Epi-Cyclophellitol Cyclosulfate, a Mechanism-Based Endoplasmic Reticulum α-Glucosidase II Inhibitor, Blocks Replication of SARS-CoV-2 and Other Coronaviruses.

The combined inhibition of endoplasmic reticulum (ER) α-glucosidases I and II has been shown to inhibit replication of a broad range of viruses that rely on ER protein quality control. We found, by screening a panel of deoxynojirimycin and cyclitol glycomimetics, that the mechanism-based ER α-glucosidase II inhibitor, 1,6-epi-cyclophellitol cyclosulfate, potently blocks SARS-CoV-2 replication in lung epithelial cells, halting intracellular generation of mature spike protein, reducing production of infectious progeny, and leading to reduced syncytium formation. Through activity-based protein profiling, we confirmed ER α-glucosidase II inhibition in primary airway epithelial cells, grown at the air-liquid interface. 1,6-epi-Cyclophellitol cyclosulfate inhibits early pandemic and more recent SARS-CoV-2 variants, as well as SARS-CoV and MERS-CoV. The reported antiviral activity is comparable to the best-in-class described glucosidase inhibitors, all competitive inhibitors also targeting ER α-glucosidase I and other glycoprocessing enzymes not involved in ER protein quality control. We propose selective blocking ER-resident α-glucosidase II in a covalent and irreversible manner as a new strategy in the search for effective antiviral agents targeting SARS-CoV-2 and other viruses that rely on ER protein quality control.

Selective labelling of GBA2 in cells with fluorescent ß-d-arabinofuranosyl cyclitol aziridines.

GBA2, the non-lysosomal ß-glucosylceramidase, is an enzyme involved in glucosylceramide metabolism. Pharmacological inhibition of GBA2 by N-alkyl iminosugars is well tolerated and benefits patients suffering from Sandhoff and Niemann-Pick type C diseases, and GBA2 inhibitors have been proposed as candidate-clinical drugs for the treatment of parkinsonism. With the ultimate goal to unravel the role of GBA2 in (patho)physiology, we sought to develop a GBA2-specific activity-based probe (ABP). A library of probes was tested for activity against GBA2 and the two other cellular retaining ß-glucosidases, lysosomal GBA1 and cytosolic GBA3. We show that ß-d-arabinofuranosyl cyclitol aziridine (ß-d-Araf aziridine) reacts with the GBA2 active site nucleophile to form a covalent and irreversible bond. Fluorescent ß-d-Araf aziridine probes potently and selectively label GBA2 both in vitro and in cellulo, allowing for visualization of the localization of overexpressed GBA2 using fluorescence microscopy. Co-staining with an antibody selective for the lysosomal ß-glucosylceramidase GBA1, shows distinct subcellular localization of the two enzymes. We proffer our ABP technology for further delineating the role and functioning of GBA2 in disease and propose the ß-d-Araf aziridine scaffold as a good starting point for the development of GBA2-specific inhibitors for clinical development.

A PIKfyve modulator combined with an integrated stress response inhibitor to treat lysosomal storage diseases.

Lysosomal degradation pathways coordinate the clearance of superfluous and damaged cellular components. Compromised lysosomal degradation is a hallmark of many degenerative diseases, including lysosomal storage diseases (LSDs), which are caused by loss-of-function mutations within both alleles of a lysosomal hydrolase, leading to lysosomal substrate accumulation. Gaucher's disease, characterized by <15% of normal glucocerebrosidase function, is the most common LSD and is a prominent risk factor for developing Parkinson's disease. Here, we show that either of two structurally distinct small molecules that modulate PIKfyve activity, identified in a high-throughput cellular lipid droplet clearance screen, can improve glucocerebrosidase function in Gaucher patient-derived fibroblasts through an MiT/TFE transcription factor that promotes lysosomal gene translation. An integrated stress response (ISR) antagonist used in combination with a PIKfyve modulator further improves cellular glucocerebrosidase activity, likely because ISR signaling appears to also be slightly activated by treatment by either small molecule at the higher doses employed. This strategy of combining a PIKfyve modulator with an ISR inhibitor improves mutant lysosomal hydrolase function in cellular models of additional LSD.

