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the rate of byproduct formation increases more sharply with the increased acid concentration, indicating
an AAcl mechanism. More importantly, the acylium ion intermediate of the
glutamyl residue has been identified in
this and other studies.
In analogy to the glutamic acid-rate
profile, the aspartimide formation in
low HF concentrations is expected to
be AAc2. As the acid concentration increases, the rate of byproduct formation changes at HF >75%. However, it
is not clear whether there is a change of
mechanism from AAc2 to AAC 1. Contrary to the case with glutamic acid, the
corresponding aspartyl acylium ion has
never been identified. Further, data obtained from other studies do not support the existence of an acylium ion intermediate ( 40). For example, Olah
(29) found that: ( l) a- or P-amino
acids, including aspartic acid, did not
give acylium ions in much stronger
acids than HF; for example, super acids
at 45° C. Under the same conditions yamino acids, such as glutamic acid, exhibited acylium ion production. 2)
Aspartimide formation is sequence dependent, while side reactions of
glutamyl peptides do not appear to be
sequence dependent. (3) Side reactions
of glutamyl peptides occur at slower
rates than the cyclization to aspartimide. These data argue against
acylium ion formation from aspartic
acid. The difference may be caused
by the greater charge separation of
the aspartyl acylium ion (+NH3CH(COOH)CH2CO+) as compared to
(+NH3CH(COOH)CH2CH2CO+) (29,
30). Since our model tetrapeptide contains both the a- and P-amino acid
linkage of aspartyl sequence, we conclude that acylium ion formation is not
likely to occur. To clarify the situation,
we reacted the [3-aspartyl peptide (4)
under conditions similar to other model
tetrapeptides (Table 5). It was anticipated that in HF, aspartimide could
only be formed by an AAc2 mechanism, in which either the a-benzyl ester
or the protonated a-benzyl ester a-carboxyl group was protonated. The formation of an acylium ion has not been
observed with a-carboxylic acids or
esters. Indeed, aspartimide was obtained from 14. These results, together
with literature data, appear to diminish
the likelihood of the AAc l mechanism
for aspartimide formation in strong
acid. An alternative explanation is
needed for the rate change of aspartimide formation in concentrated HF
solutions. A plausible explanation is
that aspartimide formation remains
AAc2 under these conditions, and the
rate change reflects the stability of the
dication 23 in the rate-determining step
Figure 12). Further, there is an increased tendency towards protonation
of the side chain carboxyl as the acidity
increases. Finally, it is clear from our
results that 0-protonation of the AspGly amide bond predominates over Nprotonation. Attack on the amide
nitrogen is only possible with the
Our results support the conclusion
the cyclohexyl ester is a suitable
protecting group for the synthesis of
Figure 12. Proposed AAc2 mechanism for aspartimide formation in strong acids.
Vol. 1. No. 1 (1988)
peptidcs containing aspartic acid. It
minimizes both base and acid catalyzed
aspartimide formation by either the
BAc2 or AAc2 mechanisms. From the
results of this work, the mechanism of
this side reaction is better understood
and can be better controlled. Furthermore. this study has provided insight
into the role of protecting groups in
aspartimide formation. It suggests a
protecting group strategy for peptide
synthesis which will minimize the formation of aspartimide.
This work was supported in part by
PHS grant DKOI260 and CA36544.
We thank Ms. Dolores Wilson and
Mrs. Rita Taylor for expert secretarial
We dedicate this paper to Professor
H. Yajima on the occasion of his retirement from Kyoto University.
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Tyrosine protecting groups: Minimization of