Synthesis of aminooxy and N-alkylaminooxy amines for use in bioconjugation.

Five Boc-protected aminooxy and N-alkylaminooxy amines have been synthesized in 60-95% overall yield using a common synthetic strategy from readily available two- and three-carbon Cbz-protected amino alcohols. The amines can be linked to biomolecules via amide formation and incorporated directly into peptoids via submonomer synthesis. Subsequent deprotection of the aminooxy and N-alkylaminooxy groups enables conjugation with desired target molecules via established chemoselective ligation methods. The range of derivatives synthesized allows different distances to be established between the conjugated molecules.

Chemoselective and microwave-assisted synthesis of glycopeptoids.

The chemoselective glycosylation of N-alkylaminooxy side chains with unprotected reducing sugars has proven useful for the synthesis of glycopeptides. Herein, we extend the N-alkylaminooxy strategy to the synthesis of glycopeptoids. A N-methylaminooxy submonomer was efficiently synthesized and incorporated into peptoids. Glycosylation of the peptoids proceeded chemoselectively and site-specifically at the N-methylaminooxy moieties. Employing microwave irradiation significantly increased the degree of glycosylation and shortened the reaction times.

Chemoselective alkylation of N-alkylaminooxy-containing peptides.

Peptides containing N-alkylaminooxy amino acids were chemoselectively alkylated with allylic, benzylic, and alpha-carbonyl bromides, N-ethylmaleimide, and hexyl acrylate in mildly acidic aqueous/organic solutions. Alkylation at the aminooxy nitrogen proceeds in good yields with excellent to complete chemoselectivity in the presence of all common amino acids except cysteine. This reaction complements the selective glycosylation and acylation of N-alkylaminooxy groups and provides an avenue for the synthesis of peptide arrays comprising a wide variety of neoglycopeptides and neolipopeptides.

2-(N-Fmoc)-3-(N-Boc-N-methoxy)-diaminopropanoic acid, an amino acid for the synthesis of mimics of O-linked glycopeptides.

Amino acids with N-alkylaminooxy side chains have proven effective for the rapid synthesis of neoglycopeptides. Chemoselective reaction of reducing sugars with peptides containing these amino acids provides glycoconjugates that are structurally similar to their natural counterparts. 2-(N-Fmoc)-3-(N-Boc-N-methoxy)-diaminopropanoic acid (Fmoc: 9-fluorenylmethoxycarbonyl; Boc: t-butyloxycarbonyl) was synthesized from Boc-Ser-OH in >40% overall yield and incorporated into peptides by standard Fmoc chemistry based solid phase peptide synthesis. The resulting peptides are efficiently glycosylated and serve as mimics of O-linked glycopeptides. The synthesis of this derivative greatly expands the availability of the N-alkylaminooxy strategy for neoglycopeptides.

A versatile set of aminooxy amino acids for the synthesis of neoglycopeptides.

Four N-alkylaminooxy amino acids have been synthesized in 22-56% overall yield from readily available amino acid precursors. Each amino acid can be efficiently incorporated into peptides using Boc-chemistry-based solid-phase peptide synthesis, and in three of the four cases the resulting peptides can be chemoselectively glycosylated at the aminooxy side chains to generate neoglycopeptides. The range of N-alkylaminooxy amino acids prepared allows attachment of sugars at two-, three-, or four-atom distances from the peptide backbone, and each ensures that attached sugars adopt cyclic conformations. These derivatives provide convenient access to arrays of biologically relevant neoglycopeptides that may be used to probe the influence of attached sugars on the structure and function of peptides and proteins.

Synthesis of N-Fmoc-O- (N'-Boc-N'-methyl)-aminohomoserine, an amino acid for the facile preparation of neoglycopeptides.

The synthesis of N-Fmoc-O-(N'-Boc-N'-methyl)-aminohomoserine in 35% overall yield from l-homoserine is described. This amino acid can be efficiently incorporated into peptides using Fmoc-chemistry-based solid-phase peptide synthesis, and the resulting peptides can be chemoselectively glycosylated at the aminooxy side chains to generate neoglycopeptides. The synthesis of this derivative greatly expands the availability of a previously developed neoglycopeptide synthesis strategy.

Dap-SL: a new site-directed nitroxide spin labeling approach for determining structure and motions in synthesized peptides and proteins.

A new approach for site-directed placement of nitroxide spin labels in chemically synthesized peptides and proteins is described. The scheme takes advantage of a novel diaminopropionic acid scaffold to independently control backbone and side chain elongation. The result is a spin-labeled side chain, referred to as Dap-SL, in which an amide bond forms a linker between the nitroxide and the peptide backbone. The method was demonstrated in a series of helical peptides. Circular dichroism and nuclear magnetic resonance showed that Dap-SL introduces only a minor perturbation in the helical structure. The electron paramagnetic resonance spectrum of the singly labeled species allowed for determination of the spin label rotational correlation time and suggests that the Dap-SL side chain is more flexible than the modified Cys side chain frequently used in site-directed spin label studies. Spectra of the doubly labeled peptides indicate a mixture of 3(10)-helix and alpha-helix, which parallels findings from previous studies. The scheme demonstrated here offers a fundamentally new approach for introducing spin labels into proteins and promises to significantly extend biophysical investigations of large proteins and receptors. In addition, the technique is readily modified for incorporation of any biophysical probe.