Christopher N. Boddy

Chemistry, Biology, and Medicine of the Glycopeptide Antibiotics

The war against infectious bacteria is not over! Although vancomycin and glycopeptide antibiotics have provided a strong last line of defence against many drug-resistant bacteria, their overuse has given rise to more dangerous strains of bacteria. An understanding of the chemistry and biology of these highly complex glycopeptides are destined to play a crucial role in the discovery of new antibiotics.

Chemie, Biologie und medizinische Anwendungen der Glycopeptid‐Antibiotika

Der Krieg gegen pathogene Bakterien ist noch nicht vorbei! Zwar bilden Vancomycin und andere Glycopeptid-Antibiotika eine letzte Verteidigungslinie gegen zahlreiche mehrfachresistente Bakterien, doch ihr leichtfertiger Gebrauch hat zur Bildung etlicher gefahrlicherer Bakterienstamme gefuhrt. Das Verstandnis der chemischen und biologischen Eigenschaften dieser hochkomplexen Glycopeptide durfte eine entscheidende Rolle fur die Entdeckung und Entwicklung neuer Antibiotika spielen.

Total Synthesis of Vancomycin—Part 1: Design and Development of Methodology

A number of valuable new synthetic strategies, such as the triazene-driven biaryl ether synthesis, have been developed during the total synthesis of vancomycin (1). Modern catalytic asymmetric reactions were employed for the construction of the required amino acid building blocks, which were then assembled to the appropriate peptide fragments, whose cyclization in the order C-O-D→AB/C-O-D→AB/C-O-D-E led to framework of the vancomycin aglycon (2). Sequential attachment of the required sugar moieties onto a suitably protected aglycon derivative, followed by deprotection, allowed the stereoselective total synthesis of the glycopeptide antibiotic vancomycin (1).

Total Synthesis of Vancomycin—Part 2: Retrosynthetic Analysis, Synthesis of Amino Acid Building Blocks and Strategy Evaluations

A number of valuable new synthetic strategies, such as the triazene-driven biaryl ether synthesis, have been developed during the total synthesis of vancomycin (1). Modern catalytic asymmetric reactions were employed for the construction of the required amino acid building blocks, which were then assembled to the appropriate peptide fragments, whose cyclization in the order C-O-D→AB/C-O-D→AB/C-O-D-E led to framework of the vancomycin aglycon (2). Sequential attachment of the required sugar moieties onto a suitably protected aglycon derivative, followed by deprotection, allowed the stereoselective total synthesis of the glycopeptide antibiotic vancomycin (1).

Probing the Ring Size of Epothilones: Total Synthesis of [14]-, [15]-, [17]-, and [18]Epothilones A

Unfamiliar rings. The pharmocophore of epothilone A does not tolerate changes in ring size. The 14-, 15-, 17-, and 18-membered ring analogues 1 (n = 1, 2, 4, 5) of epothilone and their deoxy counterparts show only low levels of activity in tubulin polymerization assays, with the exception of [18]desoxyepothilone A. These findings underscore the high specificity of the tubulin receptor.

A thioesterase from an iterative fungal polyketide synthase shows macrocyclization and cross coupling activity and may play a role in controlling iterative cycling through product offloading.

Zearalenone, a fungal macrocyclic polyketide, is a member of the resorcylic acid lactone family. Herein, we characterize in vitro the thioesterase from PKS13 in zearalenone biosynthesis (Zea TE). The excised Zea TE catalyzes macrocyclization of a linear thioester-activated model of zearalenone. Zea TE also catalyzes the cross coupling of a benzoyl thioester with alcohols and amines. Kinetic characterization of the cross coupling is consistent with a ping-pong bi-bi mechanism, confirming an acyl-enzyme intermediate. Finally, the substrate specificity of the Zea TE indicates the TE may help control iterative cycling on PKS13 by rapidly offloading the final resorcylate-containing product.

6-Deoxyerythronolide B synthase thioesterase-catalyzed macrocyclization is highly stereoselective.

Macrocyclic polyketide natural products are an indispensable source of therapeutic agents. The final stage of their biosynthesis, macrocyclization, is catalyzed regio- and stereoselectively by a thioesterase. A panel of substrates were synthesized to test their specificity for macrocyclization by the erythromycin polyketide synthase TE (DEBS TE) in vitro. It was shown that DEBS TE is highly stereospecific, successfully macrocyclizing a 14-member ring substrate with an R configured O-nucleophile, and highly regioselective, generating exclusively the 14-member lactone over the 12-member lactone.

