RESUMO
ADP-ribose (ADPr) readers are essential components of ADP-ribosylation signaling, which regulates genome maintenance and immunity. The identification and discrimination between monoADPr (MAR) and polyADPr (PAR) readers is difficult because of a lack of suitable affinity-enrichment reagents. We synthesized well-defined ADPr probes and used these for affinity purifications combined with relative and absolute quantitative mass spectrometry to generate proteome-wide MAR and PAR interactomes, including determination of apparent binding affinities. Among the main findings, MAR and PAR readers regulate various common and distinct processes, such as the DNA-damage response, cellular metabolism, RNA trafficking, and transcription. We monitored the dynamics of PAR interactions upon induction of oxidative DNA damage and uncovered the mechanistic connections between ubiquitin signaling and ADP-ribosylation. Taken together, chemical biology enables exploration of MAR and PAR readers using interaction proteomics. Furthermore, the generated MAR and PAR interaction maps significantly expand our current understanding of ADPr signaling.
Assuntos
ADP-Ribosilação , Adenosina Difosfato Ribose/química , Difosfato de Adenosina/química , Proteômica/métodos , Ubiquitina-Proteína Ligases/química , Sítio Alostérico , Animais , Anticorpos Monoclonais/química , Sítios de Ligação , Biotinilação , Comunicação Celular , Dano ao DNA , Técnicas Genéticas , Células HeLa , Humanos , Espectrometria de Massas , Camundongos , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteoma , Transdução de Sinais , UbiquitinaRESUMO
ADP-ribosylation refers to the addition of one or more ADP-ribose groups onto proteins. The attached ADP-ribose monomers or polymers, commonly known as poly(ADP-ribose) (PAR), modulate the activities of the modified substrates or their binding affinities to other proteins. However, progress in this area is hindered by a lack of tools to investigate this protein modification. Here, we describe a new method named ELTA (enzymatic labeling of terminal ADP-ribose) for labeling free or protein-conjugated ADP-ribose monomers and polymers at their 2'-OH termini using the enzyme OAS1 and dATP. When coupled with various dATP analogs (e.g., radioactive, fluorescent, affinity tags), ELTA can be used to explore PAR biology with techniques routinely used to investigate DNA or RNA function. We demonstrate that ELTA enables the biophysical measurements of protein binding to PAR of a defined length, detection of PAR length from proteins and cells, and enrichment of sub-femtomole amounts of ADP-ribosylated peptides from cell lysates.
Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , ADP-Ribosilação , Adenosina Difosfato Ribose/metabolismo , Nucleotídeos de Desoxiadenina/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , 2',5'-Oligoadenilato Sintetase/genética , Animais , Células HeLa , Humanos , Ligação Proteica , Domínios Proteicos , Células Sf9 , Ubiquitina-Proteína Ligases/genéticaRESUMO
Viruses actively reprogram the metabolism of the host to ensure the availability of sufficient building blocks for virus replication and spreading. However, relatively little is known about how picornaviruses-a large family of small, non-enveloped positive-strand RNA viruses-modulate cellular metabolism for their own benefit. Here, we studied the modulation of host metabolism by coxsackievirus B3 (CVB3), a member of the enterovirus genus, and encephalomyocarditis virus (EMCV), a member of the cardiovirus genus, using steady-state as well as 13C-glucose tracing metabolomics. We demonstrate that both CVB3 and EMCV increase the levels of pyrimidine and purine metabolites and provide evidence that this increase is mediated through degradation of nucleic acids and nucleotide recycling, rather than upregulation of de novo synthesis. Finally, by integrating our metabolomics data with a previously acquired phosphoproteomics dataset of CVB3-infected cells, we identify alterations in phosphorylation status of key enzymes involved in nucleotide metabolism, providing insight into the regulation of nucleotide metabolism during infection.
