David A. Vosburg

Dynamic thiolation–thioesterase structure of a non-ribosomal peptide synthetase

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties. Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, incorporates a given monomer unit into the growing chain. Knowledge about domain or module interactions may enable reengineering of this assembly line enzymatic organization and open avenues for the design of new bioactive compounds with improved therapeutic properties. So far, little structural information has been available on how the domains interact and communicate. This may be because of inherent interdomain mobility hindering crystallization, or because crystallized molecules may not represent the active domain orientations. In solution, the large size and internal dynamics of multidomain fragments (>35 kilodaltons) make structure determination by nuclear magnetic resonance a challenge and require advanced technologies. Here we present the solution structure of the apo-thiolation–thioesterase (T–TE) di-domain fragment of the Escherichia coli enterobactin synthetase EntF NRPS subunit. In the holoenzyme, the T domain carries the growing chain tethered to a 4′-phosphopantetheine whereas the TE domain catalyses hydrolysis and cyclization of the iron chelator enterobactin. The T–TE di-domain forms a compact but dynamic structure with a well-defined domain interface; the two active sites are at a suitable distance for substrate transfer from T to TE. We observe extensive interdomain and intradomain motions for well-defined regions and show that these are modulated by interactions with proteins that participate in the biosynthesis. The T–TE interaction described here provides a model for NRPS, PKS and FAS function in general as T–TE-like di-domains typically catalyse the last step in numerous assembly-line chain-termination machineries.

Intramolecular Hetero Diels–Alder Routes to γ-Carboline Alkaloids

Abstract Concise and efficient routes to the carboline alkaloids isocanthine (3), isocanthin-6-one (4), 1-methylisocanthine (5), and 1-methylisocanthin-6-one (6) are reported. In each case, the key synthetic step was an intramolecular hetero Diels–Alder reaction of a 1-aza-1,3-diene with an acetylenic dienophile.

Dichlorination and Bromination of a Threonyl-S-Carrier Protein by the Non-heme FeII Halogenase SyrB2

Biosynthetic tailoring of nonribosomal peptide and polyketide natural products can enhance their biological activities. Tailoring enzymes can introduce alkyl, acyl, or glycosyl groups onto natural product scaffolds and can oxidize or halogenate biosynthetic intermediates. Chlorinated and brominated molecules make up more than 95% of the more than 4500 known halogenated metabolites. Chloro and bromo substituents are frequently found on aromatic and heteroaromatic rings, and many terpene scaffolds are also brominated and chlorinated by marine microorganisms. Halogenating enzymes discovered to date fall into two categories based on their utilization either of hydrogen peroxide (haloperoxidases) or molecular oxygen (halogenases) as required oxidants. Haloperoxidases can contain either heme iron or a vanadate cofactor, thought to generate enzyme-bound hapohalite equivalents as proximal OCl or OBr. 8] The O2-utilizing halogenases are typically found embedded in biosynthetic gene clusters ; this suggests a tailoring role in specific natural product assembly. This second class of enzymes uses either FADH2 or non-heme Fe II to activate chloride or bromide oxidatively. The flavoproteins work on electron-rich aromatic and heteroaromatic substrates. 12] The Fe halogenases represent a new branch of the O2 and a-ketoglutarate-decarboxylating superfamily and are powerful enough to halogenate unactivated carbon centers on aminoacyl groups tethered to nonribosomal peptide synthetase assembly lines. Thus, the 4-Cl-l-Thr residue in the phytotoxic liACHTUNGTRENNUNGpo ACHTUNGTRENNUNGdepsipeptide syringomycin E (1) is generated by the nonheme Fe halogenase SyrB2 (Scheme 1). Chlorination occurs on the threonyl skeleton only while it is linked via a thioester to a peptidyl carrier protein domain. Remarkably, the cyclopropane ring in the amino acid coronamic acid arises by a similar g-chlorination of an l-allo-Ile-S-protein by the halogenase CmaB. The g-chloride is then displaced intramolecularly by a thioester enolate by action of CmaC. Therefore, the CmaBmediated chlorination is cryptic in cyclopropane formation. In surveying natural products in which biological chlorination is likely to have occurred at an unactivated carbon center, the remarkable functionalization of the two prochiral methyl groups of leucine to yield the regioand stereospecific generation of a trichloromethyl group in the biosynthesis of the cyanobacterial metabolites barbamide (2), dysidenin (3), and dysideathiazole (4 ; Scheme 2A) suggests analogies with the above Fe halogenases. Indeed, the barbamide biosynthetic gene cluster has been sequenced and contains two genes (barB1 and barB2) that encode proteins homologous to SyrB2, but no activity has yet been reported. barB1 and barB2 homologues have also been found in the dysidenin and dysideathiazole producers (dysB1/dysB2 ; one pair in each producer). Only a few natural products contain bromine where biological bromination might have occurred at an unactivated carbon site. One example is lyngbyaloside B (5 ; Scheme 2B), which is also of cyanobacterial origin and could arise from bromination of an unactivated carbon on a biosynthetic precursor. Whether nonheme Fe halogenases are involved in the biosynthesis of brominated natural products is unknown. To evaluate whether the non-heme Fe family of enzymes can indeed carry out bromination and iterative chlorinations at the same carbon site, we have further examined the activity of SyrB2. SyrB2 was shown to act on the threonyl group presented in thioester linkage on the peptidyl carrier protein domain of its partner protein SyrB1. 4-Cl-threonyl-S-SyrB1 was gently hydrolyzed by addition of the thioesterase TycF and detected as the isoindole adduct (Figure 1A). With an almost equimolar ratio of SyrB2/SyrB1, SyrB2 generated a new peak (Figure 1B) that coeluted with the isoindole derivative of authentic 4,4diCl-Thr, synthesized as noted in the Experimental Section. Mass analysis of the new enzymatic product confirmed both the mass and isotope ratios of the diCl-l-Thr isoindole derivative (calcd for [M+H] 392.0 (100%), 394.0 (71%); found 391.7 (100%), 394.1 (68%)). In addition, the ratio of the relative intensity of the peaks corresponding to 4-Cl-l-Thr and 4,4-diCl-lThr for the reaction run in the presence of [Cl] is doubled in the radioactivity detection channel (Figure 1B, trace c) when compared to the UV channel (Figure 1B, trace d) of the same reaction; this is in good agreement with the presence of two chloro substituents. Because the substrate for SyrB2 is a covalent aminoacyl-S protein and the chlorinated product(s) remain covalently tethered, kinetic analysis would be challenging. However, the released monoand diCl-l-Thr products were obtained at a maximum ratio of about 0.38:1 diCl-l-Thr/Cl-l-Thr (Figure 1C). When comparing the ratio of diCl-l-Thr/Cl-l-Thr in Figure 1C and B, it can be seen that the ratio is higher for panel B (1:1 diCl-l-Thr/Cl-l-Thr). This seems to be due to the difference in initial oxygen concentration prior to SyrB2 addition. In panel B, the reaction mixture was anaerobic prior to SyrB2 addition, whereas the buffer was air-saturated for panel C. The slow addition of oxygen after SyrB2 addition increases the product yield, as shown in Figure 1B. This was also observed when demonstrating the oxygen dependency of the reaction in a prior study (Figure 2B in ref. [10]), in which full [a] Dr. F. H. Vaillancourt, Prof. Dr. D. A. Vosburg, Prof. C. T. Walsh Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115 (USA) Fax: (+1)617-432-0438 E-mail : christopher_walsh@hms.harvard.edu [b] Dr. F. H. Vaillancourt Present address: Department of Biological Sciences Research and Development, Boehringer Ingelheim (Canada) Ltd Laval, QC, H7S 2G5 (Canada) [c] Prof. Dr. D. A. Vosburg Present address: Department of Chemistry, Harvey Mudd College Claremont, CA 91711 (USA) [] These authors contributed equally to this work.

