The mechanism of the α-ketoacid-hydroxylamine amide-forming ligation

Article abstract of DOI:10.1002/anie.201107198

Three-ring circus! Surprisingly complex molecular acrobatics are observed in the mechanism of the α-ketoacid-hydroxylamine amide-forming ligation reaction. Although this remarkable reaction can already be used for the chemoselective union of large, unprotected peptide fragments the elucidated mechanism provides important clues to extending its application to larger and more complex biological targets. Copyright

Full text of DOI:10.1002/anie.201107198

Angewandte
Chemie
DOI: 10.1002/anie.201107198
Reaction Mechanisms
The Mechanism of the a-Ketoacid–Hydroxylamine Amide-Forming
Ligation**
Ivano Pusterla and Jeffrey W. Bode*
Chemoselective amide-forming ligations are among the most
important and sought-after reactions in organic chemistry, as
they can provide synthetic access to large peptides and
proteins by the union of unprotected peptide fragments.[1] The
most successful example, the native chemical ligation of
peptide thioesters with peptides containing an N-terminal
cysteine residue, has revolutionized the field of synthetic
protein chemistry.[2] Our own efforts have identified the
combination of C-terminal a-ketoacids and N-terminal
hydroxylamine peptides as a remarkably selective and facile
ligation reaction for the formation of amide bonds.[3] It
proceeds in the presence of unprotected side chains, does not
require any reagents or catalysts, and produces only CO2 and
water as by-products. Recently, we demonstrated that the a-
ketoacid–hydroxylamine amide-forming ligation reaction
(KAHA ligation) can be used for the chemoselective syn-
thesis of therapeutic peptides (30 residues) without interfer-
ence from unprotected side chain functional groups.[4]
acyl phosphonates to give amides,[7] and Fang et al. have
reported the ligation of N-iodo amines and a-ketoacids.[8]
Although both type I and type II KAHA ligations give
identical amide products, their reaction conditions are quite
different: O-substituted hydroxylamines (type II) react faster
under aqueous conditions, whereas O-unsubstituted species
(type I) perform better in solvents such as DMF, DMSO, or
MeOH; water is tolerated, but detrimental to the reaction
rate. On the other hand, O-unsubstituted hydroxylamines
undergo ligation at lower concentration (10 mm), whereas
many of the substituted variants require higher concentra-
tions (50–100 mm). To explain these discrepancies and to
identify ligation partners ideally suited for the synthesis of
larger peptides and proteins, we have elucidated the mech-
anism of the type I ligation. Herein we present the results of
these studies, showing that the KAHA ligation of O-
unsubstituted hydroxylamines follows a remarkably complex
and unexpected reaction pathway.
We have identified two prototypical variants of this amide
formation: type I, the reaction of a-ketoacids with N-alkyl
hydroxylamines, in which the hydroxy group is unmodified;
and type II, the reaction of a-ketoacids with O-substituted
hydroxylamines, such as O-benzoyl (Bz) hydroxylamines[5]
and isoxazolidines (Scheme 1).[6] At least two other examples
of type II ligations have been documented: Phanstiel and co-
workers have reported that O-Bz hydroxylamines ligate with
At the outset of our studies, we considered six possible
mechanistic pathways for amide formation from an a-
ketoacid
and
an
O-unsubstituted
hydroxylamine
(Scheme 2). Path A involves nitrone II, an intermediate we
have occasionally detected, followed by decarboxylation to
give a nitrilium ion (akin to a Ritter amidation).[9] This
mechanism has also been postulated by Sucheck et al. in
related investigations.[10] Path B, which we proposed in our
original report,[3] would proceed via oxidative decarboxyla-
tion of hemiaminal I, in analogy to the known reaction of a-
ketoacids with hydrogen peroxide.[11] Paths C and C’ would
proceed via oxazetidinone III, an intermediate recently
detected by Yamamoto and Payette in an oxidative decar-
boxylation of a-hydroximino esters.[12] Paths D and E would
proceed via an oxaziridine IV, although the details of both the
decarboxylation and the formation of the oxaziridine were
unclear.
We recognized that many of these pathways could be
excluded by isotopic labeling studies. We therefore prepared
the necessary 18O-labeled substrates to perform the experi-
ments shown in Table 1. Much to our surprise, the oxygen
atom of the amide product originated from the hydroxyl-
amine (entry 3) rather than from the ketone of the a-
ketoacids (entry 2) or H218O (entry 1). An experiment with
phenyl ester 1 further disfavored the oxazetidinone pathway
(entry 4).[13] Taken together, these results excluded all of the
pathways except Path E for type I ligations and implicated
oxaziridine IV as a key intermediate in amide formation. In
contrast, an 18O label in the ketoacid was retained when O-Bz
hydroxylamines were used (entry 5), implicating pathway B
or D for type II ligations.
Scheme 1. Prototypical KAHA ligations with O-unsubstituted (type I)
