A Strategy for assessing peak purity of pharmaceutical peptides in reversed-phase chromatography methods using two-dimensional liquid chromatography coupled to mass spectrometry. Part II: Development of second-dimension gradient conditions.

The importance of therapeutic peptides continues to increase in the marketplace for treating a range of diseases including diabetes and obesity. Quality control analyses for these pharmaceutical ingredients usually depends on reversed-phase liquid chromatography, and it is critically important to ensure that no impurities coelute with the target peptide at levels that would compromise the safety or effectiveness of the drug products. This can be challenging due to the broad range of properties of impurities that can be present on one hand (e.g., amino acid substitutions, chain cleavages, etc.), and the similarity of other impurities on the other hand (e.g., d-/l-isomers). Two-dimensional liquid chromatography (2D-LC) is a powerful analytical tool that is well suited to address this particular problem; the first dimension can be used to detect impurities over a broad range in properties, while the second dimension can be used to focus specifically on those species that might coelute with the target peptide in the first dimension. While hundreds of papers have been published on the use of 2D-LC for proteomics applications, there are very few papers that have focused on its use for characterisation of therapeutic peptides. This paper is the second in a two-part series. In Part I of the series, we studied several different column / mobile phase combinations that could be useful in 2D-LC separations of therapeutic peptides, with a focus on selectivity, peak shape, and complementarity to other combinations, particularly for isomeric peptides under mass spectrometry-friendly conditions (i.e., volatile buffers). In this second part in the series, we describe a strategy to derive second-dimension (2D) gradient conditions that both, ensure elution from the 2D column, and increase the likelihood of resolving peptides with very similar properties. We find that a two-step process yields conditions that place the target peptide in the middle of the 2D chromatogram. This process begins with two scouting gradient elution conditions in the second dimension of a 2D-LC system, followed by building and refining a retention model for the target peptide using a third separation. The process is shown to be generically useful by developing methods for four model peptides, and application to a sample of degraded model peptide to demonstrate its utility for resolving impurities in a real sample.

A strategy for assessing peak purity of pharmaceutical peptides in reversed-phase chromatography methods using two-dimensional liquid chromatography coupled to mass spectrometry. Part I: Selection of columns and mobile phases.

The current study describes the development of a 2D-LC-MS-based strategy for assessing main peak purity in the analysis of pharmaceutical peptides. The focus is on 2D-LC using reversed-phase (RP) separations in both dimensions, and particularly peptide isomer selectivity, since compounds with the same mass to charge ratio are not readily differentiated by mass spectrometry and therefore must be separated chromatographically. Initially, 30 column / mobile phase combinations were evaluated for both general separation performance (i.e., selectivity and peak shape) and isomer selectivity using forcibly degraded peptide samples and mixtures of synthetic diastereomers. A ranking of more than 300 UV and MS chromatograms suggests that when developing a new method, screening a set of four columns and four volatile mobile phases with differing characteristics should be adequate to both cover the selectivity space, and yield good separation performance. When 2D-LC-MS is to be used to evaluate peak purity for a new method, our results show that a second-dimension separation comprising a C8/C18 column possessing no ionic functionality, and an acetic acid / ammonium acetate mobile phase buffered at pH 5, provides good selectivity at 25 °C for peptide isomers with a MW <10 kDa. Retention data for 29 diverse peptides (1 < MW < 14 kDa, 3.7 < pI < 12.5) measured in this study using a variety of column and mobile phase conditions (i.e., 30 in total) are consistent with the classification of these various chromatographic conditions using the previously reported Peptide RPC Column Characterisation Protocol. For the investigated peptides trifluoroacetic acid was found to reduce selectivity differences between columns of diverse properties, probably due to its potential to form ion-pairs with peptides. Trifluoroacetic acid often improves peak shape for very large peptides (i.e. MW > 10 kDa). In the current dataset which also contain smaller peptides it received the highest ranking for 40% of the column and mobile phase combinations due to better selectivity and/or peak shape. The reported work here constitutes part one of a series of two papers. The second paper focuses on the use of retention modelling for rapid and accurate selection of the shallow gradients (i.e., << 1% ACN/min) required to obtain sufficient peptide isomer retention and separation in the second dimension. The overall results presented in this series of papers provides the guidance needed to develop a 2D-LC-MS method from start to finish for the analysis of main peak purity of therapeutic peptides.

