Optical resolution of β-lactams by chiral HPLC on tris(phenylcarbamate)s of cellulose and amylose

Separation and optical resolution of 6 diastereomers and 25 racemates of β-lactams were examined by HPLC on chiral stationary phases composed of six cellulose and one amylose tris(phenylcarbamate) derivatives. Most β-lactams were optically resolved at least by one of the derivatives. The absolute configuration of β-lactams was estimated by CD spectroscopy.     

Enantioseparation of 2-aryl-1,3-dicarbonyl analogues by high performance liquid chromatography using polysaccharide type chiral stationary phase

The HPLC chiral separation of 21 kinds of 2-aryl-1,3-dicarbonyl analogues was investigated in normal phase mode with amylose tris(3,5-dimethylphenylcarbamate), amylose tris((S)-1-phenylethylcarbamate), cellulose tris(3,5-dimethylphenylcarbamate), and cellulose tris(4-methylbenzoate) chiral stationary phases, respectively. The whole set of 2-aryl-1,3-dicarbonyl analogues shows better enantioselectivity and enantioseparation on amylose tris(3,5-dimethylphenyl carbamate) (Chiralpak AD-H). The temperature dependence of enantioselectivity was studied to improve the enantioseparation. In addition, efforts are made to relate analyte structure with the quality of the achieved chiral separation.     

Enantiomeric resolution by HPLC of axial chiral amides using amylose tris[(S)-1-phenylethylcarbamate]

The enantiomeric resolution of a series of N-arylamides was examined on amylose tris[(S)-1-phenylethylcarbamate] coated onto aminopropylated 7 μm silica with 500 Å diameter pores and on naked silica 5 μm particle size with 500 Å diameter pores. The enantiomeric resolution obtained for this series was excellent on both columns. The enantioselectivity of cellulose and amylose tris (3,5-dimethylphenylcarbamate) coated onto APS-Hypersil (120 Å pore size, 5 μm particle size) was also investigated for this series of compounds. Chirality 9:109–112, 1997. © 1997 Wiley-Liss, Inc.     

Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signal-to-background ratio and improved baseline resolution

In proteomics the ability to visualize proteins from electropherograms is essential. Here a new protocol for staining and destaining gels treated with Ruthenium II tris (bathophenantroline disulfonate) is presented. The method is compared with the silver-staining procedure of Swain and Ross, the Ruthenium II tris (bathophenantroline disulfonate) stain described by Rabilloud (Rabilloud T., Strub, S. M. Luche, S., Girardet, S. L. et al., Proteomics 2001, 1, 699-704) and the SYPRO Ruby gel stain. The method offers a better signal-to-background ratio with improved baseline resolution for both sodium dodecyl sulfate-polyacrylamide gels and two-dimensional gels.     

Thiono compounds. 4. In vitro mutagenic and antineoplastic activity of TEPA and thio-TEPA

Tris (1-aziridinyl) phosphine oxide (TEPA) and tris (1-aziridinyl) phosphine sulfide (thio-TEPA) induced base pair mutations in the Ames mutagenic assay. Thio-TEPA required metabolic activation while TEPA was active without metabolic activation. Growth of a human vaginal carcinoma (A431), a human breast carcinoma (MDAMB-231), and a human cervical carcinoma (HeLa) were inhibited in soft agar in vitro at concentrations which induced mutagenesis in the Ames Assay. A fourth line, JEG choriocarcinoma, was sensitive to the antigrowth properties of both drugs at concentrations below that which induced mutagenesis. These data suggest that as more antineoplastic agents become available, and as mean survival times increase, knowledge of the relative in vitro sensitivity of a patient's neoplasm to a specific antineoplastic drug (i.e., dose required for growth inhibition) as a function of its mutagenic index might be useful for prediction of clinical remission, as well as the risk of secondary neoplasm induction.Abbreviations TEPA tris (1-aziridinyl) phosphine oxide - thio-TEPA tris (1-aziridinyl) phosphine sulfide - MEM Minimal Essential Media This study was supported by PHS grant No. CA 30321 awarded by the National Cancer Institute (DHHS).     

