On the action of sulfanilamide

Summary InE. coli, sulfanilic acid, sulfanilamide, sulfapyridine, sulfapyrimidine and sulfathiazol are antagonized by the same series of non competitive antagonists,viz., methionine, xanthine, serine, thymine and valine. This seems to indicate that the biosynthesis of these substances is successively inhibited by increasing concentrations of these sulfadrugs; the synthesis of methionine being inhibited first, that of valine only by excessive concentrations. Though the absolute concentrations vary with the drug the relative sensitivity of the five enzyme systems are very much the same. This again shows that the intrinsic toxicity of the sulfadrugs is the same.I: Ann. de l'Inst. Pasteur62, 616, 1939. II: Antonie van Leeuwenhoek7, 25, 1941. III: Ibid.7, 77, 1941. IV: Ibid7, 153, 1941. V: Ibid.7, 161, 1941. VI: Ibid.8, 10, 1942. VII: Ibid.8, 86, 1942. VIII: Ibid.8, 139, 1942. IX: Ibid.9, 115, 1943. X: Ibid.10, 1, 1944–1945. XI: Arch. of Biochemistry18. 97, 1948.     

Non-competitive antagonists for derivatives of p-aminobenzoic acid

Summary Shive's method of “inhibition analysis” was applied to the growth inhibition ofE. coli by some derivatives of p-aminobenzoic acid. In accordance with the findings ofWinkler andde Haan methionine, xanthine, serine, thymine, and valine were shown to be non-competitive antagonists of sulfanilamide forE. coli strain Bray β. It is highly probable that for this bacterium glycine should be added to this series. Methionine is the only non-competitive antagonist of the 2-chloro, 2-bromo, and 2-amino derivatives of P.A.B., provided their action is investigated at no higher concentrations than can be antagonised by P.A.B. The same complete series of non-competitive antagonists of sulfanilamide was shown to be active against the bacteriostatic action of the 2-methyl, 3-methyl, and 3-amino derivatives of P.A.B. The hypothesis is proposed that these derivatives are not competing directly with P.A.B. for an essential enzyme but that they are used by the bacterium in the formation of derivatives of pteroylglutamic acid; these may be competitive antagonists of co-enzymes, related to pteroylglutamic acid. There is some evidence in favour of this supposition. Further investigations are needed, however, before any judgment regarding its value can be given.     

Induced Resistance to Sulfonamides in Chilomonas paramecium

SUMMARY. Strains of Chilomonas paramecium differing in degree of resistance to sulfanilamide have been established through acclimatization to this sulfonamide at 50, 100 and 200 mg. %. Resistant strains differ from the normal stock in their enhanced sensitivity to p -aminobenzoic acid. In the normal stock, sulfanilamide inhibition is reversed at an SA/PABA ratio of 10,000 but not at 20,000; in the least resistant strain, at a ratio of 400,000 but not at 800,000. In resistant strains inhibition is reversed by folk acid, methionine, adenine, cytosine and thymine; in the normal stock, none of these metabolites produces reversal. In high concentrations of PABA (10–20 mg. %) growth of the normal stock is only retarded, whereas the strain least resistant to sulfanilamide fails to recover from exposure to 20 mg. % PABA. The strain most resistant to sulfanilamide is most susceptible to PABA in high concentrations. The data suggest that resistance to sulfanilamide in C. paramecium may depend mainly upon an accelerated synthesis of PABA.     

Sensitivity of Cultured Tissue of Arabidopsis thaliana Races to Sulfanilamide

Callus of Arabidopsis thaliana (L.) Heynh. was examined for the kinetics of sulfanilamide inhibition of growth. Two geographic races of this species were compared for relative sulfanilamide sensitivity (as assessed by dry weight increase on drug-containing medium) and rate of drug uptake. It was found that low concentrations of sulfanilamide in the culture medium (～ 100 μM) inhibited callus growth and that low concentrations of p-aminobenzoate (20 μM) competitively antagonized the sulfanilamide effect. Racial variation for sulfanilamide sensitivity was demonstrated. A partially drug-resistant race had greater sulfanilamide uptake (per gram fresh weight) than a sensitive race. suggesting that relative drug-sensitivity was not determined by the capacity to exclude sulfanilamide.     