Comprehensive profiling of conjugated fatty acid isomers and their lipid oxidation products by two-dimensional chiral RP×RP liquid chromatography hyphenated to UV- and SWATH-MS-detection.

This research reports on the development of a comprehensive two-dimensional liquid chromatography (2D-LC) method hyphenated to inline DAD-UV and ESI-QTOF-MS/MS-detection for the separation of conjugated polyunsaturated fatty acid isomers and structurally related (saturated, unconjugated, oxidized) compounds. In pharmaceutical lipid formulations conjugated fatty acids can be found as impurities, generated by oxidation of polyunsaturated fatty acids. Due to the structural complexity of resultant multi-component samples one dimensional liquid chromatography may be suboptimal for quality control and impurity profiling. The screened reversed-phase columns showed a lack of selectivity for the conjugated fatty acid isomers but the resolutions improved with the shape selectivity of the stationary phases (C18- < C30- < cholesteryl-ether-bonded). Further enhanced selectivity for the non-chiral conjugated FAs could be achieved with amylose/cellulose-based chiral stationary phases (CSPs) which harbor cavities for selective inclusion depending on E/Z configurations of the double bonds of the analytes. Amylose-based CSPs showed higher selectivity for conjugated fatty acids than the cellulose-based polysaccharide CSPs. Hyphenating the chiral and reversed-phase columns in a comprehensive 2D-LC-setup was favorable since they showed orthogonality and good compatibility, because both were operated under RP-conditions. The chiral dimension (1D) mainly separated the different isomers, while the reversed-phase dimension (2D) separated according to number of double bonds and degree of oxidation. Using this setup, advanced structural annotation of unknowns was possible based on UV-, MS1- and MS2-spectra. Data-independent acquisition (by SWATH) enabled differentiation of positional isomers of oxidized lipids by characteristic MS2-fragments and elucidation of co-eluted compounds by selective extracted ion chromatograms of fragment ions (MS2 EICs).

Constant pressure mode of operation in the second dimension of two-dimensional liquid chromatography: A proof of concept.

The use of two-dimensional liquid chromatography (2D-LC) continues to grow as the advantages over 1D-LC become increasingly clear in specific application areas, and the number of experienced 2D-LC users increases. As with any technique, however, there is always room for innovation that could improve the performance of 2D-LC. In recent years the technical aspects and potential benefits of a volume-based mode of operation were studied in detail for 1D-LC. The salient features of this approach that are immediately interesting for use in 2D-LC are two-fold. First, the ability to maintain a nominally constant pressure in the second dimension by dynamically adjusting the flow rate to compensate for changes in the viscosity of the fluid in the 2D flow path provides a means to more fully utilize the pressure capability of the pumping system, and accelerates separations in the second dimension (2D). Second, constant pressure operation minimizes physical stress on the system components and the 2D column. In this paper we discuss the aspects of volume-based operation of LC that are particularly relevant to 2D-LC systems. The proof-of-concept experiments illustrate the viability of the constant pressure mode of operation for the second dimension of 2D-LC. In the described separations the throughput improvement is on the order of 10%; this gain will be strongly application-dependent, and may be as large as several tens percent in some cases. Future work will involve a detailed investigation of the impact of the constant pressure mode on robustness of 2D separations.

Advances in ultra-high-pressure and multi-dimensional liquid chromatography instrumentation and workflows.

The present contribution discusses recent advances in ultra-high-pressure liquid chromatography (UHPLC) and multi-dimensional liquid chromatography (MDLC) technology. First, new developments in UHPLC column technology and system design are highlighted. The latter includes a description of a novel injector concept enabling method speed-up, emerging detectors, and instrument diagnostics approaches. Next, online MDLC workflows are reviewed and advances in modulation technology are highlighted. Finally, key applications published in 2020 are reviewed.

