John Decatur

Investigation of CO2 capture mechanisms of liquid-like nanoparticle organic hybrid materials via structural characterization

Nanoparticle organic hybrid materials (NOHMs) have been recently developed that comprise an oligomeric or polymeric canopy tethered to surface-modified nanoparticles via ionic or covalent bonds. It has already been shown that the tunable nature of the grafted polymeric canopy allows for enhanced CO(2) capture capacity and selectivity via the enthalpic intermolecular interactions between CO(2) and the task-specific functional groups, such as amines. Interestingly, for the same amount of CO(2) loading NOHMs have also exhibited significantly different swelling behavior compared to that of the corresponding polymers, indicating a potential structural effect during CO(2) capture. If the frustrated canopy species favor spontaneous ordering due to steric and/or entropic effects, the inorganic cores of NOHMs could be organized into unusual structural arrangements. Likewise, the introduction of small gaseous molecules such as CO(2) could reduce the free energy of the frustrated canopy. This entropic effect, the result of unique structural nature, could allow NOHMs to capture CO(2) more effectively. In order to isolate the entropic effect, NOHMs were synthesized without the task-specific functional groups. The relationship between their structural conformation and the underlying mechanisms for the CO(2) absorption behavior were investigated by employing NMR and ATR FT-IR spectroscopies. The results provide fundamental information needed for evaluating and developing novel liquid-like CO(2) capture materials and give useful insights for designing and synthesizing NOHMs for more effective CO(2) capture.

Alcohol-Promoted Ring-Opening Alkyne Metathesis Polymerization†

Alcohol is the answer! An inactive, air-stable, dimeric molybdenum alkylidyne complex is activated toward ring-opening alkyne metathesis polymerization (ROAMP) by the addition of methanol. The ROAMP is compatible with water and phenol-containing substrates and with the in situ photochemical generation of alkyne monomers from cyclopropenones.

Characterization of a new glycosynthase cloned by using chemical complementation.