Assuntos
Cardiovirus , Infecções por Enterovirus , Enterovirus , Picornaviridae , Humanos , Enterovirus/fisiologia , Vírus da Encefalomiocardite/fisiologia , Replicação Viral , Enterovirus Humano B/fisiologia , Células HeLaRESUMO
DNA damage triggers chromatin remodeling by mechanisms that are poorly understood. The oncogene and chromatin remodeler ALC1/CHD1L massively decompacts chromatin in vivo yet is inactive prior to DNA-damage-mediated PARP1 induction. We show that the interaction of the ALC1 macrodomain with the ATPase module mediates auto-inhibition. PARP1 activation suppresses this inhibitory interaction. Crucially, release from auto-inhibition requires a poly-ADP-ribose (PAR) binding macrodomain. We identify tri-ADP-ribose as a potent PAR-mimic and synthetic allosteric effector that abrogates ATPase-macrodomain interactions, promotes an ungated conformation, and activates the remodeler's ATPase. ALC1 fragments lacking the regulatory macrodomain relax chromatin in vivo without requiring PARP1 activation. Further, the ATPase restricts the macrodomain's interaction with PARP1 under non-DNA damage conditions. Somatic cancer mutants disrupt ALC1's auto-inhibition and activate chromatin remodeling. Our data show that the NAD+-metabolite and nucleic acid PAR triggers ALC1 to drive chromatin relaxation. Modular allostery in this oncogene tightly controls its robust, DNA-damage-dependent activation.
Assuntos
Montagem e Desmontagem da Cromatina , Dano ao DNA , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias/enzimologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Regulação Alostérica , Sítios de Ligação , Linhagem Celular Tumoral , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Humanos , Mutação , Neoplasias/genética , Neoplasias/patologia , Conformação de Ácido Nucleico , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/genética , Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose/química , Ligação Proteica , Relação Estrutura-Atividade , Fatores de TempoRESUMO
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.
Assuntos
Glutamina , Histonas , Humanos , Glutamina/química , Glutamina/metabolismo , Histonas/metabolismo , Peptídeos/química , ADP-Ribosilação , Glutamatos/metabolismoRESUMO
Nucleophilic substitution reactions are elementary reactions in organic chemistry that are used in many synthetic routes. By quantum chemical methods, we have investigated the intrinsic competition between the backside SN2 (SN2-b) and frontside SN2 (SN2-f) pathways using a set of simple alkyl triflates as the electrophile in combination with a systematic series of phenols and partially fluorinated ethanol nucleophiles. It is revealed how and why the well-established mechanistic preference for the SN2-b pathway slowly erodes and can even be overruled by the unusual SN2-f substitution mechanism going from strong to weak alcohol nucleophiles. Activation strain analyses disclose that the SN2-b pathway is favored for strong alcohol nucleophiles because of the well-known intrinsically more efficient approach to the electrophile resulting in a more stabilizing nucleophile-electrophile interaction. In contrast, the preference of weaker alcohol nucleophiles shifts to the SN2-f pathway, benefiting from a stabilizing hydrogen bond interaction between the incoming alcohol and the leaving group. This hydrogen bond interaction is strengthened by the increased acidity of the weaker alcohol nucleophiles, thereby steering the mechanistic preference toward the frontside SN2 pathway.
RESUMO
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.
Assuntos
Histidina , Técnicas de Síntese em Fase Sólida , Histidina/metabolismo , Peptídeos/química , ADP-Ribosilação , Difosfato de Adenosina/metabolismo , Adenosina Difosfato Ribose/químicaRESUMO
We report here chemoenzymatic and fully synthetic methodologies to modify aspartate and glutamate side chains with ADP-ribose at specific sites on peptides. Structural analysis of aspartate and glutamate ADP-ribosylated peptides reveals near-quantitative migration of the side chain linkage from the anomeric carbon to the 2â³- or 3â³-ADP-ribose hydroxyl moieties. We find that this linkage migration pattern is unique to aspartate and glutamate ADP-ribosylation and propose that the observed isomer distribution profile is present in biochemical and cellular environments. After defining distinct stability properties of aspartate and glutamate ADP-ribosylation, we devise methods to install homogenous ADP-ribose chains at specific glutamate sites and assemble glutamate-modified peptides into full-length proteins. By implementing these technologies, we show that histone H2B E2 tri-ADP-ribosylation is able to stimulate the chromatin remodeler ALC1 with similar efficiency to histone serine ADP-ribosylation. Our work reveals fundamental principles of aspartate and glutamate ADP-ribosylation and enables new strategies to interrogate the biochemical consequences of this widespread protein modification.
Assuntos
Ácido Aspártico , Ácido Glutâmico , Ácido Aspártico/metabolismo , Ácido Glutâmico/metabolismo , ADP-Ribosilação , Histonas/metabolismo , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/metabolismo , Peptídeos/químicaRESUMO
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.
