13C NMR studies provide a convenient way of obtaining detailed information about tautomeric and ionization states in protein-ligand complexes provided that suitably 13C-labeled molecules are available. In the present study, [4,6,8a-13C]- and [2,4a,7,9-13C]folic acid were synthesized and the 13C NMR spectra of their complexes with Lactobacillus casei dihydrofolate reductase (DHFR) were assigned and analyzed as a function of pH. From these data it was possible to determine the tautomeric and ionization states of the bound folate and to obtain further evidence about the orientation of the pteridine ring in the complexes. In the 13C spectra of the ternary complexes of the 13C-labeled folic acids with DHFR and NADP+, each labeled carbon gave rise to multiple signals, confirming our previous findings that there are three interconverting conformational forms of bound folate (forms I, IIa, and IIb) in the ternary complex (Birdsall et al., 1989b). The 13C spectra of the binary complexes of folate and DHFR also provide direct evidence for the presence of forms IIa and IIb and indirect evidence of some form I at low pH values ( < 5.0). 2D 1H-13C HMQC-NOESY experiments on ternary complexes formed using the [2,4a,7,9-13C]folic acid were used to obtain intermolecular NOEs between the folate H7 proton and protons on the protein, and these provided further characterization of the orientations of the pteridine ring in the different bound forms of folate (form IIb with its pteridine ring in the catalytically active conformation and forms I and IIa with their pteridine rings turned over by 180 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)

Halothane (1% v/v inspired) was administered for 60 min to six children of mean age 74 months (range 14-119 months). Uptake of halothane was measured from the difference in the concentration in inspired and expired gas and varied from 176 to 310 mg kg-1, depending on minute ventilation. After administration of halothane ceased, its elimination in expired gas was measured in four patients until the conclusion of anaesthesia; 32-37% of the absorbed halothane was expired 90 min after halothane administration ceased. Urinary excretion of trifluoroacetic acid, fluoride and bromide was measured for up to 1 week. Of the absorbed halothane, 11.4% (range 6.3-18.2%) was excreted in urine as trifluoroacetic acid and 0.37% (range 0.10-0.64%) as inorganic fluoride. The urinary half-life of trifluoracetic acid was 41.8 h (range 10.4-59.1 h). The quantitative and qualitative metabolism of halothane via the reductive and oxidative pathways in children are comparable to values found in adults. No differences in the metabolism of halothane by children were found which would explain the different incidence of halothane-associated hepatitis compared with adults.

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An attempt was made in children to identify a urinary halothane-cysteine conjugate which had been described previously in adult patients following administration of halothane. If this conjugate was found it would indicate that a reductive metabolite of halothane binds covalently with the sulphydryl-containing amino acid, cysteine, a reaction which could lead to hepatic injury. The potential halothane-cysteine conjugate, N-acetyl-S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine (acetyl BCFEC), was prepared and the identity of the compound established using hydrogen-1 and carbon-13 NMR spectroscopy and methane chemical ionization mass spectrometry. A measurement technique for acetyl BCFEC was developed using HPLC with u.v. detection at 200 nm. In six children after halothane anaesthesia, one child being studied twice, urine was collected for up to 1 week and analysed for acetyl BCFEC. Little or no acetyl BCFEC was detected in any of the 43 urine samples tested, indicating that in children it is not a significant urinary metabolite of halothane.

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Relative rate constants for the Birch reduction (Li/liq. NH3/ButOH) of the three isomeric hexahydrodimethanonaphthalenes (3)-(5) and the octahydro analogues (10)-(13) were obtained and compared with those obtained for the reduction of norbornadiene and norbornene from an earlier study. Diene (5) was reduced almost 2000 times more rapidly than norbornene and 20000 times more rapidly than the monoene (13). Rate-enhancement factors for dienes (3) and (4) were less substantial but meaningful: 19 for (3) [compared with (10)] and 35 for (4) [compared with (12)]. These rate enhancements were attributed to the operation of π* orbital interactions through space in diene (5) and to the presence of π* orbital interactions through four bonds in dienes (3) and (4). The existence of a linear relationship between the natural logarithm of the rate of reduction of a substrate and its LUMO energy (obtained from either gas-phase electron affinities or ab initio MO calculations) supports this conclusion. The only-fair correlation of the above relationship was attributed to the neglect of other factors, such as the electronic structure and the geometry of the anion radical, which contribute to the overall rate of the Birch reduction. These two factors were explored by using PMO theory and ab initio MO calculations. In particular, full geometry optimizations (UHF, STO-3G basis set) on the anion radicals of norbornadiene (1) (C2v symmetry constraint) and norbornene (22) (Cs symmetry constraint) were carried out, and their geometries reported. Noteworthy is the strong pyramidalization of the olehic centres of (1) and (22) in the endo direction. These pyramidalizations explain the observed stereoselective exo protonation of the anion radical of (1), and also the much faster rate of reduction of (1) compared with (5), since the pyramidalization in the anion radical of (5) is such as to hinder protonation. The geometries of anion radicals appear to have a profound effect on rates, on stereoselectivity of protonation, and on the structures of the final products, and this is discussed in detail. The synthesis of the diene (3) is also described.

Several polyhydroxy- and polyacetoxy-porphyrins have been prepared and their properties studied as part of an investigation to find suitable porphyrins for use in the detection and/or treatment of malignant tumours. Low solubility in aqueous media has so far prevented a clear assessment of their biological properties.

Eight methyl N-(lH-benzimidazol-2-yl)carbamates with1 3 various 5-substituents were synthesized, each C-enriched at carbon 2, and the carbonyl and methoxy carbons. Five were prepared by cyclization involving the appropriate 4-substituted 1,2-diaminobenzene (CgHgcO-, CH3CH2CH20-, C H~C H Z C H~S -, CgHgS-and CH3CH2CH2CH2-), and methyl-C N-[imino(methylthio)methyl-Clcarbamate-C or methyl N,N'-bis(methoxy-C-carbonyl-C)carbamimidothionate-C. The latter were prepared from commercially available and thiourea. The remaining three (5-substituents: C6H5CH(OH)-, CgHgSO-and CH3CH2CH2SO-) were prepared by side-chain reduction or oxidation. The H-and C-NMR, and the methane CI mass spectral data of the products and intermediates are presented.