Publications by authors named "Seth B Herzon"

88 Publications

Probing Microbiome Genotoxicity: A Stable Colibactin Provides Insight into Structure-Activity Relationships and Facilitates Mechanism of Action Studies.

J Am Chem Soc 2021 Sep 15;143(38):15824-15833. Epub 2021 Sep 15.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

Colibactin is a genotoxic metabolite produced by commensal-pathogenic members of the human microbiome that possess the (aka ) biosynthetic gene cluster. bacteria induce tumorigenesis in models of intestinal inflammation and have been causally linked to oncogenesis in humans. While colibactin is believed underlie these effects, it has not been possible to study the molecule directly due to its instability. Herein, we report the synthesis and biological studies of colibactin 742 (), a stable colibactin derivative. We show that colibactin 742 () induces DNA interstrand-cross-links, activation of the Fanconi Anemia DNA repair pathway, and G/M arrest in a manner similar to . The linear precursor , which mimics the biosynthetic precursor to colibactin, also recapitulates the bacterial phenotype. In the course of this work, we discovered a novel cyclization pathway that was previously undetected in MS-based studies of colibactin, suggesting a refinement to the natural product structure and its mode of DNA binding. Colibactin 742 () and its precursor will allow researchers to study colibactin's genotoxic effects independent of the producing organism for the first time.
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http://dx.doi.org/10.1021/jacs.1c07559DOI Listing
September 2021

Natural Products: An Era of Discovery in Organic Chemistry.

J Org Chem 2021 Aug;86(16):10943-10945

Yale University Department of Chemistry New Haven, Connecticut 06520, United States.

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http://dx.doi.org/10.1021/acs.joc.1c01753DOI Listing
August 2021

On the Stability and Spectroscopic Properties of 5-Hydroxyoxazole-4-carboxylic Acid Derivatives.

Org Lett 2021 07 28;23(14):5457-5460. Epub 2021 Jun 28.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

5-Hydroxyoxazole-4-carboxylic acid residues were advanced as substructures within the secondary bacterial metabolites precolibactins 969 and 795a. However, oxazoles containing both 5-hydroxy and 4-carboxy substituents are unprecedented. We have found these oxazoles are unstable with respect to hydrolytic ring opening and decarboxylation. Comparison of reported and theoretical C NMR chemical shifts between synthetic intermediates and the isolates revealed discrepancies in the oxazole region. These results suggest that precolibactins 969 and 795a may not contain 5-hydroxyoxazole-4-carboxylic acid residues.
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http://dx.doi.org/10.1021/acs.orglett.1c01796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8294165PMC
July 2021

Synthesis of the bis(cyclohexenone) core of (-)-lomaiviticin A.

Chem Sci 2020 Jul 9;11(28):7462-7467. Epub 2020 Jul 9.

Department of Chemistry, Yale University New Haven Connecticut 06520 USA

(-)-Lomaiviticin A is a complex -symmetric bacterial metabolite comprising two diazotetrahydrobenzo[]fluorene (diazofluorene) residues and four 2,6-dideoxy glycosides, α-l-oleandrose and ,-dimethyl-β-l-pyrrolosamine. The two halves of lomaiviticin A are linked by a single carbon-carbon bond oriented with respect to the oleandrose residues. While many advances toward the synthesis of lomaiviticin A have been reported, including synthesis of the aglycon, a route to the bis(cyclohexenone) core bearing any of the carbohydrate residues has not been disclosed. Here we describe a short route to a core structure of lomaiviticin A bearing two α-l-oleandrose residues. The synthetic route features a Stille coupling to form the conjoining carbon-carbon bond of the target and a double reductive transposition to establish the correct stereochemistry at this bond. Two synthetic routes were developed to elaborate the reductive transposition product to the bis(cyclohexenone) target. The more efficient pathway features an interrupted Barton vinyl iodide synthesis followed by oxidative elimination of iodide to efficiently establish the enone functionalities in the target. The bis(cyclohexenone) product may find use in a synthesis of lomaiviticin A itself.
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http://dx.doi.org/10.1039/d0sc02770gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8159427PMC
July 2020

Structure Revision of the Lomaiviticins.

J Am Chem Soc 2021 05 26;143(17):6578-6585. Epub 2021 Apr 26.

Department of Chemistry, Yale University, New Haven, Connecticut 06511, United States.

