Chemoselective amide ligations by decarboxylative condensations of N-alkylhydroxylamines and α-ketoacids

Article abstract of DOI:10.1002/anie.200503991

(Chemical Equation Presented) Additive-free: The chemoselective amide-bond-forming ligation between N-alkylhydroxylamines and α-ketoacids requires no reagents and the only by-products are water and carbon dioxide. This process proceeds on unprotected pept

Full text of DOI:10.1002/anie.200503991

Communications
Ligation Reactions
DOI: 10.1002/anie.200503991
Chemoselective Amide Ligations by
Decarboxylative Condensations of
N-Alkylhydroxylamines and a-Ketoacids**
Jeffrey W. Bode,* Ryan M. Fox, and Kyle D. Baucom
Chemoselective ligation reactions make possible covalent
bond formation between two fragments containing unpro-
tected functional groups. An ideal ligation process proceeds
[*] Prof. Dr. J. W. Bode, R. M. Fox, K. D. Baucom
Department of Chemistry and Biochemistry
University of California
Santa Barbara, CA (USA)
Fax: ( +1)805-893-4120
E-mail: bode@chem.ucsb.edu
[**] This work was supported by the Donors of the Petroleum Research
Fund (administered by the American Chemical Society), the Camille
and Henry Dreyfus Foundation (New Faculty Award to J.W.B.), and
the University of California. K.D.B. was a 2005 DeWolfe Summer
Undergraduate Fellow. We are grateful to Joshua Garretson for
preliminary efforts.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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under mild, often aqueous, conditions at low molar concen-
trations, does not require reagents or catalysts, and produces
no chemical by-products.[1] The few known reactions that
approach these criteria, including oxime formation,[2] thio-
ester-a-bromocarbonyl alkylations,[3] and the copper-pro-
moted alkyne–azide cycloaddition[4] have found diverse and
significant applications in drug discovery,[5] functionalized
polymer synthesis,[6] and the fabrication of novel nanostruc-
tures.[7] Their utility in biomolecule synthesis, however, is
limited by the fact that they produce unnatural, and often
relatively labile, covalent bonds. The development of chem-
ical ligation reactions that create amide bonds have been a
long standing goal.[8] The identification of the native chemical
ligation of C-terminal peptide thioesters and N-terminal
cysteines has revolutionized the field of synthetic protein
chemistry by making possible the coupling of large, unpro-
tected-peptide fragments.[9,10] Despite the utility of this
process, the requirement of ligation at relatively rare cysteine
residues has encouraged investigations into alternative amide
bond-forming reactions.[11,12]
Scheme 1. Reactions of N-alkylhydroxylamines with aldehydes and
ketones.
amine (2), under a variety of reaction conditions [Eq. (2),
Table 1]. Although no amide products were formed in
A significant obstacle to the development of new amide
ligations is the paucity of reaction types for amide synthesis;
nearly all known intermolecular-amide syntheses proceed by
addition–elimination reactions of activated carboxylates.
Herein we document our discovery of a novel approach to
amide synthesis by the decarboxylative condensation of N-
alkylhydroxylamines and a-keto carboxylic acids [Eq. (1);
Table 1: Reaction conditions for amide formation from hydroxylamine 1
and a-ketoacid 2.
Entry
Conditions[a]
t [h]
Yield[b] [%]
1
2
3
4
5
6
7
8
DMF, hydroxylamine free base
DMF
DMF, ketoacid sodium salt
MeOH
15
15
15
24
15
15
24
15
70
79 (88)[c]
75
72
80
DMSO
DMF/H2O (5:1)
acetate buffer (pH 4)
6n NH4Cl, 608C
72 (77)[c]
(70)[c]
68 (70)[c]
DMF = N,N-dimethylformamide]. This process proceeds in
polar protic and aprotic solvents, requires no reagents or
catalysts, produces only water and carbon dioxide as by-
products, and readily tolerates unprotected functional groups.
We provide preliminary evaluations of its application to the
chemoselective ligation of unprotected-peptide fragments.
Investigations into the mechanism and substrate scope of the
reaction reveal a rich chemistry that will lead to new solutions
for the synthesis of complex molecules and functionalized
materials.