Imaging Peptide and Protein Chirality via Amino Acid Analysis by Chiral × Chiral Two-Dimensional Correlation Liquid Chromatography.

The present contribution illustrates the utilization of a chiral × chiral two-dimensional liquid chromatography (2DLC) setup with tert-butylcarbamoyl quinine chiral stationary phase (CSP) in the first dimension (1D) and tert-butylcarbamoyl quinidine CSP in the second dimension (2D) to analyze FMOC-derivatized d and l amino acids from peptide hydrolysates. Hereby, in the 1D and 2D chiral separation dimensions factors such as selector and immobilization chemistry of the CSPs, mobile phase, temperature, column hardware dimensions, stationary phase supports, particle type and packing were identical. Orthogonality between 1D and 2D CSPs was solely based on their stereochemistry, i.e. their opposite configurations in two chiral centers of the selector molecules, which results in inversion of enantiomer elution orders in the two dimensions. Using Coreshell CSPs for fast chromatography allowed 2D-flow rates which were 60 times faster than the 1D-flow rates to enable online comprehensive two-dimensional chromatography (LC × LC). Due to very similar chemoselectivity, yet opposite elution orders of corresponding enantiomers in 1D and 2D, characteristic 2D-elution patterns for achiral and chiral components can be generated. Peaks of achiral components and impurities are lined up on the diagonal line in the 2D separation space (contour plot) and thereby removed from the chromatographic space of the target enantiomers avoiding overlaps with potential interferences. Corresponding enantiomers provide cross peaks on the 2D chromatogram. Moreover, enantioselectivity of both single CSPs is combined to result in an enhanced overall 2D enantioselectivity. The concept is illustrated for the therapeutic peptides gramicidin and bacitracin. Since all amino acids give a consistent elution order as FMOC-derivatives, all enantiomers of the same configuration are either above or below the diagonal line allowing straightforward imaging of the configuration of the amino acids in peptides by the 2D chromatogram.

Evaluation of active solvent modulation to enhance two-dimensional liquid chromatography for target analysis in polymeric matrices.

A new methodology is presented for two-dimensional liquid chromatography (2D-LC) separations of polymers. Active solvent modulation (ASM) was evaluated in its effectiveness to enhance solvent compatibility for both separation dimensions. As an example the determination of target compounds in epoxy resins was used. Ultra-high pressure size-exclusion chromatography was applied in the first dimension using THF as the solvent. The second dimension separation was operated in reversed-phase mode using an acetonitrile/water gradient. ASM prevents sample breakthrough in the second dimension and produces chromatograms that are of great peak shape and high resolution. It enables very sensitive determination of target components down to the low ppm level. The resulting high-speed 2D-LC method (10 min analysis time) showed good linearity (R2 > 0.9995) and reproducibility (as low as 0.3-0.7% peak area RSD). ASM was also applied in comprehensive 2D-LC (SECxLC) mode for characterization of molecular weight and chemical composition distribution of a polymer blend consisting of epoxy novolac and phenol novolac. The SECxLC separation was executed at short run times (20 min). ASM technology can markedly enhance productivity in 2D-LC analysis for many complex sample matrices.

Enantioselective multiple heartcut two-dimensional ultra-high-performance liquid chromatography method with a Coreshell chiral stationary phase in the second dimension for analysis of all proteinogenic amino acids in a single run.

