Form variation in Escherichia coli K1: determined by O-acetylation of the capsular polysaccharide.

The chemical basis for the alternating antigenic change called form variation noted for the Escherichia coli K1-capsular polysaccharide has been shown by 13C nuclear magnetic resonance to be a result of random O-acetylation of C7 and C9 carbons of the alpha-2-8-linked sialic acid homopolymer. A serologic method (antiserum agar) was developed to identify and isolate the form variants. The O-acetyl positive and O-acetyl negative K1 polysaccharides had unique biochemical and immunologic properties. The O-acetyl-positive variants resisted neuraminidase hydrolysis in contrast to the susceptibility of the O-acetyl negative variant to this enzyme. In addition, O-acetylation altered the antigenicity of the O-acetyl polysaccharides. When injected as whole organisms, O-acetyl positive organisms produced anti-K1 -antibodies in rabbits specific for this polysaccharide variant. O-acetyl negative organisms were comparatively less immunogenic; however, antibodies induced by these organisms reacted with both K1 polysaccharide variants. Burros, injected with either variant, produced antibodies reactive with both K1 polysaccharides.

Escherichia coli K51 and K93 capsular polysaccharides are crossreactive with the group A capsular polysaccharide of Neisseria meningitidis. Immunochemical, biological, and epidemiological studies.

Eleven Escherichia coli strains, crossreactive with the capsular polysaccharide (CPS) of Neisseria meningitidis group A (GrA), were detected among 645 stool isolates from healthy families in Cairo, Egypt. 10 of these strains were of the O107:K93:H27 or O107:K93:SP serotypes and may be considered descendents of a single bacterium or as a clone. The remaining crossreactive strain was of the O7:K51:H18 serotype. None of the 11 strains produced enterotoxins and none were enteroinvasive. The purified CPS of these E. coli strains, as well as a polysaccharide (PS) from B. pumilis, strain Sh17, precipitated with equine GrA (H49) antiserum. A partial identity between the E. coli K93, K51 and Sh17 PS on the one hand and the GrA CPS on the other was observed by double immunodiffusion when reacted against the H49 antiserum. Four K93 strains and one K51 strain were found among 320 E. coli strains from patients at the Clinical Center, National Institutes of Health, and three K93 strains were found in 105 stool samples from children in Copenhagen. The data from these three surveys suggest that these crossreactive E. coli are common organisms and could serve as a stimulus for "natural" GrA CPS antibodies. Quantitative precipitation analysis showed that K51, K93, and Sh17 PS precipitated 25, 46.8, and 50% of H49 antibodies, respectively. Absorption of H49 antiserum with the GrA CPS removed its precipitating activity with the E. coli K93, K51, and Sh17 PS. Absorption of H49 antiserum with either K51 CPS or Sh17 PS removed the homologous crossreactivity only, whereas K93 CPS absorbed both K93 and K51 reactivities. Antibodies, raised by intravenous injection of formalinized E. coli K93 or K51 cells into rabbits, precipitated with GrA CPS and were bactericidal against GrA meningococci. The crossreaction between the E. coli K93 and the GrA CPS was unexpected since these two CPS are compositionally so dissimilar.

Determination of enantiomeric homogeneity (optical purity) of cyclophosphamide by nuclear magnetic resonance spectroscopy.

The enantiomeric homogeneity of resolved samples of the chiral anticancer drug cyclophosphamide was evaluated directly by 1H and 31P nuclear magnetic resonance spectroscopy with the use of the optically active shift reagent tris-[3-(trifluoromethylhydroxymethylene)-d-camphorato]europlum(III). These measurements, in concert with optical rotatory dispersion spectroscopy, established that, for optically pure cyclophosphamide, [alphaD] = 2.3 +/- 0.2 degrees.

Solution structure of a DNA octamer containing the Pribnow box via restrained molecular dynamics simulation with distance and torsion angle constraints derived from two-dimensional nuclear magnetic resonance spectral fitting.