The discovery of this reaction stemmed from our efforts to
develop new approaches to amide and ester synthesis by
intramolecular redox reactions of aldehydes.[13] We reasoned
that intermolecular redox reactions, for example, between an
aldehyde and a hydroxylamine, could also lead to amide
formation under appropriate conditions (Scheme 1). These
studies, however, were complicated by the propensity of
aldehydes and hydroxylamines to rapidly form nitrones. In
contrast, ketones rarely condense with N-alkylhydroxyl-
amines under mild conditions. Instead, metastable hemi-
aminals are formed,[14] and we hypothesized that N-alkylhy-
droxylamines would react with a-ketoacids to produce a
hemiaminal poised for oxidative decarboxylation to give
amide products.[15]
[a] All reaction performed on a 0.2 mmol scale; [b] Yields following
chromatography; [c] HPLC yields of unpurified reaction mixtures given in
parentheses.
nonpolar solvents, we were pleased to find that simply
warming a solution of these two reactants in DMF produced
the desired amide product in > 70% yield (Table 1, entry 1).
A concern in these initial studies was the preparation and
handling of the hydroxylamine in its unstable free-base form.
Conveniently, we found that salts of the hydroxylamine are
equally, and possibly more, efficient in the amidation reaction
and as such, we selected the stable, highly crystalline
hydroxylamine oxalate salts for further studies (Table 1,
entry 2). Likewise, either the protonated ketoacids or their
carboxylate salts were suitable reactants (Table 1, entry 3).
Amide bond formation occurred in dimethyl sulfoxide
(DMSO; Table 1, entry 4), MeOH (Table 1, entry 5), aqueous
DMF (Table 1, entry 6), or as suspensions of the reactants in
aqueous buffers (Table 1, entries 7–8). Reactions were typi-
cally performed at 0.1m by using ketoacid (1.0 equiv) and
hydroxylamine oxalate (1.2 equiv) at 408C, but lower con-
centrations (0.01m, 0.005m) and other stoichiometric ratios
were also viable.
Our hypothesis was tested by mixing two readily available
substrates, phenylpyruvic acid (1) and N-phenethylhydroxyl-
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peptide 6 as a 19:1 mixture of diaste-
reomers (peptide 6 is the major form),
which demonstrates that epimerization
of the ketoacid does not occur during
the ligation reaction. Encouraged by
these results, we prepared a range of
protected- and unprotected-peptide
substrates and examined their ligations
(Table 2). To further validate the toler-
ance of this reaction to fully unpro-
tected peptides, ketoacid 7 and hydrox-
ylamine 8 were prepared and shown to
undergo efficient ligation in aqueous
DMF [Eq. (3)]. Notably, a variety of
ligation sites including Phe-Ala, Ala-
Phe, Pro-Ala, Val-Gly, and Ala-Ala
were feasible.
Several possible reaction pathways
can give rise to amide formation, and
we have initiated efforts to elucidate
the reaction mechanism. Interestingly,
although peptide substrates such as 4
and 5 do not appear to condense to
form nitrones under the reaction con-
ditions (Figure 1b), less-hindered sub-
strates, including our model reaction
[Eq. (2)], rapidly form the isomeric
nitrones cis-11 and trans-11 (Figure 2).
These nitrone intermediates were
detected by reverse-phase HPLC (Fig-
ure 2b), liquid chromatography–MS,
Figure 1. Ketoacid–hydroxylamine ligations of peptide substrates 4 and 5. a) Demonstration of preserva-
tion of stereochemistry during the reaction; b) Monitoring of the reaction by HPLC (samples taken
directly from the reaction mixture without purification or workup). A small amount of the carboxylic acid
is formed during the synthesis of ketoacid 4. Cbz=carbobenzyloxycarbonyl.
1
and in situ H and 13C NMR spectros-
copy studies of the reaction mixtures.
