Band 4, Heft 1

As a result of the selective screening worldwide during the last 18 years, approximately 250 patients with tetrahydrobiopterin deficiency were discovered. Most patients suffer from 6-pyruvoyl tetrahydropterin synthase deficiency (58%), followed by dihydropteridine reductase deficiency (35%), GTP cyclohydrolase I deficiency (3%), and “primapterinuria” (4%). The patients can be treated with neurotransmitter precursors, as well as with tetrahydrobiopterin. However, data on long term treatment are still scarce and it is therefore of great value to investigate all newborns with even mild hyperphenylalaninemia. Cloning of the enzymes involved in the biosynthesis and regeneration of tetrahydrobiopterin makes them to be easily accessible for biochemical and biological studies. So far, all proteins expressed heterologous are active in E. coli. Cloning of the wild type gene and mutant analysis of patients allow the rapid identification of the defective gene on the molecular level.

A series of 1-methyl-pterin derivatives (6-13) have been synthesized by condensation and methylation reactions. The compounds have been characterized by elemental analyses, pKa deterimations and UV spectra. Comparisons of the physical data of the corresponding N-1 and N-3 methyl-pterins indicate why the normal pterins exist in aqueous solution preferentially in the N 3 -H tautomeric configuration. The thermodynamical stability of the lactam group forces the acidic H-atom to adapt the nitrogen adjacent to the carhonyl function but no other vinylogous position. A lactam group does not participate in heteroaromatic cyclic resonance, in general.

The proposed structure of Photinuspterin isolated from the firefly Photinus pyralis has been proven by two unambiguous syntheses to be 6-(1-hydroxypropyl)-8-methylisoxanthopteIin (15), 8-MethylisoxanthopteIin (7) can be substituted in 6-position in a homolytic nucleophilie substitution reaction either by the 1-hydroxypropyl radical to give 15 directly or by the propionyl radical leading first to 8-methyl-6-propionyl-isoxanthopteIin (9) which was easily converted into 15 by sodium borohydlide reduction. These two approaches are of general nature and can be applied to synthesize a broad variety of 6-substituted 8-methylisoxanthopterins (8-19). The newly synthesized compounds have been characterized by elemental analysis. pK a determinations, UV and NMR spectra.

In order to investigate the stabilities of different tautomeric forms of pteridine derivatives with a phenacetyl side chain in the 6- or 7-position, we have performed ab initio quantum chemical geometry optimizations of both the keto form and the vinylogous amide form of 6- and 7-phenacetyl pterin. The results are in accordance with experimental expectations: the keto form is slightly more stable for the 6-substituted derivative (1 .8 kcal per mol). while the vinylogous amide form is substantially more stable for the 7-substituted compound (8 .7 kcal per mol). The vinylogous amide forms are planar molecules, and this geometry enables the interaction between the hydrogen atom at N-5 (or N-8) and the carbonyl function of the side chain. Inspection of the calculated electron densities (population analysis) underlines the suggestion that the vinylogous amide form of 7-phenacetyl pterin is stabilized by a shift of electron density from the nitrogen bonded to C-2 to the carbonyl oxygen atom of the side chain (resonance stabilization via an additional highly conjugated vinylogous amide structure).

The absolute conformation and the reactivity of atoms of the neutral form of natural tetrahydrobiopterin [(6R)-6-(L-erythro-1 ′,2′ -dihydroxypropyl)-5.6.7.8-tetrahydropterin] was determined by the Molecular-Orbital method within the MINDO/3 framework. Two different conformations (Form A and Form B) of the alkyl side chain relative to the pterin ring were obtained as the optimized form for the lowest energy conformation (ΔH= -174 kcal/lmol) of this molecular constitution with the same molecular width of 11.2 Å. Form A and B were distinguishable in the dihedral [01′ -C1′ -C6-H6J of - 88° and 30°. respectively. Net charges of atoms of Form A and B suggested that the predominant reactivity of the atoms of N1 C2. C4. O4, C4a. and C8a of the pterin ring and C1′. C2′. O1′ and O2′ of the side chain. It was also suggested that the position 2' is more active than the position 1′. in the alkyl side chain of Form A and B. toward NADP+ in the reverse reaction of sepiapterin reductase.

Spectrofluorimetric studies of the properties of 8-alkyl-pterins arc reported, together with preliminary results on the use of fluorimetry in studying the binding of these new mechanism-based substrates to dihydrofolate reductase (DHFR). The compounds fluoresce strongly in the 460-490 nm range with emission maxima for the cations being blue-shifted by ~ 5-20 nm. Quantum yields for the cations of ~ 0.14-0.23 have been measured. pK a values for the basic ionization of ~ 5.3-5.7 have been determined from fits to both the emission and excitation spectra using sample concentrations in the 1-2 µM range. Although the UV /vis spectra between pH 0 and 4 show little change. there were significant sigmoidal changes in fluorescence intensity which could he fitted to apparent ionization constant values of ~ 1.4-1.0 with fluorescence intensity decaying to almost zero at pH 0. Studies of the fluorescence for the cations and neutral forms in organic acids and polar organic solvents respectively indicate little solvent dependence. Spectral modelling using the CNDO/S-CI method confirms that the spectra are due to π→π* transitions. and that the long wavelength absorption hand of ~ 400 nm is due to transitions involving mostly the pyrazine ring and N1. Significant differences in emission wavelength and intensity between 6-methyl- and 7 -methyl-8-alkyl-pterins have been characterised with the latter exhibiting fluorescence stronger by a factor of ~ 2 and blue-shifted by ~ 20 nm which arc readily detectable visually: aqua-blue and green fluorescence respectively for the 7-methyl and 6-methyl isomers. From this understanding of the fluorescence properties of 8-alkyl-pterins and their dependence on charge state and pyrazine-ring substituents, and taking into account the fluorescence characteristics of DHFR and cofactor NADP. we have assessed the potential of fluorimetric experiments for studying both pterin dissociation constants and the form of enzyme-bound pterin

