We report an NMR chemical shift study of conformationally challenging seven-membered lactones (1–11); computed and experimental data sets are compared. The computations involved full conformational analysis of each lactone, Boltzmann-weighted averaging of the chemical shifts across all conformers, and linear correction of the computed chemical shifts. DFT geometry optimizations [M06-2X/6-31+G(d,p)] and GIAO NMR chemical shift calculations [B3LYP/6-311+G(2d,p)] provide the computed chemical shifts. The corrected-mean absolute error (CMAE), the average of the differences between the computed and experimental chemical shifts for each of the eleven lactones, is encouragingly small (0.02–0.08 ppm for 1H or 0.8–2.2 ppm for 13C). Three pairs of cis vs. trans diastereomeric lactones were used to assess the ability of the method to distinguish between stereoisomers. The experimental shifts were compared with the computed shifts for each of the two possible isomers. We introduce the use of a “match ratio”—the ratio of the larger (worse fit) to the smaller (better fit) CMAE. A greater match ratio value indicates better distinguishing ability. The match ratios are larger for proton data [2.4–4.0 (ave = 3.2)] than for carbon [1.1–2.3 (ave = 1.6)], indicating that the former provide a better basis for discriminating these diastereomers.
We report mechanistic aspects of the trapping of thermally (HDDA) generated benzyne derivatives by pendant silyl ether groups, which results in net insertion of the pair of benzyne Csp-hydribized carbon atoms into the silicon–oxygen sigma bond. Cross-over experiments using symmetrical, doubly labeled bis-silyl ether substrates established that the reaction is unimolecular in nature. Competition experiments involving either intramolecular or intermolecular dihydrogen transfer clock reactions (from within a TIPS isopropyl group or cyclooctane, respectively) vs. the silyl ether cyclization were used to gain additional insights. We evaluated effects of the steric bulk of the silyl ether trapping group and of the ring-size of the cyclic ether being formed (furan vs. pyran). These types of competition experiments allow the relative rates of various product-determining steps to be determined. This previously has only rarely been possible because aryne formation is typically rate-limiting, making it challenging to probe the kinetics of subsequent trapping reactions. Solvent effects (polarity of the medium) and computational studies were used to probe the question of stepwise vs. concerted pathways for the Si–O insertion.
The efficient dichlorination of benzynes prepared by the hexadehydro-Diels–Alder (HDDA) reaction is reported. Cycloisomerization of a triyne substrate in the presence of dilithium tetrachlorocuprate is shown to provide dichlorinated products A by capture of the benzyne intermediate. A general strategy for discerning the kinetic order of an external aryne trapping agent is presented. It merely requires measurement of the competition between bimolecular vs. unimolecular trapping events (here, dichlorination vs. IMDA reaction to give A vs. B, respectively) as a function of the concentration of the trapping agent.
Benzynes can be generated by the intramolecular thermal cycloisomerization of triynes–the title HDDA reaction. We report here that these can be trapped by cycloaddition reaction with trimethylsilyl azide (1,3-dipolar) or a furan or pyrrole (4+2 Diels–Alder).
The ene reaction is a pericyclic process in which an alkene having an allylic hydrogen atom (the ene donor) reacts with a second unsaturated species (the enophile) to form a new product with a transposed π-bond. The aromatic ene reaction, in which the alkene component is embedded in an aromatic ring, has only been reported in a few (four) instances and has proceeded in low yield (≤6%). Here we show efficient aromatic ene reactions in which a thermally generated aryne engages a pendant m-alkylarene substituent to produce a dearomatized isotoluene, itself another versatile but rare reactive intermediate. Our experiments were guided by computational studies that revealed structural features conducive to the aromatic ene process. We proceeded to identify a cascade comprising three reactions: (i) hexadehydro-Diels-Alder (for aryne generation), (ii) intramolecular aromatic ene, and (iii) bimolecular Alder ene. The power of this cascade is evident from the structural complexity of the final products, the considerable scope, and the overall efficiency of these multi-stage, reagent- and byproduct-free, single-pot transformations.
Calixarene compound 0118 is an angiostatic agent that inhibits tumor growth in mice. Although 0118 is a topomimetic of galectin-1-targeting angiostatic amphipathic peptide anginex, we had yet to prove that 0118 targets galectin-1. Galectin-1 is involved in pathological disorders like tumor endothelial cell adhesion and migration and therefore presents a relevant target for therapeutic intervention against cancer. Here, 15N-1H HSQC NMR spectroscopy demonstrates that 0118 indeed targets galectin-1 at a site away from the lectin’s carbohydrate binding site, and thereby attenuates lactose binding to the lectin. Flow cytometry and agglutination assays show that 0118 attenuates binding of galectin-1 to cell surface glycans, and the inhibition of cell proliferation by 0118 is found to be correlated with the cellular expression of the lectin. In general, our data indicate that 0118 targets galectin-1 as an allosteric inhibitor of glycan/carbohydrate binding. This work contributes to the clinical development of anti-tumor calixarene compound 0118.