Assuntos
Química Click , Histidina , Adenosina Difosfato Ribose , Catálise , TriazóisRESUMO
Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-ß1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis, showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use.
Assuntos
Antígenos de Bactérias/química , Antígenos CD1/química , Glicolipídeos/química , Glicoproteínas/química , Mycobacterium tuberculosis/química , Fosfolipídeos/química , Linfócitos T/química , Antígenos de Bactérias/imunologia , Antígenos CD1/imunologia , Linhagem Celular Transformada , Cristalografia por Raios X , Glicolipídeos/imunologia , Glicoproteínas/imunologia , Humanos , Mycobacterium tuberculosis/imunologia , Fosfolipídeos/imunologia , Linfócitos T/imunologiaRESUMO
We describe the development and optimization of a methodology to prepare peptides and proteins modified on the arginine residue with an adenosine-di-phosphate-ribosyl (ADPr) group. Our method comprises reacting an ornithine containing polypeptide on-resin with an α-linked anomeric isothiourea N-riboside, ensuing installment of a phosphomonoester at the 5'-hydroxyl of the ribosyl moiety followed by the conversion into the adenosine diphosphate. We use this method to obtain four regioisomers of ADP-ribosylated ubiquitin (UbADPr), each modified with an ADP-ribosyl residue on a different arginine position within the ubiquitin (Ub) protein (Arg42, Arg54, Arg72, and Arg74) as the first reported examples of fully synthetic arginine-linked ADPr-modified proteins. We show the chemically prepared Arg-linked UbADPr to be accepted and processed by Legionella enzymes and compare the entire suite of four Arg-linked UbADPr regioisomers in a variety of biochemical experiments, allowing us to profile the activity and selectivity of Legionella pneumophila ligase and hydrolase enzymes.
Assuntos
Adenosina Difosfato Ribose , Arginina , Adenosina Difosfato Ribose/química , Arginina/metabolismo , ADP-Ribosilação , Ubiquitina/química , Proteínas Ubiquitinadas/metabolismo , Peptídeos/químicaRESUMO
The stereoselective introduction of glycosidic bonds is of paramount importance to oligosaccharide synthesis. Among the various chemical strategies to steer stereoselectivity, participation by either neighboring or distal acyl groups is used particularly often. Recently, the use of the 2,2-dimethyl-2-(ortho-nitrophenyl)acetyl (DMNPA) protection group was shown to offer enhanced stereoselective steering compared to other acyl groups. Here, we investigate the origin of the stereoselectivity induced by the DMNPA group through systematic glycosylation reactions and infrared ion spectroscopy (IRIS) combined with techniques such as isotopic labeling of the anomeric center and isomer population analysis. Our study indicates that the origin of the DMNPA stereoselectivity does not lie in the direct participation of the nitro moiety but in the formation of a dioxolenium ion that is strongly stabilized by the nitro group.
Assuntos
Glicosídeos , Glicosídeos/química , Glicosilação , Íons , Espectrofotometria Infravermelho , EstereoisomerismoRESUMO
Mannose-6-phosphate (M6P) is recognized by the mannose-6-phosphate receptor and plays an important role in the transport of cargo to the endosomes, making it an attractive tool to improve endosomal trafficking of vaccines. We describe herein the assembly of peptide antigen conjugates carrying clusters of mannose-6-C-phosphonates (M6Po). The M6Po's are stable M6P mimics that are resistant to cleavage of the phosphate group by endogenous phosphatases. Two different strategies for the incorporation of the M6Po clusters in the conjugate have been developed: the first relies on a "post-assembly" click approach employing an M6Po bearing an alkyne functionality; the second hinges on an M6Po C-glycoside amino acid building block that can be used in solid-phase peptide synthesis. The generated conjugates were further equipped with a TLR7 ligand to stimulate dendritic cell (DC) maturation. While antigen presentation is hindered by the presence of the M6Po clusters, the incorporation of the M6Po clusters leads to increased activation of DCs, thus demonstrating their potential in improving vaccine adjuvanticity by intraendosomally active TLR ligands.