SULFONAMIDE INHIBITION IN MONOCHRYSIS LUTHERP1

The effect of sulfonamides on growth of the chry-somonad, Monochrysis lutheri, in a synthetic sea-water medium was examined over a period of 14 days. The population increased at all sulfonamide concentrations during the first several days of incubation before inhibition became apparent. Inhibitory concentrations ranged from 0.01 to 1.0 mg%. Inhibition luas most pronounced in sulfathiazole; sulfamethazine, sulfapyridine, and sulfanilamide followed in decreasing order. p-Arninobenzoic acid (0.001-1.0 mg%) competitively reversed inhibition. Folic acid, thymine, adenine, and vitamin B₁₂ neither reversed the inhibition nor spared the requirement for p-aminobenzoic acid. The significance of the inhibition pattern and the potential use of antimetabolites in the marine environment were discussed.     

Insight on specificity of uracil permeases of the NAT/NCS2 family from analysis of the transporter encoded in the pyrimidine utilization operon of Escherichia coli

The uracil permease UraA of Escherichia coli is a structurally known prototype for the ubiquitous Nucleobase‐Ascorbate Transporter (NAT) or Nucleobase‐Cation Symporter‐2 (NCS2) family and represents a well‐defined subgroup of bacterial homologs that remain functionally unstudied. Here, we analyze four of these homologs, including RutG of E. coli which shares 35% identity with UraA and is encoded in the catabolic rut (pyr imidine ut ilization) operon. Using amplified expression in E. coli K‐12, we show that RutG is a high‐affinity permease for uracil, thymine and, at low efficiency, xanthine and recognizes also 5‐fluorouracil and oxypurinol. In contrast, UraA and the homologs from Acinetobacter calcoaceticus and Aeromonas veronii are permeases specific for uracil and 5‐fluorouracil. Molecular docking indicates that thymine is hindered from binding to UraA by a highly conserved Phe residue which is absent in RutG. Site‐directed replacement of this Phe with Ala in the three uracil‐specific homologs allows high‐affinity recognition and/or transport of thymine, emulating the RutG profile. Furthermore, all RutG orthologs from enterobacteria retain an Ala at this position, implying that they can use both uracil and thymine and, possibly, xanthine as substrates and provide the bacterial cell with a range of catabolizable nucleobases.     

A mutation defective in the xanthine alternative pathway of Aspergillus nidulans: its use to investigate the specificity of uaY mediated induction.

Summary In Aspergillus nidulans uric acid can be produced from xanthine via purine hydroxylase I (xanthine dehydrogenase) or via the xanthine alternative pathway (Darlington and Scazzocchio, Biochem. Biophys. Acta, 166, 569–571; 1968). A mutation defective in the xanthine alternative pathway of Aspergillus nidulans is described. By combining this mutation with hxB-20 which results in complete loss of purine hydroxylase I and II activities, but which conserves cross-reacting material, it is possible to block completely uric acid production and thus investigate which are the effective in vivo inducers of three enzymes under the control of the positive regulatory gene uaY: adenine deaminase, purine hydroxylase I (measured as cross-reacting material) and urate oxidase. It is concluded that uric acid is the only effective physiological inducer, while its 2 and 8 thio-analogues serve as gratuitous inducers.     

Re-evaluation of superoxide scavenging capacity of xanthohumol

Abstract

The chemopreventive chalcone xanthohumol (Xh) has been reported to decrease xanthine oxidase (XOD) catalysed formation of formazan from nitroblue tetrazolium (NBT) and is discussed as a potent scavenger of superoxide. Re-evaluation of the scavenging capacity indicated that Xh disturbed detection of superoxide with NBT, in case of an insufficient NBT/Xh ratio. Xh lacked superoxide scavenging activity in contrast to the Xh-derivative 3′-hydroxy-Xh with catechol substructure, used as positive control. This was shown by the use of sufficient concentration of NBT and other detectors such as hydroxylamine, XTT, cytochrome c and hydroethidine. HPLC analysis of reaction products in a xanthine/XOD/peroxidase system demonstrated beside enhanced inhibition of NBT-formazan by Xh that NBT even prevented oxidation of Xh. p-coumaric acid or ferulic acid could replace Xh in that system, indicating that superoxide detection using NBT is likely jeopardized by interference of phenoxyl-radicals. Furthermore, this study provides evidence that Xh can moderately generate superoxide via auto-oxidation.     

