Chemoselective Amide Ligations by Decarboxylative Condensations of N ‐Alkylhydroxylamines and α‐Ketoacids (2006)

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 peptide substrates without epimerization, and as such, this process has the potential to serve as a novel chemoselective ligation for the synthesis of peptides and complex materials. Chemoselective ligation reactions make possible covalent bond formation between two fragments containing unprotected functional groups. An ideal ligation process proceeds under mild, often aqueous, conditions at low molar concentrations, 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 thioester-α-bromocarbonyl alkylations,3 and the copper-promoted alkyne–azide cycloaddition4 have found diverse and significant applications in drug discovery,5 functionalized polymer synthesis,6 and the fabrication of novel nanostructures.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 chemical 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, unprotected-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 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-alkylhydroxylamines under mild conditions. Instead, metastable hemiaminals are formed,14 and we hypothesized that N-alkylhydroxylamines would react with α-ketoacids to produce a hemiaminal poised for oxidative decarboxylation to give amide products.15 Reactions of N-alkylhydroxylamines with aldehydes and ketones. Entry Conditions[a] t [h] Yield[b] [%] 1 DMF, hydroxylamine free base 15 70 2 DMF 15 79 (88)[c] 3 DMF, ketoacid sodium salt 15 75 4 MeOH 24 72 5 DMSO 15 80 6 DMF/H2O (5:1) 15 72 (77)[c] 7 acetate buffer (pH 4) 24 (70)[c] 8 6 n NH4Cl, 60 °C 15 68 (70)[c] Ketoacid–hydroxylamine ligations of peptide substrates 4 and 5. a) Demonstration of preservation 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. Entry Ketoacid Hydroxylamine Product[a] Yield[b] [%] 1 FmocAlaPro AlaOtBu Fmoc-AlaProAla-OtBu 72 2 FmocAlaVal GlyOEt Fmoc-AlaValGly-OEt 58 3 FmocLys(Boc)-Glu(tBu)PheAla AlaOtBu Fmoc-Lys(Boc)Glu(tBu)Phe-AlaAla-OtBu[c] 80 4 H2N-LysAlaPhe AlaAsp(tBu)PheOtBu H2N-LysAlaPhe-AlaAsp(tBu)Phe-OtBu 74 5 FmocAspAlaPhe AlaAsp(tBu)PheOtBu Fmoc-AspAlaPhe-AlaAsp(tBu)PheOtBu 74 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 conditions (Figure 1 b), less-hindered substrates, 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 (Figure 2 b), liquid chromatography–MS, and in situ 1H and 13C NMR spectroscopy studies of the reaction mixtures. They could be isolated as their methyl esters by being trapped with diazomethane. Although it is clear that these nitrones can form under the reaction 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 decarboxylation of the tetrahedral intermediate 10. The question of whether the decarboxylation proceeds 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 ketoacids 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 origin of higher peptides. a) Possible reaction pathways for amide formation; b) detection of nitrone intermediates by HPLC. This chemoselective amide formation is not limited to O-unsubstituted hydroxylamines. For example, N-methoxy peptide 16 was coupled with 2-ketobutyric acid in acetonitrile in 61 % yield (Scheme 2 a).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, DMSO, or water. Cyclic hydroxylamines are exceptional substrates (Scheme 2 b); isoxazolidine 18 reacted cleanly with α-ketoacids in either nonpolar (CH2Cl2, toluene) or aqueous (0.2 M 1:1 tBuOH/H2O) conditions. The stability of the resultant ketoester product under the reaction conditions is noteworthy.20 Amide-forming ketoacid ligations with O-alkyl hydroxylamines. The coupling of α-ketoacids and hydroxylamines is a powerful, chemoselective amide bond formation that proceeds 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 molecules. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2006/z503991_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.