Release of salbutamol sulphate and ketoprofen enantiomers from matrices containing HPMC and cellulose derivatives

We studied the release of salbutamol and ketoprofen enantiomers from HPMC K100M matrices containing two types of cellulose derivatives: cellulose tris (3,5-dimethylphenylcarbamate) and cellulose tris (2,3-dichlorophenylcarbamate), chiral excipients used as stationary phases for liquid chromatography. These matrices provided an extended release of both drugs. Ketoprofen release from formulations elaborated with cellulose tris (2,3-dichlorophenylcarbamate) was by anomalous transport, because the value of n (release exponent of the diffusion equation) ranged between 0.60-0.68, whereas for all other formulations the value of exponent n ranged from 0.50-0.54. The drug thus diffuses through the matrix and is released following a quasi-Fickian diffusion mechanism (stereoselective process). The matrices preferentially retained R-salbutamol and S-ketoprofen and cellulose tris (3,5-dimethylphenylcarbamate) showed more capacity of chiral discrimination for both drugs than cellulose tris (2,3-dichlorophenylcarbamate). Moreover, we observed that stereoselectivity is dependent on the amount of chiral excipient in the formulation. Diffusion tests confirmed the chiral interaction between drugs and cellulose derivatives observed in the dissolution assays except for matrices elaborated with ketoprofen and cellulose tris (2,3-dichlorophenylcarbamate), where the low stereoselectivity observed with the matrices is due to the presence of HPMC K100M. We conclude that the inclusion of these cellulose derivatives in HPMC matrices does not result in a relevant stereoselectivity with respect to the two drugs studied.     

Stoichiometry-focused (18)F-labeling of alkyne-substituted oligodeoxynucleotides using azido([(18)F]fluoromethyl)benzenes by Cu-catalyzed Huisgen reaction

A novel method for ¹⁸F-radiolabeling of oligodeoxynucleotides (ODNs) by a Cu-catalyzed Huisgen reaction has been developed by using the lowest possible amount of the precursor biomolecule for the realization of stoichiometry-oriented PET (positron emission tomography) chemistry. Under the optimized cyclization conditions of p- or m-azido([¹⁸F]fluoromethyl)benzene and alkyne-substituted ODN (20 nmol) at 40 °C for 15 min in the presence of CuSO₄, TBTA [tris((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)amine], and sodium ascorbate (2:1:2), the synthesis of ¹⁸F-labeled ODNs with sufficiently high radioactivities of 2.1-2.5 GBq and specific radioactivities of 1800-2400 GBq/??mol have been accomplished for use in animal and human PET studies.     

Phosphorogenic and spontaneous formation of tris(bipyridine)ruthenium in peptide scaffolds

Redox‐active ruthenium complexes have been widely used in various fields; however, the harsh conditions required for their synthesis are not always conducive to their subsequent use in biological applications. In this study, we demonstrate the spontaneous formation of a derivative of tris(bipyridine)ruthenium at 37°C through the coordination of three bipyridyl ligands incorporated into a peptide to a ruthenium ion. Specifically, we synthesized six bipyridyl‐functionalized peptides with randomly chosen sequences. The six peptides bound to ruthenium ions and exhibited similar spectroscopic and electrochemical features to tris(bipyridine)ruthenium, indicating the formation of ruthenium complexes as we anticipated. The photo‐excited triplet state of the ruthenium complex formed in the peptides exhibited an approximately 1.6‐fold longer lifetime than that of tris(bipyridine)ruthenium. We also found that the photo‐excited state of the ruthenium complexes was able to transfer an electron to methyl viologen, indicating that the ruthenium complexes formed in the peptides had the same ability to transfer charge as tris(bipyridine)ruthenium. We believe that this strategy of producing ruthenium complexes in peptides under mild conditions will pave the way for developing new metallopeptides and metalloproteins containing functional metal‐complexes.     

