RESUMEN
The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used messenger RNA (mRNA) display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor-binding domain, N-terminal domain, and S2 region, distal to the angiotensin-converting enzyme 2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Pandemias/prevención & control , Péptidos/farmacologíaRESUMEN
Macrocyclisation provides a means of stabilising the conformation of peptides, often resulting in improved stability, selectivity, affinity, and cell permeability. In this work, a new approach to peptide macrocyclisation is reported, using a cyanobenzothiazole-containing amino acid that can be incorporated into peptides by both inâ vitro translation and solid phase peptide synthesis, meaning it should be applicable to peptide discovery by mRNA display. This cyclisation proceeds rapidly, with minimal by-products, is selective over other amino acids including non N-terminal cysteines, and is compatible with further peptide elaboration exploiting such an additional cysteine in bicyclisation and derivatisation reactions. Molecular dynamics simulations show that the new cyclisation group is likely to influence the peptide conformation as compared to previous thioether-based approaches, through rigidity and intramolecular aromatic interactions, illustrating their complementarity.
Asunto(s)
Aminoácidos , Péptidos , Péptidos/química , Cisteína/química , CiclizaciónRESUMEN
Herpes simplex virus (HSV-1) employs heparan sulfate (HS) as receptor for cell attachment and entry. During late-stage infection, the virus induces the upregulation of human heparanase (Hpse) to remove cell surface HS allowing viral spread. We hypothesized that inhibition of Hpse will prevent viral release thereby representing a new therapeutic strategy for HSV-1. A range of HS-oligosaccharides was prepared to examine the importance of chain length and 2-O-sulfation of iduronic moieties for Hpse inhibition. It was found that hexa- and octasaccharides potently inhibited the enzyme and that 2-O-sulfation of iduronic acid is tolerated. Computational studies provided a rationale for the observed structure-activity relationship. Treatment of human corneal epithelial cells (HCEs) infected with HSV-1 with the hexa- and octasaccharide blocked viral induced shedding of HS which significantly reduced spread of virions. The compounds also inhibited migration and proliferation of immortalized HCEs thereby providing additional therapeutic properties.
Asunto(s)
Glucuronidasa , Herpes Simple , Herpesvirus Humano 1 , Humanos , Glucuronidasa/antagonistas & inhibidores , Glucuronidasa/metabolismo , Heparitina Sulfato/farmacología , Herpes Simple/enzimología , Herpes Simple/virología , Herpesvirus Humano 1/metabolismo , Oligosacáridos/farmacología , Oligosacáridos/metabolismoRESUMEN
Propionibacterium acnes, though generally considered part of the normal flora of human skin, is an opportunistic pathogen associated with acne vulgaris as well as other diseases, including endocarditis, endophthalmitis and prosthetic joint infections. Its virulence potential is also supported by knowledge gained from its sequenced genome. Indeed, a vaccine targeting a putative cell wall-anchored P. acnes sialidase has been shown to suppress cytotoxicity and pro-inflammatory cytokine release induced by the organism, and is proposed as an alternative treatment for P. acnes-associated diseases. Here, we report the crystal structures of the surface sialidase and its complex with the transition-state mimic Neu5Ac2en. Our structural and kinetic analyses, together with insight from a glycan array screen, which probes subtle specificities of the sialidase for α-2,3-sialosides, provide a basis for the structure-based design of novel small-molecule therapeutics against P. acnes infections.
Asunto(s)
Acné Vulgar , Propionibacterium acnes , Acné Vulgar/tratamiento farmacológico , Acné Vulgar/microbiología , Humanos , Neuraminidasa , PielRESUMEN
DNA-encoded small-molecule libraries and mRNA displayed peptide libraries both use numerically large pools of oligonucleotide-tagged molecules to identify potential hits for protein targets. They differ dramatically, however, in the 'drug-likeness' of the molecules that each can be used to discover. We give here an overview of the two techniques, comparing some advantages and disadvantages of each, and suggest areas where particularly mRNA display can benefit from adopting advances developed with DNA-encoded small molecule libraries. We outline cases where chemical modification of the peptide library has already been used in mRNA display, and survey opportunities to expand this using examples from DNA-encoded small molecule libraries. We also propose potential opportunities for encoding such reactions within the mRNA/cDNA tag of an mRNA-displayed peptide library to allow a more diversity-oriented approach to library modification. Finally, we outline alternate approaches for enriching target-binding hits from a pooled and tagged library, and close by detailing several examples of how an adjusted mRNA-display based approach could be used to discover new 'drug-like' modified small peptides.
