A common mechanism for the biosynthesis of methoxy and cyclopropyl mycolic acids in Mycobacterium tuberculosis

Mycobacterium tuberculosis produces three classes of mycolic acids that differ primarily in the presence and nature of oxygen-containing substituents in the distal portion of the meromycolate branch. The methoxymycolate series has a methoxy group adjacent to a methyl branch, in addition to a cyclopropane in the proximal position. Using the gene for the enzyme that introduces the distal cyclopropane (cma1) as a probe, we have cloned and sequenced a cluster of genes coding for four highly homologous methyl transferases (mma1–4). When introduced into Mycobacterium smegmatis, this gene cluster conferred the ability to synthesize methoxymycolates. By determining the structure of the mycolic acids produced following expression of each of these genes individually and in combination, we have elucidated the biosynthetic steps responsible for the production of the major series of methoxymycolates. The mma4 gene product (MMAS-4) catalyzes an unusual S-adenosyl-l-methionine-dependent transformation of the distal cis-olefin into a secondary alcohol with an adjacent methyl branch. MMAS-3 O-methylates this secondary alcohol to form the corresponding methyl ether, and MMAS-2 introduces a cis-cyclopropane in the proximal position of the methoxy series. The similarity of these reactions and the enzymes that catalyze them suggests that some of the structural diversity of mycolic acids results from different chemical fates of a common cationic intermediate, which in turn results from methyl group addition to an olefinic mycolate precursor.

1H-1,3-Diazepines, 5H-1,3-diazepines, 1,3-diazepinones, and 2,4-diazabicyclo[3.2.0]heptenes

Tetrazolo[1,5-a] pyridines/ 2-azidopyridines 1 undergo photochemical nitrogen elimination and ring expansion to 1,3-diazacyclohepta-1,2,4,6-tetraenes 3, which react with alcohols to afford 2-alkoxy-1H-1,3-diazepines 4 (5), with secondary amines to 2-dialkylamino-5H-1,3-diazepines 16, sometimes via isolable 2-dialkylamino-1H-1,3-diazepines 15, and with water to 1,3-diazepin-2-ones 19. The latter are also obtained by elimination of isobutene or propene from 2-tert-butoxy- or 2-isopropoxy-1H-1,3-diazepines 4 or 5. 1,3-Diazepin-2-one 22B and 1,3-diazepin-4-one 24 were obtained from hydrolysis of the corresponding 4-chlorodiazepines. Diazepinones 19 undergo photochemical ring closure to diazabicycloheptenones 25 in high yields. The 2-alkoxy-1H-1,3-diazepines 4 and 5 interconvert by rapid proton exchange between positions N1 and N3. The free energies of activation for the proton exchange were measured by the Forsen - Hoffman method as DeltaGdouble dagger(298) = 16.2 +/- 0.6 kcal mol(-1) as an average for 4a - c in CD2Cl2, acetone-d(6), and methanol-d(4), and 14.1 +/- 0.6 kcal mol(-1) for 4c in acetone/D2O. The structures of 2-methoxy-5,6-bis( trifluoromethyl)-1H-1,3-diazepine 4k, 1,2-dihydro-4-diethylamino-5H-1,3-diazepin-2-one 22bB, and diazabicycloheptanone 26 were determined by X-ray crystallography. The former represents the first reported X-ray crystal structure of any monocyclic N-unsubstituted 1H-azepine.

Simulation of glassy state relaxations in polymers: A static analysis of methyl group and methoxy group rotation in poly(vinyl methyl ether)

We show that the simple quasi-static technique, also called the adiabatic mapping technique, can be used to determine the energetics of rotation of methyl and methoxy groups in amorphous poly(vinyl methyl ether) even though the latter process is too slow to be amenable to direct molecular dynamics simulation. For the methyl group rotation, we find that the mean and standard deviation of the simulated rotational barrier heights agree well with experimental data from quasi-elastic neutron scattering. In the case of the methoxy groups we find that just 4% of the groups contribute more than 90% of the observed dielectric relaxation strength. The groups which make the most contribution are those which, by virtue of their particular conformation and local environment, have two alternative positions of similar energy.