From Mechanism-Based Retaining Glycosidase Inhibitors to Activity-Based Glycosidase Profiling.

Activity-based protein profiling (ABPP) is an effective technology for the identification and functional annotation of enzymes in complex biological samples. ABP designs are normally directed to an enzyme active site nucleophile, and within the field of Carbohydrate-Active Enzymes (CAZymes), ABPP has been most successful for those enzymes that feature such a residue: retaining glycosidases (GHs). Several mechanism-based covalent and irreversible retaining GH inhibitors have emerged over the past sixty years. ABP designs based on these inhibitor chemistries appeared since the turn of the millennium, and we contributed to the field by designing a suite of retaining GH ABPs modeled on the structure and mode of action of the natural product, cyclophellitol. These ABPs enable the study of both exo- and endo-acting retaining GHs in human health and disease, for instance in genetic metabolic disorders in which retaining GHs are deficient. They are also finding increasing use in the study of GHs in gut microbiota and environmental microorganisms, both in the context of drug (de)toxification in the gut and that of biomass polysaccharide processing for future sustainable energy and chemistries. This account comprises the authors' view on the history of mechanism-based retaining GH inhibitor design and discovery, on how these inhibitors served as blueprints for retaining GH ABP design, and on some current and future developments on how cyclophellitol-based ABPs may drive the discovery of retaining GHs and their inhibitors.

Parascedosporium putredinis NO1 tailors its secretome for different lignocellulosic substrates.

Parascedosporium putredinis NO1 is a plant biomass-degrading ascomycete with a propensity to target the most recalcitrant components of lignocellulose. Here we applied proteomics and activity-based protein profiling (ABPP) to investigate the ability of P. putredinis NO1 to tailor its secretome for growth on different lignocellulosic substrates. Proteomic analysis of soluble and insoluble culture fractions following the growth of P. putredinis NO1 on six lignocellulosic substrates highlights the adaptability of the response of the P. putredinis NO1 secretome to different substrates. Differences in protein abundance profiles were maintained and observed across substrates after bioinformatic filtering of the data to remove intracellular protein contamination to identify the components of the secretome more accurately. These differences across substrates extended to carbohydrate-active enzymes (CAZymes) at both class and family levels. Investigation of abundant activities in the secretomes for each substrate revealed similar variation but also a high abundance of "unknown" proteins in all conditions investigated. Fluorescence-based and chemical proteomic ABPP of secreted cellulases, xylanases, and ß-glucosidases applied to secretomes from multiple growth substrates for the first time confirmed highly adaptive time- and substrate-dependent glycoside hydrolase production by this fungus. P. putredinis NO1 is a promising new candidate for the identification of enzymes suited to the degradation of recalcitrant lignocellulosic feedstocks. The investigation of proteomes from the biomass bound and culture supernatant fractions provides a more complete picture of a fungal lignocellulose-degrading response. An in-depth understanding of this varied response will enhance efforts toward the development of tailored enzyme systems for use in biorefining.IMPORTANCEThe ability of the lignocellulose-degrading fungus Parascedosporium putredinis NO1 to tailor its secreted enzymes to different sources of plant biomass was revealed here. Through a combination of proteomic, bioinformatic, and fluorescent labeling techniques, remarkable variation was demonstrated in the secreted enzyme response for this ascomycete when grown on multiple lignocellulosic substrates. The maintenance of this variation over time when exploring hydrolytic polysaccharide-active enzymes through fluorescent labeling, suggests that this variation results from an actively tailored secretome response based on substrate. Understanding the tailored secretomes of wood-degrading fungi, especially from underexplored and poorly represented families, will be important for the development of effective substrate-tailored treatments for the conversion and valorization of lignocellulose.

Human milk oligosaccharide 2'-fucosyllactose protects against high-fat diet-induced obesity by changing intestinal mucus production, composition and degradation linked to changes in gut microbiota and faecal proteome profiles in mice.