The lomaiviticins are dimeric genotoxic metabolites that contain unusual diazocyclopentadiene functional groups and 2-4 deoxyglycoside residues. Because only 6 of 19 carbon atoms in the monomeric aglycon unit are proton-attached, their structure determination by NMR spectroscopic analysis is difficult. Prior structure elucidation efforts established that the two halves of the lomaiviticins are joined by a single carbon-carbon bond appended to an oxidized cyclohexenone ring. This ring was believed to comprise a 4,5-dihydroxycyclohex-2-ene-1-one. The bridging bond was positioned at C6. This structure proposal has not been tested because no lomaiviticin has been prepared by total chemical synthesis or successfully analyzed by X-ray crystallography. Here, we disclose microED studies which establish that (-)-lomaiviticin C contains a 4,6-dihydroxy-cyclohex-2-ene-1-one residue, that the bridging carbon-carbon bond is located at C5, and that the orientation of the cyclohexenone ring and configuration of the secondary glycoside are reversed, relative to their original assignment. High-field (800 MHz) NMR analysis supports the revised assignment and suggests earlier efforts were misled by a combination of a near-zero coupling constant and a coupling interpreted as a coupling. DFT calculations of the expected C chemical shifts and C-H coupling constants provide further robust support for the structure revision. Because the interconversion of lomaiviticins A, B, and C has been demonstrated, these findings apply to each isolate. These studies clarify the structures of this family of metabolites and underscore the power of microED analysis in natural product structure determination.
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http://dx.doi.org/10.1021/jacs.1c01729DOI Listing
May 2021

General Method for the Synthesis of α- or β-Deoxyaminoglycosides Bearing Basic Nitrogen.

J Am Chem Soc 2021 02 8;143(7):2777-2783. Epub 2021 Feb 8.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

The introduction of glycosides bearing basic nitrogen is challenging using conventional Lewis acid-promoted pathways owing to competitive coordination of the amine to the Lewis acid promoter. Additionally, because many aminoglycosides lack a C2 substituent, diastereomeric mixtures of -glycosides are often produced. Herein, we present a method for the synthesis of α- or β- 2,3,6-trideoxy-3-amino- and 2,4,6-trideoxy-4-amino -glycosides from a common precursor. Our strategy proceeds by the reductive lithiation of thiophenyl glycoside donors and trapping of the resulting anomeric anions with 2-methyltetrahydropyranyl peroxides. We apply this strategy to the synthesis of α- and β-forosamine, pyrrolosamine, acosamine, and ristosamine derivatives using primary and secondary peroxides as electrophiles. α-Linked products are obtained in 60-96% yield and with >50:1 selectivity. β-Linked products are obtained in 45-94% yield and with 1.7->50:1 stereoselectivity. Contrary to donors bearing an equatorial amine substituent, donors bearing an axial amine substituent favored β-products at low temperatures. This work establishes a general strategy to synthesize -glycosides bearing a basic nitrogen.
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http://dx.doi.org/10.1021/jacs.0c11262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935422PMC
February 2021

Metric-Based Analysis of Convergence in Complex Molecule Synthesis.

Acc Chem Res 2021 02 1;54(4):903-916. Epub 2021 Feb 1.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

