Proteasome Subunit Selective Activity-Based Probes Report on Proteasome Core Particle Composition in a Native Polyacrylamide Gel Electrophoresis Fluorescence-Resonance Energy Transfer Assay

Most mammalian tissues contain a single proteasome species: constitutive proteasomes. Tissues able to express, next to the constitutive proteasome catalytic activities (β1c, β2c, β5c), the three homologous activities, β1i, β2i and β5i, may contain numerous distinct proteasome particles: immunoproteasomes (composed of β1i, β2i and β5i) and mixed proteasomes containing a mix of these activities. This work describes the development of new subunit-selective activity-based probes and their use in an activity-based protein profiling assay that allows the detection of various proteasome particles. Tissue extracts are treated with subunit-specific probes bearing distinct fluorophores and subunit-specific inhibitors. The samples are resolved by native polyacrylamide gel electrophoresis, after which fluorescence-resonance energy transfer (FRET) reports on the nature of proteasomes present

Reagent Controlled Stereoselective Synthesis of α‑Glucans

The development of a general glycosylation method that allows for the stereoselective construction of glycosidic linkages is a tremendous challenge. Because of the differences in steric and electronic properties of the building blocks used, the outcome of a glycosylation reaction can vary greatly when switching form one glycosyl donor–acceptor pair to another. We here report a strategy to install <i>cis</i>-glucosidic linkages in a fully stereoselective fashion that is under direct control of the reagents used to activate a single type of donor building block. The activating reagents are tuned to the intrinsic reactivity of the acceptor alcohol to match the reactivity of the glycosylating agent with the reactivity of the incoming nucleophile. A protecting group strategy is introduced that is based on the sole use of benzyl-ether type protecting groups to circumvent changes in reactivity as a result of the protecting groups. For the stereoselective construction of the α-glucosyl linkages to a secondary alcohol, a per-benzylated glusosyl imidate donor is activated with a combination of trimethylsilyltriflate and DMF, while activation of the same imidate donor with trimethylsilyl iodide in the presence of triphenylphosphine oxide allows for the stereoselective <i>cis</i>-glucosylation of primary alcohols. The effectiveness of the strategy is illustrated in the modular synthesis of a <i>Mycobacterium tuberculosis</i> nonasaccharide, composed of an α-(1–4)-oligoglucose backbone bearing different α-glucosyl branches

2,2-Dimethyl-4-(4-methoxy-phenoxy) butanoate and 2,2-Dimethyl-4-azido Butanoate: Two New Pivaloate-ester-like Protecting Groups

The title compounds were developed to extend the available orthogonalities within the class of protecting groups removed by assisted cleavage. The mild, complementary (oxidative vs reductive) reaction conditions for the removal, together with their pivaloate-like character, were exploited, in combination with a levulinoyl-ester functioning as a third orthogonal protecting group, in the assembly of a <i>Streptococcus mutans</i> hexasaccharide built up from a oligorhamnose backbone featuring β-glucosyl appendages

Synthetic Studies on the Preparation of Alanyl Epoxysulfones as Cathepsin Cysteine Protease Electrophilic Traps

A Darzens reaction between <i>tert</i>-butoxycarbonyl alaninal and chloromethyl phenyl sulfone afforded chlorohydrins, which were converted into epoxysulfones by reaction with sodium <i>tert</i>-butoxide. Epoxysulfone <b>10</b> and chloroketone <b>14</b> derived from chlorohydrins by oxidation proved to be inhibitors of cathepsins H, S, and C as determined by competitive activity-based protein profiling

Branching of poly(ADP-ribose): Synthesis of the Core Motif

The synthesis of the core motif of branched poly­(adenosine diphosphate ribose) (poly­(ADPr)) is described, and structural analysis reasserted the proposed stereochemistry for branching. For the synthesis, a ribose trisaccharide was first constructed with only α-<i>O</i>-glycosidic linkages. Finally, the adenine nucleobase was introduced via a Vorbrüggen-type glycosylation reaction. The orthogonality of the selected protecting groups was demonstrated, allowing for the construction of branched poly­(ADPr) oligomers in the near future

Reagent Controlled Stereoselective Synthesis of α‑Glucans

The development of a general glycosylation method that allows for the stereoselective construction of glycosidic linkages is a tremendous challenge. Because of the differences in steric and electronic properties of the building blocks used, the outcome of a glycosylation reaction can vary greatly when switching form one glycosyl donor–acceptor pair to another. We here report a strategy to install <i>cis</i>-glucosidic linkages in a fully stereoselective fashion that is under direct control of the reagents used to activate a single type of donor building block. The activating reagents are tuned to the intrinsic reactivity of the acceptor alcohol to match the reactivity of the glycosylating agent with the reactivity of the incoming nucleophile. A protecting group strategy is introduced that is based on the sole use of benzyl-ether type protecting groups to circumvent changes in reactivity as a result of the protecting groups. For the stereoselective construction of the α-glucosyl linkages to a secondary alcohol, a per-benzylated glusosyl imidate donor is activated with a combination of trimethylsilyltriflate and DMF, while activation of the same imidate donor with trimethylsilyl iodide in the presence of triphenylphosphine oxide allows for the stereoselective <i>cis</i>-glucosylation of primary alcohols. The effectiveness of the strategy is illustrated in the modular synthesis of a <i>Mycobacterium tuberculosis</i> nonasaccharide, composed of an α-(1–4)-oligoglucose backbone bearing different α-glucosyl branches