A chromogenic cephalosporin for β-lactamase inhibitor screening assays

Production of β-lactamases is the primary mechanism of antibiotic resistance employed by gram-negative pathogens. Chromogenic β-lactams are important reagents for detection and assay of β-lactamases, but limited commercial availability and exorbitant pricing of these compounds are prohibitive. Here we describe a straightforward synthesis of a chromogenic cephalosporin for β-lactamase assay that gives an overall yield of 74%. On hydrolysis, its λ(max) undergoes a bathochromic shift that is easy to see and measure spectrophotometrically with a Δε(442 nm) of 14,500cm(-1)M(-1). This compound was shown to be a substrate for a variety of β-lactamases.     

Flow injection chemiluminescent determination of tetracycline using a tris(2,2'-bipyridine)ruthenium(II)-cerium(IV) sulphate system.

A flow-injection chemiluminescence method for the determination of tetracycline was developed. The method is based on an enhancement by tetracycline of the chemiluminescence light emission of tris(2,2'-bipyridine)ruthenium(II). In sulphuric acid medium, the chemiluminescence is generated by the continuous oxidation of tris(2,2'-bipyridine)ruthenium(II) by cerium (IV) sulphate. The light-emission intensity is greatly enhanced in the presence of tetracycline. Under the optimum conditions, the calibration curve is linear over the range 3.75 x 10(-8) g/mL-1.5 x 10(-5) g/mL for tetracycline with the linear equation: deltaINT = 205.898 x C - 20.442 (R2 = 0.9974). The detection limit is 3.27 x 10(-8) g/mL. The proposed method was also successfully used to determine tetracycline in pharmaceutical formulation (mean recovery of tetracycline, 100.7%).     

Ubiquitously expressed hematological and neurological expressed 1 downregulates Akt-mediated GSK3β signaling, and its knockdown results in deregulated G2/M transition in prostate cells

As the molecular mechanism of β-catenin deregulation is not well understood, and stabilized β-catenin is known to translocate into the nucleus and activate genes for proliferation, a novel regulatory factor, hematological and neurological expressed 1 (HN1), for Akt-GSK3β-β-catenin axis is reported here. In our studies, HN1 gene structure was characterized. HN1 expression was found to be epidermal growth factor-responsive in PC-3 cells, and protein expression was also upregulated in PC-3 and LNCaP but not in DU145 cells. Additionally, HN1 was found to be downregulated by the specific AKT inhibitor wortmannin but not with PI3K or MAPK inhibitors, LY294002 and PD98059, respectively, in PC-3 and MCF-7 cells. Further, siRNA-mediated knockdown of HN1 resulted in considerable increase in Akt((S473)) and GSK3β((S9),(Y216)) phosphorylations; moreover, subsequent accumulation of β-catenin, increase in c-myc expression, and nuclear accumulation of cyclin D1 were observed in PC-3 cells. Knockdown of HN1 also resulted in prolongation of G(1) phase in cell cycle, increasing tetraploidy, presumably because of cells escaping from abnormal mitosis in PC-3 cells. Consistently, overexpression of HN1 reversed the cell-cycle-specific observations, resulted in accumulation of cells in G(2)/M, and reduced the proliferation rate, which were investigated using flow cytometry and methylthiazol tetrazolium assays. As activating mutations of β-catenin have been demonstrated in late-stage tumors, and β-catenin stabilization was correlated with poor prognosis in previous reports, epidermal growth factor-upregulated HN1 expression might have a role in deregulating the AKT-GSK3β((S9))-mediated signaling as a novel compensating mechanism.     