Convergent syntheses are characterized by the coupling of two or more synthetic intermediates of similar complexity, often late in a pathway. At its limit, a fully convergent synthesis is achieved when commercial or otherwise readily available intermediates are coupled to form the final target in a single step. Of course, in all but exceptional circumstances this level of convergence is purely hypothetical; in practice, additional steps are typically required to progress from fragment coupling to the target. Additionally, the length of the sequence required to access each target is a primary consideration in synthetic design.In this Account, we provide an overview of alkaloid, polyketide, and diterpene metabolites synthesized in our laboratory and present parameters that may be used to put the degree of convergence of each synthesis on quantitative footing. We begin with our syntheses of the antiproliferative, antimicrobial bacterial metabolite (-)-kinamycin F () and related dimeric structure (-)-lomaiviticin aglycon (). These synthetic routes featured a three-step sequence to construct a complex diazocyclopentadiene found in both targets and an oxidative dimerization to unite the two halves of (-)-lomaiviticin aglycon (). We then follow with our synthesis of the antineurodegenerative alkaloid (-)-huperzine A (). Our route to (-)-huperzine A () employed a diastereoselective three-component coupling reaction, followed by the intramolecular α-arylation of a β-ketonitrile intermediate, to form the carbon skeleton of the target. We then present our syntheses of the hasubanan alkaloids (-)-hasubanonine (), (-)-delavayine (), (-)-runanine (), (+)-periglaucine B (), and (-)-acutumine (). These alkaloids bear a 7-azatricyclo[4.3.3.0]dodecane (propellane) core and a highly oxidized cyclohexenone ring. The propellane structure was assembled by the addition of an aryl acetylide to a complex iminium ion, followed by intramolecular 1,4-addition. We then present our synthesis of the guanidinium alkaloid (+)-batzelladine B (), which contains two complex polycyclic guanidine residues united by an ester linkage. This target was logically disconnected by an esterification to allow for the independent synthesis of each guanidine residue. A carefully orchestrated cascade reaction provided (+)-batzelladine B () in a single step following fragment coupling by esterification. We then discuss our synthesis of the diterpene fungal metabolite (+)-pleuromutilin (). The synthesis of (+)-pleuromutilin () proceeded via a fragment coupling involving two neopentylic reagents and employed a nickel-catalyzed reductive cyclization reaction to close the eight-membered ring, ultimately providing access to (+)-pleuromutilin (), (+)-12--pleuromutilin (), and (+)-12--mutilin (). Finally, we discuss our synthesis of (-)-myrocin G (), a tricyclic pimarane diterpene that was assembled by a convergent annulation.In the final section of this Account, we present several paramaters to analyze and quantitatively assess the degree of convergence of each synthesis. These parameters include: (1) the number of steps required following the point of convergence, (2) the difference in the number of steps required to prepare each coupling partner, (3) the percentage of carbons (or, more broadly, atoms) present at the point of convergence, and (4) the complexity generated in the fragment coupling step. While not an exhaustive list, these parameters bring the strengths and weaknesses each synthetic strategy to light, emphasizing the key contributors to the degree of convergence of each route while also highlighting the nuances of these analyses.
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http://dx.doi.org/10.1021/acs.accounts.0c00817DOI Listing
February 2021

Enantioselective Synthesis of Euonyminol.

J Am Chem Soc 2021 01 7;143(2):699-704. Epub 2021 Jan 7.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

We describe an enantioselective total synthesis of the nonahydroxylated sesquiterpenoid euonyminol, the dihydro-β-agarofuran nucleus of the macrocyclic terpenoid alkaloids known as the cathedulins. Key features of the synthetic sequence include a highly diastereoselective intramolecular alkene oxyalkylation to establish the C10 quaternary center, an intramolecular aldol-dehydration to access the tricyclic scaffold of the target, a tandem lactonization-epoxide opening to form the -C2-C3 vicinal diol residue, and a late-stage diastereoselective α-ketol rearrangement. The synthesis provides the first synthetic access to enantioenriched euonyminol and establishes a platform to synthesize the cathedulins.
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http://dx.doi.org/10.1021/jacs.0c12998DOI Listing
January 2021

Synthesis and Biological Evaluation of (2,2')-Lomaiviticin A.

J Am Chem Soc 2021 01 7;143(2):1126-1132. Epub 2021 Jan 7.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

(-)-Lomaiviticin A () is a genotoxic -symmetric metabolite that arises from the formal dimerization of two bis(glycosylated) diazotetrahydrobenzo[]fluorenes. Here we present a synthesis of the monomer and its coupling to form (2,2')-lomaiviticin A (), an unnatural diastereomer of . (2,2')-Lomaiviticin A () is significantly less genotoxic, a result we attribute to changes in the orientation of the diazofluorene and carbohydrate residues, relative to . These data bring the importance of the configuration of the conjoining bond to light and place the total synthesis of itself within reach.
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http://dx.doi.org/10.1021/jacs.0c11960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8174553PMC
January 2021

Employing chemical synthesis to study the structure and function of colibactin, a "dark matter" metabolite.

Nat Prod Rep 2020 11;37(11):1532-1548

Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA. and Department of Pharmacology, Yale School of Medicine, New Haven, Connecticut 06520, USA.

Covering: 2015 to 2020 The field of natural products is dominated by a discovery paradigm that follows the sequence: isolation, structure elucidation, chemical synthesis, and then elucidation of mechanism of action and structure-activity relationships. Although this discovery paradigm has proven successful in the past, researchers have amassed enough evidence to conclude that the vast majority of nature's secondary metabolites - biosynthetic "dark matter" - cannot be identified and studied by this approach. Many biosynthetic gene clusters (BGCs) are expressed at low levels, or not at all, and in some instances a molecule's instability to fermentation or isolation prevents detection entirely. Here, we discuss an alternative approach to natural product identification that addresses these challenges by enlisting synthetic chemistry to prepare putative natural product fragments and structures as guided by biosynthetic insight. We demonstrate the utility of this approach through our structure elucidation of colibactin, an unisolable genotoxin produced by pathogenic bacteria in the human gut.
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http://dx.doi.org/10.1039/d0np00072hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7700718PMC
November 2020

Macrocyclic colibactins.