Assuntos
Antígenos/metabolismo , Manosefosfatos/metabolismo , Peptídeos/metabolismo , Receptores Toll-Like/metabolismo , Antígenos/química , Humanos , Ligantes , Manosefosfatos/química , Estrutura Molecular , Peptídeos/química , Receptores Toll-Like/químicaRESUMO
Synthetic vaccines, based on antigenic peptides that comprise MHC-I and MHC-II T-cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity inâ vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Toll-like receptor 2 (TLR2). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam3 CysSK4 . A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini-UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T-cell therapy. Homogeneous mini-UPam-SP conjugates have been prepared in good yields by stepwise solid-phase synthesis that employed a mini-UPam building block pre-prepared in solution and the standard set of Fmoc-amino acids. The immunogenicity of the novel mini-UPam-SP conjugates was demonstrated by using the cancer patient's T-cells.
Assuntos
Antígenos de Neoplasias/química , Vacinas Anticâncer/imunologia , Ligantes , Receptor 2 Toll-Like/química , Vacinas Sintéticas/imunologia , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/química , Linhagem Celular , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Desenho de Fármacos , Humanos , Interleucina-8/metabolismo , Lipopeptídeos/síntese química , Lipopeptídeos/química , Lipopeptídeos/imunologia , Lipoilação , Ativação Linfocitária , Receptor 2 Toll-Like/metabolismo , Vacinas Sintéticas/químicaRESUMO
Wall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram-positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP) residues that are decorated with d-alanine and N-acetyl-d-glucosamine (GlcNAc) modifications, in a seemingly random manner. These WTA-modifications play an important role in shaping the interactions of WTA with the host immune system. Due to the structural heterogeneity of WTAs, it is impossible to isolate pure and well-defined WTA molecules from bacterial sources. Therefore, here synthetic chemistry to assemble a broad library of WTA-fragments, incorporating all possible glycosylation modifications (α-GlcNAc at the RboP C4; ß-GlcNAc at the RboP C4; ß-GlcNAc at the RboP C3) described for S. aureus WTAs, is reported. DNA-type chemistry, employing ribitol phosphoramidite building blocks, protected with a dimethoxy trityl group, was used to efficiently generate a library of WTA-hexamers. Automated solid phase syntheses were used to assemble a WTA-dodecamer and glycosylated WTA-hexamer. The synthetic fragments have been fully characterized and diagnostic signals were identified to discriminate the different glycosylation patterns. The different glycosylated WTA-fragments were used to probe binding of monoclonal antibodies using WTA-functionalized magnetic beads, revealing the binding specificity of these WTA-specific antibodies and the importance of the specific location of the GlcNAc modifications on the WTA-chains.
Assuntos
Infecções Estafilocócicas , Ácidos Teicoicos , Parede Celular/metabolismo , Glicosilação , Humanos , Staphylococcus aureus/metabolismoRESUMO
ADP-ribosylation (ADPr), as a post-translational modification, plays a crucial role in DNA-repair, immunity and many other cellular and physiological processes. Serine is the main acceptor for ADPr in DNA damage response, whereas the physiological impact of less common ADPr-modifications of cysteine and threonine side chains is less clear. Generally, gaining molecular insights into ADPr recognition and turn-over is hampered by the availability of homogeneous, ADP-ribosylated material, such as mono-ADP-ribosylated (MARylated) peptides. Here, a new and efficient solid-phase strategy for the synthesis of Ser-, Thr- and Cys-MARylated peptides is described. ADP-ribosylated cysteine, apart from being a native post-translational modification in its own right, proved to be suitable as a stabile bioisostere for ADP-ribosylated serine making it a useful tool to further biochemical research on serine ADP-ribosylation. In addition, it was discovered that the Streptococcus pyogenes encoded protein, SpyMacroD, acts as a Cys-(ADP-ribosyl) hydrolase.