OBJECTIVE: To decipher the mechanisms by which the major human milk oligosaccharide (HMO), 2'-fucosyllactose (2'FL), can affect body weight and fat mass gain on high-fat diet (HFD) feeding in mice. We wanted to elucidate whether 2'FL metabolic effects are linked with changes in intestinal mucus production and secretion, mucin glycosylation and degradation, as well as with the modulation of the gut microbiota, faecal proteome and endocannabinoid (eCB) system. RESULTS: 2'FL supplementation reduced HFD-induced obesity and glucose intolerance. These effects were accompanied by several changes in the intestinal mucus layer, including mucus production and composition, and gene expression of secreted and transmembrane mucins, glycosyltransferases and genes involved in mucus secretion. In addition, 2'FL increased bacterial glycosyl hydrolases involved in mucin glycan degradation. These changes were linked to a significant increase and predominance of bacterial genera Akkermansia and Bacteroides, different faecal proteome profile (with an upregulation of proteins involved in carbon, amino acids and fat metabolism and a downregulation of proteins involved in protein digestion and absorption) and, finally, to changes in the eCB system. We also investigated faecal proteomes from lean and obese humans and found similar changes observed comparing lean and obese mice. CONCLUSION: Our results show that the HMO 2'FL influences host metabolism by modulating the mucus layer, gut microbiota and eCB system and propose the mucus layer as a new potential target for the prevention of obesity and related disorders.

Conformational and Electronic Variations in 1,2- and 1,5a-Cyclophellitols and their Impact on Retaining α-Glucosidase Inhibition.

Glycoside hydrolases (glycosidases) take part in myriad biological processes and are important therapeutic targets. Competitive and mechanism-based inhibitors are useful tools to dissect their biological role and comprise a good starting point for drug discovery. The natural product, cyclophellitol, a mechanism-based, covalent and irreversible retaining ß-glucosidase inhibitor has inspired the design of diverse α- and ß-glycosidase inhibitor and activity-based probe scaffolds. Here, we sought to deepen our understanding of the structural and functional requirements of cyclophellitol-type compounds for effective human α-glucosidase inhibition. We synthesized a comprehensive set of α-configured 1,2- and 1,5a-cyclophellitol analogues bearing a variety of electrophilic traps. The inhibitory potency of these compounds was assessed towards both lysosomal and ER retaining α-glucosidases. These studies revealed the 1,5a-cyclophellitols to be the most potent retaining α-glucosidase inhibitors, with the nature of the electrophile determining inhibitory mode of action (covalent or non-covalent). DFT calculations support the ability of the 1,5a-cyclophellitols, but not the 1,2-congeners, to adopt conformations that mimic either the Michaelis complex or transition state of α-glucosidases.

Detection of Sulfoquinovosidase Activity in Cell Lysates Using Activity-Based Probes.

The sulfolipid sulfoquinovosyl diacylglycerol (SQDG), produced by plants, algae, and cyanobacteria, constitutes a major sulfur reserve in the biosphere. Microbial breakdown of SQDG is critical for the biological utilization of its sulfur. This commences through release of the parent sugar, sulfoquinovose (SQ), catalyzed by sulfoquinovosidases (SQases). These vanguard enzymes are encoded in gene clusters that code for diverse SQ catabolic pathways. To identify, visualize and isolate glycoside hydrolase CAZY-family 31 (GH31) SQases in complex biological environments, we introduce SQ cyclophellitol-aziridine activity-based probes (ABPs). These ABPs label the active site nucleophile of this enzyme family, consistent with specific recognition of the SQ cyclophellitol-aziridine in the active site, as evidenced in the 3D structure of Bacillus megaterium SQase. A fluorescent Cy5-probe enables visualization of SQases in crude cell lysates from bacteria harbouring different SQ breakdown pathways, whilst a biotin-probe enables SQase capture and identification by proteomics. The Cy5-probe facilitates monitoring of active SQase levels during different stages of bacterial growth which show great contrast to more traditional mRNA analysis obtained by RT-qPCR. Given the importance of SQases in global sulfur cycling and in human microbiota, these SQase ABPs provide a new tool with which to study SQase occurrence, activity and stability.