Authors:
Seth B Herzon

Nat Chem 2020 11 21;12(11):1005-1006. Epub 2020 Sep 21.

Department of Chemistry, Yale University, New Haven, CT, USA.

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http://dx.doi.org/10.1038/s41557-020-00551-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935421PMC
November 2020

Development of a Convergent Enantioselective Synthetic Route to (-)-Myrocin G.

J Org Chem 2020 07 2;85(14):8952-8989. Epub 2020 Jul 2.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

Myrocins are a family of antiproliferative antibiotic fungal metabolites possessing a masked electrophilic cyclopropane. Preliminary chemical reactivity studies imputed the bioactivity of these natural products to a DNA cross-linking mechanism, but this hypothesis was not confirmed by studies with native DNA. We recently reported a total synthesis of (-)-myrocin G (), the putative active form of the metabolite myrocin C (), that featured a carefully orchestrated tandem fragment coupling-annulation cascade. Herein, we describe the evolution of our synthetic strategy toward and report the series of discoveries that prompted the design of this cascade coupling. Efforts to convert the diosphenol (-)-myrocin G () to the corresponding 5-hydroxy-γ-lactone isomer myrocin C () are also detailed. We present a preliminary evaluation of the antiproliferative activities of (-)-myrocin G () and related structures, as well as DNA cross-linking studies. These studies indicate that myrocins do not cross-link DNA, suggesting an alternative mode of action potentially involving a protein target.
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http://dx.doi.org/10.1021/acs.joc.0c00891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7520802PMC
July 2020

New Leads for the Treatment of Multidrug Resistant .

Authors:
Seth B Herzon

ACS Cent Sci 2020 Jun 12;6(6):833-835. Epub 2020 Jun 12.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

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http://dx.doi.org/10.1021/acscentsci.0c00684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318060PMC
June 2020

Structure and bioactivity of colibactin.

Bioorg Med Chem Lett 2020 08 23;30(15):127280. Epub 2020 May 23.

Department of Chemistry, Yale University, New Haven, CT 06520, United States; Department of Pharmacology, Yale School of Medicine, New Haven, CT 06520, United States. Electronic address:

Colibactin is a secondary metabolite produced by certain strains of bacteria found in the human gut. The presence of colibactin-producing bacteria has been correlated to colorectal cancer in humans. Colibactin was first discovered in 2006, but because it is produced in small quantities and is unstable, it has yet to be isolated from bacterial cultures. Here we summarize advances in the field since ~2017 that have led to the identification of the structure of colibactin as a heterodimer containing two DNA-reactive electrophilic cyclopropane residues. Colibactin has been shown to form interstrand cross-links by alkylation of adenine residues on opposing strands of DNA. The structure of colibactin contains two thiazole rings separated by a two-carbon linker that is thought to exist as an α-aminoketone following completion of the biosynthetic pathway. However, synthetic studies have now established that this α-aminoketone is unstable toward aerobic oxidation; the resulting oxidation products are in turn unstable toward nucleophilic cleavage under mild conditions. These data provide a simple molecular-level explanation for colibactin's instability and potentially also explain the observation that cell-to-cell contact is required for genotoxic effects.
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http://dx.doi.org/10.1016/j.bmcl.2020.127280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309967PMC
August 2020

Depurination of Colibactin-Derived Interstrand Cross-Links.

Biochemistry 2020 02 13;59(7):892-900. Epub 2020 Feb 13.

Department of Chemistry , Yale University , New Haven , Connecticut 06520 , United States.

Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer. Evidence suggests that colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we find that colibactin-derived ICLs are unstable toward depurination and elimination of the 3' phosphate. This ICL degradation pathway leads progressively to single strand breaks (SSBs) and subsequently DSBs. The spontaneous conversion of ICLs to DSBs is consistent with the finding that nonhomologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein refine our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.
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http://dx.doi.org/10.1021/acs.biochem.9b01070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521609PMC
February 2020

Fragment Coupling Reactions in Total Synthesis That Form Carbon-Carbon Bonds via Carbanionic or Free Radical Intermediates.

Angew Chem Int Ed Engl 2021 01 24;60(3):1116-1150. Epub 2020 Sep 24.