and O-substituted (type II) hydroxylamines. Bz=benzyl.
[*] I. Pusterla, Prof. Dr. J. W. Bode
Laboratorium fꢀr Organische Chemie, Departement Chemie und
Angewandte Biowissenschaften, ETH-Zꢀrich
Wolfgang Pauli Strasse 10, 8093 Zꢀrich (Switzerland)
E-mail: bode@org.chem.ethz.ch
Homepage: http://www.bode.ethz.ch
[**] This work was supported by the Swiss National Science Foundation
(200021_131957).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201107198.
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Scheme 2. Possible mechanistic pathways for type I KAHA ligations.
Table 1: Exclusion of pathways and mechanistic probes.
Entry
Label transfer
0%
Path excluded
A
1
2
3
0%
B, C, C’, D
>80%
A, B, C, C’, D
4
5
–
C
>80%
A, C, C’, E
A
survey of the literature
revealed that at least one a-oxazir-
idinyl acid has been described,[14] and
we sought to prepare and isolate such
an intermediate to probe its ability to
undergo an amide-forming oxidative
decarboxylation. Potassium carbox-
ylate 2 was prepared as shown in
Scheme 3 and proved to be a stable,
easily handled solid. Upon treatment
of this salt with 1.0 equivalent of
trifluoacetic acid (TFA), clean decar-
Scheme 3. Synthesis and rearrangement of a-oxaziridinyl acid 2. mCPBA=m-chloroperoxybenzoic
boxylation occurred to give amide 3. acid, TMS=trimethylsilyl, TFA=trifluoroacetic acid.
514
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Chemie
Eyring analysis (see Supporting Information) revealed a
highly ordered transition state with an activation entropy
(DS°) of À36.5 calmolÀ1 KÀ1).
The finding that a-oxaziridinyl acids are the probable
intermediates in the ligation leaves the question of how they
are formed under the reaction conditions. Direct cyclization
of hemiaminal I, as shown in Path E, seems unfeasible.
Furthermore, we have noted that amide formation also
proceeds cleanly from nitrone II and that the rate of amide
formation from the nitrone does not change, even in the
presence of an additional 20 equivalents of H2O. Also, both
E- and Z-nitrones undergo the rearrangement, albeit at
slightly different rates. These results imply that oxaziridine IV
arises through the formation and rearrangement of nitrone II.
Although such rearrangements are known under photochem-
ical conditions,[15] this ligation proceeds cleanly in the dark.
We therefore postulated a mechanism for the nitrone–
oxaziridine rearrangement unique to the a-nitrone acids:
attack of the carboxylate to give an a-lactone[16] followed by
opening of this intermediate by the hydroxylamine to give
oxaziridine IV. Decarboxylation of this intermediate gives the
observed amide product (Scheme 4).
Scheme 5. Overall mechanism for type I KAHA ligation.
ates; nitrones and oxaziridines can arise only from a-
ketoacids and hydroxylamines. However, this mechanism
also points to a fundamental limitation of the type I ligation.
It is unlikely that it will be fully compatible with the aqueous
conditions usually required for protein synthesis and semi-
synthesis. These studies therefore suggest that type II liga-
tions with O-substituted hydroxylamines, which may follow a
different mechanism and do not proceed via a nitrone
intermediate, are likely to be better suited for this purpose.
We have adjusted our investigations accordingly and will
report a new class of ligation partners that better tolerate
aqueous conditions and operate at low reaction concentra-
tions in due course.
Scheme 4. Proposed pathway from nitrone to oxaziridine via an a-
lactone intermediate.
Although not common intermediates, a-lactones have
been implicated in related a-carboxyl displacements, includ-
ing the facile hydrolysis of a,a-dichloro acids.[17] Extensive
mechanistic studies carried out by Williams and co-workers
have shown that a-lactones are intermediates in the addition
of aqueous bromide to disodium dimethylmaleate and
dimethylfumarate.[18] Importantly, both the formation of a-
lactone V (3-exo-trig) and its opening to give oxazidirine IV
(3-exo-tet) are favored by Baldwinꢀs rules; the direct for-
mation from the nitrone (3-endo-tet) is not.[19]
Received: October 11, 2011
Published online: November 29, 2011
These investigations support the overall mechanism for
amide formation from a-ketoacids and unsubstituted hydrox-
ylamines shown in Scheme 5. This mechanism requires the
formation of a nitrone—a process that we had previously
considered to be a non-productive pathway and sought to
avoid—and is likely the reason why water is often detrimental
to this particular ligation. The precise mechanism of the
ligation with O-substituted hydroxylamines remains open.
Either direct oxidative decarboxylation (Path B) or formation
of the oxaziridine by nucleophilic displacement at nitrogen to
give the same oxaziridine intermediate IV (Path D) are
possibilities. Further studies to determine the exact mecha-
nistic pathway are underway.
Keywords: amides · isotopic labeling · ligation reactions ·
peptides · reaction mechanisms
.
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