New Method Aids Nucleotide Synthesis Coenzyme synthesis shows value of alkoxyvinyl phosphates as good phosphorylating agents 141ST
ACS
NATIONAL
MEETING
Organic Chemistry
A phosphorylating technique developed at Yale University offers chemists a new route to nucleotides. As an example of how valuable the method can be, Yale's Dr. Harry Wasserman and Dr. David Cohen have used it to make the coenzyme, flavin adenine dinucleotide. The method has grown out of the reaction of alkoxy acetylenes with two moles of a carboxylic acid. The products in this reaction are normally the anhydride of the acid and alkyl acetate. But Dr. Wasserman and Dr. Peter Wharton found a few years ago that they could control the reaction so as to stop it at an intermediate stage —reacting just one molecule of the acid. Thus, they were able to prepare 1-alkoxyvinyl esters of carboxylic acids. The 1-alkoxyvinyl esters are very reactive toward nucleophilic reagents. Extending the reaction to phosphoric acid esters, Dr. Wasserman and Dr. Cohen reacted diesters of phosphoric acid with ethoxy acetylene. The free acid group of the diester added to the triple bond of the acetylene in the same way as the carboxylic acids did. With the diphenyl ester of phosphoric acid, the product is diphenyl 1-ethoxyvinyl phosphate, which can be isolated and stored at low temperatures. The dibenzyl analog, on the other hand, is less stable and more difficult to isolate. IR Helps. The alkoxyvinyl phosphates show a characteristic infrared absorption in the 5- to 6-micron range. Both the diphenyl and dibenzyl esters have sharp peaks at 5.74 and at 5.95 microns. These typical bands are useful in showing the presence of alkoxyvinyl phosphates when they cannot readily be isolated in the pure state. The IR bands are handy also in following the reaction of alkoxyvinyl phosphates with various nucleophilic compounds. In reactions with methanol, phenol, thymidine, diesters of phosphoric acid, cyclohexylamine, and
benzoic acid, the intensity of the peaks decreases, depending on how much of the nucleophile is added. In these reactions, the alkoxyvinyl phosphate acts as a phosphorylating agent, transferring the diester phosphate group from the alkoxyvinyl fragment to the nucleophile. And it is this property that makes these compounds so useful. Dr. Wasserman and Dr. Cohen find that a monoester of phosphoric acid will also add to ethoxyacetylene to produce active phosphorylating agents. Adenosine-5' phosphate as the pyridinium or triethylammonium salt reacts to give 1-ethoxyvinyl adenosine-5' phosphate. This product was not isolated, but in methanol solution it slowly yielded the methyl ester of adenosine monophosphate. Next,
they made a dithymidine phosphate, showing that the method can link nucleotides. Now Dr. Wasserman and his coworker were ready to try forming the pyrophosphate linkage of a dinucleotide. The procedure was to react one nucleoside phosphate with ethoxyacetylene, as before, and then to react the product (without isolating it) with a second, different phosphate. By using adenosine-5' phosphate in the first stage and riboflavin^ phosphate in the second, they synthesized flavin adenine dinucleotide ( F A D ) . The method avoids the formation of by-products such as riboflavin--^,5r cyclic phosphate and needs no protecting groups on the reactants. The yields of FAD were between 10 and 15%. From the work done so far, Dr. Wasserman concludes that ethoxyacetylene is a versatile reagent for activating mono- and dialkyl phosphates. Its use in nucleotide synthesis is an important new tool in a very active field.