They could be isolated as their methyl
Ketoacid–hydroxylamine amide ligations occur for a wide
range of simple and complex substrates. We have focused our
initial investigations on the explora-
tion of the suitability of this process
for peptide ligations. Peptide ketoacid
4 was prepared as a > 15:1 mixture of
diastereomers (with 4 being the major
diastereomer) by a variant of Wasser-
manꢀs ylide method[16,17] and reacted
with (S)-alanine hydroxylamine oxa-
late salt 5 (1.2 equiv).[18] The ligation
reaction occurred cleanly over the
course of 12 h (Figure 1) to give
esters by being trapped with diazomethane. Although it is
clear that these nitrones can form under the reaction
Table 2: Ketoacid–hydroxylamine peptide ligations of selected protected- and unprotected-peptide substrates.
Entry
Ketoacid
Hydroxylamine
Product[a]
Yield[b] [%]
1
2
3
4
5
FmocAlaPro
FmocAlaVal
FmocLys(Boc)-Glu(tBu)PheAla
H2N-LysAlaPhe
FmocAspAlaPhe
AlaOtBu
GlyOEt
AlaOtBu
AlaAsp(tBu)PheOtBu
AlaAsp(tBu)PheOtBu
Fmoc-AlaProAla-OtBu
Fmoc-AlaValGly-OEt
72
58
80
74
74
Fmoc-Lys(Boc)Glu(tBu)Phe-AlaAla-OtBu[c]
H2N-LysAlaPhe-AlaAsp(tBu)Phe-OtBu
Fmoc-AspAlaPhe-AlaAsp(tBu)PheOtBu
[a] All reaction performed at 0.02–0.1m in DMF or DMSO containing ca. 5% H2O at 408C for 10–24 h using 1 equiv ketoacid and 1.2–2 equiv
hydroxylamine oxalates; [b] Yields of pure products following preparative TLC or RP-HPLC. The reported yields include the preparation of the ketoacids
by oxidation of the appropriate cyanoylide followed by coupling with the hydroxylamine; [c] 0.01m, 48 h.
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conditions, we do not currently believe that they are involved
in product formation; all attempts to trap the postulated
nitrilium ion 12 by the addition of nucleophiles (MeOH as the
reaction solvent, thiophenol, cysteine, glycine) afforded only
the usual amide product. Thus, although the rapid formation
of nitrone 11 may contribute to the efficiency of the reaction,
conversion to the product is likely to proceed through
This chemoselective amide formation is not limited to O-
unsubstituted hydroxylamines. For example, N-methoxy pep-
tide 16 was coupled with 2-ketobutyric acid in acetonitrile in
61% yield (Scheme 2a).[19] Unexpectedly, and in contrast to
the O-unsubstituted hydroxylamines, these substrates
required added acid and showed increased efficiency in
acetronitrile, but were slower in the presence of DMF,
decarboxylation of the tetrahedral intermediate 10. T he DMSO, or water. Cyclic hydroxylamines are exceptional
question of whether the decarboxylation pro-
ceeds through a stepwise or concerted pathway
remains to be addressed, although the isolation
of small amounts of nitrone 15 suggests that a
stepwise reaction is at least a possibility. We
note, however, that decarboxylated nitrones
are never isolated when peptide-derived keto-
acids and hydroxylamines are employed.
Although the details remain to be elucidated,
the available evidence points to a distinct
mechanistic manifold that may have further
implications including a role in the prebiotic
Scheme 2. Amide-forming ketoacid ligations with O-alkyl hydroxylamines.
origin of higher peptides.
substrates (Scheme 2b); isoxazolidine 18 reacted cleanly with
a-ketoacids in either nonpolar (CH2Cl2, toluene) or aqueous
(0.2m 1:1 tBuOH/H2O) conditions. The stability of the
resultant ketoester product under the reaction conditions is
noteworthy.[20]
The coupling of a-ketoacids and hydroxylamines is a
powerful, chemoselective amide bond formation that pro-
ceeds in the presence of reactive functional groups, requires
no reagents or catalysts, and produces only water and CO2 as
by-products. This reaction should be useful for diverse
applications that require the coupling of unprotected mole-
cules.
Received: November 9, 2005
Published online: January 17, 2006
Keywords: amides · chemoselectivity · ketoacids ·
.
ligation reactions · peptides
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