replacement of intein fused to the target protein by hydrazides displayed on the
surface of Qds (Scheme 2.24). The reaction using hydrazide-Qd was very efficient, as all the Qds were modified with luci-
ferase in 2 hrs at room temperature.
2.3.5
Introduction of chemical tags for site-specific labelling on peptides or proteins
2.3.5.1 Introduction of Small Functional Group Tags
Introduction of Ketone or Aldehyde Tags
To achieve site-specific labelling of proteins, approaches have been developed to
introduce aldehydes/ketones and aminooxy or hydrazide functionalities to proteins through genetic encoding, enzymatic
labelling, and chemical reactions [259, 260]. Sodium periodate oxidation has been used frequently in the past to generate
aldehyde functionalities on glycans or n-terminal serines or threonines [9]. The newly generated aldehyde groups on the
proteins or glycoproteins can then be labelled selectively via oxime/hydrozone ligation to introduce functional molecules,
such as Peg, fluorophores, and other proteins or peptides [261, 262]. Introduction of ketone groups using a cell's own
machinery has been well utilised by the bertozzi group in labelling cell surface proteins [30]. using a metabolic labelling
approach, monosaccharides with modified functional handles could enter the metabolic pathway to generate glycoproteins
bearing these handles, including, but not limited to, keto, azido, alkynyl, alkanyl, crotonoyl, and thiol groups [263-266]. It
has been proven as a valuable approach to labelling cell surface proteins, specifically at the sialic acid residues of the glycan,
in live mammalian cells and in living organisms.
Schultz and co-workers demonstrated the first genetic encoding of keto amino acids
p
-acetyl-L-phenylalanine and
m
-acetyl-
L-phenylalanine into proteins expressed in
E. coli
via amber suppression method, and applied these groups for selective label-
ling of proteins with fluorescent dyes both
in vitro
and in living
E. coli
[267-270]. In mammalian cells, ye et al. incorporated
unnatural amino acids
p
-acetyl-L-phenylalanine and
p
-benzoyl-L-phenylalanine (bzp) into chemokine receptor CCR5 and
rhodopsin at three specific sites with high efficiency, allowing their modification with different reagents [271]. More recently,
bertozzi and co-workers incorporated aldehyde tags via genetic introduction of formylglycine to the target protein, allowing
further site-specific modification of the protein through reactions of aldehyde, such as oxime/hydrazone ligation [259, 272].
In an improved procedure of site-directed spin labelling, genetically encoded unnatural amino acid
p
-acetyl-L-phenylalanine
was introduced to mutants of T4 lysozyme, allowing further labelling with hydroxylamine reagents [273]. Robust protein
farnesyltransferase was able to incorporate an aldehyde-containing substrate analogue to a protein, which was subsequently
immobilised onto aminooxy-functionalised agarose beads or labelled with a fluorophore [274]. An F-18 radiolabel was intro-
duced to a mouse hormone protein leptin via the oxime ligation, wherein the aminooxy group was incorporated at the C-terminus
of leptin using expressed protein ligation, followed by aniline-catalysed oximation reaction with [F-18]fluorobenzaldehyde.
After modification, the hormone was biologically active both
in vitro
and
in vivo
and was applied to positron emission tomog-
raphy (PeT) in ob/ob mice [257]. In a different approach, the aminooxy group was efficiently introduced to protein through
chemical substitution of C-terminal thioacid [275] by a 1,2-bis(oxyamino)ethane under mild conditions (pH 7.4 buffer) [276].