Synthesis and Macrodomain Binding of Gln-carba-ADPr-peptide.

Mono-ADP-ribosylation is a dynamic post-translational modification (PTM) with important roles in cell signalling. This modification occurs on a wide variety of amino acids, and one of the canonical modification sites within proteins is the side chain of glutamic acid. Given the transient nature of this modification (acylal linkage) and the high sensitivity of ADP-ribosylated glutamic acid, stabilized isosteres are required for structural and biochemical studies. Here, we report the synthesis of a mimic of ADP-ribosylated peptide derived from histone H2B that contains carba-ADP-ribosylated glutamine as a potential mimic for Glu-ADPr. We synthesized a cyclopentitol-ribofuranosyl derivative of 5'-phosphoribosylated Fmoc-glutamine and used this in the solid-phase synthesis of the carba-ADPr-peptide mimicking the ADP-ribosylated N-terminal tail of histone H2B. Binding studies with isothermal calorimetry demonstrate that the macrodomains of human MacroD2 and TARG1 bind to carba-ADPr-peptide in the same way as ADPr-peptides containing the native ADP-riboside moiety connected to the side chain of glutamine in the same peptide sequence.

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

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.

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

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.

Synthesis and application of bacterial exopolysaccharides.

Exopolysaccharides are produced and excreted by bacteria in the generation of biofilms to provide a protective environment. These polysaccharides are generally generated as heterogeneous polymers of varying length, featuring diverse substitution patterns. To obtain well-defined fragments of these polysaccharides, organic synthesis often is the method of choice, as it allows for full control over chain length and the installation of a pre-determined substitution pattern. This review presents several recent syntheses of exopolysaccharide fragments of Pseudomonas aeruginosa and Staphylococcus aureus and illustrates how these have been used to study biosynthesis enzymes and generate synthetic glycoconjugate vaccines.

ß-l-Arabinofurano-cyclitol Aziridines Are Covalent Broad-Spectrum Inhibitors and Activity-Based Probes for Retaining ß-l-Arabinofuranosidases.

GH127 and GH146 microorganismal retaining ß-l-arabinofuranosidases, expressed by human gut microbiomes, feature an atypical catalytic domain and an unusual mechanism of action. We recently reported that both Bacteroides thetaiotaomicron BtGH146 and Bifidobacterium longum HypBA1 are inhibited by ß-l-arabinofuranosyl cyclophellitol epoxide, supporting the action of a zinc-coordinated cysteine as a catalytic nucleophile, where in most retaining GH families, an aspartate or glutamate is employed. This work presents a panel of ß-l-arabinofuranosyl cyclophellitol epoxides and aziridines as mechanism-based BtGH146/HypBA1 inhibitors and activity-based probes. The ß-l-arabinofuranosyl cyclophellitol aziridines both inhibit and label ß-l-arabinofuranosidase efficiently (however with different activities), whereas the epoxide-derived probes favor BtGH146 over HypBA1. These findings are accompanied by X-ray structural analysis of the unmodified ß-l-arabinofuranosyl cyclophellitol aziridine in complex with both isozymes, which were shown to react by nucleophilic opening of the aziridine, at the pseudoanomeric carbon, by the active site cysteine nucleophile to form a stable thioether bond. Altogether, our activity-based probes may serve as chemical tools for the detection and identification of low-abundance ß-l-arabinofuranosidases in complex biological samples.

Synthetic Dual Cysteine-ADP Ribosylated Peptides from the Androgen Receptor are Recognized by the DTX3L/PARP9 Complex.