Development of calix[4]arene-functionalized microcantilever array sensing system for the rapid, sensitive and simultaneous detection of metal ions in aqueous solutions

The work described in this thesis was conducted with the aim of: 1) investigating the binding capabilities of calix[4]arene-functionalized microcantilevers towards specific metal ions and 2) developing a new16-microcantilever array sensing system for the rapid, and simultaneous detection of metal ions in fresh water. Part I of this thesis reports on the use of three new bimodal calix[4]arenes (methoxy, ethoxy and crown) as potential host/guest sensing layers for detecting selected ions in dilute aqueous solutions using single microcantilever experimental system. In this work it was shown that modifying the upper rim of the calix[4]arenes with a thioacetate end group allow calix[4]arenes to self-assemble on Au(111) forming complete highly ordered monolayers. It was also found that incubating the microcantilevers coated with 5 nm of Inconel and 40 nm of Au for 1 h in a 1.0 M solution of calix[4]arene produced the highest sensitivity. Methoxy-functionalized microcantilevers showed a definite preference for Ca²⁺ ions over other cationic guests and were able to detect trace concentration as low as 10⁻¹² M in aqueous solutions. Microcantilevers modified with ethoxy calix[4]arene displayed their highest sensitivity towards Sr²⁺ and to a lesser extent Ca²⁺ ions. Crown calix[4]arene-modified microcantilevers were however found to bind selectively towards Cs⁺ ions. In addition, the counter anion was also found to contribute to the deflection. For example methoxy calix[4]arene-modified microcantilever was found to be more sensitive to CaCl₂ over other water-soluble calcium salts such as Ca(NO₃)₂ , CaBr₂ and CaI₂. These findings suggest that the response of calix[4]arene-modified microcantilevers should be attributed to the target ionic species as a whole instead of only considering the specific cation and/or anion. Part II presents the development of a 16-microcantilever sensor setup. The implementation of this system involved the creation of data analysis software that incorporates data from the motorized actuator and a two-axis photosensitive detector to obtain the deflection signal originating from each individual microcantilever in the array. The system was shown to be capable of simultaneous measurements of multiple microcantilevers with different coatings. A functionalization unit was also developed that allows four microcantilevers in the array to be coated with an individual sensing layer one at the time. Because of the variability of the spring constants of different cantilevers within the array, results presented were quoted in units of surface stress unit in order to compare values between the microcantilevers in the array.

A new low temperature synthesis route of fraipontite (Zn,Al)3(Si,Al)2O5(OH)4

A low temperature synthesis method based on the decomposition of urea at 90°C in water has been developed to synthesise fraipontite. This material is characterised by a basal reflection 001 at 7.44 Å. The trioctahedral nature of the fraipontite is shown by the presence of a 06l band around 1.54 Å, while a minor band around 1.51 Å indicates some cation ordering between Zn and Al resulting in Al-rich areas with a more dioctahedral nature. TEM and IR indicate that no separate kaolinite phase is present. An increase in the Al content however, did result in the formation of some SiO2 in the form of quartz. Minor impurities of carbonate salts were observed during the synthesis caused by to the formation of CO32- during the decomposition of urea.

Raman spectroscopic characteristics of phthalocyanine and naphthalocyanine in sandwich-type phthalocyaninato and porphyrinato rare earth complexes Part 5. – Raman spectroscopic characteristics of naphthalocyanine in mixed [tetrakis(4-tert-butylphenyl)porphyrinato]-(naphthalocyaninato) rare earth double-deckers

Raman spectra were recorded in the range 400–1800 cm−1 for a series of 15 mixed \[tetrakis(4-tert-butylphenyl)porphyrinato](2,3-naphthalocyaninato) rare earth double-deckers M(TBPP)(Nc) (M = Y; La–Lu except Pm) using laser excitation at 632.8 and 785 nm. Comparisons with bis(naphthalocyaninato) rare earth counterparts reveal that the vibrations of the metallonaphthalocyanine M(Nc) fragment dominate the Raman features of M(TBPP)(Nc). When excited with radiation of 632.8 nm, the most intense vibration appears at about 1595 cm−1, due to the naphthalene stretching. These complexes exhibit the marker Raman band for Nc•− as a medium-intense band in the range 1496–1507 cm−1, attributed to the coupling of pyrrole and aza stretching, while the marker Raman band of Nc2− in intermediate-valence Ce(TBPP)(Nc) appears as a strong band at 1493 cm−1 and is due to the isoindole stretchings. By contrast, when excited with radiation of 785 nm that is in close resonance with the main Q absorption band of the naphthalocyanine ligand, the ring radial vibrations at ca 680 and 735 cm−1 for MIII(TBPP)(Nc) are selectively intensified and are the most intense bands. For the cerium double-decker, the most intense vibration also acting as the marker Raman band of Nc2− appears at 1497 cm−1 with contributions from both pyrrole CC and aza CN stretches. The same vibrational modes show weak to medium intensity scattering at 1506–1509 cm−1 for MIII(TBPP)(Nc) and this is the marker Raman band of Nc•− when thus excited. The scatterings due to the Nc breathings, ring radial vibration, aza group stretchings, naphthalene stretchings, benzoisoindole stretchings and the coupling of pyrrole CC and aza CN stretchings in MIII(TBPP)(Nc) are all slightly blue shifted along with the decrease in rare earth ionic radius, confirming the effects of increased ring–ring interactions on the Raman characteristics of naphthalocyanine in the mixed ring double-deckers.