Department of Chemistry, Yale University, 225 Prospect St, New Haven, CT, USA.

Fragment coupling reactions that form carbon-carbon bonds are valuable transformations in synthetic design. Advances in metal-catalyzed cross-coupling reactions in the early 2000s brought a high level of predictability and reliability to carbon-carbon bond constructions involving the union of unsaturated fragments. By comparison, recent years have witnessed an increase in fragment couplings proceeding via carbanionic and open-shell (free radical) intermediates. The latter has been driven by advances in methods to generate and utilize carbon-centered radicals under mild conditions. In this Review, we survey a selection of recent syntheses that have implemented carbanion- or radical-based fragment couplings to form carbon-carbon bonds. We aim to highlight the strategic value of these disconnections in their respective settings and to identify extensible lessons from each example that might be instructive to students.
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http://dx.doi.org/10.1002/anie.201913645DOI Listing
January 2021

Addendum: Synthesis and reactivity of precolibactin 886.

Nat Chem 2019 12;11(12):1167

Department of Chemistry, Yale University, New Haven, CT, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41557-019-0383-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523535PMC
December 2019

Synthesis and reactivity of precolibactin 886.

Nat Chem 2019 10 23;11(10):890-898. Epub 2019 Sep 23.

Department of Chemistry, Yale University, New Haven, CT, USA.

The clb gene cluster encodes the biosynthesis of metabolites known as precolibactins and colibactins. The clb pathway is found in gut commensal Escherichia coli, and clb metabolites are thought to initiate colorectal cancer via DNA crosslinking. Here we report confirmation of the structural assignment of the complex clb product precolibactin 886 via a biomimetic synthetic pathway. We show that an α-ketoimine linear precursor undergoes spontaneous cyclization to precolibactin 886 on HPLC purification. Studies of this α-ketoimine and the related α-dicarbonyl revealed that these compounds are unexpectedly susceptible to nucleophilic cleavage under mildly basic conditions. This cleavage pathway forms other known clb metabolites or biosynthetic intermediates and explains the difficulties in isolating fully mature biosynthetic products. This cleavage also accounts for a recently identified colibactin-adenine adduct. The colibactin peptidase ClbP deacylates synthetic precolibactin 886 to form a non-genotoxic pyridone, which suggests precolibactin 886 lies off the path of the major biosynthetic route.
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http://dx.doi.org/10.1038/s41557-019-0338-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761996PMC
October 2019

Structure elucidation of colibactin and its DNA cross-links.

Science 2019 09 8;365(6457). Epub 2019 Aug 8.

Department of Chemistry, Yale University, New Haven, CT 06520, USA.

Colibactin is a complex secondary metabolite produced by some genotoxic gut strains. The presence of colibactin-producing bacteria correlates with the frequency and severity of colorectal cancer in humans. However, because colibactin has not been isolated or structurally characterized, studying the physiological effects of colibactin-producing bacteria in the human gut has been difficult. We used a combination of genetics, isotope labeling, tandem mass spectrometry, and chemical synthesis to deduce the structure of colibactin. Our structural assignment accounts for all known biosynthetic and cell biology data and suggests roles for the final unaccounted enzymes in the colibactin gene cluster.
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http://dx.doi.org/10.1126/science.aax2685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6820679PMC
September 2019

Programmable Synthesis of 2-Deoxyglycosides.

J Am Chem Soc 2019 05 9;141(20):8098-8103. Epub 2019 May 9.

Department of Chemistry , Yale University , New Haven , Connecticut 06520 , United States.

Control of glycoside bond stereochemistry is the central challenge in the synthesis of oligosaccharides. 2-Deoxyglycosides, which lack a C2 substituent to guide stereoselectivity, are among the most difficult classes of glycoside bond constructions. Here we present a method to synthesize 2-deoxysaccharides with specified glycoside bond stereochemistry using a nucleophilic carbohydrate residue and the synthetic equivalent of an alcohol electrophile. Because the configuration of the nucleophile can be precisely controlled, both α- and β-glycosides can be synthesized from the same starting material in nearly all cases examined. Stereoselectivities in these reactions are often greater than 50:1 and yields typically exceed 70%. This strategy is amenable to the stereocontrolled syntheses of trisaccharide diastereomers, and a tetrasaccharide. This method may be extensible to other classes of carbohydrates.
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http://dx.doi.org/10.1021/jacs.9b03982DOI Listing
May 2019

Synthesis of Myrocin G, the Putative Active Form of the Myrocin Antitumor Antibiotics.