A multiple heartcut (MHC) 2D-UHPLC method with UV detection has been developed for the enantioselective analysis of complex amino acid mixtures in a single run. The MHC method is based on an achiral gradient RPLC separation with 1.8 µm C18 phase (100 × 2.1 mm ID column) in the first dimension (1D) and enantioselective isocratic separation on a tert-butylcarbamoylquinine-based 2.7 µm Coreshell particle column (50 × 3 mm ID) in the second dimension (2D). Pre-column derivatization has been performed with Sanger's reagent (2,4-dinitrofluorobenzene) yielding chromogenic 2,4-dinitrophenylated amino acids (DNP-AAs). Heartcuts of 40 µL fractions of the 1D peaks were sampled into the 2D system via a two-position four-port dual valve connected to two loop decks each equipped with six 40 µL parking loops. Using this setup, 25 amino acids (20 proteinogenic plus allo-Thr, allo-Ile, homoserine (Hse), Orn, ß-Ala) have been analyzed enantioselectively in a fully automated manner with a single chiral column within 130 min total run time (1D and 2D). All 2D separations together took 101.5 min (29 cuts with 3.5 min run time each) and thus the total analysis time was quite efficiently utilized. Faster separations were restricted by some software constraints which did not allow to adjust run times in 2D individually. The practical utility of this enantioselective MHC method is documented by application for the absolute configuration determination of the amino acids in gramicidin and bacitracin. Further optimizations should lead to a generic enantioselective amino acid analyzer for the quality control of synthetic peptides and the structural characterization of non-ribosomal peptides.

Active Solvent Modulation: A Valve-Based Approach To Improve Separation Compatibility in Two-Dimensional Liquid Chromatography.

Two-dimensional liquid chromatography (2D-LC) is increasingly being viewed as a viable tool for solving difficult separation problems, ranging from targeted separations of structurally similar molecules to untargeted separations of highly complex mixtures. In spite of this performance potential, though, many users find method development challenging and most frequently cite the "incompatibility" between the solvent systems used in the first and second dimensions as a major obstacle. This solvent strength related incompatibility can lead to severe peak distortion and loss of resolution and sensitivity in the second dimension. In this paper, we describe a novel approach to address the incompatibility problem, which we refer to as Active Solvent Modulation (ASM). This valve-based approach enables dilution of 1D effluent with weak solvent prior to transfer to the 2D column but without the need for additional instrument hardware. ASM is related to the concept we refer to as Fixed Solvent Modulation (FSM), with the important difference being that ASM allows toggling of the diluent stream during each 2D separation cycle. In this work, we show that ASM eliminates the major drawbacks of FSM including complex elution solvent profiles, baseline disturbances, and slow 2D re-equilibration and demonstrate improvements in 2D separation quality using both simple small molecule probes and degradants of heat-treated bovine insulin as case studies. We believe that ASM will significantly ease method development for 2D-LC, providing a path to practical methods that involve both highly complementary 1D and 2D separations and sensitive detection.

Multiple heart-cutting two dimensional liquid chromatography mass spectrometry: Towards real time determination of related impurities of bio-pharmaceuticals in salt based separation methods.

Many of the chromatographic methods used in industry to determine related impurities in bio pharmaceuticals employ salt containing mobile phases. "Salty" mobile phases often provide superior chromatographic performance but are not compatible with mass spectrometry (MS) detection. Peak tracking necessary for method development is therefore often based on peak areas and the chemist's experience/intuition. In addition, MS characterization of impurities usually is done by offline fraction collection, which apart from being time consuming often suffers from poor recovery or the degradation of impurities collected. The recent development of multiple heart-cutting (MHC) two-dimensional liquid chromatography (2D-LC) provides a way to address these problems. This study shows how MHC 2D-LC-MS can be used to obtain almost real time MS data for bovine insulin related impurities present at low level (<<0.03%). High quality MS spectra were obtained even for a first dimension using a mobile phase containing high concentrations of sodium, sulphate and phosphate. Thereby MHC 2D-LC-MS offers a possibility to eliminate the guesswork currently associated with peak tracking during method development. Furthermore, in contrast to current characterization methods involving fraction collection, solvent reduction/exchange etc., MS determination is done directly, which markedly shortens the workflow (from days to hours) and reduces the risk for poor recovery and degradation.

Loop-based multiple heart-cutting two-dimensional liquid chromatography for target analysis in complex matrices.

