Mono-ADP-Ribosylation of Peptides: An Overview of Synthetic and Chemoenzymatic Methodologies

Abstract

Adenosine diphosphate (ADP)-ribosylation is a ubiquitous post-translational modification that regulates vital biological processes like histone reorganization and DNA-damage repair through the modification of various amino acid residues. Due to advances in mass-spectrometry, the collection of long-known ADP-ribose (ADPr) acceptor sites, e. g. arginine, cysteine and glutamic acid, has been expanded with serine, tyrosine and histidine, among others. Well-defined ADPr-peptides are valuable tools for investigating the exact structures, mechanisms of action and interaction partners of the different flavors of this modification. This review provides a comprehensive overview of synthetic and chemoenzymatic methodologies that enabled the construction of peptides mono-ADP-ribosylated on various amino acids, and close mimetics thereof.

Keywords: chemoenzymatic synthesis; glycosylation; mono-ADP-ribosylated peptides; pyrophosphate; solid-phase peptide synthesis.

Figure 1

Schematic overview of ADP‐ribosylation where NAD⁺ is consumed and the ADPr moiety is covalently attached to a defined nucleophilic side chain (X=O, N or S) in an α‐selective manner. Numbering denotation and nomenclature used to discriminate between the two ribofuranosides is included. ADPr=Adenosine diphosphate ribose, NAD=Nicotinamide adenine dinucleotide and NAM=nicotinamide.

Figure 2

The catalytic triad of diphtheria toxin (left) and cholera toxin (right), which are both highly conserved among ADP‐ribosylating bacterial toxins, including their essential interactions with the NAD⁺ substrate.

Figure 3

Toxin mediated ADP‐ribosylation of synthetic peptides. Modification of arginine in synthetically derived angiotensine peptides (1A–D) and kemptide (1E) by Kharadia & Graves (Top). ADP‐ribosylation of Cys‐351 in G_i3α(335–354): H‐VFDRVTDVIIKNNLKE‐C‐GLY‐OH (3) by Scheuring & Schramm (bottom). BSA=bovine serum albumin, DTT=dithiothreitol, CHAPS=3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate and NAD⁺=nicotinamide adenosine dinucleotide.

Scheme 1

Semisynthesis‐based strategies for the preparation of ADP‐ribosylated serine containing peptides that are compatible with protein ligation reactions by Mohapatra et al. A. Preparation of histones H2B and H3 with an ADP‐modification on the N‐terminal tail (serine 6 and 10 respectively). B. Preparation of histone 1.2 ADP‐ribosylated on Ser‐150, where the synthetic peptide sequences are H3(1–20): H2N‐ARTKQTARKSTGGKAPRKQL‐NHNH2, H2B(1–16): H2N‐PEPKSAPAPKKGSKK‐NHNH2 and H1.2(143–162): Thz‐GGATPKKSAKKTPKKAKKKPA‐SEA. GP buffer=guanidine HCl (6 M)+sodium phosphate (0.1 M). MESNa=sodium 2‐mercaptoethanesulfonate, Thz=thiazolidine, SEA=bis(2‐sulfanylethyl)amide, SPPS=solid‐phase peptide synthesis, TCEP=tris(2‐carboxyethyl)phosphine and TFET=trifluoroethanethiol.

Scheme 2

An SPPS‐based approach towards ADP‐ribosylated peptides on asparagine and glutamine by Van der Heden van Noort et al. Sequence A=Ac‐VANIEV‐OMe and sequence B=Ac‐PQPSKSA‐OMe, where the modification site is highlighted (bold). Ac=acetyl, Bz=benzoyl, Bn=benzyl, Dmab=4‐(N‐[1‐(4,4‐dimethyl‐2,6‐dioxocyclohexylidene)‐3‐methylbutyl]amino)benzyl, DCI=dicyanoimidazole, EDC=1‐Ethyl‐3‐(3‐dimethyl‐aminopropyl)carbodiimide, Fmoc=fluorenylmethoxycarbonyl, i‐Bu=iso‐butyryl, Osu=O‐succinimide, Pyr=pyridine, SPPS=solid‐phase peptide synthesis, TBDPS=tert‐butylchlorodiphenylsilane, TEA=triethylamine, TFA=trifluoroacetic acid, Trt=trityl and Z=benzyloxycarbonyl.