A Concise Synthesis of Fumagillol

A 13-step synthesis of (±)-fumagillol (1), the direct precursor of the potent angiogenesis inhibitors TNP-470 and fumagillin, from crotonaldehyde, diethylamine, and acrolein (see the scheme) has been achieved. The synthesis features a remarkable hetero-Claisen rearrangement. Small-molecule inhibitors of angiogenesis are promising chemotherapeutic agents for the treatment of cancer and inflammatory diseases.

Cooperative Loading and Release Behavior of a Metal–Organic Receptor

In order to design artificial chemical systems that are capable of achieving complex functions, it is useful to design synthetic receptors that mimic their biological counterparts. Biological functions are underpinned by properties that include specific binding with high affinity and selectivity, cooperativity, and release triggered by external stimuli. Here we show that a metal-organic receptor constructed through subcomponent self-assembly can selectively and cooperatively load and release oxocarbon anions. The flexible coordination spheres of its cadmium(II) centers allow the receptor to dynamically adjust its structure upon exchanging four triflate or triflimide counterions for two oxocarbon anions, resulting in strong cooperativity and very tight binding, with an apparent association constant for C5O5(2-) of 5 × 10(10) M(-1). Substituting the cadmium(II) ions for copper(I) by switching solvent prompted a structural reorganization and release of the oxocarbon anions. Its cooperative behavior allows the receptor to carry a greater payload than would be possible in a noncooperative analogue.

A Concise Route to Isocanthin-6-one

Abstract An efficient, four-step route to isocanthin-6-one ( 4 ) is reported. The key step is an intramolecular hetero Diels-Alder reaction.

A concise, biomimetic total synthesis of (+)-davanone.

A concise, biomimetic synthesis of the antifungal and antispasmodic natural product (+)-davanone is described. The key stereoselective reactions are a Sharpless asymmetric epoxidation, a thiazolium-catalyzed esterification, and a palladium-mediated cyclization. All carbons are derived from isoprene units and no protecting groups are used, permitting an atom- and redox-economical synthesis.

Solvent-Free Synthesis and Fluorescence of a Thiol-Reactive Sensor for Undergraduate Organic Laboratories

A green organic laboratory experiment was developed in which students synthesize a sensor for thiols using a microscale, solventless Diels-Alder reaction at room temperature or 37 °C. The molecular probe is easily purified by column chromatography in a Pasteur pipet and characterized by thin-layer chromatography and NMR spectroscopy. The thiol-reactive sensor becomes intensely fluorescent upon exposure to thiols from N-acetylcysteine, bovine serum albumin, or human hair (pretreated with a reducing agent to reveal cysteine thiols in α-keratin). This fluorescence is observable even with micrograms of probe.

Divergent Diels-Alder reactions in the biosynthesis and synthesis of endiandric-type tetracycles: A computational study

Endiandric acids and related polyketide natural products arise from polyene precursors and occur naturally as fused and bridged tetracycles. In some cases, the intramolecular Diels-Alder reactions that produce fused and bridged tetracycles result from a diene tether that may act as either a 4π or 2π component in the cycloaddition. To examine the preference for fused or bridged products, we applied density functional theory (using the M06-2X and B3LYP functionals) to reactants with various substituents for both fused and bridged transition states. Fused products were generally preferred except when disfavored by extreme steric hindrance (e.g., a tert-butyl group). These computational results are consistent with experimental data and suggest the existence of as-yet undiscovered natural products.