Biomimetic α-selective ribosylation enables two-step modular synthesis of biologically important ADP-ribosylated peptides

Abstract

The α-type ADP-ribosylated peptides represent a class of important molecular tools in the field of protein ADP-ribosylation, however, they are difficult to access because of their inherent complicated structures and the lack of effective synthetic tools. In this paper, we present a biomimetic α-selective ribosylation reaction to synthesize a key intermediate, α-ADP-ribosyl azide, directly from native β-nicotinamide adenine dinucleotide in a clean ionic liquid system. This reaction in tandem with click chemistry then offers a two-step modular synthesis of α-ADP-ribosylated peptides. These syntheses can be performed open air in eppendorf tubes, without the need for specialized instruments or training. Importantly, we demonstrate that the synthesized α-ADP-ribosylated peptides show high binding affinity and desirable stability for enriching protein partners, and reactivity in post-stage poly ADP-ribosylations. Owing to their simple chemistry and multidimensional bio-applications, the presented methods may provide a powerful platform to produce general molecular tools for the study of protein ADP-ribosylation.

Fig. 1. ADP-ribosylations and strategies for synthesizing α-type ADP-ribosylated peptides.

a The enzymatic synthesis route for ADP-ribosylations. b The general chemical structure of α-ADP-ribosylated proteins. c The chemical synthesis route for ADP-ribosylated peptides. d The bioconjugation method for syntheses of ADP-ribosylated peptides. e The biomimetic route for syntheses of ADP-ribosylated peptides that was shown in this work. ADP adenosine diphosphate, ARTs ADP-ribosyltransferases, Pg protecting group, ADPr ADP-ribose, IL ionic liquid.

Fig. 2. The biomimetic α-selective ribosylation reactions are developed by using ionic-liquid-based screening.

a Synergistic effects in enzymatic ADP-ribosylation reactions. b Cations and anions of ionic liquids for screening. c Biomimetic transformations of β-NAD⁺ to α-type ADP-ribosyl azide. d Results of screening on 54 ionic liquids; black bars show the ionic liquids with high α-selectivity (the ratios of α/β-isomer more than 10:1). Experiments were performed in triplicates. Data (available at 10.6084/m9.figshare.12375032) are reported as mean ± s.d. (n = 3), and black dots indicate value of each sample. e Best reaction system for the synthesis of α-ADPr-N₃ (1). f Biomimetic synthesis of α-ADPPr-N₃ from β-NADP. g Biomimetic synthesis of 8-Br-α-ADPr-N₃ from 8-Br-β-NAD⁺. h Biomimetic synthesis of α-ADPr-N₃ from cyclic ADP-ribose (cADPR).

Fig. 3. Modular synthesis of α-type ADP-ribosylated peptides in two steps for functional investigations.

a The concise synthetic route involving two reaction steps for the preparation of ADP-ribosylated peptides. b The optimized catalytic system for the click reaction of α-ADPr-N₃. c The programmable steps for preparations of ADP-ribosylated peptides. d Replacing the highly labile ester-ADPr linkage with the triazolyl-ADPr linkage. e Results of the synthesis of ADP-ribosylated peptides. f The chemical structure of peptide 1–4. g The R substituent groups in peptide 1–4. h Biological assays to determine the activities of peptide 1–4.

Fig. 4. The triazolyl-linked ADP-ribosylation represents a good mimic of the natural ADP-ribosylation.

a α-ADP-ribosylated peptide-1 labeled mH2A1.1 in a concentration-dependent manner. Data are reported as mean ± s.d. (n = 3). b β-ADP-ribosylated peptide-2 labeling of mH2A1.1 was much weaker and inefficient compared with peptide-1. c Competition between α-ADP-ribosylated peptide-1 and peptide-3 in labeling mH2A1.1. Data are reported as mean ± s.d. (n = 3). d β-ADP-ribosylated peptide-4 failed to compete with peptide-1 in labeling mH2A1.1. e Peptide-1 selectively labeled mH2A1.1 but not mH2A1.1 G224E mutant or mH2A1.2. f Peptide-1 enriched endogenous mH2A1.1 and PARP9 from HeLa S3 cell lysate. g The α-ADP-ribosylated peptide-3 serves as a substrate of PARP-1-induced poly ADP-ribosylation. In a, c, e, f, and g, the blottings represent three independent experiments. In b and d, the blottings represent two independent experiments. In a and c, curves were normalized between 100% and 0% at the highest and lowest luminescence. See “Photo-cross-linking and visualization of the biotinylated proteins” in the “Methods” section for more details of the photo-cross-linking-based assays. The uncropped blots and protein markers are provided in Supplementary Fig. 9 in the Supplementary Information. Source data of uncropped versions of gels or blots in Fig. 4 are provided as a Source Data file (10.6084/m9.figshare.12375032).