Sialic acid and N-acyl sialic acid analog production by fermentation of metabolically and genetically engineered Escherichia coli

Sialic acid is the terminal sugar found on most glycoproteins and is crucial in determining serum half-life and immunogenicity of glycoproteins. Sialic acid analogs are antiviral therapeutics as well as crucial tools in bacterial pathogenesis research, immunobiology and development of cancer diagnostic imaging. The scarce supply of sialic acid hinders production of these materials. We have developed an efficient, rapid and cost effective fermentation route to access sialic acid. Our approach uses low cost feedstock, produces an industrially relevant amount of sialic acid and is scalable to manufacturing levels. We have also shown that precursor directed biosynthesis can be used to produce a N-acyl sialic acid analog. This work demonstrates the feasibility of engineering manufacturing-friendly bacteria to produce complex, unavailable small molecules.

Examining the Role of Hydrogen Bonding Interactions in the Substrate Specificity for the Loading Step of Polyketide Synthase Thioesterase Domains

The final step in polyketide synthase-mediated biosynthesis of macrocyclic polyketides is thioesterase (TE)-catalyzed cyclization of a linear polyketide acyl chain. TEs are highly specific in the chemistry they catalyze. Understanding the molecular basis for substrate specificity of TEs is crucial for engineering these enzymes to macrocyclize non-native linear substrates. We investigated the role of hydrogen bonding interactions in the substrate specificity of formation of an acyl-enzyme intermediate for the TE from the 6-deoxyerythronolide B biosynthetic pathway. Thirteen single site-directed mutants were constructed, via removal of side chain hydrogen bonding groups from the binding cavity. Specificity constants for four different substrates with and without hydrogen bond donors and acceptors were determined for the five active mutants. The relative magnitude of specificity constants for substrates did not change for the mutant TEs. Circular dichroism spectroscopy was used to show that the majority of the catalytically inactive mutants did not fold. Two mutations were identified that enabled mutant TEs to form a folded but catalytically inactive tertiary structure. Our data do not support a role for hydrogen bonding in mediating substrate specificity of bacterial polyketide synthase TEs. The highly conserved polar residues in the binding cavity appear to stabilize the unusual substrate channel, which passes through the enzyme. We propose that hydrophobic interactions between the binding cavity and substrate drive substrate specificity, as is seen in many protein-carbohydrate recognition events. This hypothesis is in agreement with high-resolution structural data for nonhydrolyzable acyl-enzyme intermediates from the picromycin TE.

Biomimetic transannular oxa-conjugate addition approach to the 2,6-disubstituted dihydropyran of laulimalide yields an unprecedented transannular oxetane.

2,6-Disubstituted dihydropyrans are a common feature in many bioactive polyketides, including the anticancer marine polyketide laulimalide. While much of the uncharacterized biosynthetic pathway for laulimalide can be confidently postulated, the biosynthetic origins of the trans 2,6-disubstituted dihydropyran cannot. We hypothesize that a transannular oxa-conjugate addition in a macrocyclic laulimalide precursor could be the origin of the 2,6-dihydropyran. To test this hypothesis, we constructed a model containing the key functional groups for oxa-conjugate addition-mediated dihydropyran formation. Under acid-mediated conditions, the model under went regiospecific oxa-conjugate addition producing a stable trans oxetane as the only regioisomer. The desired, more stable dihydropyran was not detected. This unprecedented regiospecificity is unexpected due to the ring strain of the oxetane and the anticipated facile ring opening retro-oxa-conjugate addition. The oxetane is stable to acid and basic conditions, as are a number of literature acyclic oxetanes that could undergo similar retro-oxa-conjugate addition. While the source of the oxetane kinetic stability is yet to be characterized, it may enable general oxetane construction via oxa-conjugate addition. The more stable dihydropyran regioisomer could not be generated due to poor geometrical orbital alignment and hard-soft incompatibility between the hard oxygen nucleophile and the soft activated polyenoate electrophile. These factors disfavor the breaking of conjugation by oxa-conjugate addition. Based on these results we propose that dihydropyran formation does not occur on completed polyketide macrocycles as we had proposed but rather during polyketide biosynthesis on the growing polyketide chain.