Androgen signaling in prostate cancer cells involves multisite cysteine ADP-ribosylation of the androgen receptor (AR) by PARP7. The AR modification is read by ADP-ribosyl binding macrodomains in PARP9, but the reason that multiple cysteines are modified is unknown. Here, we use synthetic peptides to show that dual ADP-ribosylation of closely spaced cysteines mediates recognition by the DTX3L/PARP9 complex. Mono and dual ADP-ribosylated cysteine peptides were prepared using a novel solid-phase synthetic strategy utilizing a key, Boc-protected, ribofuranosylcysteine building block. This synthetic strategy allowed us to synthesize fluorescently labeled peptides containing a dual ADP-ribosylation motif. It was found that the DTX3L/PARP9 complex recognizes the dual ADP-ribosylated AR peptide (Kd = 80.5 nM) with significantly higher affinity than peptides with a single ADP-ribose. Moreover, oligomerization of the DTX3L/PARP9 complex proved crucial for ADP-ribosyl-peptide interaction since a deletion mutant of the complex that prevents its oligomer formation dramatically reduced peptide binding. Our data show that features of the substrate modification and the reader contribute to the efficiency of the interaction and imply that multivalent interactions are important for AR-DTX3L/PARP9 assembly.

Trans-cyclosulfamidate mannose-configured cyclitol allows isoform-dependent inhibition of GH47 α-d-mannosidases through a bump-hole strategy.

Class I inverting exo-acting α-1,2-mannosidases (CAZY family GH47) display an unusual catalytic itinerary featuring ring-flipped mannosides, 3S1 → 3H4‡ → 1C4. Conformationally locked 1C4 compounds, such as kifunensine, display nanomolar inhibition but large multigene GH47 mannosidase families render specific "isoform-dependent" inhibition impossible. Here we develop a bump-and-hole strategy in which a new mannose-configured 1,6-trans-cyclic sulfamidate inhibits α-d-mannosidases by virtue of its 1C4 conformation. This compound does not inhibit the wild-type GH47 model enzyme by virtue of a steric clash, a "bump", in the active site. An L310S (a conserved residue amongst human GH47 enzymes) mutant of the model Caulobacter GH47 awoke 574 nM inhibition of the previously dormant inhibitor, confirmed by structural analysis of a 0.97 Å structure. Considering that L310 is a conserved residue amongst human GH47 enzymes, this work provides a unique framework for future biotechnological studies on N-glycan maturation and ER associated degradation by isoform-specific GH47 α-d-mannosidase inhibition through a bump-and-hole approach.

Fluorescence polarisation activity-based protein profiling for the identification of deoxynojirimycin-type inhibitors selective for lysosomal retaining alpha- and beta-glucosidases.

Lysosomal exoglycosidases are responsible for processing endocytosed glycans from the non-reducing end to produce the corresponding monosaccharides. Genetic mutations in a particular lysosomal glycosidase may result in accumulation of its particular substrate, which may cause diverse lysosomal storage disorders. The identification of effective therapeutic modalities to treat these diseases is a major yet poorly realised objective in biomedicine. One common strategy comprises the identification of effective and selective competitive inhibitors that may serve to stabilize the proper folding of the mutated enzyme, either during maturation and trafficking to, or residence in, endo-lysosomal compartments. The discovery of such inhibitors is greatly aided by effective screening assays, the development of which is the focus of the here-presented work. We developed and applied fluorescent activity-based probes reporting on either human GH30 lysosomal glucosylceramidase (GBA1, a retaining ß-glucosidase) or GH31 lysosomal retaining α-glucosidase (GAA). FluoPol-ABPP screening of our in-house 358-member iminosugar library yielded compound classes selective for either of these enzymes. In particular, we identified a class of N-alkyldeoxynojirimycins that inhibit GAA, but not GBA1, and that may form the starting point for the development of pharmacological chaperone therapeutics for the lysosomal glycogen storage disease that results from genetic deficiency in GAA: Pompe disease.

The development of a broad-spectrum retaining ß-exo-galactosidase activity-based probe.