NMR; protein; lectin; glycan; galactose
Release of lipopolysaccharide (LPS) endotoxin from Gram negative bacterial membranes triggers macrophages to produce large quantities of cytokines that can lead to septic shock and eventual death. Agents that bind to and neutralize LPS may provide a means to clinically prevent septic shock upon bacterial infection. Previously, we reported the design of antibacterial helix peptide SC4 and β-sheet-forming βpep peptides that neutralize LPS in vitro. We hypothesized that the ability of these and other such peptides to neutralize LPS rested in the common denominator of positively charged amphipathic structure. Here, we describe the design and synthesis of non-peptide, calixarene-based helix/sheet topomimetics that mimic the folded conformations of these peptides in their molecular dimensions, amphipathic surface topology, and compositional properties. From a small library of topomimetics, we identified several compounds that neutralize LPS in the 10−8 M range, making them as effective as bactericidal/permeability increasing (BPI) protein and polymyxin B. In an endotoxemia mouse model, three of the most in vitro effective topomimetics are shown to be at least partially protective against challenges of LPS from different bacterial species. NMR studies provide mechanistic insight by suggesting the site of molecular interaction between topomimetics and the lipid A component of LPS, with binding being mediated by electrostatic and hydrophobic interactions. This research contributes to the development of pharmaceutical agents against endotoxemia and septic shock.
Previously, we reported on the anti-tumor activities of two designed
calixarene-based topomimetics (PTX008 and PTX009) of the amphipathic,
angiostatic peptide Anginex. Here, we chemically modified the hydrophobic and
hydrophilic faces of PTX008 and PTX009, and discovered new calixarene compounds
that are more potent, cytotoxic anti-tumor agents. One of them, PTX013, is
particularly effective at inhibiting the growth of several human cancer cell
lines, as well as drug resistant cancer cells. Mechanistically, PTX013 induces
cell cycle arrest in sub-G1 and G0/G1 phases of e.g. SQ20B cells, a
radio-resistant human head and neck carcinoma model. In the syngeneic B16F10
melanoma tumor mouse model, PTX013 (0.5 mg/Kg) inhibits tumor growth by about
50-fold better than parent PTX008. A preliminary pharmacodynamics study strongly
suggests that PTX013 exhibits good in vivo exposure and a relatively long
half-life. Overall, this research contributes to the discovery of novel
therapeutics as potentially useful agents against cancer in the clinic.
Calixarenes; Galectin-1; Structure-activity relationships; Therapeutics
A concise total synthesis of the plant alkaloid (±)-leuconolam (1) has been achieved. A regio- and diastereoselective Lewis-acid mediated allylative cyclization was used to establish, simultaneously, two adjacent tetrasubstituted carbon centers. Furthermore, an essential arene cross-coupling to a hindered haloalkene was enabled by the use of a novel 2-anilinostannane.
Flash nanoprecipitation (FNP) is a process that, through rapid mixing, stabilizes an insoluble low molecular weight compound in a nano-sized, polymer-stabilized delivery vehicle. The polymeric components are typically amphiphilic diblock copolymers (BCPs). In order to fully exploit the potential of FNP, factors affecting particle structure, size, and stability must be understood. Here we show that polymer type, hydrophobicity and crystallinity of the small molecule, and small molecule loading levels all affect particle size and stability. Of the four block copolymers (BCP) that we have studied here, poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (PEG-b-PLGA) was most suitable for potential drug delivery applications due to its ability to give rise to stable nanoparticles, its biocompatibility, and its degradability. We found little difference in particle size when using PLGA block sizes over the range of 5 to 15kDa. The choice of hydrophobic small molecule was important, as molecules with a calculated water-octanol partition coefficient (clogP) below 6 gave rise to particles that were unstable and underwent rapid Ostwald ripening. Studies probing the internal structure of nanoparticles were also performed. Analysis of differential scanning calorimetry (DSC), cryogenic transmission electron microscopy (cryo-TEM), and 1H-NMR experiments support a three-layer core-shell-corona nanoparticle structure.
flash nanoprecipitation; insoluble drugs; drug delivery; paclitaxel; core-shell particles
The hexadehydro-Diels–Alder (HDDA) cascade enables the synthesis of complex benzenoid products with various substitution patterns via aryne intermediates. The first stage of this cascade involves generation of a highly reactive ortho-benzyne intermediate by a net [4+2] cycloisomerization of a triyne substrate. The benzyne can be rapidly ‘trapped’ either intra- or intermolecularly with a myriad of nucleophilic or π-bond-donating reactants. As a representative example of a general procedure to synthesize highly substituted benzenoids, this protocol describes the synthesis of a typical triyne substrate and its use as the reactant in an HDDA cascade to form a phthalide. The synthetic procedure detailed herein (four chemical reactions) takes 16–20 h of active effort over a several day period for preparation of the triyne precursor and ~2 h of active effort over a 3-day period for generation and trapping of the benzyne and isolation of the phthalide product.