The DNA octamer [d(GTATAATG].[(CATATTAC)], containing the prokaryotic upstream consensus recognition sequence, has been examined via proton homonuclear two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered correlation (2QF-COSY) spectra. All proton resonances, except those of H5' and H5" protons, were assigned. A temperature dependence study of one-dimensional nuclear magnetic resonance (NMR) spectra, rotating frame 2D NOE spectroscopy (ROESY), and T1 rho measurements revealed an exchange process that apparently is global in scope. Work at lower temperatures enabled a determination of structural constraints that could be employed in determination of a time-averaged structure. Simulations of the 2QF-COSY cross-peaks were compared with experimental data, establishing scalar coupling constant ranges of the individual sugar ring protons and hence pucker parameters for individual deoxyribose rings. The rings exhibit a dynamic equilibrium of N and S-type conformers with 80 to 100% populations of the latter. A program for iterative complete relaxation matrix analysis of 2D NOE spectral intensities, MARDIGRAS, was employed to give interproton distances for each mixing time. According to the accuracy of the distance determination, upper and lower distance bounds were chosen. The distance bounds define the size of a flat-well potential function term, incorporated into the AMBER force-field, which was employed for restrained molecular dynamics calculations. Torsion angle constraints in the form of a flat-well potential were also constructed from the analysis of the sugar pucker data. Several restrained molecular dynamics runs of 25 picoseconds were performed, utilizing 184 experimental distance constraints and 80 torsion angle constraints; three different starting structures were used: energy minimized A-DNA, B-DNA, and wrinkled D-DNA, another member of the B-DNA family. Convergence to similar structures obtained with root-mean-square deviations between resulting structures of 0.37 to 0.92 A for the central hexamer of the octamer. The average structure from the nine different molecular dynamics runs was subjected to final restrained energy minimization. The resulting final structure was in good agreement with the structures derived from different molecular dynamics runs and exhibited a substantial improvement in the 2D NOE sixth-root residual index in comparison with the starting structures. An approximation of the structure in the terminal base-pairs, which displayed experimental evidence of fraying, was made by maintaining the structure of the inner four base-pairs and performing molecular dynamics simulations with the experimental structural constraints observed for the termini.(ABSTRACT TRUNCATED AT 400 WORDS)

31P-NMR studies of Mycoplasma gallisepticum cells using a continuous perfusion technique.

31P-NMR studies of Mycoplasma gallisepticum cells have been carried out using a continuous perfusion technique; these are the first such studies with this organism. Using this technique, glucose metabolism was monitored in the intact organisms, and cell extracts were prepared to identify the intermediates. Under glycolytic conditions, high levels of fructose-1,6-diphosphate were observed, indicating that this sugar may play a key role in the regulation of metabolism. The level of phosphoenolpyruvate was low under normal glycolytic conditions, and did not increase during starvation. From the position of the internal inorganic phosphate peak, the intracellular pH was estimated. The cells were found to maintain an intracellular pH of approximately 7.1 over an investigated external pH range of 6.6-8.6.

31P nuclear magnetic resonance spectroscopic observation of the intracellular transformations of oncostatic cyclophosphamide metabolites.

31P NMR spectroscopy was used to directly monitor, for the first time, the intracellular chemistry of the ultimate active metabolite of cyclophosphamide, namely, phosphoramide mustard. These NMR studies utilized a human histiocytic lymphoma cell line (U937), embedded in agarose gel threads, and perfused with medium containing synthetically derived metabolites (4-hydroxycyclophosphamide, aldophosphamide, and phosphoramide mustard). Metabolites 2 or 3 or both readily crossed the cell membrane; in contrast, the membrane was relatively impermeable to 4. Intracellular concentrations of 4 could, therefore, be attributed primarily to the intracellular fragmentation of 3. Signals suggestive of either carboxyphosphamide or 4-ketophosphamide were not detected. Spectral data were used to calculate a rate constant of (5.4 +/- 0.3) X 10(-3) min-1 for the intracellular disappearance of 4 at 23 degrees C. The intracellular pH was determined to be 7.1 from the chemical shift of the internal inorganic phosphate signal.