Mapping the Reactivity and Selectivity of 2‑Azidofucosyl Donors for the Assembly of <i>N</i>‑Acetylfucosamine-Containing Bacterial Oligosaccharides

The synthesis of complex oligosaccharides is often hindered by a lack of knowledge on the reactivity and selectivity of their constituent building blocks. We investigated the reactivity and selectivity of 2-azidofucosyl (FucN₃) donors, valuable synthons in the synthesis of 2-acetamido-2-deoxyfucose (FucNAc) containing oligosaccharides. Six FucN₃ donors, bearing benzyl, benzoyl, or <i>tert</i>-butyldimethylsilyl protecting groups at the C3-<i>O</i> and C4-<i>O</i> positions, were synthesized, and their reactivity was assessed in a series of glycosylations using acceptors of varying nucleophilicity and size. It was found that more reactive nucleophiles and electron-withdrawing benzoyl groups on the donor favor the formation of β-glycosides, while poorly reactive nucleophiles and electron-donating protecting groups on the donor favor α-glycosidic bond formation. Low-temperature NMR activation studies of Bn- and Bz-protected donors revealed the formation of covalent FucN₃ triflates and oxosulfonium triflates. From these results, a mechanistic explanation is offered in which more reactive acceptors preferentially react via an S_N2-like pathway, while less reactive acceptors react via an S_N1-like pathway. The knowledge obtained in this reactivity study was then applied in the construction of α-FucN₃ linkages relevant to bacterial saccharides. Finally, a modular synthesis of the <i>Staphylococcus aureus</i> type 5 capsular polysaccharide repeating unit, a trisaccharide consisting of two FucNAc units, is described

Stereoselectivity of Conformationally Restricted Glucosazide Donors

Glycosylations of 4,6-tethered glucosazide donors with a panel of model acceptors revealed the effect of acceptor nucleophilicity on the stereoselectivity of these donors. The differences in reactivity among the donors were evaluated in competitive glycosylation reactions, and their relative reactivities were found to be reflected in the stereoselectivity in glycosylations with a set of fluorinated alcohols as well as carbohydrate acceptors. We found that the 2-azido-2-deoxy moiety is more β-directing than its C-2-<i>O</i>-benzyl counterpart, as a consequence of increased destabilization of anomeric charge development by the electron-withdrawing azide. Additional disarming groups further decreased the α-selectivity of the studied donors, whereas substitution of the 4,6-benzylidene acetal with a 4,6-di-<i>tert</i>-butyl silylidene led to a slight increase in α-selectivity. The C-2-dinitropyridone group was also explored as an alternative for the nonparticipating azide group, but this protecting group significantly increased β-selectivity. All studied donors exhibited the same acceptor-dependent selectivity trend, and good α-selectivity could be obtained with the weakest acceptors and most reactive donors

Systematic Analyses of Substrate Preferences of 20S Proteasomes Using Peptidic Epoxyketone Inhibitors

Cleavage analyses of 20S proteasomes with natural or synthetic substrates allowed to infer the substrate specificities of the active sites and paved the way for the rational design of high-affinity proteasome inhibitors. However, details of cleavage preferences remained enigmatic due to the lack of appropriate structural data. In a unique approach, we here systematically examined substrate specificities of yeast and human proteasomes using irreversibly acting α′,β′epoxyketone (ep) inhibitors. Biochemical and structural analyses provide unique insights into the substrate preferences of the distinct active sites and highlight differences between proteasome types that may be considered in future inhibitor design efforts. (1) For steric reasons, epoxyketones with Val or Ile at the P1 position are weak inhibitors of all active sites. (2) Identification of the β2c selective compound Ac-LAE-ep represents a promising starting point for the development of compounds that discriminate between β2c and β2i. (3) The compound Ac-LAA-ep was found to favor subunit β5c over β5i by three orders of magnitude. (4) Yeast β1 and human β1c subunits preferentially bind Asp and Leu in their S1 pockets, while Glu and large hydrophobic residues are not accepted. (5) Exceptional structural features in the β1/2 substrate binding channel give rise to the β1 selectivity of compounds featuring Pro at the P3 site. Altogether, 23 different epoxyketone inhibitors, five proteasome mutants, and 43 crystal structures served to delineate a detailed picture of the substrate and ligand specificities of proteasomes and will further guide drug development efforts toward subunit-specific proteasome inhibitors for applications as diverse as cancer and autoimmune disorders

Synthetic α- and β‑Ser-ADP-ribosylated Peptides Reveal α‑Ser-ADPr as the Native Epimer

A solid-phase methodology to synthesize oligopeptides, specifically incorporating serine residues linked to ADP-ribose (ADPr), is presented. Through the synthesis of both α- and β-anomers of the phosphoribosylated Fmoc-Ser building block and their usage in our modified solid-phase peptide synthesis protocol, both α- and β-ADPr peptides from a naturally Ser-ADPr containing H2B sequence were obtained. With these, and by digestion studies using the human glycohydrolase, ARH3 (hARH3), compelling evidence is obtained that the α-Ser-ADPr linkage comprises the naturally occurring configuration