Peptide cyclization and cyclodimerization by Cu(I)-mediated azide-alkyne cycloaddition

Abstract

Head-to-tail cyclodimerization of resin-bound oligopeptides bearing azide and alkyne groups occurs readily by 1,3-dipolar cycloaddition upon treatment with Cu(I). The process was found to be independent of peptide sequence, sensitive to the proximity of the alkyne to the resin, sensitive to solvent composition, facile for alpha- and beta-peptides but not for gamma-peptides, and inhibited by the inclusion of tertiary amide linkages. Peptides shorter than hexamers were predominantly converted to cyclic monomers. Oligoglycine and oligo(beta-alanine) chains underwent oligomerization by 1,3-dipolar cycloaddition in the absence of a copper catalyst. These results suggest that cyclodimerization depends on the ability of the azido-alkyne peptide to form in-frame hydrogen bonds between chains in order to place the reacting groups in close proximity and lower the entropic penalty for dimerization. The properties of the resin and solvent are crucial, giving rise to a productive balance between swelling and interstrand H-bonding. These findings allow for the design of optimal substrates for triazole-forming ring closure and for the course of the reaction to be controlled by the choice of conditions.

Figure 1

On-resin peptide cyclodimerization of two sequences originally reported by Finn et al. Amino acids are represented by their single-letter codes in bold print (X = propargylglycine).

Figure 2

Solution-phase cyclization of an alkyne- and azide-derivatized peptide.

Figure 3

Test of the dependence of sequence length on cyclization. RP-HPLC chromatograms of the crude products are shown. No linear monomers were observed, verified by co-injection of peptides before cyclization (not shown); “x” indicates peak due to a small amount of a chromophore with high extinction coefficient.

Figure 4

Sequences prepared to determine the efficacy of cyclodimerization when the distance between alkyne and solid support (Rink amide MBHA resin) is increased.

Figure 5

(Top) Mixed peptide sequences with sub-stoichiometric quantities of azide or alkyne groups. (Bottom) Summary of the results from exposure to cyclization reaction conditions.

Figure 6

Oligomers of β-Ala, γ-Abu, and Gly tested for on-resin CuAAC cyclization. The observed products of standard Cu^I treatment are indicated in parentheses.

Figure 7

Resin-bound peptoid sequences subjected to standard CuAAC conditions and subsequent cleavage with TFA. All returned cyclic monomers in clean fashion, with the exception of 61, which gave cyclic monomer + oligomers.

Figure 8

Peptides used to test the dependence of backbone amide methylation on cyclodimerization, and their results after applying standard reaction conditions.

Figure 9

IR spectra of protected resin-bound nonapeptide 39 swollen overnight in either CH₂Cl₂ (blue), DMSO (green), or the standard reaction solvent system of 4:1 MeCN/DMSO (red).

Figure 10

(Left) Potential hydrogen patterns formed by head-to-tail alignments of oligo(Gly), oligo(β-Ala), and oligo(γ-Abu), placing inter-strand alkyne and azide moieties in close proximity to each other. The shaded bar represents the polystyrene chains to which the peptides are attached. (Right) Proposed CuAAC cyclodimerization pathway directed by head-to-tail H-bonding.

Figure 11

Cartoon representations of proposed hydrogen-bonded alignments formed by resin-43 (Figure 4).

Figure 12

Preparative-scale syntheses of a representative cyclic dimer and monomer.

Figure 13

Incorporation of alkyne and azide groups during solid-phase peptide synthesis.