Scheme 3

ADP‐ribosylation of asparagine, glutamine and citrulline via an updated methodology by Kistemaker et al. Activator is tert‐butyldimethylsilyl trifluoromethanesulfonate (TBSOTf) for asparagine and citrulline, and HClO4‐SiO2 for glutamine. Citrulline products were obtained as anomeric mixture: *(α/β=34 : 66), **(α/β=60 : 40) and ***(α/β=62 : 38). Ac=acetyl, Asn=asparagine, Bz=benzoyl, Bn=benzyl, Cit=citrulline, CSO=(1S)‐(+)‐(10‐camphorsulfonyl)‐oxaziridine, DBU=1,8‐diazabicyclo‐[5.4.0]undec‐7‐ene, ETT=5‐ethylthiotetrazole, Fmoc=fluorenylmethoxycarbonyl, Gln=glutamine, HFIP=hexafluoro‐iso‐propanol, i‐Bu=iso‐butyryl, NMI=1‐methylimdazole, Ph=phenyl, PMB=para‐methoxybenzyl, SPPS=solid‐phase peptide synthesis, TBDPS=tert‐butylchlorodiphenylsilane, t‐Bu=tert‐butyl,TFE=trifluoroethanol and TIPS=triisopropylsilane.

Scheme 4

Synthesis of an H2B_4–14 peptide fragment (full sequence=AcNH‐PAKSAPAPKKG‐NH₂) with an α‐ or β‐configured ADP‐ribosylated serine residue by Voorneveld et al. Ac=acetyl, Bn=benzyl, Bz=benzoyl, CSO=(1S)‐(+)‐(10‐camphorsulfonyl)‐oxaziridine, DBU=1,8‐diazabicyclo[5.4.0]undec‐7‐ene, ETT=5‐ethylthiotetrazole, Fmoc=fluorenylmethoxycarbonyl, i‐Bu=iso‐butyryl, PMB=para‐methoxybenzyl,NMI=1‐methylimdazole, Ph=phenyl, SPPS=solid‐phase peptide synthesis, t‐Bu=tert‐butyl, TFA=trifluoroacetic acid, TFE=trifluoroethanol, TIPS=triisopropylsilane and TMSOTf=trimethylsilyl trifluoromethanesulfonate.

Scheme 5

An improved and versatile methodology for synthetic oligopeptides ADP‐ribosylated on serine, threonine or cysteine side chains by Voorneveld et al. CSO was a suitable oxidation agent for peptides A–F, while the milder t‐BuOOH was used for biotinylated peptide G. Ac=acetyl, Boc=tert‐butyloxycarbonyl, CSO=(1S)‐(+)‐(10‐camphorsulfonyl)‐oxaziridine, DBU=1,8‐diazabicyclo[5.4.0]undec‐7‐ene, DMBA=1,3‐dimethylbarbituric acid, ETT=5‐ethylthiotetrazole, Fm=9‐fluorenylmethyl, Fmoc=fluorenylmethoxycarbonyl, Ph=phenyl, PMB=para‐methoxybenzyl, SPPS=solid‐phase peptide synthesis, i‐Pr=iso‐propyl, TBAF=tetrabutylammonium fluoride, TBDPS=tert‐butylchlorodiphenylsilane, TBS=tert‐butylsilyl, and TFA=trifluoroacetic acid.

Scheme 6

A chemical methodology for the synthesis of full length histones H3 and H2B ADP‐ribosylated on serine 10 and 6, respectively. The N‐tail fragments were obtained via solid‐phase peptide synthesis with key building block 70 and conjugated to the respective truncated recombinant C‐terminus through native chemical ligation. Sequence of synthetic fragments H3(1–14)=ARTKQTARKSTGGK and H2B(1–16)=PEPAKSAPAPKKGSKK. Acac=acetylacetone, Bn=benzyl, Boc=tert‐butyloxycarbonyl, Cbz=benzyl chlorocarbamate, CSO=(1S)‐(+)‐(10‐camphorsulfonyl)‐oxaziridine, DBU=1,8‐diazabicyclo[5.4.0]undec‐7‐ene, DMBA=1,3‐dimethylbarbituric acid, DMNB=4,5‐dimethoxy‐2‐nitrobenzyl, DMTCl=4,4‐dimethoxytritylchloride, ETT=5‐(Ethylthio)‐1H‐tetrazole, Fmoc=fluorenylmethoxycarbonyl, i‐Pr=iso‐propyl, Me=methyl, MPAA=4‐Mercaptophenylacetic acid, Ph=phenyl, SPPS=solid‐phase peptide synthesis, TCEP=tris(2‐carboxyethyl)phosphine, TFET=trifluoroethanethiol and TFA=trifluoroacetic acid.

