4.2 Bioorthogonal Chemistry
109
N
N
Ph
N
NH
O
Ph
PhH
80
o
C, 22 h
EtO 2 C
Cl
N
NH
MeO
Et 3 N, CHCl 3
60
o C, 16 h
Ph
N
N
EtO 2 C
OMe
DCM
rt, 0.5 h
79 %
over 3 steps
TFA
N
N
Ph
N N
CO 2 Et
MeO
Scheme 4.7 The use of a solid support in the NI-mediated synthesis of pyrazoles
Lin introduced the concept of protein conjugation via NI cycloaddition in 2008
[35]. A 2,5-tetrazole moiety was incorporated into lysozyme and green fluorescent
protein through simple amidation chemistry, before the formation of a pyrazoline
adduct via treatment of the proteins with acrylamide following liberation of the
NI. Later the same year, in a separate report, Lin employed the 2,5-tetrazole as the
ligating agent. Incorporation of O-allyltyrosine into Z-domain protein furnished the
augmented dipolarophile, which was rapidly modified when exposed to the relevant
NI (Scheme 4.8) [32]. One of the immediately apparent advantages of photoclick
O
O
N N
CO 2 Me
N
N
N N
CO 2 Me
h
PBS buffer, DMSO
rt, 10 min
Z-domain
protein
Z-domain
protein
fluorescent
Scheme 4.8 Incorporation of a dipolarophile within a protein and its subsequent ligation using NI
chemistry