Preparation of a Rigid and Nearly Coplanar Bis-tetracene Dimer through an Application of the CANAL Reaction.

A rigid tetracene dimer with a substantial interchromophore distance has been prepared through an application of the recently developed catalytic arene-norbornene annulation (CANAL) reaction. An iterative cycloaddition route was found to be unsuccessful, so a shorter route was adopted whereby fragments were coupled in the penultimate step to form a 13:1 mixture of two diastereomers, the major of which was isolated and crystallized. Constituent tetracene moieties are linked with a rigid, well-defined bridge and feature a near-co-planar mutual orientation of the acenes.

Targeting the Conformational Change in ArnA Dehydrogenase for Selective Inhibition of Polymyxin Resistance.

Polymyxins are important last resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. However, pathogens have acquired resistance to polymyxins through a pathway that modifies lipid A with 4-amino-4-deoxy-l-arabinose (Ara4N). Inhibition of this pathway is, therefore, a desirable strategy to combat polymyxin resistance. The first pathway-specific reaction is an NAD+-dependent oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcA) catalyzed by the dehydrogenase domain of ArnA (ArnA_DH). We present the crystal structure of Salmonella enterica serovar typhimurium ArnA in complex with UDP-GlcA showing that binding of the sugar nucleotide is sufficient to trigger a conformational change conserved in bacterial ArnA_DHs but absent in its human homologs, as confirmed by structure and sequence analysis. Ligand binding assays show that the conformational change is essential for NAD+ binding and catalysis. Enzyme activity and binding assays show that (i) UDP-GlcA analogs lacking the 6' carboxylic acid bind the enzyme but fail to trigger the conformational change, resulting in poor inhibition, and (ii) the uridine monophosphate moiety of the substrate provides most of the ligand binding energy. Mutation of asparagine 492 to alanine (N492A) disrupts the ability of ArnA_DH to undergo the conformational change while retaining substrate binding, suggesting that N492 is involved in sensing the 6' carboxylate in the substrate. These results identify the UDP-GlcA-induced conformational change in ArnA_DH as an essential mechanistic step in bacterial enzymes, providing a platform for selective inhibition.

Identifying signatures of proteolytic stability and monomeric propensity in O-glycosylated insulin using molecular simulation.

Insulin has been commonly adopted as a peptide drug to treat diabetes as it facilitates the uptake of glucose from the blood. The development of oral insulin remains elusive over decades owing to its susceptibility to the enzymes in the gastrointestinal tract and poor permeability through the intestinal epithelium upon dimerization. Recent experimental studies have revealed that certain O-linked glycosylation patterns could enhance insulin's proteolytic stability and reduce its dimerization propensity, but understanding such phenomena at the molecular level is still difficult. To address this challenge, we proposed and tested several structural determinants that could potentially influence insulin's proteolytic stability and dimerization propensity. We used these metrics to assess the properties of interest from [Formula: see text] aggregate molecular dynamics of each of 12 targeted insulin glyco-variants from multiple wild-type crystal structures. We found that glycan-involved hydrogen bonds and glycan-dimer occlusion were useful metrics predicting the proteolytic stability and dimerization propensity of insulin, respectively, as was in part the solvent-accessible surface area of proteolytic sites. However, other plausible metrics were not generally predictive. This work helps better explain how O-linked glycosylation influences the proteolytic stability and monomeric propensity of insulin, illuminating a path towards rational molecular design of insulin glycoforms.

Using Structurally Well-Defined Norbornyl-Bridged Acene Dimers to Map a Mechanistic Landscape for Correlated Triplet Formation in Singlet Fission.

Structurally well-defined TIPS-acetylene substituted tetracene (TIPS-BT1') and pentacene (TIPS-BP1') dimers utilizing a [2.2.1] bicyclic norbornyl bridge have been studied-primarily using time-resolved spectroscopic methods-to uncover mechanistic details about primary steps in singlet fission leading to formation of the biexcitonic 1TT state as well as decay pathways to the ground state. For TIPS-BP1' in room-temperature toluene, 1TT formation is rapid and complete, occurring in 4.4 ps. Decay to the ground state in 100 ns is the primary loss pathway for 1TT in this system. For TIPS-BT1', the 1TT is also observed to form rapidly (with a time constant of 5 ps), but in this case it occurs in concert with establishment of an excited-state equilibrium ( K ∼ 1) with the singlet exciton state S1 at an energy of 2.3 eV above the ground state. The equilibrated states survive for 36 ns and are lost to ground state through both radiative and nonradiative pathways via the S1 and nonradiative pathways via the 1TT. The rapidity of 1TT formation in TIPS-BT1' is at first glance surprising. However, our analysis suggests that the few-parameter rate constant expression of Marcus theory explains both individual and comparative findings in the set of systems, thus establishing benchmarks for diabatic coupling and reorganization energy needed for efficient 1TT formation. Finally, a comparison of TIPS-BT1' with previous results obtained for a close constitutional isomer (TIPS-BT1) differing in the placement of TIPS-acetylene side groups suggests that the magnitude of exchange interaction in the correlated triplet manifold plays a critical role dictating 1TT yield in the tetracenic systems.