Diazabicyclooctanes (DBOs): a potent new class of non-β-lactam β-lactamase inhibitors

The β-lactams have been among the most successful classes of antibacterial agents for the past half century. However, a disturbing increase in resistance to β-lactams has been noted among Gram-negative bacteria, which is attributable to β-lactamase enzymes not within the spectrum of currently marketed β-lactams or β-lactam/β-lactamase inhibitor combinations. Diazabicyclooctanes (DBOs) were first investigated as β-lactam mimics in the mid-1990s by chemists at Hoechst Marion Roussel (now part of Sanofi-Aventis) and proved to be a rich source of β-lactamase inhibitors (BLI). Two members of this novel series of highly potent, broad spectrum BLIs are now in clinical development and their properties are reviewed here.     

Production of (2S,3S)-2,3-butanediol and (3S)-acetoin from glucose using resting cells of Klebsiella pneumonia and Bacillus subtilis

Production of highly pure (2S,3S)-2,3-butanediol ((2S,3S)-2,3-BD) and (3S)-acetoin ((3S)-AC) in high concentrations is desirable but difficult to achieve. In the present study, glucose was first transformed to a mixture of (2S,3S)-2,3-BD and meso-2,3-BD by resting cells of Klebsiella pneumoniae CICC 10011, followed by biocatalytic resolution of the mixture by resting cells of Bacillus subtilis 168. meso-2,3-BD was transformed to (3S)-AC, leaving (2S,3S)-2,3-BD in the reaction medium. Using this approach, 12.5 g l(-1) (2S,3S)-2,3-BD and 56.7 g l(-1) (3S)-AC were produced. Stereoisomeric purity of (2S,3S)-2,3-BD and enantiomeric excess of (3S)-AC was 96.9 and 96.2%, respectively.     

Enantiomeric discrimination of pyrethroic acid esters on polysaccharide derived chiral stationary phases

Enantiomeric separation of pyrethroic acid methyl and ethyl esters was examined on cellulose-based chiral stationary phases (CSPs): chiralcel OD (cellulose tris(3,5-dimethylphenyl carbamate)) and chiralcel OF (cellulose tris(4-chlorophenyl carbamate)). The good resolution of pyrethroic acid esters was achieved on chiralcel OD and OF. Separation factors ranged from 1.19-5.12 for Chiralcel OD and 1.00-1.59 for chiralcel OF. Hexane/2-propanol (100:0.15, v/v %) was used as the eluent. The resolution capability of CSPs was greater chiralcel OD than chiralcel OF in the case of the pyrethroic acid esters. The flow rate was 0.8 ml/min and detection was set at 230 nm. The results of the chromatographic data and molecular mechanics suggest that steric effect was a major factor in the enantioseparation. Furthermore, the hydrogen bond between analytes and CSP played an important role in the chiral recognition.     

Resolution of enantiomers by HPLC on tris(4-alkoxyphenylcarbamate)s of cellulose and amylose

Ten phenylcarbamate derivatives of cellulose and amylose having alkoxy groups such as ethoxy, isopropoxy, and isobutoxy at 4-position, and methyl groups at 3- and 5-positions and methoxy group at 4-position were synthesized and their chiral recognition abilities as stationary phases for high-performance liquid chromatography were investigated and compared to those with tris(4-methoxyphenylcarbamate)s of cellulose and amylose. In 4-alkoxy derivatives of cellulose, chiral recognition ability increased as the bulkiness of 4-alkoxy groups increased. 4-Isopropoxy and 4-isobutoxy derivatives showed high chiral recognition. On the other hand, chiral discrimination of amylose 4-alkoxy derivatives scarcely depended on the bulkiness of the alkoxy group, and 4-methoxy and 4-isopropoxy derivatives showed high chiral recognition. 3,5-Dimethyl-4-methoxyphenylcarbamates of cellulose and amylose possessed higher chiral recognition ability than the corresponding 4-methoxy derivatives. © 1993 Wiley-Liss, Inc.     

