Chemistry Reference
In-Depth Information
N
3
N
3
32
33
NO
2
NH[CH
2
]
4
CH
NO
2
NH[CH
2
]
4
CH
CO
2
-
CO
2
-
NH
3
+
NH
3
+
SCHEME 3.16.
mechanistic shift might affect the interpretation of PAL studies is provided by the
PAL labeling of antibodies for the photo-precursor of aryl nitrenes.
95-97
In these
studies, rabbit antibodies for
32
(Scheme 3.16). This antibody-hapten complex was then irradiated and covalent
bonding between the two was determined to have occurred adjacent to the antibody
hypervariable region of the heavy protein chain possibly at cysteine
91
and alanine
93
to the extent on about 13%.
96
The exact sites of the cross-links were not rigorously
established, and might not be completely accurate, since cross-linking to alanine is
surprising. The azide
32
were generated and bound to their hapten
is a nitrenium ion precursor, if proton sources are
available,
52
and alanine is not a nucleophilic amino acid that one would expect
to react with nitrenium ions. Therefore, one must assume that (i) proton sources are
not readily available in this bonding site, or (ii) the amino acid residue to which
cross-linking occurs is misassigned. Another problem arises in an extension of this
study in which the modified azide
32
33
was reacted with the same rabbit antibody used
with
on the assumption that the slightly different orientation of the nitrene (azide)
group might facilitate bonding to different amino acid residues in the same antibody
binding site.
98
In apparent confirmation of this hypothesis, the ratio of heavy to light
antibody chain attachments was found to be quite different, 2.5-5.0
96,97
with
32
32
and
1.71 with
. Unfortunately, no detailed analysis of the amino acid residues to which
33
had become attached was reported. Since
33
is not a nitrenium ion precursor, the
assumption that the two nitrenes will have comparable reactivities is not justified.
From these results, it appears that
33
, and
that both the differences in reactivity, as well as, the positioning of the reactive
nitrogen species governs the cross-linking processes with the antibody active site.
Since nitrenium ion formation will only occur with appropriately subsituted aryl
azides if the precursor nitrenes have access to fairly acidic proton sources such as
water, alcohols, amine salts, and thiols, and so on, studies with nitrenium ion PAL
agents in lipids environments are particularly interesting. In several studies, neuronal
plasma membrane gangliosides have been modified with the appropriate nitrophenyl
azide PAL units on both the hydrophobic and hydrophilic ends,
33
is reacting more indiscriminately than
32
99-101
and
,
102
34
35
respectively (Scheme 3.17). The PAL unit
might be expected to become
embedded in the hydrophobic core of the membrane where proton sources are
scarce, and thus, react mostly via conventional nitrene chemistry rather than
nitrenium ion chemistry. Nitrenes in such a hydrophobic environment might become
cross-linked to membrane-embedded proteins or perhaps even unsaturated hydro-
carbon chains via hydrogen atom abstraction and radical coupling. Alternatively, the
34