Scheme 7

The first approach towards a tandem Cys‐ADP‐ribosylated oligopeptide derived from the androgen receptor (AR) by Wijngaarden et al. Boc=tert‐butyloxycarbonyl, CSO=(1S)‐(+)‐(10‐camphorsulfonyl)‐oxaziridine, DBU=1,8‐diazabicyclo[5.4.0]undec‐7‐ene, DMAP=4‐Dimethylaminopyridine, ETT=5‐ethylthio‐tetrazole, Fm=9‐fluorenylmethyl, Fmoc=fluorenylmethoxycarbonyl, i‐Pr=iso‐propyl, Ph=phenyl, PMB=para‐methoxybenzyl, PyAOP=(7‐azabenzotriazol‐1‐yloxy)tripyrrolidinophosphonium hexafluorophosphate, TBDPS=tert‐butylchlorodiphenylsilane, Tf=trifluoromethanesulfonyl, TFA=trifluoroacetic acid, THT=tetrahydrothiophene, TIS=triisopropylsilane, and Trt=trityl.

Scheme 8

Exploitation of a Steglich esterification in the preparation of oligopeptides ADP‐ribosylated on glutamic or aspartic acid residues. The aspartic and glutamic acid esters migrate between the 1’’‐, 2’’‐ and 3’’‐OH after removal of the isopropylidene functionality. Ac=acetyl, Boc=tert‐butyloxycarbonyl, CSO=(1S)‐(+)‐(10‐camphorsulfonyl)‐oxaziridine, DBU=1,8‐diazabicyclo[5.4.0]undec‐7‐ene, DMBA=1,3‐Dimethylbarbituric acid, DMAP=4‐Dimethylaminopyridine, EDC=1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide, ETT=5‐ethylthio‐tetrazole, Fm=9‐fluorenylmethyl, Fmoc=fluorenylmethoxycarbonyl, Me=methyl, i‐Pr=iso‐propyl, Pyr=pyridine TBDPS=tert‐butylchlorodiphenylsilane and TFA=trifluoroacetic acid.

Scheme 9

The use of a Lewis acid promoted conjugation between ornithine and isothiourea in the synthesis of arginine‐linked ADPr oligopeptides and full‐length ubiquitin as reported by Voorneveld et al. Alloc=N‐allyloxycarbonyl, Boc=tert‐butyloxycarbonyl, CSO=(1S)‐(+)‐(10‐camphorsulfonyl)‐oxaziridine, DBU=1,8‐diaza bicyclo[5.4.0]undec‐7‐ene, DMBA=1,3‐dimethylbarbituric acid, Et=ethyl, i‐Pr=iso‐propyl, PMB=para‐methoxybenzyl, SPPS=solid‐phase peptide synthesis, TBAF=tetrabutylammonium fluoride, TBDPS=tert‐butylchlorodiphenylsilane, TFA=trifluoroacetic acid and Ub=ubiquitin.

Scheme 10

A synthetic approach towards three distinct ADP‐ribosylated histidine isomers where the key building blocks 146–148 were obtained via a base‐assisted Mukaiyama‐like glycosylation reaction with histidine. HPF1(221–233): T‐F‐Pra‐G‐A‐G‐L‐V‐V‐P‐V‐D‐K. Boc=tert‐butyloxycarbonyl, Bz=benzoyl, CSO=(1S)‐(+)‐(10‐camphorsulfonyl)‐oxaziridine, DBU=1,8‐diazabicyclo[5.4.0]undec‐7‐ene, ETT=5‐ethyl‐thio‐tetrazole, Fm=9‐fluorenylmethyl, Fmoc=fluorenylmethoxycarbonyl, i‐Pr=iso‐propyl, PMB=para‐methoxybenzyl, Pyr=pyridine, TBDPS=tert‐butylchlorodiphenylsilane, Tf=trifluoromethanesulfonyl and TFA=trifluoroacetic acid.

Scheme 11

Site‐specific conjugation of ADP‐ribose to aminooxy or N‐methyl aminooxy functionalized histone H2B tail fragments by Moyle and Muir. Boc‐SPPS was applied for the assembly of 164 and 165, while Fmoc‐based conditions were used for 166. H2B_3–19 sequence=PAK SAPAP KKGSK KAVT. Boc=tert‐butyloxycarbonyl, Fmoc=fluorenylmethoxycarbonyl and SPPS=solid‐phase peptide synthesis.