J Am Chem Soc 2018 11 16;140(47):16058-16061. Epub 2018 Nov 16.

Department of Chemistry , Yale University , New Haven , Connecticut 06520 , United States.

The antiproliferative antimicrobial fungal metabolites known as the myrocins have been proposed to cross-link DNA by double nucleotide addition. However, the nature of the DNA-reactive species is ambiguous, as myrocins have been isolated as functionally distinct 5-hydroxy-γ-lactone and diosphenol isomers. Based on literature precedent, we hypothesized that the diosphenol 7 (assigned here the trivial name myrocin G) is the biologically active form of the representative isolate (+)-myrocin C (1). To probe this, we developed a short enantioselective route to 7. A powerful fragment-coupling reaction that forms the central ring of the target in 38% yield and in a single step was developed. In support of our hypothesis, 7 was efficiently transformed to the bis(sulfide) 6, a product previously isolated from reactions of 1 with excess benzenethiol. This work provides the first direct access to the diosphenol 7, sets the stage for elucidating the mode of interaction of the myrocins with DNA, and provides a foundation for the synthesis of other pimarane diterpenes.
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http://dx.doi.org/10.1021/jacs.8b10891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001008PMC
November 2018

Model Colibactins Exhibit Human Cell Genotoxicity in the Absence of Host Bacteria.

ACS Chem Biol 2018 12 20;13(12):3286-3293. Epub 2018 Nov 20.

Department of Microbial Pathogenesis , Yale School of Medicine , New Haven , Connecticut 06536 , United States.

Colibactins are genotoxic secondary metabolites produced in select Enterobacteriaceae, which induce downstream DNA double-strand breaks (DSBs) in human cell lines and are thought to promote the formation of colorectal tumors. Although key structural and functional features of colibactins have been elucidated, the full molecular mechanisms regulating these phenotypes remain unknown. Here, we demonstrate that free model colibactins induce DSBs in human cell cultures and do not require delivery by host bacteria. Through domain-targeted editing, we demonstrate that a subset of native colibactins generated from observed module skipping in the nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) biosynthetic assembly line share DNA alkylation phenotypes with the model colibactins in vitro. However, module skipping eliminates the strong DNA interstrand cross-links formed by the wild-type pathway in cell culture. This product diversification during the modular NRPS-PKS biosynthesis produces a family of metabolites with varying observed mechanisms of action (DNA alkylation versus cross-linking) in cell culture. The presence of membranes separating human cells from model colibactins attenuated genotoxicity, suggesting that membrane diffusion limits colibactin activity and could account for the reported bacterium-human cell-to-cell contact phenotype. Additionally, extracellular supplementation of the colibactin resistance protein ClbS was able to intercept colibactins in an Escherichia coli-human cell transient infection model. Our studies demonstrate that free model colibactins recapitulate cellular phenotypes associated with module-skipped products in the native colibactin pathway and define specific protein domains that are required for efficient DNA interstrand cross-linking in the native pathway.
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http://dx.doi.org/10.1021/acschembio.8b00714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001666PMC
December 2018

Characterization of Natural Colibactin-Nucleobase Adducts by Tandem Mass Spectrometry and Isotopic Labeling. Support for DNA Alkylation by Cyclopropane Ring Opening.

Biochemistry 2018 11 31;57(45):6391-6394. Epub 2018 Oct 31.

Department of Chemistry , Yale University , New Haven , Connecticut 06520 , United States.

Colibactins are genotoxic secondary metabolites whose biosynthesis is encoded in the clb gene cluster harbored by certain strains of gut commensal Escherichia coli. Using synthetic colibactin analogues, we previously provided evidence that colibactins alkylate DNA by addition of a nucleotide to an electrophilic cyclopropane intermediate. However, natural colibactin-nucleobase adducts have not been identified, to the best of our knowledge. Here we present the first identification of such adducts, derived from treatment of pUC19 DNA with clb E. coli. Previous biosynthetic studies established cysteine and methionine as building blocks in colibactin biosynthesis; accordingly, we used cysteine (Δ cysE) and methionine (Δ metA) auxotrophic strains cultured in media supplemented with l-[U-C]Cys or l-[U-C]Met to facilitate the identification of nucleobases bound to colibactins. Using MS and MS analysis, in conjunction with the known oxidative instability of colibactin cyclopropane-opened products, we were able to characterize adenine adducts derived from cyclopropane ring opening. This study provides the first reported detection of nucleobase adducts derived from clb E. coli and lends support to our earlier model suggesting DNA alkylation by addition of a nucleotide to an electrophilic cyclopropane.
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http://dx.doi.org/10.1021/acs.biochem.8b01023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997931PMC
November 2018

Cobalt bis(acetylacetonate)--butyl hydroperoxide-triethylsilane: a general reagent combination for the Markovnikov-selective hydrofunctionalization of alkenes by hydrogen atom transfer.