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Biblioteca de Péptidos , Bibliotecas de Moléculas Pequeñas , ADN/química , Descubrimiento de Drogas/métodos , ARN Mensajero/genética , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Thiols are a functional group commonly used for selective reactions in a biochemical setting because of their high nucleophilicity. Phosphorus nucleophiles can undergo some similar reactions to thiols, but remain underexploited in this setting. In this work we show that phosphine nucleophiles react cleanly and quickly with a dehydroalanine electrophile, itself generated from cysteine, to give a stable adduct in a peptide context. NMR reveals the product to be a phosphonium ion and indicates some backbone conformational constraint, possibly arising from transient carbonyl coordination. The reaction proceeded quickly, with a pseudo-first order rate constant of 0.126 min-1 at 1 mM peptide (80% conversion in 10 min), and with no detectable side products on the peptide. A broad peptide sequence scope and water-soluble phosphines with alkyl as well as aromatic groups were all shown to react efficiently. Phosphine addition proved to be efficient on nisin as a model Dha-containing biologically-derived peptide and on an mRNA-displayed peptide, as well as on TCEP-modified agarose for peptide capture from solution. This reaction thus presents a promising approach for modification of peptides for cargo attachment or altered physical properties in peptide discovery.
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Fosfinas , Alanina/análogos & derivados , Secuencia de Aminoácidos , Fosfinas/química , Compuestos de SulfhidriloRESUMEN
O-GlcNAc transferase (OGT) is the only enzyme that catalyzes the post-translational modification of proteins at Ser/Thr with a single ß-N-acetylglucosamine (O-GlcNAcylation). Its activity has been associated with chronic diseases such as cancer, diabetes and neurodegenerative disease. Although numerous OGT substrates have been identified, its accepted substrate scope can still be refined. We report here an attempt to better define the peptide-recognition requirements of the OGT active site by using mRNA display, taking advantage of its extremely high throughput to assess the substrate potential of a library of all possible nonamer peptides. An antibody-based selection process is described here that is able to enrich an OGT substrate peptide from such a library, but with poor absolute recovery. Following four rounds of selection for O-GlcNAcylated peptides, sequencing revealed 14â peptides containing Ser/Thr, but these were shown by luminescence-coupled assays and peptide microarray not to be OGT substrates. By contrast, subsequent testing of an N-terminal tag approach showed exemplary recovery. Our approach demonstrates the power of genetically encoded libraries for selection of peptide substrates, even from a very low initial starting abundance and under suboptimal conditions, and emphasizes the need to consider the binding biases of antibodies and both C- and N-terminal tags in profiling peptide substrates by high-throughput display.
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N-Acetilglucosaminiltransferasas/metabolismo , Fragmentos de Péptidos/metabolismo , Análisis por Matrices de Proteínas/métodos , ARN Mensajero/metabolismo , Dominio Catalítico , Humanos , Técnicas In Vitro , Luminiscencia , N-Acetilglucosaminiltransferasas/genética , ARN Mensajero/genética , Especificidad por SustratoRESUMEN
Targeting chemokine signaling is an attractive avenue for the treatment of inflammatory disorders. Tyrosine sulfation is an important post-translational modification (PTM) that enhances chemokine-receptor binding and is also utilized by a number of pathogenic organisms to improve the binding affinity of immune-suppressive chemokine binding proteins (CKBPs). Here we report the display selection of tyrosine-sulfated cyclic peptides using a reprogrammed genetic code to discover high-affinity ligands for the chemokine CCL11 (eotaxin-1). The selected cyclic sulfopeptides possess high affinity for the target chemokine (as well as one or more of the related family members CCL2, CCL7 and CCL24) and inhibit CCL11 activation of CC chemokine receptor 3 (CCR3). This work demonstrates the utility of exploiting native PTMs as binding motifs for the generation of new leads for medicinal chemistry.
