Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Mar 14;140(10):3768-3774.
doi: 10.1021/jacs.8b00046. Epub 2018 Mar 1.

A Versatile Approach to Noncanonical, Dynamic Covalent Single- and Multi-Loop Peptide Macrocycles for Enhancing Antimicrobial Activity

James F Reuther  1 , Andrew C Goodrich  2 , P Rogelio Escamilla  1 , Tiffany A Lu  1 , Valarie Del Rio  1 , Bryan W Davies  2 , Eric V Anslyn  1
Affiliations

A Versatile Approach to Noncanonical, Dynamic Covalent Single- and Multi-Loop Peptide Macrocycles for Enhancing Antimicrobial Activity

James F Reuther et al. J Am Chem Soc. .

Abstract

Peptide oligomers offer versatile scaffolds for the formation of potent antimicrobial agents due to their high sequence versatility, inherent biocompatibility, and chemical tunability. Though many methods exist for the formation of peptide-based macrocycles (MCs), increasingly pervasive in commercial antimicrobial therapeutics, the introduction of multiple looped structures into a single peptide oligomer remains a significant challenge. Herein, we report the utilization of dynamic hydrazone condensation for the versatile formation of single-, double-, and triple-loop peptide MCs using simple dialdehyde or dihydrazide small-molecule cross-linkers, as confirmed by MALDI-TOF MS, HPLC, and SDS-PAGE. Furthermore, incorporation of aldehyde-containing side chains onto peptides synthesized from hydrazide C-terminal resins resulted in tunable peptide MC assemblies formed directly upon resin cleavage post solid-phase peptide synthesis. Both of these types of dynamic covalent assemblies produced significant enhancements to overall antimicrobial properties when introduced into a known antimicrobial peptide, buforin II, when compared to the original unassembled sequence.

PubMed Disclaimer

Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
MALDI-TOF MS of macrocycles (MCs; a and b) and multi-loop macrocycles (MLCs; c) with two loops formed upon reaction of DiAl/Hy and TetAl/Hy peptides, respectively, with small-molecule cross-linkers. These small molecules include flexible, aliphatic dihydrazides and rigid aromatic dialdehydes all showing efficient cyclization. All reactions were conducted at c = 1.0 mM in either 1:1 water:acetonitrile (a and c) or 1:1 PBS buffer (pH = 7.4):acetonitrile (b).
Figure 2
Figure 2
Analytical HPLC chromatograms monitored at 254 nm absorbance of linear DiAl1 (a), DiAl4 (c), TetAl1 (e), and TetAl2 (g) along with the peptides macrocyclized with ADH (b and h) or SDH (c and f) showing quantitative conversion to the corresponding DC-MC or DC-MLC when reacting for 2 h at c = 1.0 mM in 1:1 water:acetonitrile.
Figure 3
Figure 3
DC peptides with aldehyde side chains synthesized on hydrazide C-terminal resins (HyRes) assemble into various macrocyclic and oligomeric structures (shown experiments were from c = 3.3 mM solutions), as characterized by MALDI-TOF MS, based on the number of amino acids between DC functionalities. The four peptide synthesized, HyRes1–4, are ordered by increasing number of AA spacing (2, 4, 6, and 10, respectively). The adopted DC assemblies include intramolecular macrocycles (green sphere), linear/macrocyclic dimers (red cube), linear/macrocyclic trimers (blue triangle), and macrocyclic tetramers (purple hexagon).
Figure 4
Figure 4
MALDI-TOF MS (a) and analytical HPLC traces (b; monitored at 254 nm) of DC assemblies for antimicrobial buforin II peptides with two, three, and four aldehydes or hydrazides incorporated, including MC assemblies with DiAl buforin (1–2), MLC assemblies with TetAl buforin (3,4), DiAl + DiHy buforin intermolecular MC (5), and TriAl + TriHy buforin zipper assembly (6).
Figure 5
Figure 5
Tricine SDS-PAGE of antimicrobial buforin DC peptides and assemblies after reacting for 3 days at 3.0 mM: DiAl (A), DiHy (B), MC(DiAl+SDH) (C), MC(DiAl+ADH) (D), MC(DiHy+TDA) (E), MC(DiHy+ThDA) (F), DiAl+DiHy (G; oligomer and MC), TriAl (H), TriHy (I), TetAl (J), MLC(TetAl+SDH) (K), Z(TriAl +TriHy) (L), and HyRes buforin (M; oligomers and MC).
Figure 6
Figure 6
MIC values (μM) of buforin DC assemblies, DC peptides, and buforin II versus wild-type E. coli W3110, drug-resistant A. baumannii 5075, and colistin-resistant E. coli WD101 conducted in Mueller-Hinton broth showing a turn-on in antimicrobial activity upon DC assembly. with the crude reactions of TriAl+TriHy buforin, DiAl +DiHy buforin, and HyRes buforin performing the best. Arrows indicate activities greater than 128 μM which were not measured.
Scheme 1
Scheme 1
(a) Reversible Condensation of Hydrazides onto Aldehydes and (b) Solid-Phase Copper-Catalyzed Azide–Alkyne Cycloaddition (SP-CuAAC) Incorporation of Unnatural, Dynamic Covalent (DC) Aldehyde and Hydrazide Functionalities into Peptide Oligomers Containing Unnatural Azidolysine (AzK) Amino Acid Residues
See this image and copyright information in PMC

References

    1. Cabello FC. Environ Microbiol. 2006;8:1137–1144. - PubMed
    1. Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A, Schaberle TF, Hughes DE, Epstein S, Jones M, Lazarides L, Steadman VA, Cohen DR, Felix CR, Fetterman KA, Millett WP, Nitti AG, Zullo AM, Chen C, Lewis K. Nature. 2015;517:455–459. - PMC - PubMed
    1. Wright GD. Nat Rev Microbiol. 2007;5:175–186. - PubMed
    1. Loose C, Jensen K, Rigoutsos I, Stephanopoulos G. Nature. 2006;443:867–869. - PubMed
    1. Peschel A, Sahl H-G. Nat Rev Microbiol. 2006;4:529–536. - PubMed

Publication types

MeSH terms