Loop-based multiple heart-cutting (MHC) two-dimensional liquid chromatography (2D-LC) is presented as a solution to quantify target components in complex matrices, such as additives in polymers, at very high chromatographic resolution. The determination of hexabromocyclododecane (HBCD) in polystyrene (PS) is described. One dimensional ((1)D) LC analysis with UV detection did not allow quantitation of the main isomers of HBCD due to peak overlap with polymer components. MHC 2D-LC analysis provided the separation power, accuracy, and repeatability needed for quantitative analysis of the additives of interest. Heart-cuts from peaks of the (1)D-chromatogram or entire regions of interest are sampled into loops, where they remain parked until their sequential reinjection onto the second dimension ((2)D) column. A column set consisting of phenyl ((1)D) and C18 ((2)D) stationary phases gave baseline separation in (2)D between HBCD and PS background. Linearity for spiked polymer samples was achieved over a range of 0.02-1.00 wt % HBCD relative to the amount of polymer. The limit of quantitation was estimated at 0.01 wt % HBCD in PS. A peak area RSD of 0.7% obtained for ten replicates of a real sample demonstrated excellent repeatability of the analysis. MHC 2D-LC is an elegant solution for quantitative analyses of difficult-to-separate samples when conventional (1)D separation fails.

Instrument contributions to resolution and sensitivity in ultra high performance liquid chromatography using small bore columns: comparison of diode array and triple quadrupole mass spectrometry detection.

UHPLC with DAD-UV detection or in combination with mass spectrometry (MS) has proven to be a robust and widely applicable platform for high sensitivity analyses of many types of chemical compounds. The majority of users employ narrow bore columns with 2.1mm internal diameter (ID) typically exhibiting very high efficiencies (>200,000 plates/m). This ultimately sets stringent demands upon the chromatographic system as the separation efficiency can be compromised by external contributions to dispersion caused by connection capillaries, auto-sampler and/or the detection device. Sample limited applications often use reduced column diameters down to capillary- or even nano-column format. Capillary (ID≤0.5mm) or small-bore columns (ID≤1mm) can be a good compromise between system robustness and enhanced sensitivity. Yet in this case, extra-column dispersion gains additional importance due to reduced peak volumes. To design an optimized system configuration for specific column dimensions and applications it is crucial to understand the dispersion contributions of individual extra-column components. This was subject to many studies done within our group and by others. Here, we employed a fully optimized UHPLC/UV system to investigate the contribution to peak dispersion obtained from columns ranging from capillary to narrow bore (0.3, 0.5, 1, 2.1mm) using a set of small molecules that were analyzed in gradient mode. Further UV detection was replaced by a triple quadrupole (QQQ) MS in order to evaluate its contribution to band broadening. In this context the impact of column-ID upon MS sensitivity when interfaced with an Agilent Jet Stream source was investigated. Data obtained from our test suite of compounds shows mostly mass-sensitive behavior of this advanced electrospray technology.

Practical observations on the performance of bare silica in hydrophilic interaction compared with C18 reversed-phase liquid chromatography.

The kinetic performance of a bare silica and C18 phase prepared from the same sub-2µm and 3.5µm base materials were compared in the HILIC and RP mode using both charged and neutral solutes. The HILIC column was characterised using the neutral solute 5-hydroxymethyluridine, the weak base cytosine, and the strong base nortriptyline, the latter having sufficient retention also in the RP mode to allow comparison of performance. Naphthalene was also used as a simple neutral substance to evaluate the RP column alone. The retention factors of all substances were adjusted to give similar values (k'∼5.5) at their respective optimum linear velocities. Reduced van Deemter b-coefficients (determined by curve fitting and by the peak parking method, using a novel procedure involving switching to a dummy column) were significantly lower in HILIC for all substances compared with those found under RP conditions. Against expectation, c-coefficients were always lower in RP when compared with HILIC using sub-2µm particles. While measurement of these coefficients is complicated by retention shifts caused by the influence of high pressure and by frictional heating effects, broadly similar results were obtained on larger particle (3.5µm) phases. The mechanism of the separations was further investigated by examining the effect of buffer concentration on retention. It was concluded that HILIC can sometimes show somewhat inferior performance to RP for fast analysis at high mobile phase velocity, but clearly shows advantages when high column efficiencies, using longer columns at low flow velocity, are employed. The latter result is attributable to the lower viscosity of the mobile phase in HILIC and the reduced pressure requirement as well as the lower b-coefficients.