Synergistic effects between aminoethyl-chitosans and β-lactams against methicillin-resistant Staphylococcus aureus (MRSA)

Two kinds of aminoethyl-chitosans (AEC), AEC90 and AEC50, which had degrees of deacetylation of 90% and 50%, respectively, were prepared and their synergistic effects in combination with β-lactams including ampicillin, penicillin, and oxacillin against two standard methicillin-resistant Staphylococcus aureus (MRSA) strains and twelve clinical isolated MRSA strains were investigated. When AECs and β-lactams were combined, synergistic effects were observed with fractional inhibitory concentration (FIC) indices of 0.252–0.508, and the MICs of β-lactams in the presence of AECs were dramatically reduced.     

Differential gene expression in planktonic and biofilm cells of multiple antibiotic-resistant Salmonella Typhimurium and Staphylococcus aureus

This study was designed to evaluate gene expression patterns of the planktonic and biofilm cells of Staphylococcus aureus and Salmonella Typhimurium in trypticase soy broth adjusted to pH 5.5 and pH 7.3. The planktonic and biofilm cells of multiple antibiotic-resistant S. aureus (S. aureus(R) ) and S. Typhimurium (S. Typhimurium(R) ) were more resistant to β-lactams than those of antibiotic-susceptible S. aureus (S. aureus(S) ) and S. Typhimurium (S. Typhimurium(S) ) at pH 5.5 and pH 7.3. The relative gene expression levels of norB, norC, and mdeA genes were increased by 7.0-, 4.7-, and 4.6-fold, respectively, in the biofilm cells of S. aureus(S) grown at pH 7.3, while norB, norC, mdeA, sec, seg, sei, sel, sem, sen, and seo genes were stable in the biofilm cells of S. aureus(R) . This study provides useful information for understanding gene expression patterns in the planktonic and biofilm cells of antibiotic-resistance pathogens exposed to acidic stress.     

C4-Phenylthio β-lactams: Effect of the chirality of the β-lactam ring on antimicrobial activity

C4-phenylthio β-lactams are a new family of antibacterial agents that have activity against two phylogenetically distant bacteria – Mycobacterium tuberculosis (Mtb) and Moraxella catarrhalis (M. cat). These compounds are effective against β-lactamase producing Mtb and M. cat unlike the clinically relevant β-lactam antibiotics. The structure-activity relationship for the C4 phenylthio β-lactams has not yet been completely defined. Earlier efforts in our laboratories established that the C4-phenylthio substituent is essential for antimicrobial activity, while the N1 carbamyl substituent plays a more subtle role. In this present study, we investigated the role that the stereochemistry at C4 plays in these compounds’ antibacterial activity. This was achieved by synthesizing and testing the antimicrobial activity of diastereomers with a chiral carbamyl group at N1. Our findings indicate that a strict stereochemistry for the C4-phenylthio β-lactams is not required to obtain optimal anti-Mtb and anti-M. cat activity. Furthermore, the structure–bioactivity profiles more closely relate to the electronic requirement of the phenylthiogroup. In addition, the MICs of Mtb are sensitive to growth medium composition. Select compounds showed activity against non-replicating and multi-drug resistant Mtb.     

Preparative scale enantioseparation of 1-cyclohexyl-1-phenylethyl hydroperoxide and 1,2,3,4-tetrahydro-1-naphthyl hydroperoxide on a modified cellulose stationary phase

The analytical and preparative scale optical resolution of 1-cyclohexyl-1-phenylethyl hydroperoxide and 1,2,3,4-tetrahydro-1-napthyl hydroperoxide has been achieved by chiral HPLC on a cellulose tris(3,5-dimethylphenyl carbamate) stationary phase coated on silica gel. The method has been used to obtain several hundred milligrams of highly enriched enantiomers (%ee >98) which were characterized by [α]_D and circular dichroism spectra, respectively. Configurational assignments were achieved for 1,2,3,4-tetrahydro-1-naphthyl hydroperoxide enantiomers. © 1995 Wiley-Liss, Inc.