Solid-Phase Synthesis and Biological Evaluation of Peptides ADP-Ribosylated at Histidine

Abstract

The transfer of an adenosine diphosphate (ADP) ribose moiety to a nucleophilic side chain by consumption of nicotinamide adenine dinucleotide is referred to as ADP-ribosylation, which allows for the spatiotemporal regulation of vital processes such as apoptosis and DNA repair. Recent mass-spectrometry based analyses of the "ADP-ribosylome" have identified histidine as ADP-ribose acceptor site. In order to study this modification, a fully synthetic strategy towards α-configured N(τ)- and N(π)-ADP-ribosylated histidine-containing peptides has been developed. Ribofuranosylated histidine building blocks were obtained via Mukaiyama-type glycosylation and the building blocks were integrated into an ADP-ribosylome derived peptide sequence using fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis. On-resin installation of the ADP moiety was achieved using phosphoramidite chemistry, and global deprotection provided the desired ADP-ribosylated oligopeptides. The stability under various chemical conditions and resistance against (ADP-ribosyl) hydrolase-mediated degradation has been investigated to reveal that the constructs are stable under various chemical conditions and non-degradable by any of the known ADP-ribosylhydrolases.

Keywords: ADP-Ribosylation; Glycosylation; Histidine; Peptides; Solid-Phase Synthesis.

Figure 1

A) Schematic overview of mono‐ADP‐ribosylation where an ADPr moiety of NAD⁺ is covalently attached to a nucleophilic side chain (X=O, N or S) of the target protein in an α‐selective manner. B) Schematic overview of the ADP‐ribosylation of histidine residues including the uncertainties surrounding the nature of this specific modification. The identity of the transferase and hydrolase involved in the construction and degradation of the PTM remain unknown and a referred to as PARP “X” and ARH “Y” respectively. NAM=nicotinamide and AMP=adenosine monophosphate.

Figure 2

Retrosynthetic analysis of N(τ)‐ and N(π)‐ADP‐ribosylated histidine peptides. Key reactions of the novel methodology are highlighted.

Scheme 1

Protecting group manipulations of ribosylated histidine analogues. Reagents and conditions: a) LiOH, THF/H₂O (3 : 1), 0 °C, 1.5 h (46 % from 11, 47 % from 12). b) Pd(PPh₃)₄, DCM, rt, 1 h (78 % from 13, 89 % from 14). c) HCl, HFIP, 0 °C, 10 min (36 %).

Figure 3

Schematic representation of the three ribosylated histidine analogues 15 16 and 17 depicted in their presumed ³E or E₃ envelope conformation, with the most relevant observed proton‐proton interactions observed in NOESY measurements highlighted in red and blue. The H1′→H2′ interaction in structure 15 has been omitted for clarity.

Figure 4

Chemical structures of the α‐configured 1,4‐ and 1,5‐disubstituted 1,2,3‐triazole‐based isosteres, here referred to as ADPr‐Trz that mimic their N(τ)‐ and N(π)‐ADP ribosylated histidine counterparts, respectively.

Scheme 2

Incorporation of ribosylated histidine building blocks 15, 16 and 18 and in a peptide fragment originating from HPF1 a) HF‐pyridine, pyridine, rt, 2x 45 min. b) (FmO)₂N(i‐Pr)₂, ETT, MeCN, rt, 30 min. c) CSO, MeCN, rt, 30 min. d) DBU, DMF, rt, 2x 15 min. e) 24, ETT, MeCN, rt, 30 min. f) CSO, MeCN, rt, 30 min. g) DBU, DMF, rt, 2x 10 min. h) TFA, DCM, rt, 1 h (28 % for 26, 35 % for 27 and 28 % for 28 over 8 steps).

Figure 5

A) Chemical stability of peptide 28 under basic conditions (NaOH, 0.1 M). Samples were extracted at different time points (2, 4, 24, 48, 72 and 169 h) and quenched with TFA prior to LC–MS injection. Peptide degradation was quantified by analyzing the UV‐trace (260 nm) using Xcalibur software. Including an exponential one‐phase decay trendline (Y=42.2*e^(−0.1024*X)+57.8, R²=0.999). B) Enzymatic hydrolysis of interglycosidic linkages in ADP‐ribosylated histidine peptides 26–28 and 1,2,3‐triazole‐based isosteres 19 and 20. Enzymatic turnover of the various peptides was assessed by monitoring AMP release directly (NudT16) or converting released ADPr via NudT5 to AMP. AMP was measured using the AMP‐Glo assay (Promega). Samples are background corrected and normalized to NudT16 activity The data represents mean values±SD measured in triplicates. Abbreviations: CdrNADAR, NADAR from Clostridium drakei; SauMacroD, zinc‐containing MacroD from Staphylococcus aureus; TaqDarG, DarG from Thermus aquaticus.