Synthesis of New 5″-Sulfonylamido Derivatives of 3″-Azido-3″-Deoxythymidine (AZT)

Abstract

A series of 5′-N-methanesulfonyl derivatives of 3′-azido-5′-(alkylamino)-3′,5′-dideoxythymidine was synthesised. The first step of the synthesis involved the reaction of 1-(2,5-dideoxy-5-O-tosyl-β-D-threo-pentofuranosyl)thymine 1 with an appropriate amine to give 1-[5-(alkylamino)-2,5-dideoxy-β-D-threo-pentofuranosyl]thymines 2a-e and 1-(2,5-dideoxy-β-threo-pent-4-enofuranosyl)thymine 3 as a by-product. Compounds 2a-e were treated with an excess of methanesulfonyl chloride to yield intermediates 1-[5-(dimethylamino)-3-O-methanesulfonyl-2,3,5-trideoxy-β-D-threo-pentofuranosyl]-thymine 4a and 1-[5-(N-alkyl-N-methanesulfonyl)-3-O-methanesulfonyl-2,3,5-trideoxy-β-D-threo-penfuranosyl]thymines 4b-e. The reaction of 4a-e with lithium azide in dimethyl-formamide afforded the final compounds 1-[3-azido-5-(N-methyl-N-methanesulfonyl)-2,3,5-trideoxy-β-D-erythro-penofuranosyl]thymine 5a and 1-[3-azido-5-(N-alkyl-N-methanesulfonyl)-2,3,5-trideoxy-β-D-erythro-penofuranosyl]thymines 5b-e. The independent synthesis of 4′,5′-unsaturated product 3 was also described.     

Elimination of overgrowth in delayed-incubation membrane filter test for total coliforms by m-ST holding medium.

Two holding media were compared for their effects on total coliform recovery by the delayed-incubation membrane filter procedure. LES-MF holding medium contains tryptone, m-Endo broth, dipotassium hydrogen phosphate, sodium benzoate, sulfanilamide, para-aminobenzoic acid, and cycloheximide (pH 7.0). m-ST holding medium contains ethanol, sodium monophosphate, dipotassium hydrogen phosphate, sulfanilamide, and Tris (pH 8.6). In tests with natural water and wastewater samples from various sources, recovery with LES-MF and m-ST were similar after a 1-day holding period. With LES-MF, however, after a 2- or 3-day holding period, coliform bacteria frequently were partially or totally overgrown by noncoliforms, causing significant reductions in coliform counts. No significant overgrowth was observed with m-ST. We propose that m-ST be used for all holding periods longer than 1 day.     

Elimination of overgrowth in delayed-incubation membrane filter test for total coliforms by m-ST holding medium

Two holding media were compared for their effects on total coliform recovery by the delayed-incubation membrane filter procedure. LES-MF holding medium contains tryptone, m-Endo broth, dipotassium hydrogen phosphate, sodium benzoate, sulfanilamide, para-aminobenzoic acid, and cycloheximide (pH 7.0). m-ST holding medium contains ethanol, sodium monophosphate, dipotassium hydrogen phosphate, sulfanilamide, and Tris (pH 8.6). In tests with natural water and wastewater samples from various sources, recovery with LES-MF and m-ST were similar after a 1-day holding period. With LES-MF, however, after a 2- or 3-day holding period, coliform bacteria frequently were partially or totally overgrown by noncoliforms, causing significant reductions in coliform counts. No significant overgrowth was observed with m-ST. We propose that m-ST be used for all holding periods longer than 1 day.     

