Purification and properties of a β-galactosidase with potential application as a digestive supplement

Functional-based screening of crude β-galactosidase activities from 42 yeast strains resulted in the selection of a single enzyme of potential interest as a digestive supplement. β-Galactosidase produced by Kluyveromyces marxianus DSM5418 was purified to homogeneity by a combination of gel filtration, ion-exchange, and hydroxylapatite chromatographies. The denatured (123 kDa) and native molecular masses (251 kDa) suggest that the enzyme is a homodimer. The optimum pH and temperature of the purified enzyme were 6.8 and 37°C, respectively. The unpurified β-galactosidase in particular displayed a high level of stability when exposed to simulated intestinal conditions in vitro for 4 h. Matrix-assisted laser desorption ionization mass sectrometry analysis revealed that the enzyme's trypsin-generated peptide mass fingerprint shares several peptide fragment hits with β-galactosidases from Kluyveromyces lactis. This confirms the enzyme's identity and indicates that significant sequence homology exists between these enzymes.     

Kinetic resolution of atropisomeric amides

We have demonstrated the feasibility of the kinetic resolution of atropisomeric amides using the commercially avaliable AD-mix. To our knowledge, this methodology represents the first catalytic kinetic resolution of such compounds. Relative rates of up to 32 have been found for the kinetic resolution processes. We have also determined the barriers to rotation and half-lives of some of these amides. The half-lives range from 7 to 135 h at room temperature.     

Stereoselective synthesis of 3-substituted 2-aminocyclopentanecarboxylic acid derivatives and their incorporation into short 12-helical beta-peptides that fold in water

A stereoselective synthetic route is reported for the introduction of side chains at the 3-position of trans-2-aminocyclopentanecarboxylic acid (ACPC). Ring opening of the aziridine 2-benzyloxymethyl-6-azabicyclo[3.1.0]hexane with selected nucleophiles occurs in a regioselective manner and provides ACPC precursors with functional groups at the 3-position, trans to the 2-amino group. Oligomers composed of the 3-substituted ACPC residues maintain the 12-helical conformation displayed by the nonsubstituted analogues, as shown by their similar circular dichroism signatures. The added diversity of the new residues provides good dispersion of NMR signals, allowing the assignment of nearly all the NOE signals of a selected hexamer in aqueous solution. The NOEs between protons on nonadjacent residues are characteristic of the 12-helix. 3-Substituted ACPC residues allow one to arrange specific functional groups in a geometrically defined fashion, which should facilitate the design of beta-peptides for biological applications.     

Preparation and biological evaluation of the new chlorin photosensitizer T3,4BCPC for detection and treatment of tumors

The new water-soluble photosensitizer 5,10,15,20-tetrakis[3,4-bis(carboxymethyleneoxy)phenyl]chlorin (T3,4BCPC) has been prepared, characterized and labeled with 99mTc radionuclide. The radiotracer was evaluated for tissue distribution in Wistar rats. Accumulation of administrated activities in the liver, kidney, bladder and large intestine at 4 h post-injection indicated that the labeled ligand was largely eliminated through the renal and partly through the hepatobiliary system. In vivo biodistribution studies of the labeled compound were carried out in rodent and murine tumor models in comparison with other tumor-seeking radiopharmaceuticals such as 99mTc(V)-dimercaptosuccinic acid (DMSA), 201thallous chloride (TlCl) and 99mTc-citrate using a gamma camera computer system. In N-nitrosomethylurea (NMU)-induced rat mammary tumors, the labeled ligand showed a five-fold tumor to muscle (T/M) ratio compared to 99mTc(V)-DMSA (3-fold) and 201TlCl (3-fold). In the case of C(3)H/J virus-induced spontaneous mammary tumors, the differences were not marked. However, in the transplanted rat C(6)-glioma, the T/M ratio of the labeled compound was appreciably higher (four-fold) than that noted with 99mTc(V)-DMSA (two-fold), 201TlCl (three-fold) and 99mTc-citrate (more than three-fold). These findings suggest that the radiolabeled T3,4BCPC may have potential for the detection of cancer. In order to ascertain the efficacy of the compound for photodynamic therapy applications, a preclinical PDT study was carried out in fibrosarcoma-bearing mice after injecting 5.0 mg/kg body weight of the T3,4BCPC. A laser dose of 20 mW for 60 s resulted in 80% destruction of tumors. These data suggest that this molecule could be useful for PDT of cancer. The labeled agent could also be useful in monitoring the progression/regression of tumors before, during, and after chemotherapy, radiation therapy or PDT.     