Despite their fundamental role in biological processes and potential use as therapeutics, it still remains difficult to synthesize carbohydrates. In the past two decades, there has been tremendous progress in the chemical synthesis of complex carbohydrates. However, chemical synthesis is still limited by the need for differentially protected intermediates and reactant-dependent coupling yields and stereocontrol. Enzymes, with their control of both regioand stereochemistry, provide an obvious alternative to traditional small-molecule chemistry for the synthesis of oligosaccharides. 9] Recently, Withers and co-workers demonstrated that retaining glycosidases can be engineered to glycosynthases simply by mutating the nucleophilic Glu residue at the base of the active site to a small hydrophobic residue and using an a-glycosyl fluoride as the donor substrate. This strategy is based on extensive characterization of the mechanism of retaining glycosidases. Mutation of the active-site nucleophile to a small residue both accommodates the glycosyl fluoride donor and inactivates the hydrolytic activity of the enzyme, allowing the reaction to proceed in the reverse direction. This approach was first demonstrated with the Agrobacterium sp. b-glucosidase/galactosidase (Abg). The active-site nucleophile Glu358 was mutated to Ala. This Abg:E358A variant was shown to accept both galactosyl fluoride and glucosyl fluoride as donors to form glycosidic bonds with several monoand disaccharides. This result opened a new route for carbohydrate synthesis, and already several retaining glycosidases have been successfully converted to glycosynthases with this strategy. Directed evolution would offer an obvious route to improve the activity and alter the substrate selectivity of these enzymes, except that there is no intrinsic way to screen or select for glycosynthase activity. Mayer et al. developed a coupled enzyme assay using an endo-cellullase that can be used to screen for glycosynthase mutants with improved activity. This screen, however, can only be used for glycosynthases that synthesize products that are substrates of the endo-cellulase. Screens only allow relatively small libraries, about 10 variants, to be assayed. Thus, our laboratory applied “chemical complementation”, a general, high-throughput assay for enzyme catalysis of bond formation and cleavage reactions, to the directed evolution of glycosynthases. In chemical complementation, glycosynthase activity is linked to reporter gene transcription and hence cell survival through covalent coupling of a methoACHTUNGTRENNUNGtrexACHTUNGTRENNUNGate ACHTUNGTRENNUNG(Mtx)-disaccharide-fluoride donor and a dexa ACHTUNGTRENNUNGmethaACHTUNGTRENNUNGsoneACHTUNGTRENNUNG(Dex)-disaccharide acceptor, such that Dex-tetrasaccharide-Mtx effectively reconstitutes the transcriptional activator and increases transcription of a downstream reporter gene. Use of the reporter gene LEU2 allows for a growth selection in the ACHTUNGTRENNUNGabsence of leucine (Figure 1). Using the LEU2 selection, we previously demonstrated that chemical complementation can be used to read-out glycosynthase activity, and a Humicola insolens Cel7B:E197S variant with a fivefold increase in glycosynthase activity was selected from a Glu197 saturation library. Having established chemical complementation as a selection for glycosynthase activity, we then sought a glycosynthase that would provide a robust scaffold for the directed evolution of glycosynthase variants with altered substrate specificities. In our initial publication, the H. insolens CeL7B:E197A glycosynthase was employed because it was the only reported endoglycosynthase at that time. However, this enzyme has poor expression properties and does not present an obvious scaffold for protein engineering. Thus, we sought to develop an endo-glycosynthase derived from a family 5 glycosidase. The in vitro activities and substrate specificities of many family 5 glycosidases have been extensively characterized, and several of these enzymes have shown good expression in E. coli. Moreover, family 5 retaining glycosidases are monomeric triose-phosphate isomerase (TIM) barrel enzymes, an appealing scaffold for enzyme engineering given that TIM barrels arguably are a “privileged” scaffold for enzyme catalysis of diverse chemical transformations. In this paper, we report the cloning and characterization of a new glycosynthase from a family 5 glycosidase using a chemical complementation LEU2 enrichment assay. Given that not all retaining glycosidases provide efficient glycosynthases upon mutation of the active-site nucleophile, we adapted our LEU2 selection as an enrichment assay to clone the new glycosynthase. Using this assay, the family 5 TIM-barrel glycosynthase was cloned by screening the activesite E:G and E:S variants of known family 5 glycosidases. Specifically, three family 5 glycosidases, Clostridium cellulolyticum Cel5A, Clostridium thermocellum CelG and Clostridium cellulolyticum Cel5N, were tested. Among these genes, Cel5A and CelG have been overexpressed and purified from E. coli, while the catalytic domains of CelG and Cel5N share high sequence identity. The high-resolution structure of the Cel5A catalytic domain shows a classic TIM barrel fold, with Glu170 as the [a] Dr. H. Tao, P. Peralta-Yahya, Dr. J. Decatur, Prof. V. W. Cornish Department of Chemistry, Columbia University New York, NY 10027 (USA) Fax: (+1)212-932-1289 E-mail : vc114@columbia.edu [b] Dr. H. Tao Current address: Genomics Institute of the Novartis Research Foundation San Diego, CA 92121 (USA) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author: Experimental procedures and NMR data.

Identification and quantitation of 3,4-methylenedioxy-N-methylamphetamine (MDMA, ecstasy) in human urine by 1H NMR spectroscopy. Application to five cases of intoxication

Identification of 3,4-methylenedioxy-N-methylamphetamine (MDMA, ecstasy) in five cases of intoxication using nuclear magnetic resonance (NMR) spectroscopy of human urine is reported. A new water suppression technique PURGE (Presaturation Utilizing Relaxation Gradients and Echoes) was used. A calibration curve was obtained using spiked samples. The method gave a linear response (correlation coefficient of 0.992) over the range 0.01-1mg/mL. Subsequently, quantitation of the amount of MDMA present in the samples was performed. The benefit and reliability of NMR investigations of human urine for cases of intoxication with MDMA are discussed.