On the action of sulfanilamide

Summary The better activity (in vitro) of various sulfanilamide compounds as compared with sulfanilamide itself is only quantitative,i.e., an equal activity is obtained with lower concentrations. It is shown, that the activity of the studied drugs is so narrowly related to their adsorption in the bacteria (B. coli), that probably the varying activity of the studied compounds is due to differences in adsorbability. For different drugs the adsorbed amount was equal for concentrations with equal activity.The concentration of p. aminobenzoic acid which re-establishes growth — in cultures containing the studied compounds in concentrations of equal activity — was equal in all cases. This fact corroborates the hypothesis, that activity and adsorption are correlated and shows, that the mechanism of action (in vitro) is the same in all cases.Ninth communication: K. C.Winkler, Antonie van Leeuwenhoek9, 115, 1943.     

Chlorination studies. IV. The reaction of aqueous hypochlorous acid with pyrimidine and purine bases

The labile intermediates and stable end products formed by the reaction of aqueous HOCl with thymine, uracil, 5-Br-uracil, N,N-dimethyluracil and 6-methyluracil have been identified. The purine ring system of guanine, adenine and xanthine was more resistant to attack by aqueous HOCl and reaction times of one week resulted in the formation of parabanic acid. Caffeine and theophylline under similar reaction conditions yielded N,N-dimethylparabanic acid.     

Enzymatic Synthesis of 5-Methyluridine from Adenosine and Thymine with High Efficiency

5-Methyluridine (5MU) was synthesized efficiently from adenosine, thymine, and phosphate by a combination of adenosine deaminase (ADA), purine nucleoside phosphorylase (PUNP), pyrimidine nucleoside phosphorylase (PYNP), and xanthine oxidase (XOD). Adenosine was converted into inosine first by ADA. 5MU and hypoxanthine were synthesized from inosine and thymine by PUNP and PYNP. The hypoxanthine formed was converted into urate via xanthine by XOD. After inosine was completely consumed, an equilibrium state, in which 5MU, thymine, ribose-1-phosphate, and phosphate were involved, was achieved. At the equilibrium state, the maximum yield of 5MU was obtained. The yield of 5MU was 74%, when the initial concentrations of adenosine, thymine, and phosphate were 5 mM each. On the other hand, in the absence of ADA or XOD the yield of 5MU was 1.8%. Several kinds of nucleosides were also synthesized with high yield by the same method.     

3,5,2',4'-Tetrahydroxychalcone, a new non-purine xanthine oxidase inhibitor

Xanthine oxidase is a key enzyme that catalyses hypoxanthine and xanthine to uric acid and the overproduction of uric acid will lead to hyperuricemia which is an important cause of gout. In the present study, three chalcone derivatives were synthesized and evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds, only Compound 1, 3,5,2′,4′-tetrahydroxychalcone, exhibited a significant inhibitory activity on xanthine oxidase with an IC₅₀ value of 22.5 μM. Lineweaver–Burk transformation of the inhibition kinetics data demonstrated that it was a competitive inhibitor of xanthine oxidase and Ki value was 17.4 μM. In vivo, intragastric administration of Compound 1 was able to significantly reduce serum uric acid levels and inhibited hepatic xanthine oxidase activities of hyperuricemic mice in a dose-dependent manner. Acute toxicity study in mice showed that Compound 1 was very safe at a dose of up to 5 g/kg. These results suggest that Compound 1 is a novel competitive xanthine oxidase inhibitor and is worthy of further development.     

Reactions of Some 2,3-Anhydro Pyrimidine Nucleosides with Dilithium Tetrahalocuprates

Abstract

The reaction of 1-(2,3-anhydro-5-0-trityl-β-D-lyxofuranosyl)-2-0-methyluracil (2a) and its thymine analogue (2b) with dilithium tetrahalocuprates (Li₂CuX₄) revealed an excellent to perfect regioselectivity, yielding 2,2′-anhydro-3′-halonucleosides (3a-d), while the same reactions with 2,3-anhdro uracil and thymine nucleosides (5a,b) gave arabinosyl (6a-d) and xylosyl halohydrins (7a-d) with respective product ratios of 7:3 to 8:2 which were estimated after mesylation to 8a-d and 9a-d.     