A link between acute rheumatic fever (ARF) and autoimmune disorders has been postulated. To investigate this relationship. we performed radioimmunoassay measurements of serum neoplerin levels in patients with ARF. Twenty-five young men with ARF were included in our study. Patients were investigated on 3-7 days after the onset of symptoms and on the 30th-35th days after onset. Serum neopterin levels were elevated (>8.7 nmol/L) in 17 out of 25 patients with ARF. Mean neopterin concentration was significantly higher in ARF patients (10.9± 3.9 nmol/L) compared to healthy controls (5.2± 2.1 nmol/L; p<0.001). After treatment of patients .with antibiotics and indometacin, neopterin levels were normal in 16 of 23 patients. There was an association between higher neopterin concentration and development of aortic incompetence during follow-up (p<0.05). Also a significant correlation between neopterin and haemoglobin levels was observed (R= - 0.40. p = 0.(2). The increased neopterin levels indicate enhanced cellular immune activation in patients with ARF. The data suggest that serum neopterin might he a predictive marker of aortic valve lesions in patients with ARF.

Sepiapterin reductase, an enzyme that catalyses the synthesis of tetrahydrobiopterin (BH 4 ). was partially purified from Drosophila melanogaster using ammonium sulfate fractionation. Affi-gel blue chromatography and hydroxyapatite chromatography. The molecular weight of the enzyme determined by Ultrogel AcA44 column was 39,000. When the enzyme was subjected to polyacrylamide gel electrophoresis in SDS. a 38.000 MW species was found to be the major protein band. The Km values for sepiapterin and NADPH were determined to he 75.4 µM. and 14 µM, respectively. The optimal temperature and pH for the reaction were 30°C and pH 5.7-6.7. The half-life of the activity was 30 minutes when treated at 48°C The enzyme was markedly inhibited by tri-and tetravalent cations. Fe 3+ . Sn 4+ and divalent cation. Cd 2+ . It was found that pyrimidodiazcpine (a homopterin) and 2.4-diamino-6.7-diisopropyl caused the reduction of sepiapterin reductase activity by about 50% at the concentration of 0.1 mM. Among the neurotransmitters and their precursors tested. 3 mM concentrations of melatonin and N-acetylserotonin inhibited the enzyme activity completely. In addition to sepiapterin. the enzyme uses rather broad spectrum of carbonyl compounds as substrate including menadione. p-nitrohenzaldehyde, and various dicarhonyl compounds

This report describes the effects of the natural diastereoisomer of 5-formyltetrahydrofolate (6S)- and of the mixture of both the natural and unnatural diastereoisomers (6RS)-5-HCO-H 4 PteGlu (folinate) on the cytotoxicity of 5-fluoro-2′-deoxyuridine (FdUrd) and 5-fluorouracil (FUra) in MCF-7 and MDA-MB 231. human breast cancer cell lines. Continuous exposure of exponentially growing cells to various concentrations of FUra for 72 h caused a 50% inhibition of growth with lC 50 at concentrations of 3.40± 0.16 and 29± 1.40 11M in MCF-7 and MDA-MB 231. respectively. Preincubation of the cells for 24 h with (6S)-5-CHO-H 4 PteGlu (5 µM ) prior to addition of FUra decreased the IC 50 values by 2.4-2.6-fold in both cell lines. A quantitatively similar effect was achieved by preincubation with ]() µM of (6RS)-5-HCO-H 4 PteGlu. The cytotoxicity of FdUrd was markedly more pronounced than that of FUra exhibiting IC 50 , values at a range of 0.045- 0.050 µM for both cell lines. Preincubation with (6S)-5-HCO-H 4 PteGlu increased the susceptibility to the drug by about 3.5-fold. The optimal dose of (6S)-5-CHO-H 4 PteGlu for achieving the lowest IC 50 value for FdURd in MCF-7 cells was 5 µM. whereas that in MDA-MB 231 was 20 µM. Similar increase of the cytotoxicity of FdUrd was obtained with about a 2-fold higher concentration of (6RS)-5-HCO-H 4 PteGlu. Cells pretreated lor 24 h with FUra or FdUrd respectively. exhibited enhanced incorporation of [ 3 H] thymidine into the TCA-precipitable fraction . Presence of (6S)-5-HCO-H 4 PteGlu (10 µM) doubled the amount of incorporated thymidine. which reflects depletion of intracellular thymidylate pools due to enhanced formation and stabilization of the complex formed between thymidylate synthase. flouorodeoxydylate and 5.1.0-methylenetetrahydrofolate.

The regulation of methionine synthase. serine hydroxymethyltransferase. methylenetetrahydrofolate oxidorelluctase and dihydrofolate reductase in Aspergillus nidulans have been found to be controlled by endogenous pools methionine and homocysteine. Mutants impaired in sulfur metaholite repression. which have highly elevated pools of these amino acids. also show elevated levels of folate metabolizing enzymes mentioned above. This effect was found to he the result of the accumulation of endogenous homocysteine. which induces folate enzymes. High concentration (up to 5 mM) or methionine in growth medium leads to repression of these enzymes. It appears. therefore. that the levels of folate metaholizing enzymes arc determined by the ratio of cellular levels or methionine and homocysteine.