The mixed ionocovalent bonding in small insulating clusters

We report a selfconsistent tight binding calculation of the electronic and structural properties of small insulating clusters (up to 12 atoms): these include alkaline-earth oxides (MgO and CaO) and NaCl clusters. Interatomic distance contractions are found in most cases, as in small metallic clusters. In addition, new effects take place in some of the compounds; these are a reduction of the ionic charge and, for the first time, the prediction of a dimerization of the interatomic distances. The microscopic processes responsible for their occurence and size dependence are dicussed with analytical arguments. It is shown that for these small clusters the equilibrium configuration and the electronic structure are fixed by a competition between covalent and electrostatic interactions, especially in the oxides.     

Preparation of diastereomeric 5,8-methanobenzoxazino[2,1–6]- and [2,3–6]-1,3-benzoxazin-4-ones by reaction of norbornane/ene-condensed dihydro-1,3-oxazines with salicyl chloride

Norbornane and norbornene-condensed dihydro-1,3-oxazines 1–6 were converted with salicyl chloride to 5,8-methanobenzoxazino[2,1–6]- and -[2,3-b]-1,3-benzoxazin-4-ones 7–12. The addition takes place to the C ? N bond: after acylation, the intermediate is stabilized through cyclization to the aryl-substituted carbon by hydrogen chloride elimination. Diastereomers containing the oxazine rings in isomeric positions could be isolated in two cases. This is the first example of the isolation of diastereomers in such a salicyl chloride reaction. In contrast with earlier findings with reactions of related systems, no addition to the C ? C bond could be observed. The steric structures of the compounds were elucidated by ir, ¹H- and ¹³C-nmr spectroscopy.     

Reversible storage of molecular hydrogen by sorption into multilayered TiO2 nanotubes

The sorption of hydrogen between the layers of the multilayered wall of nanotubular TiO2 was studied in the temperature range of -195 to 200 degrees C and at pressures of 0 to 6 bar. Hydrogen can intercalate between layers in the walls of TiO2 nanotubes forming host-guest compounds TiO2 x xH2, where x < or = 1.5 and decreases at higher temperatures. The rate of hydrogen incorporation increases with temperature and the characteristic time for hydrogen sorption in TiO2 nanotubes is several hours at 100 degrees C. The rate of intercalate formation is limited by the diffusion of molecular hydrogen inside the multilayered walls of the TiO2 nanotube. 1H NMR-MAS and XRD data confirm the incorporation of hydrogen between the layers in the walls of TiO2 nanotubes. The nature and possible applications of the observed intercalates are considered.     

A one-pot multicomponent coupling reaction for the stereocontrolled synthesis of (Z)-trisubstituted allylic alcohols

In this Communication, we outline a new one-pot, multicomponent coupling reaction that allows easy access to (Z)-trisubstituted allylic alcohols. Our strategy is based on E to Z isomerization of the 1-bromo-1-dialkylvinylborane upon reaction with dialkylzinc reagents, and subsequent transmetalation to give (Z)-trisubstituted vinylzinc species. In situ trapping of the reactive vinylzinc intermediates with aldehydes furnished a series of (Z)-trisubstituted allylic alcohols. This method represents a viable alternative to the Still-Gennari modification of the HWE olefination reaction, and it has the advantage that it allows coupling of larger fragments.     

Mechanistic studies of the addition of carbonyl compounds to tetramesityldigermene

The mechanism of the addition of carbonyl compounds to digermenes was investigated by examining the reaction between tetramesityldigermene and two mechanistic probes: trans-2-phenylcyclopropane carbaldehyde and trans,trans-2-methoxy-3-phenylcyclopropane carbaldehyde. In each reaction, two diastereomers of the formal [2+2] cycloadduct between the aldehyde and the digermene were obtained. These findings, together with the results of a recent theoretical study, indicate that the addition of carbonyl compounds to digermenes proceeds via a mechanism involving a zwitterionic intermediate.     

Optimization of catalyst enantioselectivity and activity using achiral and meso ligands

The optimization of asymmetric catalysts for enantioselective synthesis has conventionally revolved around the synthesis and screening of enantiopure ligands. In contrast, we have optimized an asymmetric reaction by modification of a series of achiral ligands. Thus, employing (S)-3,3'-diphenyl BINOL [(S)-Ph(2)-BINOL] and a series of achiral diimine and diamine activators in the asymmetric addition of alkyl groups to benzaldehyde, we have observed enantiomeric excesses between 96% (R) and 75% (S) of 1-phenyl-1-propanol. Some of the ligands examined have low-energy chiral conformations that can contribute to the chiral environment of the catalyst. These include achiral diimine ligands with meso backbones that adopt chiral conformations, achiral diimine ligands with backbones that become axially chiral on coordination to metal centers, achiral diamine ligands that form stereocenters on coordination to metal centers, and achiral diamine ligands with pendant groups that have axially chiral conformations. Additionally, we have structurally characterized (Ph(2)-BINOLate)Zn(diimine) and (Ph(2)-BINOLate)Zn(diamine) complexes and studied their solution behavior.