Molecular Modeling Study on Dapsone and Sulfonamides Comparing Structures and Properties with Respect to Anti-Leprosy Activity

Despite the very close structural relationship between dapsone (4,4′-diaminodiphenyl sulfone, 4,4′ sulphonyldianiline, diaphenyl sulphone, DDS) and sulfanilamide (p-aminobenzene sulfonamide), being the prototype of all other sulfonamides, only dapsone shows remarkable efficient pharmacological activity against Mycobacterium leprae. Cells of certain micro-organism need para-aminobenzoic acid (PABA), the latter playing the role of natural substrate to biosynthesis of folic acid. Sufones and sulfonamides show competitive antagonism as chemical analogs of PABA. It is most surprising that, despite of sharing this molecular mechanism, only dapsone shows anti-leprosy activity in vivo. The study was accomplished using molecular mechanics (SYBYL) and semiempirical methods (MOPAC). The calculations of aromaticity, charges, protonation by MOPAC, and of lipophilicity by our empirical program LIPOP(hilicity) give evidence that dapsone is more lipophilic (log P values 0.97) than sulfanilamide (-0.67). The extremely lipophilic cell wall of Mycobacterium leprae contributes to the surprising difference in anti-leprosy activity. Sulfonamides are more or less deprotonated (45 to 99 %) at physiological pH units, whereas dapsone is totally undissociated. This results in different permeability rates into the bacterial cells in vivo. Compared to other sulfones and sulfonamides, the unique combination of high lipophilicity and low ionic dissociation favors anti-leprotic potency in dapsone. On principle, amide groups do not hinder activity, but cause acidity and subsequently dissociation.     

On the action of sulfanilamide

Summary Studying the action of sulfanilamide on bacterial nitrogen metabolism, it was shown that: a. Sulfanilamide does not alter the rate of gelatin-hydrolysis by papain or by the proteinase ofB. pyocyaneum andB. prodigiosum. b. Sulfanilamide does not influence the synthesis of aspartic acid from fumaric acid and ammonium chloride by restingB. coli. c. Addition of single amino acids does not counteract sulfanilamide. d. Addition of single amino acids merely accelerates growth slightly; a marked acceleration was obtained only by adding various amino acids simultaneously. e. The addition of such an optimal mixture of amino acids did not exert any influence on the action of sulfanilamide on growth. As the growth acceleration shows that the bacteria are saved an important output of energy in synthesis as a result of the supply of the amino acids, we conclude that sulfanilamide action cannot be due to interference with the synthesis of amino acids from inorganic nitrogen (f.i. NH₂ + pyruvate).Considering these facts, we expect sulfanilamide to pursuit its action on bacterial growth by interfering with protein anabolism, anywhere in the synthesis of protein from amino acids.     

Synthesis of 4-Deoxy-4-C-hydroxymethyl-α-L-Lyxo-Pyranosyl Thymine

Abstract

The synthesis of 1-[4-deoxy-4-C-hydroxymethyl-α-L-lyxopyranosyl]thymine has been accomplished by two synthetic routes both starting from methyl 2, 3-O-isopropylidene-β-D-ribopyranoside. The first route makes use of a ring opening, ring closure reaction sequence to increase the proportion of the desired L-isomers. The second route utilizes the soft nucleophilic character of malonyl anions and ozonolytic cleavage of enol ether to introduce the branched chain. The newly obtained pyranosyl nucleoside obtains a ⁴C₁ conformation with an equatorially oriented thymine moiety.     

On the action of sulfanilamide

Summary A sulfanilamide activating principle was found to be present in red cells of the horse. This activator substance is active in the rather high dilution of 0.5% haemolysed red cells.The substance or substances are present in the red cells, not in their cell membranes. They seem to be of a protein nature or adsorbed to the protein (haemoglobin).In some media no sulfanilamide action is obtained without the activator. In other media sulfanilamide action, though clearly present, is markedly enhanced. So it must be emphasized, that the substance under discussion is an activator and not a conditio sine qua non for the sulfanilamide action and its characteristics.The substance is activating sulfanilamide against streptococci, staphylococci andB. coli.The substance is not present in human blood or in the red cells of sheep, rabbits or mice.Sixth communication: K. C.Winkler, Antonie van Leeuwenhoek8, 10, 1942.