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Quimiocina CCL11/antagonistas & inhibidores , Descubrimiento de Drogas , Péptidos/farmacología , ARN Mensajero/efectos de los fármacos , Quimiocina CCL11/genética , Quimiocina CCL11/metabolismo , Humanos , Estructura Molecular , Péptidos/química , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Targeting of proteins in the histone modification machinery has emerged as a promising new direction to fight disease. The search for compounds that inhibit proteins that readout histone modification has led to several new epigenetic drugs, mostly for proteins involved in recognition of acetylated lysines. However, this approach proved to be a challenging task for methyllysine readers, which typically feature shallow binding pockets. Moreover, reader proteins of trimethyllysine K36 on the histone H3 (H3K36me3) not only bind the methyllysine but also the nucleosomal DNA. Here, we sought to find peptide-based binders of H3K36me3 reader PSIP1, which relies on DNA interactions to tightly bind H3K36me3 modified nucleosomes. We designed several peptides that mimic the nucleosomal context of H3K36me3 recognition by including negatively charged Glu-rich regions. Using a detailed NMR analysis, we find that addition of negative charges boosts binding affinity up to 50-fold while decreasing binding to the trimethyllysine binding pocket. Since screening and selection of compounds for reader domains is typically based solely on affinity measurements due to their lack of enzymatic activity, our case highlights the need to carefully control for the binding mode, in particular for the challenging case of H3K36me3 readers.
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Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Nucleosomas/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Electricidad Estática , TermodinámicaRESUMEN
Genetic code reprogramming is a powerful approach to controlled protein modification. A remaining challenge, however, is the generation of vacant codons. We targeted the initiation machinery of E. coli, showing that restriction of the formyl donor or inhibition of the formyl transferase during in vitro translation is sufficient to prevent formylation of the acylated initiating tRNA and thereby create a vacant initiation codon that can be reprogrammed by exogenously charged tRNA. Our approach conveniently generates peptides and proteins tagged N-terminally with non-canonical functional groups at up to 99 % reprogramming efficiency, in combination with decoding the AUG elongation codons either with native methionine or with further reprogramming with azide- and alkyne-containing cognates. We further show macrocyclization and intermolecular modifications with these click handles, thus emphasizing the applicability of our method to current challenges in peptide and protein chemistry.
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Escherichia coli/metabolismo , ARN de Transferencia/metabolismo , Acilación , Escherichia coli/genética , Código Genético , Modelos Moleculares , Conformación Molecular , Péptidos/genética , Péptidos/metabolismo , Proteínas/genética , Proteínas/metabolismo , ARN de Transferencia/genéticaRESUMEN
Retaining glycosidases are an important class of enzymes involved in glycan degradation. To study better the role of specific enzymes in deglycosylation processes, and thereby the importance of particular glycosylation patterns, a set of potent inhibitors, each specific to a particular glycosidase, would be an invaluable toolkit. Towards this goal, we detail here a more in-depth study of a prototypical macrocyclic peptide inhibitor of the model retaining glycosidase human pancreatic α-amylase (HPA). Notably, incorporation of l-DOPA into this peptide affords an inhibitor of HPA with potency that is tenfold higher (Ki =480â pm) than that of the previously found consensus sequence. This represents a first successful step in converting a recently discovered natural-product-derived motif, already specific for the catalytic side-chain arrangement conserved in the active sites of retaining glycosidases, into a tuneable retaining glycosidase inhibition warhead.
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Inhibidores Enzimáticos/metabolismo , Flavonoles/química , alfa-Amilasas Pancreáticas/metabolismo , Péptidos/metabolismo , Plantas/química , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Dicroismo Circular , Inhibidores Enzimáticos/química , Flavonas/química , Humanos , Cinética , Levodopa/química , Simulación de Dinámica Molecular , alfa-Amilasas Pancreáticas/antagonistas & inhibidores , Péptidos/química , Plantas/metabolismo , Trisacáridos/químicaRESUMEN
Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalyzed by the Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct (1)H NMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme. Kinetic isotope effects arising from deuterium-for-hydrogen substitution at carbons 1 and 4 provide evidence for separate first-irreversible and overall rate-determining steps in the hydration reaction, with two potential mechanisms proposed to explain these results. Based on the positioning of catalytic residues in the enzyme active site, the lack of efficient turnover of a 2-deoxy-2-fluoro-substrate, and several unsuccessful attempts at confirmation of a simpler mechanism involving a covalent glycosyl-enzyme intermediate, the most plausible mechanism is one involving an intermediate bearing an epoxide on carbons 1 and 2.