Scheme 12

Preparation of triazole‐linked ADPr peptides and full‐length ubiquitin via a CuAAC between alkyne modified ADPr 173 and the target sequences 174, equipped with an azide click handle. β‐azidoalanine (n=1) and β‐azidohomoalanine (n=2) are abbreviated as A(N₃) and Aha(N₃) respectively. Ac=acetyl, DBU=1,8‐diazabicyclo[5.4.0]undec‐7‐ene, DCI=dicyanoimidazole, i‐Pr=iso‐propyl, NaAsc=sodium ascorbate, PA=propargyl alcohol, Ph=phenyl, PMB=para‐methoxybenzyl, Pyr=pyridine, t‐Bu=tert‐butyl, TBDPS=tert‐butylchlorodiphenylsilane, TFA=trifluoroacetic acid, THPTA=tris(hydroxypropyl‐triazolylmethylamine) and Ub=ubiquitin.

Scheme 13

Modular synthesis of ADP‐ribosylated histone H2B fragments via a CuAAC between ADPr‐N₃ analogues and the target peptide with a propargylglycine at the modification site by Li and Zhang and coworkers. H2B_4–19 sequence=P‐A‐K‐S‐A‐P‐A‐P‐K‐K‐G‐S‐K‐K‐A‐V‐T. Ac=acetyl, DEOA=diethanol‐amine, NaAsc=sodium ascorbate, Ph=phenyl, and THPTA=tris(hydroxypropyltriazolylmethylamine).

Scheme 14

An alternative route towards a‐ and b‐configured azido‐ADPr analogues and their implementation via a late‐stage Cu(I)‐catalyzed conjugation in peptide fragments HPF1(221–233): T‐F‐Pra‐G‐A‐G‐L‐V−V‐P‐V‐D‐K and PARP1(529–553): G‐G‐A‐A‐V‐D‐P‐D‐S‐G‐L‐E‐Pra‐S−A. The α/β assignment involves the anomeric centre highlighted by *. Ac=acetyl, Bz=benzoyl, DBU=1,8‐Diazabicyclo(5.4.0)undec‐7‐ene, Fm=9‐fluorenylmethyl, i‐Bu=iso‐butyl, i‐Pr=iso‐propyl, NaAsc=sodium ascorbate, PMB=para‐methoxybenzyl, Pra=propargylglycine, p‐TsOH=para‐toluenesulfonic acid, SPPS=solid‐phase peptide synthesis, TBDPS=tert‐butylchlorodiphenylsilane, TBS=tert‐butylsilyl, TMSOTf=trimethylsilyl trifluoromethanesulfonate, and THPTA=tris(hydroxypropyltriazolylmethylamine).

Scheme 15

An SPPS‐based approach towards a complete set of 1,4‐ and 1,5‐triazolyl linked ADP‐ribosylated peptide fragments by exploiting both Cu(I)‐ and Ru(II)‐catalyzed click chemistry. HPF1(221–233): T‐F‐H‐G‐A‐G‐L‐V‐V‐P‐V‐D‐K, where the site of modification is depicted bold. Bz=benzoyl, Boc=tert‐butyloxycarbonyl, Bn=benzyl, Cp*=cyclopentadienyl, CSO=(1S)‐(+)‐(10‐camphorsulfonyl)‐oxaziridine, DBU=1,8‐Diazabicyclo(5.4.0)undec‐7‐ene, ETT=5‐(Ethylthio)‐1H‐tetrazole, Fm=9‐fluorenylmethyl, Fmoc=fluorenylmethoxycarbonyl, i‐Bu=iso‐butyl, i‐Pr=iso‐propyl, PMB=para‐methoxybenzyl, Pyr=pyridine, TBDPS=tert‐butylchlorodiphenylsilane, and TFA=trifluoroacetic acid.

Scheme 16

The use of an Au(II)‐mediated glycosylation reaction in the synthesis of ‐configured thio‐furanoside on serine and its ensuing incorporation in the N‐terminus fragment of H2B(4–14): P‐A‐K‐S‐A‐P‐A‐P‐K‐K‐G, where the site of the modification in highlighted bold. Boc=tert‐butyloxycarbonyl, Bu=butyl, DBU=1,8‐Diazabicyclo(5.4.0)undec‐7‐ene, DMBA=1,3‐dimethylbarbituric acid, ETT=5‐(Ethylthio)‐1H‐tetrazole, Fm=9‐ fluorenylmethyl, Fmoc=fluorenylmethoxycarbonyl, i‐Pr=isoPr, m‐CPBA=meta‐Chloroperoxybenzoic acid, MsCl=methanesulfonyl chloride, Mtt=4‐methyltrityl, PMB=para‐methoxylbenzyl, TBAF=tetrabutylammonium fluoride, TBDPS=tert‐butylchlorodiphenylsilane, TBS=tert‐butylsilyl, Tf=triflyl and TFA=trifluoroacetic acid.