OH i OssfJ-o-C+L j
©if
W i
* _
Adenine
+fC = C~0£t Dimethylsulfsxidc
AtJenasine—5' Phosphate
Adenine
FLAVIN ADENINE DINUCLEOTIDE
PHOSPHORYLATING. Reaction sequence illustrates new phosphorylating technique used by Yale chemists to make the coenzyme, flavin adenine dinucleotide APRIL
9, 1962
C&EN
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Ferrocene Gives Three Diaryl Derivatives 141ST
ACS
NATIONAL
MEETING
Organic Chemistry
The reaction of ferrocene with aryldiazonium salts produces all three of the possible diaiylferrocenes, rather than just the l,l'-diaryl derivative previously reported. Moreover, the simple free radical mechanism usually proposed for the reaction is very likely not correct, says Dr. Myron Rosenblum of Brandeis University. Instead, he proposes one involving the internal rearrangement of a ferrocene-diazonium salt complex. Dr. Rosenblum and co-workers—Dr. W. G. Howells, Dr. A. K. Banerjee, and C. Bennett—prepared the monoaryl and the 1,1'-, 1,2-, and 1,3-diaryl derivatives of ferrocene. In the present work, they reacted ferrocene with phenyl, p-methoxyphenyl, or p-acetylphenyl diazonium salts. They separated the various isomers by careful chromatography on alumina columns and proved the structures by a combination of infrared data and synthesis. In the chromatography, they noted that the relative adsorption of the isomeric diaiylferrocenes is 1,2 —'CH2CH2CH2CH2—CH2CH2- -j~ CH2 = CHCH3 —> CH3
I CH3
I
—CH2CH- + CH2 = CHCH 5 -* CH3
1
Olefins Copolymerize By Free Radicals
CH3
1
—CH2CHGH2CHCH3
I
—CH3—CH- -j- CH2 = CH2 —>• CH3
I
—CH,—CH—CH2— CH,141ST
ACS
NATIONAL
MEETING
Polymer Chemistry
Copolymers of ethylene and a-olefins have been made by free radical copolymerization by Dr. George A. Mortimer, Luigi Boghetich, and Greg W. Daves of Monsanto's plastics division at Texas City, Tex. Although ethylene alone polymerizes this way in commercial
Other things being equal, as the concentration of the a-olefins increases, the over-all rate of polymerization decreases. This effect is less pronounced at 220° C. than at 130° C. Also, the polymer molecular weight decreases, and both short-chain branching and unsaturation increase as the olefin concentration is increased. All these effects are more pronounced With butene and octene than with propylene.
Sulfur Analogs of Inositol Prepared 141ST
ACS
NATIONAL
MEETING
Carbohydrate Chemistry
The first sulfur analogs of inositol have been prepared and characterized by Dr. G. E. McCasland, Dr. Arthur Furst, and Stanley Furuta at the Institute of Chemical Biology, University of San Francisco. They have made two cyclohexanetetroldithiols in a sevenstep synthesis starting with quebrachitol from rubber latex. The interest in derivatives of inositol —or specifically r?i J/o-inositol (one of the eight diastereoisomers that occur in muscle tissue)—stems from the fact that for many years it was classified as a vitamin. More recently, however, evidence shows higher animals can synthesize mt/o-inositol, and its classification as a vitamin became doubtful. Then, in 1957, Dr. Harry Eagle at the National Institutes of Health showed that 7??j/o-inositol is essential for growing both normal and malignant human cells in tissue culture. This opened up the possibility that inositol derivatives could be found which would be lethal to malignant cells while being tolerated by normal cells. Partly from this impetus, several research groups around the world are carrying on a program of sustained research in cyclitol stereochemistry. Starting with quebrachitol from rubber latex, Dr. McCasland and his co-workers go through an inositol to an inositol diacetone ketal. They then convert this ketal via its di-p-toluene sulfonate to l,2-anhydro-«//o-inositol3,4,5,6-diacetone ketal. Reaction with carbon disulfide converts this to two diastereoisomeric trithiocarbonate diketals [5,6-thiocarbonyldithio-l,2,3,4-di- (isopropylidenedioxy) -cyclohexanes]. The product, when purified and slowly crystallized, gives two visibly different kinds of crystals: pale yellow plates and bright yellow rods. Using the technique originated by Pasteur, the workers separate these by careful hand picking. Reduction of the two trithiocarbonate diketals with lithium aluminum hydride gives the corresponding tetroldithiol diketals. Finally, hydrolysis with aqueous acetic acid gives two diastereoisomers of 5,6dimercapto-l,2,3,4-cyclohexanetetrol.
OUR BENT?
