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
[**] This work was supported by the Swiss National Science Foundation
(200021_131957).
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2012, 51, 513 –516
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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