Chemistry Reference
In-Depth Information
OH
HO
HO
OH
O
HO
OH
O
O
HO
O
O
2
N
OH
AcHN
O
O
O
-
O
2
C
HO
O
HN
N
3
HO
HN
HO
HO
O
H
O
O
AcHN
HO
HO
34
HO
H
N
3
O
2
N
HN
HO
HO
OH
O
HO
OH
O
O
HO
O
OH
AcHN
O
O
O
-
O
2
C
HO
O
HO
HN
HO
HO
O
H
O
O
AcHN
HO
HO
35
HO
H
SCHEME 3.17.
PAL unit
would position the nitrophenyl azide unit on the surface of the
membrane where proton sources should be abundant, and therefore, it might be
expected to form nitrenium ions readily and become cross-linked to proteins and
saccarides lining the membrane surface. In these studies, both
35
were labeled
with tritium in the terminal sugar unit, and cross-linking was measured by incorpo-
ration of radioactivity into membrane components. Consistent with this model shown
in Figure 3.4,
34
and
35
35
was found to afford much more efficient cross-linking than did
.
102
Furthermore, in the case of
34
34
, the amount of cross-linking was found to be
dependent on the incubation time prior to irradiation.
99
Thus, incubation of
34
with
the cells for only 2 h afforded stable incorporation of
into the cell membrane, but
very few proteins became radioactive on irradiation. On the other hand, when
34
was
incubated for 24 h before irradiation, it underwent metabolism to several derivatives
and migrated from the surface membrane of the cell into the interior of the cell where
irradiation afforded many radioactive proteins.
A similar strategy has been used to study the structure of critical membrane-bound
enzymes such as cytochrome
c
oxidase, blue unit in Figure 3.4, which is bound to a
mitochondrial inner membrane.
103
To function effectively, the cytochrome c subunits
must be bound together with two high-affinity functional and two low-affinity
structural molecules of cardiolipin, a diphosphatidylglycerol. This lipid contains
four hydrophobic octadec-9,12-dienoic acid ester chains and a hydrophilic trigly-
cerol diphosphate head group. Both the polar head group and lipophilic fatty acid
chains have been modified with aryl azide PAL agents. Two of the four hydrophobic
fatty acid chains have been appended to 4-azido-2-nitrophenyl amine PAL units that
will afford nitrenium ion in protic environments, but the availability of proton
sources in a hydrophobic interior membrane environment is problematic. Alterna-
tively, the polar head group has been appended to a 3-azido-4-nitrophenyl carbox-
amide PAL agent, which is probably not a nitrenium ion precursor, but is probing the
34