Prediction of drug absorption using multivariate statistics.

Literature data on compounds both well- and poorly-absorbed in humans were used to build a statistical pattern recognition model of passive intestinal absorption. Robust outlier detection was utilized to analyze the well-absorbed compounds, some of which were intermingled with the poorly-absorbed compounds in the model space. Outliers were identified as being actively transported. The descriptors chosen for inclusion in the model were PSA and AlogP98, based on consideration of the physical processes involved in membrane permeability and the interrelationships and redundancies between available descriptors. These descriptors are quite straightforward for a medicinal chemist to interpret, enhancing the utility of the model. Molecular weight, while often used in passive absorption models, was shown to be superfluous, as it is already a component of both PSA and AlogP98. Extensive validation of the model on hundreds of known orally delivered drugs, "drug-like" molecules, and Pharmacopeia, Inc. compounds, which had been assayed for Caco-2 cell permeability, demonstrated a good rate of successful predictions (74-92%, depending on the dataset and exact criterion used).

NMR spectroscopic studies of intermediary metabolites of cyclophosphamide. 2. Direct observation, characterization, and reactivity studies of iminocyclophosphamide and related species.

4-Hydroxy-5,5-dimethylcyclophosphamide (6) was synthesized as a stable (to fragmentation) analogue of 4-hydroxycyclophosphamide (1). In anhydrous Me2SO-d6 (less than or equal to 0.03 mol % water), cis- and trans-6 were observed by multinuclear NMR spectroscopy to equilibrate with alpha, alpha-dimethylaldophosphamide (7) and 5,5-dimethyliminocyclophosphamide (8). Identification of 8 was based on 1H, 13C, and 31P chemical shifts, selective INEPT and two-dimensional NMR correlation experiments, and temperature-dependent equilibria data. The interconversion of cis-/trans-6 and -7 was also observed in lutidine buffer; 8 was not detected under the aqueous conditions. In Me2SO-d6, hydroxy metabolite 1 underwent dehydration to give iminocyclophosphamide (5), as evidenced by chemical shift data and a selective INEPT experiment. Concentrations of cis-/trans-1, aldophosphamide (2), and 5 were found to be temperature-dependent with higher temperatures favoring 2 and 5 in a reversible manner, thus indicating that 1/2/5 were intercoverting. The addition of small amounts of water to Me2SO-d6 solutions of imine 5 resulted in the immediate disappearance of its NMR signals. The role of imine 5 in the conversion of 1 to C-4 substituted analogues of 1 was elucidated for the formation of 4-cyanocyclophosphamide (3a) from 1 and sodium cyanide in lutidine buffer.

Synthesis of 3-hydroxycyclophosphamide and studies related to its possible role in the metabolism of cyclophosphamide.

Hydrogenolysis of 3-(benzyloxy)cyclophosphamide (10) using Pd/C catalyst and ethyl acetate as solvent leads to the formation of 3-hydroxycyclophosphamide (3, approximately 20%) and cyclophosphamide (1, approximately 10%), accompanied by regioselective hydrogen-exchange reactions at the C-4 and C-5 positions in 3 and 1. A variety of oxidizing reagents and liver microsomal incubation failed to provide evidence (31P NMR) for conversion of 1 into 3, whereas identical incubation of 3 led to its reduction to 1. Compound 3 is stable at pH 6.5-8.2, 37 degrees C, and exhibits anticancer activity comparable to 1 when tested against L1210 leukemia in mice. Data are discussed with regard to a previously reported suggestion that metabolism of 1 may involved oxidation to give 3 followed by rearrangement of 3 to 2.