Acid ß-galactosidase (GLB1) and galactocerebrosidase (GALC) are retaining exo-ß-galactosidases involved in lysosomal glycoconjugate metabolism. Deficiency of GLB1 may result in the lysosomal storage disorders GM1 gangliosidosis, Morquio B syndrome, and galactosialidosis, and deficiency of GALC may result in Krabbe disease. Activity-based protein profiling (ABPP) is a powerful technique to assess the activity of retaining glycosidases in relation to health and disease. This work describes the use of fluorescent and biotin-carrying activity-based probes (ABPs) to assess the activity of both GLB1 and GALC in cell lysates, culture media, and tissue extracts. The reported ABPs, which complement the growing list of retaining glycosidase ABPs based on configurational isomers of cyclophellitol, should assist in fundamental and clinical research on various ß-galactosidases, whose inherited deficiencies cause debilitating lysosomal storage disorders.

Selection and enrichment of microbial species with an increased lignocellulolytic phenotype from a native soil microbiome by activity-based probing.

Multi-omic analyses can provide information on the potential for activity within a microbial community but often lack specificity to link functions to cell, primarily offer potential for function or rely on annotated databases. Functional assays are necessary for understanding in situ microbial activity to better describe and improve microbiome biology. Targeting enzyme activity through activity-based protein profiling enhances the accuracy of functional studies. Here, we introduce a pipeline of coupling activity-based probing with fluorescence-activated cell sorting, culturing, and downstream activity assays to isolate and examine viable populations of cells expressing a function of interest. We applied our approach to a soil microbiome using two activity-based probes to enrich for communities with elevated activity for lignocellulose-degradation phenotypes as determined by four fluorogenic kinetic assays. Our approach efficiently separated and identified microbial members with heightened activity for glycosyl hydrolases, and by expanding this workflow to various probes for other function, this process can be applied to unique phenotype targets of interest.

Re-Exploring the Anthracycline Chemical Space for Better Anti-Cancer Compounds.

The anthracycline anti-cancer drugs are intensely used in the clinic to treat a wide variety of cancers. They generate DNA double strand breaks, but recently the induction of chromatin damage was introduced as another major determinant of anti-cancer activity. The combination of these two events results in their reported side effects. While our knowledge on the structure-activity relationship of anthracyclines has improved, many structural variations remain poorly explored. Therefore, we here report on the preparation of a diverse set of anthracyclines with variations within the sugar moiety, amine alkylation pattern, saccharide chain and aglycone. We assessed the cytotoxicity in vitro in relevant human cancer cell lines, and the capacity to induce DNA- and chromatin damage. This coherent set of data allowed us to deduce a few guidelines on anthracycline design, as well as discover novel, highly potent anthracyclines that may be better tolerated by patients.

Four of a Kind: A Complete Collection of ADP-Ribosylated Histidine Isosteres Using Cu(I)- and Ru(II)-Catalyzed Click Chemistry.

Adenosine diphosphate ribosylation (ADP-ribosylation) is a crucial post-translational modification involved in important regulatory mechanisms of numerous cellular pathways including histone maintenance and DNA damage repair. To study this modification, well-defined ADP-ribosylated peptides, proteins, and close analogues thereof have been invaluable tools. Recently, proteomics studies have revealed histidine residues to be ADP-ribosylated. We describe here the synthesis of a complete set of triazole-isosteres of ADP-ribosylated histidine to serve as probes for ADP-ribosylating biomachinery. By exploiting Cu(I)- and Ru(II)-catalyzed click chemistry between a propargylglycine building block and an α- or ß-configured azidoribose, we have successfully assembled the α- and ß-configured 1,4- and 1,5-triazoles, mimicking N(τ)- and N(π)-ADP-ribosylated histidine, respectively. The ribosylated building blocks could be incorporated into a peptide sequence using standard solid-phase peptide synthesis and transformed on resin into the ADP-ribosylated fragments to provide a total of four ADP-ribosyl triazole conjugates, which were evaluated for their chemical and enzymatic stability. The 1,5-triazole analogues mimicking the N(π)-substituted histidines proved susceptible to base-induced epimerization and the ADP-ribosyl α-1,5-triazole linkage could be cleaved by the (ADP-ribosyl)hydrolase ARH3.