HDDA; benzyne; Cadiot-Chodkiewicz; cascade; organic synthesis
The results of several experiments designed to probe the energetic viability of a reaction path for generation of penostatins I (3) and F (4) via spontaneous [3,3]-sigmatropic rearrangement are reported. In particular, the enolate derived from the 2-vinyl-6-acyldihydropyran 8-cis gave cyclooctadienone 12 via facile anionic oxy-Claisen rearrangement, demonstrating the feasibility of such an event.
We have developed shelf- and air-stable ortho-stannylated aniline reagents that can directly be coupled with alkenyl and aryl halides via Stille cross-coupling. We report i) the efficient preparation of o-(tributylstannyl)aniline (2a) and o-(trimethylstannyl)aniline (2b), ii) the comparison of the reactivities of 2a and 2b with those of related organostannanes in cross-coupling reaction with an alkenyl halide, and iii) the cross-coupling of 2a and 2b with a series of arylhalides and triflate.
Cross-coupling; Ortho-metalation; Aniline; Organostannanes
Drug nanocarrier clearance by the immune system must be minimized to achieve targeted delivery to pathological tissues. There is considerable interest in finding in vitro tests that can predict in vivo clearance outcomes. In this work, we produce nanocarriers with dense PEG layers resulting from block copolymer-directed assembly during rapid precipitation. Nanocarriers are formed using block copolymers with hydrophobic blocks of polystyrene (PS), poly-ε-caprolactone (PCL), poly-D,L-lactide (PLA), or poly-lactide-co-glycolide (PLGA), and hydrophilic blocks of polyethylene glycol (PEG) with molecular weights from 1.5 kg/mol to 9 kg/mol. Nanocarriers with paclitaxel prodrugs are evaluated in vivo in Foxn1nu mice to determine relative rates of clearance. The amount of nanocarrier in circulation after 4 h varies from 10% to 85% of initial dose, depending on the block copolymer. In vitro complement activation assays are conducted in an effort to correlate the protection of the nanocarrier surface from complement binding and activation and in vivo circulation. Guidelines for optimizing block copolymer structure to maximize circulation of nanocarriers formed by rapid precipitation and directed assembly are proposed, relating to the relative size of the hydrophilic and hydrophobic block, the hydrophobicity of the anchoring block, the absolute size of the PEG block, and polymer crystallinity. The in vitro results distinguish between the poorly circulating PEG5k-PCL9k and the better circulating nanocarriers, but could not rank the better circulating nanocarriers in order of circulation time. Analysis of PEG surface packing on monodisperse 200 nm latex spheres indicates that the sizes of the hydrophobic PCL, PS, and PLA blocks are correlated with the PEG blob size, and possibly the clearance from circulation. Suggestions for next step in vitro measurements are made.
nanocarrier; polyethylene glycol; complement activation; in vivo circulation; nanoparticle; block copolymer; polycaprolactone; polylactic acid; stealth
A convergent total synthesis of peloruside A (1) is described. The key strategic features are a diastereoselective lactonization to generate a C5-C9 valerolactone from the C2-symmetric ketone 4, which comprises C1–C9 of 1, and a relay ring closing metathesis (RRCM) reaction to produce a dehydrovalerolactone 20, which embodies C13–C19. A new isomer of 1, the valerolactone iso-peloruside A (iso-1), was identified.
aldol reaction; lactones; metathesis; relay ring closing metathesis (RRCM); total synthesis
Phomopsichalasin was isolated and assigned structure 1 over 15 years ago. Analysis of its proton NMR data led us to hypothesize that not all aspects of the relative configuration of this structure were correct. We have used both empirical and computational methods to propose an alternative structure. Diaporthichalasin was reported several years ago, and its structure was assigned as 7, a diastereomer of structure 1, and confirmed by a single crystal X-ray study. We have shown that diaporthichalasin and phomopsichalasin are identical; i.e., both have structure 7. Additional aspects of NMR interpretation that provide guidance for avoiding some of the pitfalls that can lead to incorrect structure assignments are discussed. These recommendations/reminders include i) the use of complementary solvents for acquiring NMR data that break accidental chemical shift degeneracy, ii) the importance of assigning coupling constants as extensively as possible, and iii) exercising caution when interpreting correlations in 2D spectra where overlapping resonances are involved.