Helical (5Z, 11E)-Dibenzo[a,e]cyclooctatetrene: A Spring-Loaded Monomer†

The introduction of a kink into an annulene cycle through having one of its double bonds in the trans configuration has severe consequences on the structure and stability of the resulting annulene. Modeling indicates that the p systems of either the [6]-annulene 1 or [8]-annulene 2 should wind into a helical conformation. The computed strain energy for 1 is circa 107 kcalmol , and for 2 is approximately 21 kcal mol . Molecule 1 is probably too unstable at room temperature to allow it to be isolated and studied. Herein we present a method to create the [8]-annulene 3, a benzannulated version of 2. Single crystal X-ray diffraction analysis of 3 reveals that it is a helix. The strain of this ring structure makes it functional as a monomer for ring opening metathesis polymerization (ROMP), yielding a new type of living, regioregular oligophenylene vinylene. Scheme 1 shows the method used to prepare 3, in which ophthaldehyde reacts with the 1,2-bis(triphenylphosphonium ylide) at room temperature. The isolated yield of 3 on a 20 mmol scale is about 20%. Annulene 3 is stable for weeks. The strain energy we calculate using density functional theory (DFT) for 3 relative to the cis,cis-dibenzo[a,e]cyclooctatetraene is about 18 kcalmol . The stabilization owing to benzannulation, along with the steric protection provided by the aromatic rings, allows us to isolate 3. Previous studies on the synthesis of dibenzo[a,e]cyclooctatetraene have not reported the presence of 3, and focus exclusively on the cis,cis isomer. Using H NMR spectroscopy, we observe only a trace amount of cis,cisdibenzo[a,e]cyclooctatetraene. The only compounds we could isolate from the reaction mixture were annulene 3, a small amount of isomerized cis,cis-dibenzo[a,e]cyclooctatetraene, and a substance which has been difficult to characterize and that we tentatively assign as the dimer 4 (Scheme 1). We suspect that earlier published work also likely produced 3, but in those experiments 3 had isomerized during workup and/or purification. Emphasizing both the kinetic stability and the thermodynamic instability of 3, we found that either UV irradiation, iodine, or traces of acid readily (and quantitatively) convert 3 into the more stable cis,cis-dibenzo[a,e]cyclooctatetraene. Wittig and co-workers synthesized dibenzo[a,e]cyclooctatetraene by a synthetic route that involved multiple Hofmann eliminations, and found that the material contained anomalous IR stretches. These resonances would be reasonable for a strained, trans double bond, such as the one found in trans-cyclooctene, but the structure was assigned to the more stable cis,cis-dibenzo[a,e]cyclooctatetraene. Subsequent studies, which prepared dibenzo[a,e]cyclooctatetraene by different methods, did not observe these anomalous IR stretching vibrations. Further comScheme 1. Double Wittig reaction to produce 3 and 4. Reaction conditions: a) LiOEt in EtOH, DMF, slow addition over 6 h.

Stereoselective synthesis of 2,3,7-trimethylcyclooctanone and related compounds and determination of their relative and absolute configurations by the M alpha NP acid method

The copper/chiral phosphoramidite (L(1))-catalyzed conjugate addition of dimethylzinc to cycloocta-2,7-dienone 4, followed by the methylation of the intermediate enolate, yielded a single isomer of 7,8-dimethylcyclooct-2-enone (+)-5. Compound (+)-5 was subjected to the second conjugate addition with ent-L(1) giving only one stereoisomer of 2,3,7-trimethylcyclooctanone (+)-6, which was converted to 2,3,7-trimethylcyclooctanol 7. To determine the relative and absolute configurations of these compounds, the (1)H NMR anisotropy method using (S)-(+)-2-methoxy-2-(1-naphthyl)propionic acid {(S)-(+)-MalphaNP acid} 1 was applied. Racemic alcohol (+/-)-7 was esterified with (S)-(+)-MalphaNP acid 1 yielding diastereomeric esters, which were efficiently separated by HPLC on silica gel affording the first-eluted MalphaNP ester (-)-10a and the second-eluted one (-)-10b. The relative and absolute configurations of ester (-)-10a were determined to be (S;1R,2S,3R,7S) by analyzing the (1)H and (13)C NMR spectra of (-)-10a and (-)-10b, especially their HSQC-TOCSY and NOESY spectra, and by applying the MalphaNP anisotropy method. The alcohol 7 formed from (+)-6 was similarly esterified with (S)-(+)-MalphaNP acid 1 yielding an MalphaNP ester, which was identical with (-)-10a, and the relative and absolute configurations of 2,3,7-trimethylcyclooctanone (+)-6 were determined to be (2S,3R,7S).