31P NMR studies of the kinetics of bisalkylation by isophosphoramide mustard: comparisons with phosphoramide mustard.

31P nuclear magnetic resonance spectroscopy was used to measure the pKa (4.28 +/- 0.2) of isophosphoramide mustard (IPM) at 20 degrees C and to study the kinetics and products of the decomposition of IPM at a solution pH value of ca. 7.4 and at temperatures between 20 and 47 degrees C in the presence of nucleophilic trapping agents. At 37 degrees C, the half-life for the first alkylation was ca. 77 min and ca. 171 min for the second alkylation; these data may be compared with those for phosphoramide mustard (Engle, T.W.; Zon, G.; Egan, W.J. Med. Chem. 1982, 25, 1347), wherein the half-lives for the first and second alkylations are approximately the same (18 min). The rate of fragmentation of aldoifosfamide to IPM and acrolein was also studied by NMR spectroscopy (pH 7.0; 37 degrees C; 0.07 M phosphate); under the noted conditions, the half-life of aldoifosfamide was found to be ca. 60 min.

NMR spectroscopic studies of intermediary metabolites of cyclophosphamide. A comprehensive kinetic analysis of the interconversion of cis- and trans-4-hydroxycyclophosphamide with aldophosphamide and the concomitant partitioning of aldophosphamide between irreversible fragmentation and reversible conjugation pathways.

Multinuclear (31P, 13C, 2H, and 1H) Fourier-transform NMR spectroscopy, with and without isotopically enriched materials, was used to identify and quantify, as a function of time, the following intermediary (short-lived) metabolites of the anticancer prodrug cyclophosphamide (1, Scheme I): cis-4-hydroxycyclophosphamide (cis-2), its trans isomer (trans-2), aldophosphamide (3), and its aldehyde-hydrate (5). Under a standard set of reaction conditions (1 M 2,6-dimethylpyridine buffer, pH 7.4, 37 degrees C), the stereospecific deoxygenation of synthetic cis-4-hydroperoxycyclophosphamide (cis-12, 20 mM) with 4 equiv of sodium thiosulfate (Na2S2O3) afforded, after approximately 20 min, a "pseudoequilibrium" distribution of cis-2, 3, 5, and trans-2, i.e., the relative proportions of these reactants (57:4:9:30, respectively) remained constant during their continual disappearance. NMR absorption signals indicative of "iminophosphamide" (8) and enol 6 were not detected (less than 0.5-1% of the synthetic metabolite mixture). A computerized least-squares fitting procedure was applied to the individual 31P NMR derived time courses for conversion of cis-2, 3 plus 5 (i.e., "3"), and trans-2 into acrolein and phosphoramide mustard (4), the latter of which gave an expected array of thiosulfate S-alkylation products (e.g., 16) and other phosphorus-containing materials derived from secondary decomposition reactions. This kinetic analysis gave the individual forward and reverse rate constants for the apparent tautomerization processes, viz., cis-2 in equilibrium "3" in equilibrium trans-2, as well as the rate constant (k3) for the irreversible fragmentation of 3. The values of k3 at pH 6.3, 7.4, and 7.8 were equal to 0.030 +/- 0.004, 0.090 +/- 0.008, and 0.169 +/- 0.006 min-1, respectively. Replacement of the HC(O)CH2 moiety n 3 with HC(O)CD2 led to a primary kinetic isotope effect (kH/kD = 5.6 +/- 0.4) for k3. The apparent half-lives (tau 1/2) for cis-2, "3", and trans-2 under the standard reaction conditions, at "pseudoequilibrium" (constant ratio of cis-2/"3"/trans-2), were each equal to approximately 38 min, which is considerably shorter than the widely cited colorimetrically derived half-lives reported by earlier investigators. The values of tau 1/2 for cis-2, "3", and trans-2 were affected by pH in the same manner as that found for k3 but were relatively insensitive to the presence of either K+, Na+, Ca2+, or Mg2+. The presence of certain primary amines led to marked decreases in tau 1/2 and, in some cases, the formation of acyclic adducts of aldehyde 3.(ABSTRACT TRUNCATED AT 400 WORDS)

The management of carcinoma of the larynx in a prominent patient, or did Morell Mackenzie really cause World War I?.