Modular Synthesis of Rigid Polyacene Dimers for Singlet Fission.

An improved, modular synthesis of rigid, geometrically well-defined, alkyne-substituted tetracene (1) and pentacene (2) dimers is reported. The synthesis is rooted in sequential Diels-Alder reactions of a norbornyl tetraene with triisopropylsilylacetylene-substituted (TIPS-acetylene) quinone dienophiles. The incorporation of solubilizing and stabilizing TIPS-acetylene groups early in the synthesis affords a mild and reliable route, providing access, for the first time, to norbornyl-bridged pentacene dimers. A preliminary exploration of the excited state behavior of these molecules is also described.

Synthesis of Geometrically Well-Defined Covalent Acene Dimers for Mechanistic Exploration of Singlet Fission.

We report the first synthesis of norbornyl-bridged acene dimers (2 and 3) with well-defined and controlled spatial relationships between the acene chromophore subunits. We employ a modular 2-D strategy wherein the central module, common to all our compounds, is a norbornyl moiety. The acenes are attached to this module using the Diels-Alder reaction, which also forms one of the acene rings. Manipulation of the Diels-Alder adducts provides the desired geometrically defined bis-acenes. The modular nature of this synthesis affords flexibility and allows for the preparation of a variety of acene dimers, including functionalized tetracene dimers.

The Conversion of tert-Butyl Esters to Acid Chlorides Using Thionyl Chloride.

The reaction of tert-butyl esters with SOCl2 at room temperature provides acid chlorides in unpurified yields of 89% or greater. Benzyl, methyl, ethyl, and isopropyl esters are essentially unreactive under these conditions, allowing for the selective conversion of tert-butyl esters to acid chlorides in the presence of other esters.

Doubly Vinylogous Aldol Reaction of Furoate Esters with Aldehydes and Ketones.

The use of bulky Lewis acids, aluminum tris(2,6-diphenylphenoxide) (ATPH) and aluminum tris(2,6-di-2-naphthylphenoxide) (ATNP), in the doubly vinylogous aldol reaction between methyl-5-methyl-2-furoate and aldehydes or ketones is described. These reactions proceed smoothly and in high yields with both enolizable and non-enolizable substrates. This C-C bond-forming reaction enables a new bond construction for the synthesis of functionalized furans.

Regioselective Ring Opening of Di-isopropylsilylenes Derived from 1,3-Diols with Alkyl Lithium Reagents.

The selective alkyl lithium-induced ring opening of 1,3-di-isopropylsilylenes is described. The reaction affords a differentially substituted 1,3-diol bearing a silane that resides at the oxygen in the more sterically demanding position. The reaction can be highly selective with a regiochemical preference up to >50:1 and likely proceeds via an alkoxy-silane intermediate. This intermediate can by trapped by methyl iodide to provide the corresponding silyl methyl ether, wherein the silane again resides at the oxygen in the more sterically demanding position.

Synthesis of N-Vinyl Nitrones via 1,4-Conjugate Elimination.

A number of structurally and electronically diverse N-vinyl nitrones have been synthesized by a two-step method. The sequence consists of condensation of an α-chloroaldehyde or an α-phenoxy- or α-acetoxy ketone with a substituted benzyl hydroxylamine to provide the corresponding nitrone. Treatment of these species with a base induces a 1,4-elimination to provide the desired N-vinyl nitrone in good to excellent yields.

Select steroid hormone glucuronide metabolites can cause toll-like receptor 4 activation and enhanced pain.

We have recently shown that several classes of glucuronide metabolites, including the morphine metabolite morphine-3-glucuronide and the ethanol metabolite ethyl glucuronide, cause toll like receptor 4 (TLR4)-dependent signaling in vitro and enhanced pain in vivo. Steroid hormones, including estrogens and corticosterone, are also metabolized through glucuronidation. Here we demonstrate that in silico docking predicts that corticosterone, corticosterone-21-glucuronide, estradiol, estradiol-3-glucuronide and estradiol-17-glucuronide all dock with the MD-2 component of the TLR4 receptor complex. In addition to each docking with MD-2, the docking of each was altered by pre-docking with (+)-naloxone, a TLR4 signaling inhibitor. As agonist versus antagonist activity cannot be determined from these in silico interactions, an in vitro study was undertaken to clarify which of these compounds can act in an agonist fashion. Studies using a cell line transfected with TLR4, necessary co-signaling molecules, and a reporter gene revealed that only estradiol-3-glucuronide and estradiol-17-glucuronide increased reporter gene product, indicative of TLR4 agonism. Finally, in in vivo studies, each of the 5 drugs was injected intrathecally at equimolar doses. In keeping with the in vitro results, only estradiol-3-glucuronide and estradiol-17-glucuronide caused enhanced pain. For both compounds, pain enhancement was blocked by the TLR4 antagonist lipopolysaccharide from Rhodobacter sphaeroides, evidence for the involvement in TLR4 in the resultant pain enhancement. These findings have implications for several chronic pain conditions, including migraine and temporomandibular joint disorder, in which pain episodes are more likely in cycling females when estradiol is decreasing and estradiol metabolites are at their highest.