o-Benzynes (arynes) are among the most versatile of all reactive (short-lived) intermediates in organic chemistry. These species can be trapped to give products that are valuable from the perspective of both fine (pharmaceuticals) and commodity (agrochemicals, dyes, polymers, etc.) chemicals. Here we show a fundamentally new strategy that unites a de novo generation of benzynes, through the title hexadehydro-Diels–Alder (HDDA) reaction, with their in situ elaboration into structurally complex benzenoid products. In the HDDA reaction a 1,3-diyne is engaged in a [4+2] cycloisomerization with a third (pendant) alkyne–the diynophile–to produce the highly reactive benzyne intermediate. The metal- and reagent-free reaction conditions for this simple, thermal transformation are notable. The subsequent and highly efficient trapping reactions increase the power of the overall process. Finally, we provide examples of how this de novo benzyne generation approach allows new modes of intrinsic reactivity to be revealed.
The spirotetronate okilactomycin D (7) has been efficiently synthesized by a route featuring a substrate-controlled, diastereoselective (8:1) intramolecular Diels-Alder (IMDA) reaction of 11. The assigned absolute configuration of (−)-7 was confirmed.
Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable copolymer that is also acceptable for use in a variety of biomedical applications. Typically, a random PLGA polymer is synthesized in a bulk batch polymerization using a tin-based catalyst at high temperatures. This methodology results in relatively broad polydispersity indexes (PDIs) due to transesterification, and the polymer product is often discolored. We report here the use of 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU), a known, effective, and convenient organocatalyst for the ring-opening polymerization of cyclic esters, to synthesize random copolymers of lactide and glycolide. The polymerization kinetics of the homo- and copolymerizations of lactide and glycolide were explored via NMR spectroscopy. A novel strategy that employs a controlled addition of the more reactive glycolide monomer to a solution containing the lactide monomer, the poly(ethylene glycol) (PEG) macroinitiator, and DBU catalyst was developed. Using this tactic (semi-batch polymerization), we synthesized a series of block copolymers that exhibited excellent correlation of the expected and observed molecular weights and possessed narrow PDIs. We also measured the thermal properties of these block copolymers and observed trends based on the composition of the block copolymer. We also explored the need for experimental rigor in several aspects of the preparations and have identified a set of convenient reaction conditions that provide polymer products that retain the aforementioned desirable characteristics. These polymerizations proceed rapidly at room temperature and without the need for tin-based catalysts to provide PEG-b-PLGAs suitable for use in biomedical investigations.
Thermal generation of acylketenes in diol-containing substrates results in dual macrocyclization/pyran-hemiketal formation. This transformation expands the scope of acylketene macrolactonizations and their application to the synthesis of complex macrolides. Triol and even tetrol substrates also have been closed in highly regioselective fashion. Additionally, the challenging macrolactonization of a tertiary alcohol was achieved.
acylketenes; regioselective macrolactonization; concerted addition
Various functionalized steroidal side chains were conveniently accessed by a modified Julia olefination strategy using a common sulfone donor and an appropriate α-branched aldehyde acceptor. For the coupling of these hindered classes of reaction partners (and in contrast to typically observed trends), the benzothiazolyl(BT)-sulfone anion gave superior outcomes compared to the phenyltetrazolyl(PT)-sulfone anion.
A variety of unnatural bile acid derivatives (9a–9f) were synthesized and used to examine the specificity with which the sea lamprey (Petromyzon marinus) olfactory system detects these compounds. These compounds are analogs of petromyzonol sulfate (PS, 1), a component of the sea lamprey migratory pheromone. Both the stereochemical configuration at C5 (i.e., 5α vs. 5β) and the extent and sites of oxygenation (hydroxylation or ketonization) of the bile acid derived steroid skeleton were evaluated by screening the compounds for olfactory activity using electro-olfactogram recording. 5β-Petromyzonol sulfate (9a) elicited a considerable olfactory response at sub-nanomolar concentration. In addition, less oxygenated systems (i.e., 9b–9e) elicited olfactory responses, albeit with less potency. The sea lamprey sex pheromone mimic 9f (5β-3-ketopetromyzonol sulfate) was also examined and found to produce a much lower olfactory response. Mixture studies conducted with 9a and PS (1) suggest that stimulation is occurring via similar modes of activation, demonstrating a relative lack of specificity for recognition of the allo-configuration (i.e., 5α) in sea lamprey olfaction. This attribute could facilitate design of pheromone analogs to control this invasive species.
steroid synthesis; olfactory SAR; pheromone; bile acid sulfates