Chemistry Reference
In-Depth Information
DCC
tail fragments
(
4
-
22
)
SH
S
H
SH
S
+
S
SH
+
1
/
2
O
2
, -H
2
O
S
S
dithiol DCC
anchor fragment
(
F
,
G
,
H
,
I
or
J
)
Zn
II
Zn
II
Zn
II
Zn
II
Zn
II
Zn
II
BcII
BcII
BcII
DCC anchor fragments
CO
2
H
CO
2
H
CO
2
H
CO
2
H
CO
2
H
HS
HS
SH
SH
HS
HS
HS
HS
SH
G
SH
F
H
I
J
SH
SH
SH
R
3
N
N
X
N
R
HS
SH
H
N
H
R
2
16
(77) R
=
NH
2
17
(106) R
=
CO
2
H
18
(94) R
=
SH
N
N
R
1
13
(111)
14
(152)
15
(150)
4
(110) R
1
=
R
2
=
R
3
=
H, X
=
CH
5
(154) R
1
=
CO
2
H, R
2
=
R
3
=
H, X
=
CH
6
(154) R
1
=
R
3
=
H, R
2
=
CO
2
H, X
=
CH
7
(154) R
1
=
R
2
=
H, R
3
=
CO
2
H, X
=
CH
8
(125) R
1
=
NH
2
, R
2
=
R
3
=
H, X
=
CH
9
(125) R
1
=
R
3
=
H, R
2
=
NH
2
, X
=
CH
10
(125) R
1
=
R
2
=
H, R
3
=
NH
2
, X
=
CH
11
(155) R
1
=
CO
2
H, R
2
=
R
3
=
H, X
=
N
12
(155) R
1
=
R
2
=
H, R
3
=
CO
2
H, X
=
N
SH
HO
2
C
SH
SH
SH
H
2
N
CO
2
H
19
(76)
20
(168)
21
(124)
22
(121)
Scheme 7.4
Anchor fragments equipped with two thiol moieties (
F
-
J
) and thiol partner frag-
ments
(4-22)
for DCC utilizing thiol-disulfide exchange and targeting metallo-
β
-lactamase.
The composition of the DCL accessible with thiol-disulfide exchange is complicated by
the non-orthogonal nature of thiol/dithiol reactivity, which permits more permutations of
reaction fragment combinations and also the potential for oligomerization compared with
orthogonal reversible reactions such as hydrazone exchange. Schofield and co-workers
minimized this complication by using controlled reaction conditions together with fast MS
analysis as described next. The DCC experiments were prepared in an oxygen-free atmo-
sphere (
<
1 ppm O
2
) to limit the occurrence of thiol-disulfide exchange prior to the anchor
fragments binding to the active site zinc cations of BcII. Individual thiols
(4-17, 19-22)
and
dithiol
(18)
stock solutions (100 mM) were freshly dissolved in DMSO and then diluted to
75 M with aqueous NH
4
OAc (15 mM, pH 7.5). Similarly, solutions of the anchor frag-
ments (
F-J
) were prepared at a final concentration of 100 MinNH
4
OAc (15 mM, pH
7.5). The fragment solutions were combined as appropriate with the target enzyme BcII to
give a final
in situ
DCL that consisted of BcII (15 M), anchor fragment dithiol (one each of
F-J
,30M) and complementary fragments (
4-24
10 M each), in 15 mMNH
4
OAc at pH
7.5 (Scheme 7.4). An aliquot of each DCL experiment was placed in a 96-well microtitre