The influence of fate on history is a fascinating subject. This paper reviews the death of the Crown Prince of Prussia from carcinoma of the larynx. This was a critical turning point in history. The roles of the European physicians involved in his care are reviewed. It was not until after the death of Queen Victoria that the true character of these physicians was revealed.

O-Methylhydroxylamine as a new trapping reagent for quantitative studies of 4-hydroxycyclophosphamide and aldophosphamide.

31P- and 1H-NMR spectroscopy were used to demonstrate that the primary metabolites of the anticancer drug cyclophosphamide (4-hydroxycyclophosphamide and its acyclic tautomer, aldophosphamide) are quantitatively converted by O-methylhydroxylamine, at pH 7.4 and 37 degrees, into the E and Z isomers of aldophosphamide O-methyl oxime. These trapping products are readily extracted from aqueous media with either chloroform or ethyl acetate, are stable at pH 6-8 toward oxime hydrolysis and elimination of phosphoramide mustard (a secondary metabolite of cyclophosphamide), and showed no evidence for transoximination with either ketone or aldehyde acceptors. All of these features support the use of aldophosphamide O-methyl oxime in quantitative studies related to cyclophosphamide metabolism.

Synthesis, hydrolytic behavior, and anti-HIV activity of selected acyloxyalkyl esters of trisodium phosphonoformate (foscarnet sodium).

The synthesis and anti-HIV activity of selected (acyloxy)alkyl esters of trisodium phosphonoformate (foscarnet sodium) are described. The conversion of bis(trimethylsilyl) (alkoxycarbonyl)phosphonates 11a-d to the corresponding disilver salts 12a-d and their subsequent reaction with iodoalkyl acrylates 4a-c gave the desired bis(acyloxyalkyl) phosphonates 6-9(a-c). Of the analogs tested, only the dichlorophenyl analog 9a showed a dose-dependent inhibition of HIV activity in H9 cells. Using 31P-NMR, bioreversibility has been investigated in an attempt to rationalize these results.

Synthesis and evaluation of 3-halocyclophosphamides and analogous compounds as novel anticancer "pro-prodrugs".

3-Fluoro-, 3-chloro-, and 3-bromocyclophosphamide were prepared from the reaction of trifluoromethylhypofluorite, sodium hypochlorite, and bromine with the anticancer drug cyclophosphamide. Treatment of cis- and trans-4-phenylcyclophosphamide and 5,6-benzocyclophosphamide with sodium hypochlorite afforded cis- and trans-3-chloro-4-phenylcyclophosphamide and 3-chloro-5,6-benzocyclophosphamide, respectively. 31P-NMR spectroscopy was used to study the reactivity of these compounds: the fluoro derivative was reduced to cyclophosphamide on incubation with mouse liver slices, and the reactivity order for sulfhydryl-induced reduction of the 3-halocyclophosphamides was Br approximately equal to Cl much greater than F. Compared with the therapeutic efficacy of cyclophosphamide against L-1210 and P-388 cancers in mice, 3-fluoro- and 3-chlorocyclophosphamide were less active, although the fluoro derivative was more efficacious than the 3-chloro compound. The individual R and S enantiomers of 3-chlorocyclophosphamide, prepared from (S)- and (R)-cyclophosphamide, respectively, showed no significant difference in therapeutic activity in the P-388 test system.

Mass-spectral studies of isomeric D-ribofuranosylribitol disaccharides from the capsular polysaccharides of Haemophilus influenzae type b and Escherichia coli K 100.

