Dec. 5, 1957
RELATION OF STRUCTURE TO
PROPERTIES OF
DIPHOSPHOPYRIDINE NUCLEOTIDE 6173
[CONTRIBUTION FROM THE MCCOLLUM-FRATT INSTITUTE,JOHNS HOPKINSUNIVERSITYAND THE NATIONAL INSTITUTE OF NEUROLOGICAL DISEASESAND BLINDNESS, NATIONAL INSTITUTES OF HEALTH,PUBLIC HEALTHSERVICE,DEPARTMENT OF HEALTH,EDUCATION AND WELFARE]
Relationship of Structure to Properties of Diphosphopyridine Nucleotide and Other Pyridinium Compounds BY MARVINR. LAMBORG,~ ROBERTMAINBURTONAND NATHAN0. KAPLAN RECEIVED JANUARY 26,1957 A number of 3- or 4-substituted 1-methylpyridinium compounds were synthesized as model systems for studying t h e chemical reactions of diphosphopyridine nucleotide.8 These compounds were classsed according to their ability to undergo the following reactions: (1) reaction with aqueous potassium cyanide, (2) reaction with alcoholic potassium cyanide, (3) chemical reduction by sodium hydrosulfite and (4) oxidation by purified rabbit liver aldehyde oxidase. I t was shown that the cyanide addition reaction was, in several cases, strongly dependent upon the dielectric constant of the solvent, suggesting a bimolecular nucleophilic addition reaction. Spectrophotometric evidence based on the cyanide addition reaction is presented to indicate a physical and chemical function for the ARPPR moiety of DPN. Methods.-All 1-methylpyridinium iodide compounds Introduction studied were synthesized in the following general manner: 1-Alkyl pyridinium compounds have been exten- 0.02 mole of the pyridine compound was dissolved in 5 to 10 sively employed as models for the study of the ml. of methanol (alternatively benzene), 0.03 mole of methyl chemical properties of DPN. Karrer, et aZ.14,6aiodide was added and the mixture was refluxed for 5-10 Recrystallization was effected from hot methanol and Rafter and Colowick5bstudied the reduction of hours. after treatment with animal charcoal. some of these compounds by sodium hydrosulfite. Aqueous cyanide addition reactions were carried out acMeyerhof, et al.,e Colowick, et al.,7and San Pietros cording to the method of Colowick, et aL7 Alcoholic cyaanalyzed the potassium cyanide addition reaction nide addition reactions were carried out in the same manner that methanol replaced water as solvents. Themethand KnOxg and Hurwitz'O have investigated the except anol was saturated with potassium cyanide (approx. 0.7 M oxidation of 1-methyl-3-carbamoylpyridiniumio- a t 25'). dide (1-methylnicotinamide iodide) by an aldeReductions of the pyridinium compounds were carried hyde oxidase obtained from rabbit liver. This out in sodium carbonate solution with sodium dithionite (hydrosulfite) as described by Karrer and Blumerla except paper presents data with 1-methylpyridinium com- that extracting solvent, chloroform, was not removed pounds, DPN and D P N derivatives in the three prior the to recording the spectrum of these compounds. This type reactions, which permits some evaluation of precaution was taken since it was found that one of these the physical and chemical effects of the carbamoyl compounds, trigonelline (1-methylnicotinic acid), was particularly labile, yielding a product with an absorption maxiand ARPPR groups in the DPN molecule. mum at 290 mfi.
Experimental Materials.-Various pyridine derivatives were purchased from Distillation Products Industries, Aldrich Chemical Co. and Nutritional Biochemicals Corp. 1-Methylnicotinic acid (trigonelline) was purchased from Nutritional Biochemicals Corp. Methyl iodide was purchased from Fischer Scientific Co. and redistilled prior to use. Aldehyde oxidase was prepared according to the method of Hurwitz.Io Twice recrystallized yeast alcohol dehydrogenase was obtaiiied from Worthington Biochemical Corp. This was diluted 1/26 (v./v.) with potassium phosphate buffer (0.1 M ; p H 7 . 5 ) . APDPN was prepared according to the method of Kaplan and Ciotti." (1) Contribution KO.189 of the McCollum-Pratt Institute. This investigation was aided by grants from the American Cancer Society, as recommended by the Committee on Growth of the National Research Council and the National Cancer Institute, National Institutes of Health (Grant No. '2-2374). A preliminary report of this work has been presented a t the 130th meeting of the American Chemical Society, September, 1956, Atlantic City, New Jersey. (2) Predoctoral Fellow of the National Cancer Institute, National Institutes of Health, U. S. Public Health Service, 1956-1957. (3) The following abbreviations are used: D P N , diphosphopyridine nucleotide; ARPPR, the non-nicotinamide moiety of DPN, adenosinediphosphate ribosyl; NR, nicotinamide riboside; N M N , nicotinamide mononucleotide; APDPN, the 3-acetylpyridine analog of DPN. (4) (a) P. Karrer and 0. Warburg, Biochem. Z . , 286, 297 (1936); (b) P. Karrer, G. Schwarzenbach, F. Benz and U. Solmssen, Hclv. Chim. A c t a , 19, 811 (1936). (5) (a) P. Karrer and F. J. Stare, ibid., 20, 418 (1937); (b) G. W. Rafter and S. P. Colowick, J. Biol. Chem., 209, 773 (1954). (6) 0. Meyerhof, P. Ohlmeyer and W. Mohle, Biochem. Z.,297, 113 (1938). (7) S. P. Colowick, N. 0. Kaplan and M. M. Ciotti, J. Biol. Chem., 191, 447 (1951). (8) A. San Pietro, ibid , 217, 579 (1955). ('3) W. E. Knox, ibid., 163, 699 (1946). (10) J. Hurwitz, i b i d . , 212, 757 (1955). (11) N. 0. Kaplnn and M. M. Ciotti. i b i d . . 221, 823 (1950).
Oxidation of the pyridinium compounds by the aldehyde oxidase was measured by the reduction of 2,6-dichloroindophenol a t 610 mp. The oxidations were carried out in air. Corrections were made for non-enzymatic chemical oxidation of the dye. All spectrophotometric measurements were made with the models DU or DK-2 Beckman spectrophotometers using 3-ml. quartz cells with a light path of 1 cm. The Beckman model G pH meter was used for all pH nieasurements.
Results and Discussion A compilation of the 1-methylpyridinium iodide compounds studied is listed in Table 11. It can be seen from the data that most of these compounds fit in one of three general categories. I. Compounds which are capable of aqueous potassium cyanide addition reactions, sodium hydrosulfite reduction and oxidation by aldehyde oxidase. 11. Compounds which will not give an aqueous cyanide addition product but will form an addition product with alcoholic potassium cyanide. This group of compounds can in general be reduced and they are poorlyoxidized.l3 111. Compounds which will not give a cyanide addition reaction, cannot be reduced b y the method employed and are poorly oxidized. 1-Methylnicotinamide iodide and the monosubstituted amides undergo only partial aqueous cyanide addition. As expected from the inductive effect of the alkyl group the substituted amides yielded less addition product a t equilibrium than 1(12) P. Karrer and F. Blumer, Haiv. Chim. Acta, 30, 1157 (1947). (13) 1-Methyl-3-butoxycarbonylpyridinium iodide is an unexplained exceptiou to this generalization.
MARVINR.LAMBORG, ROBERTM. BURTON AND NATHAN 0. KAPLAN
6174
VOl. 79
TABLE I ANALYTICAL DATA R--..R~-QN+-cH~ Ic
R
I