Beilstein J Org Chem 2018 28;14:2259-2265. Epub 2018 Aug 28.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

We show that cobalt bis(acetylacetonate) [Co(acac)], -butyl hydroperoxide (TBHP), and triethylsilane (EtSiH) constitute an inexpensive, general, and practical reagent combination to initiate a broad range of Markovnikov-selective alkene hydrofunctionalization reactions. These transformations are believed to proceed by cobalt-mediated hydrogen atom transfer (HAT) to the alkene substrate, followed by interception of the resulting alkyl radical intermediate with a SOMOphile. In addition, we report the first reductive couplings of unactivated alkenes and aryldiazonium salts by an HAT pathway. The simplicity and generality of the Co(acac)-TBHP-EtSiH reagent combination suggests it as a useful starting point to develop HAT reactions in complex settings.
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http://dx.doi.org/10.3762/bjoc.14.201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122341PMC
August 2018

Antibacterial properties and clinical potential of pleuromutilins.

Nat Prod Rep 2019 01 6;36(1):220-247. Epub 2018 Jul 6.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA. and Department of Pharmacology, Yale School of Medicine, New Haven, Connecticut 06520, USA.

Covering: up to 2018Pleuromutilins are a clinically validated class of antibiotics derived from the fungal diterpene (+)-pleuromutilin (1). Pleuromutilins inhibit bacterial protein synthesis by binding to the peptidyl transferase center (PTC) of the ribosome. In this review we summarize the biosynthesis and recent total syntheses of (+)-pleuromutilin (1). We review the mode of interaction of pleuromutilins with the bacterial ribosome, which involves binding of the C14 extension and the tricyclic core to the P and A sites of the PTC, respectively. We provide an overview of existing clinical agents, and discuss the three primary modes of bacterial resistance (mutations in ribosomal protein L3, Cfr methylation, and efflux). Finally we collect structure-activity relationships from publicly available reports, and close with some forward looking statements regarding future development.
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http://dx.doi.org/10.1039/c8np00042eDOI Listing
January 2019

A convergent approach to batzelladine alkaloids. Total syntheses of (+)-batzelladine E, (-)-dehydrobatzelladine C, and (+)-batzelladine K.

Tetrahedron 2018 Jun 18;74(26):3188-3197. Epub 2018 Apr 18.

Department of Chemistry, Yale University, New Haven, CT 06520, United States.

We recently reported a convergent strategy to access the polycyclic guanidinium alkaloid (+)-batzelladine B via an aldol addition-retro-aldol-aza-Michael addition cascade. Here we describe the application of this approach toward the total syntheses of (+)-batzelladine E, (-)-dehydrobatzelladine C, and (+)-batzelladine K. The identification of suitable methods to functionalize a common tropane core by electrophilic alkynylation and nucleophilic 1,2-addition were essential to generalizing this approach. We provide evidence for the intermediacy of an acylallene species in the cascade reaction.
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http://dx.doi.org/10.1016/j.tet.2018.04.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8078486PMC
June 2018

Directed C-H Bond Oxidation of (+)-Pleuromutilin.

J Org Chem 2018 07 1;83(13):6843-6892. Epub 2018 May 1.

Antibiotics derived from the diterpene fungal metabolite (+)-pleuromutilin (1) are useful agents for the treatment Gram-positive infections in humans and farm animals. Pleuromutilins elicit slow rates of resistance development and minimal cross-resistance with existing antibiotics. Despite efforts aimed at producing new derivatives by semisynthesis, modification of the tricyclic core is underexplored, in part due to a limited number of functional group handles. Herein, we report methods to selectively functionalize the methyl groups of (+)-pleuromutilin (1) by hydroxyl-directed iridium-catalyzed C-H silylation, followed by Tamao-Fleming oxidation. These reactions provided access to C16, C17, and C18 monooxidized products, as well as C15/C16 and C17/C18 dioxidized products. Four new functionalized derivatives were prepared from the protected C17 oxidation product. C6 carboxylic acid, aldehyde, and normethyl derivatives were prepared from the C16 oxidation product. Many of these sequences were executed on gram scales. The efficiency and practicality of these routes provides an easy method to rapidly interrogate structure-activity relationships that were previously beyond reach. This study will inform the design of fully synthetic approaches to novel pleuromutilins and underscores the power of the hydroxyl-directed iridium-catalyzed C-H silylation reaction.
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http://dx.doi.org/10.1021/acs.joc.8b00462DOI Listing
July 2018

DNA Repair: Unconventional Lesions Require Unconventional Repair.