Assuntos
ADP-Ribosilação , Serina , Reparo do DNA , Peptídeos , Processamento de Proteína Pós-Traducional , Serina/metabolismoRESUMO
Legionnaires' disease is caused by infection with the intracellularly replicating Gram-negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host proteins by generating a phosphoribosyl linkage between substrate proteins and ubiquitin by making use of an ADPribosylated ubiquitin (UbADPr ) intermediate. The family of SidE effector enzymes that catalyze this reaction is counteracted by Legionella hydrolases, which are called Dups. This unusual ubiquitination process is important for Legionella proliferation and understanding these processes on a molecular level might prove invaluable in finding new treatments. Herein, a modular approach is used for the synthesis of triazole-linked UbADPr , and analogues thereof, and their affinity towards the hydrolase DupA is determined and hydrolysis rates are compared to natively linked UbADPr . The inhibitory effects of modified Ub on the canonical eukaryotic E1-enzyme Uba1 are investigated and rationalized in the context of a high-resolution crystal structure reported herein. Finally, it is shown that synthetic UbADPr analogues can be used to effectively pull-down overexpressed DupA from cell lysate.
Assuntos
ADP-Ribosilação , Legionella pneumophila/enzimologia , Doença dos Legionários/microbiologia , Ubiquitina/química , Ubiquitina/metabolismo , Proteínas de Bactérias/metabolismo , Células HEK293 , Humanos , Hidrolases/metabolismo , Legionella pneumophila/crescimento & desenvolvimento , Enzimas Ativadoras de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
We have quantum chemically studied the Lewis acid-catalyzed epoxide ring-opening reaction of cyclohexene epoxide by MeZH (Z = O, S, and NH) using relativistic dispersion-corrected density functional theory. We found that the reaction barrier of the Lewis acid-catalyzed epoxide ring-opening reactions decreases upon ascending in group 1 along the series Cs+ > Rb+ > K+ > Na+ > Li+ > H+. Our activation strain and Kohn-Sham molecular orbital analyses reveal that the enhanced reactivity of the Lewis acid-catalyzed ring-opening reaction is caused by the reduced steric (Pauli) repulsion between the filled orbitals of the epoxide and the nucleophile, as the Lewis acid polarizes the filled orbitals of the epoxide more efficiently away from the incoming nucleophile. Furthermore, we established that the regioselectivity of these ring-opening reactions is, aside from the "classical" strain control, also dictated by a hitherto unknown mechanism, namely, the steric (Pauli) repulsion between the nucleophile and the substrate, which could be traced back to the asymmetric orbital density on the epoxide. In all, this work again demonstrates that the concept of Pauli-lowering catalysis is a general phenomenon.
RESUMO
The assembly of complex bacterial glycans presenting rare structural motifs and cis-glycosidic linkages is significantly obstructed by the lack of knowledge of the reactivity of the constituting building blocks and the stereoselectivity of the reactions in which they partake. We here report a strategy to map the reactivity of carbohydrate building blocks and apply it to understand the reactivity of the bacterial sugar, caryophyllose, a rare C12-monosaccharide, containing a characteristic tetrasubstituted stereocenter. We mapped reactivity-stereoselectivity relationships for caryophyllose donor and acceptor glycosides by a systematic series of glycosylations in combination with the detection and characterization of different reactive intermediates using experimental and computational techniques. The insights garnered from these studies enabled the rational design of building blocks with the required properties to assemble mycobacterial lipooligosaccharide fragments of M. marinum.
Assuntos
Lipopolissacarídeos/metabolismo , Mycobacterium marinum/metabolismo , Proteínas de Bactérias/metabolismo , Teoria da Densidade Funcional , Glicosilação , Lipopolissacarídeos/química , EstereoisomerismoRESUMO
Bioorthogonal chemistry allows the selective modification of biomolecules in complex biological samples. One application of this methodology is in two-step activity-based protein profiling (ABPP), a methodology that is particularly attractive where direct ABPP using fluorescent or biotinylated probes is ineffective. Herein we describe a set of norbornene-modified, mechanism-based proteasome inhibitors aimed to be selective for each of the six catalytic sites of human constitutive proteasomes and immunoproteasomes. The probes developed for ß1i, ß2i, ß5c, and ß5i proved to be useful two-step ABPs that effectively label their developed proteasome subunits in both Raji cell extracts and living Raji cells through inverse-electron-demand Diels-Alder (iEDDA) ligation. The compound developed for ß1c proved incapable of penetrating the cell membrane, but effectively labels ß1c in vitro. The compound developed for ß2c proved not selective, but its azide-containing analogue LU-002c proved effective in labeling of ß2c via azide-alkyne click ligation chemistry both in vitro and inâ situ. In total, our results contribute to the growing list of proteasome activity tools to include five subunit-selective activity-based proteasome probes, four of which report on proteasome activities in living cells.