The vinylogous aldol reaction of unsaturated esters and enolizable aldehydes using the novel Lewis acid aluminum tris(2,6-di-2-naphthylphenoxide).

The synthesis of the novel Lewis acid, aluminum tris(2,6-di-2-naphthylphenoxide) (ATNP), and its use in the vinylogous aldol reaction between methyl crotonate and enolizable aldehydes are described. ATNP is related to Yamamoto's Lewis acid, aluminum tris(2,6-diphenylphenoxide) (ATPH), but the 2-naphthyl groups more effectively block the α-position of aldehydes, enabling the selective enolization of crotonate esters in the presence of enolizable aldehydes. Vinylogous aldol reactions then proceed smoothly and in high yields with a variety of substrates.

Toward the synthesis of (+)-peloruside A via an intramolecular vinylogous aldol reaction.

The use of the intramolecular vinylogous aldol reaction for the preparation of an advanced intermediate for the synthesis of peloruside A is described. The reaction was applied to compound 19 and proceeds in high yield and good levels of diastereoselectivity. Application of the Achmatowicz reaction to this intermediate provided the corresponding pyranone, a late stage intermediate well positioned for conversion to the natural product.

Total synthesis of dermostatin A.

The concise total synthesis of dermostatin A is described. Highlights include a two-directional application of the asymmetric acetate aldol method developed in our lab, a novel diastereotopic-group-selective acetal isomerization for terminus differentiation, and a selective cross-metathesis reaction between a terminal olefin and a trienal. A study of the scope and viability of similar cross-metathesis reactions is also described. The synthesis is convergent and utilizes fragments of roughly equal complexity.

New inhibitors of colony spreading in Bacillus subtilis and Bacillus anthracis.

We have recently characterized sliding motility in Bacillus subtilis strains that lack functional flagella, and here describe the discovery of inhibitors of colony spreading in these strains as well as the aflagellate pathogen, Bacillus anthracis. Aflagellate B. subtilis strains were used to screen for new types of antibacterials that might inhibit colony spreading on semi-solid media. From a diverse set of organic structures, p-nitrophenylglycerol (NPG), an agent used primarily in clinical laboratories to control Proteus swarming, was found to inhibit colony spreading. The four stereoisomers of NPG were synthesized and tested, and only the 1R,2S-(1R-anti) and 1R,2R-(1R-syn) NPG isomers had significant activity in a quantitative colony-spreading assay. Twenty-six NPG analogs and related structures were synthesized and tested to identify more active inhibitors. p-Methylsulfonylphenylglycerol (p-SPG), but not its ortho or meta analogs, was found to be the most effective of these compounds, and synthesis and testing of all four p-SPG stereoisomers showed that the 1R-anti-isomer was the most active with an average IC(50) of 16 µM (3-5 µg mL(-1)). For B. anthracis, the colony-spreading IC(50) values for 1R-anti-SPG and 1R-anti-NPG are 12 µM (2-4 µg mL(-1)) and >150 µM, respectively. For both Bacillus species tested, 1R-anti-SPG inhibits colony spreading of surface cultures on agar plates, but is not bacteriostatic or bacteriocidal in liquid cultures. Work is in progress to find the cellular target(s) of the NPG/SPG class of compounds, since this could lead to an understanding of the mechanism(s) of colony spreading as well as design and development of more potent inhibitors for the control of B. anthracis surface cultures.

Synthesis of methyl-1-(tert-butoxycarbonylamino)-2-vinylcyclopropanecarboxylate via a Hofmann rearrangement utilizing trichloroisocyanuric acid as an oxidant.

A trichloroisocyanuric acid (TCCA) mediated Hofmann rearrangement was utilized to synthesize methyl-1-(tert-butoxycarbonylamino)-2-vinylcyclopropanecarboxylate. A variety of functional groups are tolerated in this reaction including vinyl, cyclopropyl, pyridyl, aryl, benzyl, and nitro groups.

Alpha-arylation of 3-aryloxindoles.

A versatile method for the synthesis of 3,3-diaryloxindoles via Pd-catalyzed alpha-arylations or an S(N)Ar reaction is described. The reaction proceeds using mild base, is tolerant of a variety of functional groups, and is capable of preparing hindered all-carbon quaternary centers.

Application of the intramolecular Yamamoto vinylogous aldol reaction to the synthesis of macrolides.

An intramolecular version of the Yamamoto vinylogous aldol reaction, a method that employs the bulky Lewis acid ATPH to control the site of aldolization, is described. This macrocyclization process is effective for the construction of 10-, 12-, and 14-membered macrolides. The yields are high (70-90%), and the reaction can proceed with excellent remote stereocontrol (dr > or = 20:1) with chiral substrates.