Authors:
Seth B Herzon

Biochemistry 2018 02 6;57(7):1057-1058. Epub 2018 Feb 6.

Department of Chemistry, Yale University , New Haven, Connecticut 06511, United States.

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http://dx.doi.org/10.1021/acs.biochem.7b01241DOI Listing
February 2018

ClbS Is a Cyclopropane Hydrolase That Confers Colibactin Resistance.

J Am Chem Soc 2017 12 28;139(49):17719-17722. Epub 2017 Nov 28.

Department of Microbial Pathogenesis, Yale School of Medicine , New Haven, Connecticut 06536, United States.

Certain commensal Escherichia coli contain the clb biosynthetic gene cluster that codes for small molecule prodrugs known as precolibactins. Precolibactins are converted to colibactins by N-deacylation; the latter are postulated to be genotoxic and to contribute to colorectal cancer formation. Though advances toward elucidating (pre)colibactin biosynthesis have been made, the functions and mechanisms of several clb gene products remain poorly understood. Here we report the 2.1 Å X-ray structure and molecular function of ClbS, a gene product that confers resistance to colibactin toxicity in host bacteria and which has been shown to be important for bacterial viability. The structure harbors a potential colibactin binding site and shares similarity to known hydrolases. In vitro studies using a synthetic colibactin analog and ClbS or an active site residue mutant reveal cyclopropane hydrolase activity that converts the electrophilic cyclopropane of the colibactins into an innocuous hydrolysis product. As the cyclopropane has been shown to be essential for genotoxic effects in vitro, this ClbS-catalyzed ring-opening provides a means for the bacteria to circumvent self-induced genotoxicity. Our study provides a molecular-level view of the first reported cyclopropane hydrolase and support for a specific mechanistic role of this enzyme in colibactin resistance.
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http://dx.doi.org/10.1021/jacs.7b09971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202678PMC
December 2017

Development of a Modular Synthetic Route to (+)-Pleuromutilin, (+)-12-epi-Mutilins, and Related Structures.

J Am Chem Soc 2017 11 2;139(45):16377-16388. Epub 2017 Nov 2.

Department of Chemistry, Yale University , New Haven, Connecticut 06520, United States.

We describe the development of an enantioselective synthetic route to (+)-pleuromutilin (1), (+)-12-epi-mutilin, and related derivatives. A key hydrindanone was prepared in three steps and 48% overall yield from cyclohex-2-en-1-one. 1,4-Hydrocyanation provided a nitrile (53%, or 85% based on recovered starting material) that was converted to the eneimide 57 in 80% yield by the 1,2-addition of methyllithium to the nitrile function, cyclization, and in situ acylation with di-tert-butyldicarbonate. The eneimide 57 was employed in a 2-fold neopentylic coupling reaction with an organolithium reagent derived from the alkyl iodides (R)- or (S)-30, which contain the C11-C13 atoms of the target, to provide diastereomeric diketones in 60% or 48% yield (for coupling with (R)- or (S)-30, respectively). The diketone derived from (S)-30 contains the (S)-C12 stereochemistry found in pleuromutilin and was elaborated to an alkynylaldehyde. Nickel-catalyzed reductive cyclization of this alkynylaldehyde, to construct the eight-membered ring of the target, unexpectedly provided a cyclopentene (67%), which arises from participation of the C12-α-olefin in the transformation. The diketone derived from the enantiomeric C12-fragment (R)-30 underwent reductive cyclization to provide the desired product in 60% yield. This was elaborated to 12-epi-mutilin by a four-step sequence (39% overall). Installation of the glycolic acid residue followed by C12 epimerization (Berner et al. Monatsh. Chem. 1986, 117, 1073) generated (+)-pleuromutilin (1). (+)-12-epi-Pleuromutilin and (+)-11,12-di-epi-pleuromutilin were prepared by related sequences. This work establishes a convergent entry to the pleuromutilins and provides a foundation for the production of novel antibiotics to treat drug-resistant and Gram-negative infections.
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http://dx.doi.org/10.1021/jacs.7b09869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7024634PMC
November 2017
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