Influence of phase type and solute structure on changes in retention with pressure in reversed-phase high performance liquid chromatography.

The influence of pressure on the retention of several types of solute, including acids, bases and neutrals, was studied by the use of restriction capillaries added to the end of various monomeric and polymeric octadecylsilyl-modified 5µm particle size columns. Although it appeared that certain polymeric columns could give somewhat greater increases in retention with pressure, differences in behaviour between these different C18 columns were rather small. Differences in solute molecular size were most important in determining increases in retention with pressure. However, solute structure such as polarity and planarity were also influential. A prototype C30 column gave interesting selectivity changes between planar and non-planar solutes as a function of pressure. Considerable selectivity differences with pressure were shown when diverse mixtures of solutes were analysed. For the solutes studied, only minor effects of increased pressure on column efficiency and peak shape were noted.

Multidimensional nano-HPLC coupled with tandem mass spectrometry for analyzing biotinylated proteins.

Multidimensional high-performance liquid chromatography (HPLC) is a key method in shotgun proteomics approaches for analyzing highly complex protein mixtures by complementary chromatographic separation principles. Here, we describe an integrated 3D-nano-HPLC/nano-electrospray ionization quadrupole time-of-flight mass spectrometry system that allows an enzymatic digestion of proteins followed by an enrichment and subsequent separation of the created peptide mixtures. The online 3D-nano-HPLC system is composed of a monolithic trypsin reactor in the first dimension, a monolithic affinity column with immobilized monomeric avidin in the second dimension, and a reversed phase C18 HPLC-Chip in the third dimension that is coupled to a nano-ESI-Q-TOF mass spectrometer. The 3D-LC/MS setup is exemplified for the identification of biotinylated proteins from a simple protein mixture. Additionally, we describe an online 2D-nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS setup for the enrichment, separation, and identification of cross-linked, biotinylated species from chemical cross-linking of cytochrome c and a calmodulin/peptide complex using a novel trifunctional cross-linker with two amine-reactive groups and a biotin label.

A comparison of overload behaviour for some sub 2 µm totally porous and sub 3 µm shell particle columns with ionised solutes.

The overloading performance of some 2.7 µm shell and sub 2 µm totally porous columns, including one pair manufactured from similar materials with similar bonding chemistries, was compared using strongly acidic and basic probe compounds. In general, the capacity of shell particles was not greatly reduced, despite containing a smaller porous volume. Nevertheless, at low pH, both types of column were overloaded by only small concentrations of ionised solute. Considerable improvement could be gained by increasing the buffer concentration, although sensitivity in mass spectrometric detection may be compromised. The capacity of columns of different internal diameter may not be directly compared merely by scaling the injection volumes, as it is possible that the sample is not homogeneously distributed across the column radius, especially in larger diameter columns, where the sample may travel preferentially through a central core of the packing. A totally porous charged surface hybrid phase gave much improved loading properties of the basic probe in low ionic strength mobile phases such as formic acid, often used in mass spectrometry. However, its relative advantage over conventional phases was reduced as the mobile phase ionic strength was increased. Furthermore, acidic compounds may give tailing on this phase. At pH 7, all columns tested showed evidence of interaction with ionised silanols; peak shapes improved as the buffer concentration was increased. Column efficiency first increased and then decreased as solute concentration was increased at constant buffer concentration, which can be attributed to the decreasing proportion of solute molecules retained by the ion exchange process.

Development of screening assays and discovery of initial inhibitors of pneumococcal peptidoglycan deacetylase PgdA.