Asunto(s)
Proteínas Bacterianas/química , Clostridium perfringens/enzimología , Glicósido Hidrolasas/química , Proteínas Bacterianas/metabolismo , Medición de Intercambio de Deuterio , Glicósido Hidrolasas/metabolismo , Hidrólisis , Resonancia Magnética Nuclear Biomolecular , Especificidad por Sustrato/fisiologíaRESUMEN
Over the sixty years since Koshland initially formulated the classical mechanisms for retaining and inverting glycosidases, researchers have assembled a large body of supporting evidence and have documented variations of these mechanisms. Recently, however, researchers have uncovered a number of completely distinct mechanisms for enzymatic cleavage of glycosides involving elimination and/or hydration steps. In family GH4 and GH109 glycosidases, the reaction proceeds via transient NAD(+)-mediated oxidation at C3, thereby acidifying the proton at C2 and allowing for elimination across the C1-C2 bond. Subsequent Michael-type addition of water followed by reduction at C3 generates the hydrolyzed product. Enzymes employing this mechanism can hydrolyze thioglycosides as well as both anomers of activated substrates. Sialidases employ a conventional retaining mechanism in which a tyrosine functions as the nucleophile, but in some cases researchers have observed off-path elimination end products. These reactions occur via the normal covalent intermediate, but instead of an attack by water on the anomeric center, the catalytic acid/base residue abstracts an adjacent proton. These enzymes can also catalyze hydration of the enol ether via the reverse pathway. Reactions of α-(1,4)-glucan lyases also proceed through a covalent intermediate with subsequent abstraction of an adjacent proton to give elimination. However, in this case, the departing carboxylate "nucleophile" serves as the base in a concerted but asynchronous syn-elimination process. These enzymes perform only elimination reactions. Polysaccharide lyases, which act on uronic acid-containing substrates, also catalyze only elimination reactions. Substrate binding neutralizes the charge on the carboxylate, which allows for abstraction of the proton on C5 and leads to an elimination reaction via an E1cb mechanism. These enzymes can also cleave thioglycosides, albeit slowly. The unsaturated product of polysaccharide lyases can then serve as a substrate for a hydration reaction carried out by unsaturated glucuronyl hydrolases. This hydration is initiated by protonation at C4 and proceeds in a Markovnikov fashion rather than undergoing a Michael-type addition, giving a hemiketal at C5. This hemiketal then undergoes a rearrangement that results in cleavage of the anomeric bond. These enzymes can also hydrolyze thioglycosides efficiently and slowly turn over substrates with inverted anomeric configuration. The mechanisms discussed in this Account proceed through transition states that involve either positive or negative charges, unlike the exclusively cationic transition states of the classical Koshland retaining and inverting glycosidases. In addition, the distribution of this charge throughout the substrate can vary substantially. The nature of these mechanisms and their transition states means that any inhibitors or inactivators of these unusual enzymes probably differ from those presently used for Koshland retaining or inverting glycosidases.
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Glicósido Hidrolasas/metabolismo , Glicósidos/metabolismo , Glicósidos/químicaRESUMEN
Natural and synthetic unsaturated glucuronides were tested as substrates for Clostridium perfringens unsaturated glucuronyl hydrolase to probe its mechanism and to guide inhibitor design. Of the natural substrates, a chondroitin disaccharide substrate with sulfation of the primary alcohol on carbon 6 of its N-acetylgalactosamine moiety was found to have the highest turnover number of any substrate reported for an unsaturated glucuronyl hydrolase, with kcat =112 s(-1) . Synthetic aryl glycoside substrates with electron-withdrawing aglycone substituents were cleaved more slowly than those with electron-donating substituents. Similarly, an unsaturated glucuronyl fluoride was found to be a particularly poor substrate, with kcat /Km =44 nM(-1) s(-1) -a very unusual result for a glycoside-cleaving enzyme. These results are consistent with a transition state with positive charge at carbon 5 and the endocyclic oxygen, as anticipated in the hydration mechanism proposed. However, several analogues designed to take advantage of strong enzyme binding to such a transition state showed little to no inhibition. This result suggests that further work is required to understand the true nature of the transition state stabilised by this enzyme.