Two isomeric-D-ribofuranosylribitols, derived from capsular polysaccharides of Haemophilus influenzae type b and Escherichia coli K 100, were methylated or acetylated, and the products analyzed by gas-liquid chromatography-mass spectrometry. The marked difference in the mass spectra of the methyl ethers of these disaccharides allowed clear distinction between 1- and 2-O-D-ribofuranosylribitol was characteristic for this disaccharide; its isomer, the (1 leads to 2)-linked species, has a base peak at m/e 57. The difference in the base peaks is attributable to fragmentation of the methylated ribitol, as both spectra display common ions characteristic of the methylated D-ribofuranosyl group. For the acetylated disaccharides, the mass spectra displayed common ions characteristic of the acetylated D-ribofuranosyl group. However, no ions similar to those found for the methylated ribitol allowed ready differentiation between the two acetates. Instead, their spectra displayed similar ions, differing somewhat in relative abundance; the M-1 ion, m/e 577, was obtained for both. Comparison of the relative abundance of m/e 139, 259, and 303 in the spectra of the two acetates did allow distinction between them.

Structural studies of the Haemophilus influenzae type e capsular polysaccharide.

The structure of the Haemophilus influenzae type e capsular polysaccharide has been determined by a combination of chemical and spectroscopic methods. The structure of the repeating unit of the polymer was found to be leads to 3)-beta-D-GlcNAc-(1 leads to 4)-beta-D-ManANAc-(1 leads to ; both sugars were present in the pyranoid form.

A conformational study of alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) by NMR nuclear Overhauser effect spectroscopy (NOESY) and molecular dynamics calculations.

The conformational preference of the disaccharide alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) (1) about the glycosidic torsion angles, phi and psi, was studied by NMR NOESY spectroscopy and molecular mechanics calculations. The NOE data were consistent with either of two distinct conformations close to minima on a calculated phi/psi potential energy surface. Starting from the lowest energy conformation, a 1-ns molecular dynamics (MD) trajectory was computed in vacuo, from which the NOE curves were simulated and compared to the experimentally observed NOESY data.

Structural and immunological studies of the Escherichia coli K7 (K56) capsular polysaccharide.

The structure of the Escherichia coli K7 capsular polysaccharide has been investigated by a combination of chemical and spectroscopic methods. The Structure of the repeating unit of the polymer was found to be goes to 3)-beta-D-ManNAcA-(1 leads to 4)-beta-D-Glc-(1 goes to ; the O-6 atom of the D-glucosyl residue in the repeating unit is acetylated. The K7 polysaccharide is cross-reactive with the Streptococcus pneumoniae type 3 polysaccharide, the structure of which had previously been determined; our n.m.r. studies of the S. pneumoniae type 3 polysaccharide are in accord with this structure. The E. coli K7 and K56 capsular antigens have been shown by serology and 13C-n.m.r. spectroscopy to be identical.

New n.m.r.-spectroscopic approaches for structural studies of polysaccharides: application to the Haemophilus influenzae type a capsular polysaccharide.

The extension of several modern nuclear magnetic resonance (n.m.r.) spectroscopic techniques to polysaccharides is discussed and illustrated, using the native Haemophilus influenzae type a capsular polysaccharide. These techniques provide for the unambiguous assignment of all n.m.r. resonances (1H, 13C, and 31P) via high-sensitivity homonuclear and 1H-detected heteronuclear correlations, and they are capable of locating the intersaccharide linkages (both O-linked and phosphoric diester-linked) and appended groups (e.g. O-acetyl groups). To illustrate the power and sensitivity of these methods, a 10-mg sample of the H. Influenzae type a polysaccharide (repeat unit mol. wt. = 376) was studied. The combined acquisition time for the two-dimensional 1H-13C correlation data (one-bond and multiple-bond), the 1H-31P correlation data, and the 1H-1H (homonuclear Hartmann-Hahn) data was approximately 18 h.