The essential cell wall peptidoglycan is the target of several components of the innate immune system and its disruption results in lysis of invading bacteria. The pathogen Streptococcus pneumoniae produces a peptidoglycan N-acetylglucosamine deacetylase, PgdA, to modify the peptidoglycan structure. The activity of PgdA contributes to the bacteria's resistance to lysozyme, which is an important antimicrobial factor of the human innate immune system. In this study we report on the activity of PgdA against natural and artificial substrates. We have also established a virtual high-throughput screening and a new enzyme assay to search for compounds inhibiting PgdA. Two compounds with IC(50) values in the micromolar range have been identified and they could serve as leads for the search of inhibitors of PgdA, an important pneumococcal virulence factor.

Synthesis of CDP-activated ribitol for teichoic acid precursors in Streptococcus pneumoniae.

Streptococcus pneumoniae has unusually complex cell wall teichoic acid and lipoteichoic acid, both of which contain a ribitol phosphate moiety. The lic region of the pneumococcal genome contains genes for the uptake and activation of choline, the attachment of phosphorylcholine to teichoic acid precursors, and the transport of these precursors across the cytoplasmic membrane. The role of two other, so far uncharacterized, genes, spr1148 and spr1149, in the lic region was determined. TarJ (spr1148) encodes an NADPH-dependent alcohol dehydrogenase for the synthesis of ribitol 5-phosphate from ribulose 5-phosphate. TarI (spr1149) encodes a cytidylyl transferase for the synthesis of cytidine 5'-diphosphate (CDP)-ribitol from ribitol 5-phosphate and cytidine 5'-triphosphate. We also present the crystal structure of TarI with and without bound CDP, and the structures present a rationale for the substrate specificity of this key enzyme. No transformants were obtained with insertion plasmids designed to interrupt the tarIJ genes, indicating that their function could be essential for cell growth. CDP-activated ribitol is a precursor for the synthesis of pneumococcal teichoic acids and some of the capsular polysaccharides. Thus, all eight genes in the lic region have a role in teichoic acid synthesis.

Attenuation of penicillin resistance in a peptidoglycan O-acetyl transferase mutant of Streptococcus pneumoniae.

The level of penicillin resistance in clinical isolates of Streptococcus pneumoniae depends not only on the reduced affinity of penicillin binding proteins (PBPs) but also on the functioning of enzymes that modify the stem peptide structure of cell wall precursors. We used mariner mutagenesis in search of additional genetic determinants that may further attenuate the level of penicillin resistance in the bacteria. A mariner mutant of the highly penicillin-resistant S. pneumoniae strain Pen6 showed reduction of the penicillin minimum inhibitory concentration (MIC) from 6 to 0.75 microg ml(-1). Decrease in penicillin MIC was also observed upon introduction of the mutation (named provisionally adr, for attenuator of drug resistance) into representatives of major epidemic clones of penicillin-resistant pneumococci. Attenuation of resistance levels was specific for beta-lactams. The adr mutant has retained unchanged (low affinity) PBPs, unaltered murM gene and unchanged cell wall stem peptide composition, but the mutant became hypersensitive to exogenous lysozyme and complementation experiments showed that both phenotypes--reduced resistance and lysozyme sensitivity--were linked to the defective adr gene. DNA sequence comparison and chemical analysis of the cell wall identified adr as the structural gene of the pneumococcal peptidoglycan O-acetylase.

Determination of ionisation constants of organic bases in aqueous methanol solutions using capillary electrophoresis.

The pKa of eight organic bases was determined in aqueous and aqueous methanol solutions of 0-70% (v/v) methanol using capillary electrophoresis. The bases investigated include compounds commonly used to test the activity of RP columns in HPLC. The variation of pKa with temperature in aqueous methanol solutions was also investigated and found to closely resemble temperature coefficients reported for bases in purely aqueous solutions. pKa values determined by CE were compared to those reported using NMR spectroscopy. The good agreement of the results is evidence that either technique is suitable to perform pKa measurements.