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Proteínas Bacterianas/metabolismo , Glicósido Hidrolasas/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Clostridium perfringens/enzimología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Glucurónidos/síntesis química , Glucurónidos/química , Glucurónidos/metabolismo , Glicósido Hidrolasas/antagonistas & inhibidores , Cinética , Unión Proteica , Especificidad por Sustrato , TermodinámicaRESUMEN
Messenger RNA (mRNA) display is being increasingly adopted for peptide drug candidate discovery. While many conditions have been reported for the affinity enrichment step and in some cases for peptide modification, there is still limited understanding about the versatility of peptide-puromycin-mRNA/cDNA (complementary DNA) complexes. This work explores the chemical stability of mRNA/cDNA hybrid complexes under a range of different fundamental chemical conditions as well as with peptide modification conditions reported in an mRNA display setting. We further compare the stability of full complexes originating from two different mRNA display systems (RaPID and cDNA-TRAP). Overall, these complexes were found to be stable under a broad range of conditions, with some edge conditions benefitting from encoding directly in cDNA rather than mRNA. This should allow for more and broader exploitation of late-stage peptide modification chemistry in mRNA display, with confidence regarding the stability of encoding, and potentially better hit-finding campaigns as a result.
RESUMEN
The human complement pathway plays a pivotal role in immune defence, homeostasis, and autoimmunity regulation, and complement-based therapeutics have emerged as promising interventions, with both antagonistic and agonistic approaches being explored. The classical pathway of complement is initiated when the C1 complex binds to hexameric antibody platforms. Recent structural data revealed that C1 binds to small, homogeneous interfaces at the periphery of the antibody platforms. Here, we have developed a novel strategy for complement activation using macrocyclic peptides designed to mimic the interface between antibodies and the C1 complex. In vitro selection utilizing the RaPID system identified a cyclic peptide (cL3) that binds to the C1 complex via the globular head domains of C1q. Notably, when immobilized on surfaces, cL3 effectively recruits C1 from human serum, activates C1s proteases, and induces lysis of cell-mimetic lipid membranes. This represents the first instance of a peptide capable of activating complement by binding C1 when immobilized. Further characterization and synthesis of deletion mutants revealed a critical cycle size of cL3 essential for C1 binding and efficient complement activation. Importantly, cL3 also demonstrated the ability to inhibit complement-mediated lysis without affecting C1 binding, highlighting its potential as a therapeutic modality to prevent complement-dependent cytotoxicity whilst promoting cellular phagocytosis and cell clearance. In summary, this study introduces the concept of "Peptactins" - peptide-based activators of complement - and underscores the potential of macrocyclic peptides for complement modulation, offering potential advantages over traditional biologicals in terms of size, production, and administration.
RESUMEN
Peptide display technologies are a powerful method for discovery of new bioactive sequences, but linear sequences are often very unstable in a biological setting. Macrocyclisation of such peptides is beneficial for target affinity, selectivity, stability, and cell permeability. However, macrocyclisation of a linear hit is unreliable and requires extensive structural knowledge. Genetically encoding macrocyclisation during the discovery process is a better approach, and so there is a need for diverse cyclisation options that can be deployed in the context of peptide display techniques such as mRNA display. In this work we show that meta-cyanopyridylalanine (mCNP) can be ribosomally incorporated into peptides, forming a macrocycle in a spontaneous and selective reaction with an N-terminal cysteine generated from bypassing the initiation codon in translation. This reactive amino acid can also be easily incorporated into peptides during standard Fmoc solid phase peptide synthesis, which can otherwise be a bottleneck in transferring from peptide discovery to peptide testing and application. We demonstrate the potential of this new method by discovery of macrocyclic peptides targeting influenza haemagglutinin, and molecular dynamics simulation indicates the mCNP cross-link stabilises a beta sheet structure in a representative of the most abundant cluster of active hits. Cyclisation by mCNP is also shown to be compatible with thioether macrocyclisation at a second cysteine to form bicycles of different architectures, provided that cysteine placement reinforces selectivity, with this bicyclisation happening spontaneously and in a controlled manner during peptide translation. Our new approach generates macrocycles with a more rigid cross-link and with better control of regiochemistry when additional cysteines are present, opening these up for further exploitation in chemical modification of in vitro translated peptides, and so is a valuable addition to the peptide discovery toolbox.
