Chemistry Reference
In-Depth Information
OH
OH
H
O
HO
O
S
HO
S
H
HO
S
C 91
OH
OH
OH
S
S
H
74
O
H
HO
S
S
(33 eq.)
C
75
OH
λ
max
365 nm, DPAP, 5 min
DMSO (5% v/v)-phosphate buffer (pH 7.4)
OH
H
O
H
HO
HS
C 34
S
S
OH
75
BSA,
72
OH
O
O
HO
O
O
HO
2
C
NH
HO
OH
HN
S
HO
CO
2
H
O
HO
N
S
S
HN
N
SH
S
S
S
HO
76
HN
(160 eq.)
S
CO
2
H
S
O
λ
max
365 nm, DPAP, 5 min
DMSO (5% v/v)-phosphate buffer (pH 7.4)
S
OH
O
O
OH
S
S
HO
HO
S
HN
H
S
77
O
CO
2
H
OH
HO
HO
O
OH
O
HO
SCHEME 3.25
Glycosylation and fluorescein labelling of bovine serum albumin (BSA) via
sequential TYC and TEC.
so-called copper-free SPAAC approach developed by Bertozzi, Boons [3] and their
coworkers. Actually, this turned out to be the case. In fact, it was demonstrated that
a dibenzocyclooctyne derivative reacted with BSA in the
presence
and also in the
absence
of light. It was inferred that this side reaction might constitute a limitation
on the use of cycloalkynes as partners of SPAAC in
in vivo
experiments due to the
presence of cysteine-containing proteins.
Synthesis of glycoprotein
77
. A solution of
74
(3.5 mg, 15.0
mol) and DPAP
(0.3 mg, 1.3
mol) in DMSO (150
L) was slowly added to a solution of commercial
BSA
72
(30 mg, 0.46
mol) in 20 mM phosphate buffer at pH 7.4 (3 mL). The
mixture was irradiated (
max
365 nm) at room temperature for 5 minutes under
magnetic stirring, then filtered twice through Ultrafree-MC microcentrifuge filter
(nominal MW limit of 5000 Da) for 30 minutes at 5000 rpm. A solution of
76
(34 mg, 73
L) was added
to a solution of the protein glycoconjugate
75
in 20 mM phosphate buffer at pH
7.4 (3 mL). The mixture was irradiated again (
mol) and DPAP (1.9 mg, 7.3
mol) in DMSO (150
max
365 nm) at room temperature
for 10 minutes under magnetic stirring, then filtered twice through Ultrafree-MC
microcentrifuge filter (nominal MW limit of 5000 Da) for 30 minutes at 5000 rpm to
give
77
.
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