RESUMEN
Influenza A viruses pose a serious pandemic risk, while generation of efficient vaccines against seasonal variants remains challenging. There is thus a pressing need for new treatment options. We report here a set of macrocyclic peptides that inhibit influenza A virus infection at low nanomolar concentrations by binding to hemagglutinin, selected using ultrahigh-throughput screening of a diverse peptide library. The peptides are active against both H1 and H5 variants, with no detectable cytotoxicity. Despite the high sequence diversity across hits, all tested peptides were found to bind to the same region in the hemagglutinin stem by HDX-MS epitope mapping. A mutation in this region identified in an escape variant confirmed the binding site. This stands in contrast to the immunodominance of the head region for antibody binding and suggests that macrocyclic peptides from in vitro display may be well suited for finding new druggable sites not revealed by antibodies. Functional analysis indicates that these peptides stabilize the prefusion conformation of the protein and thereby prevent virus-cell fusion. High-throughput screening of macrocyclic peptides is thus shown here to be a powerful method for the discovery of novel broadly acting viral fusion inhibitors with therapeutic potential.
Asunto(s)
Virus de la Influenza A , Anticuerpos Antivirales/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Hemaglutininas , Virus de la Influenza A/química , Biblioteca de PéptidosRESUMEN
Unsaturated glucuronyl hydrolases (UGLs) from GH family 88 of the CAZy classification system cleave a terminal unsaturated sugar from the oligosaccharide products released by extracellular bacterial polysaccharide lyases. This pathway, which is involved in extracellular bacterial infection, has no equivalent in mammals. A novel mechanism for UGL has previously been proposed in which the enzyme catalyzes hydration of a vinyl ether group in the substrate, with subsequent rearrangements resulting in glycosidic bond cleavage. However, clear evidence for this mechanism has been lacking. In this study, analysis of the products of UGL-catalyzed reactions in water, deuterium oxide, and dilute methanol in water, in conjunction with the demonstration that UGL rapidly cleaves thioglycosides and glycosides of inverted anomeric configuration (substrates that are resistant to hydrolysis by classical glycosidases), provides strong support for this new mechanism. A hydration-initiated process is further supported by the observed UGL-catalyzed hydration of a C-glycoside substrate analogue. Finally, the observation of a small ß-secondary kinetic isotope effect suggests a transition state with oxocarbenium ion character, in which the hydrogen at carbon 4 adopts an axial geometry. Taken together, these observations validate the novel vinyl ether hydration mechanism and are inconsistent with either inverting or retaining direct hydrolase mechanisms at carbon 1.
Asunto(s)
Clostridium perfringens/enzimología , Glicósido Hidrolasas/metabolismo , Glicósidos/metabolismo , Conformación de Carbohidratos , Clostridium perfringens/metabolismo , Glicósidos/química , Cinética , Especificidad por Sustrato , Tioglicósidos/química , Tioglicósidos/metabolismoRESUMEN
New methodology for the preparation of a variety of aminoiminohextitols is described. Key in the synthesis is the application of a diastereoselective Strecker reaction and the extension of our carbamate annulation methodology to protected and functionalized alkenylamines. Insight into the effects that the substitution patterns of the alkenylamines have on the diastereoselectivity of the iodocyclization and carbamate annulation is discussed. An evaluation of the glycosidase inhibitory activity of the aminoiminohexitols and derivatives is also presented, with the previously undisclosed D-talo isomer showing good selective inhibition of ß-D-glucosidase.