FLEXIBILITY IN GEL TIMES
A
B
C
D
E
F
( R E P R E S E N T A T I V E RESINS)
The significant difference in gel times c LUPERSOL® Methyl Ethyl Ketone peroxide will give the flexibility you require for an application. The gel times charted abov were run at 30°C on commercially avail able polyester resins. W R I T E FOR DATA S H E E T S or Consult CHEMICAL MATERIALS CATALOG, Page 516
W A L L A C E & TIERIMAN I N C . LUC1D0L DIVISION
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APRIL
9,
1962
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51
New Syntheses May Cut Cost of Indoleacids Reaction of indole with lactones gives high yields of 3-indoleacids; second synthesis also promising 141ST
ACS
NATIONAL
MEETING
Agricultural and Food Chemistry
A new way to make 3-indolealkanoic acids, considered among the most active plant growth regulators known, points to their far greater commercial use. The new synthesis involves treating indole with lactones at controlled temperatures in the presence of alkali hydroxide. Since the reactants are commercially available, the indoleacids can now be made at relatively low cost. Additional compounds of this class may have even greater use in gardening and agriculture, and they can also be made by the new process, says Dr. Henry E. Fritz of Union Carbide Olefins. While study and use of these compounds go back to 1934, their high cost and unavailability have resulted in restricted use. Previous syntheses #re long and tedious, particularly for the higher homologs, or give very low yields. The new synthesis, by contrast, is a one-step reaction of lactones with indole in the presence of base (at 200° to 300° C ) , and gives yields ranging from 41 to 82%. The efficiency of the reaction, based on indole, ranges from 61 to 96%. The reaction mixture consists of 1.0 mole of indole, 1.05 moles of lactone, and 1.1 moles of base. Although Dr. Fritz uses potassium hydroxide, sodium hydroxide and sodium methoxide also give good results. No solvent was used in the work; tetralin, methylnaphthalene, or diethylbenzene can be used, however. The purity of the crude a c i d s isolated by adding water to the reaction mixture and acidifying the aqueous phase—ranges from 85 to 98%, depending on the reaction conditions and reactants used. In general, the acids with the longest carbon chain in the alkanoic acid portion are obtained in highest purity. The acids with fewer than five carbons in the acid chain come out in lower purity, and are more susceptible to oxidation. The Union Carbide Olefins work 52
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has also led to good yields of 3-indolebutyric acid from indole, butyrolactone, and potassium hydroxide. Highest yields (67 to 82%) result from reactions between 220° and 290° C. for 20 hours with an equimolar quantity of alkali. Both 1- and 3indole propionic acids were also made in high yields from indole, propiolactone, and potassium hydroxide by varying the reaction temperature. New Uses. The 3-indoleacetic and 3-indolebutyric acids give many varied responses in plants, such as inducement of parthenocarpic development, enlargement of cells, stimulation of root formation, increased fruit production, and control of flowering. Nurseries use them to promote root formation on plant cuttings. An increased commercial use appears likely, since research shows that these compounds affect the rootings of many important trees, such as citrus, fig, and date, as well as many other plants, including orchid, potato, carrot, sugar cane, and grapevine. Other studies indicate that they retard tobacco mosaic, decrease loss due to frost damage, form seedless tomatoes, control beet leafhopper, increase cotton production, and hasten bark renewal of trees. Recent work by others shows that the higher homologs and their derivatives, particularly those containing an even number of carbon atoms in the acid side chain (3-indolecaproic acid, for instance), are as biologically active as the acetic and butyric acid derivatives. Dr. Fritz says his work shows that the higher homologs are more oxidatively stable. Second Synthesis. Indoleacetic acid in high yields results from a second new reaction of indole with potassium glycolate at 250° C , according to Dr. Herbert E. Johnson of Union Carbide Chemicals and Dr. Donald G. Crosby of the Pesticide Research Laboratory, University of California. This method provides the material and its simple derivatives at a potentially low cost and in yields of about 90%. The starting indole is readily available through the cyclization of o-ethyl
aniline. The a-hydroxy acids, particularly glycolic and lactic acids, are low cost products, Dr. Johnson points out. The method leads to synthesis of a wide variety of substituted indoleacetic acids; the only limits are those imposed by the reaction conditions. Further derivatives of these substituted materials can be made to permit a greater degree of selectivity in growth regulation. There are other than agricultural uses for the products, Dr. Johnson says. Methyl indoleacetate has been converted in high yield to indolepyruvic acid by others (Japanese patent 4274). The chemical may replace tryptophan nutritionally, he adds. In addition, indole derivatives in general are achieving a place of major importance in the pharmaceutical industry, and the new method will provide materials for additional areas of study.
Glass Electrode Potential Analyzed 141ST
ACS
NATIONAL
MEETING
Colloid and S u r f a c e C h e m i s t r y
First steps toward putting a number on ionic interactions that cause a potential to come from glass electrodes have been taken at Eastern Pennsylvania Psychiatric Institute. Work by Dr. George Eisenman and Dr. George Karreman shows that in certain glasses potential is primarily due to the movement of ions; in others it is primarily due to potentials set up when the ion comes to equilibrium on exchange sites on the glass. The voltage set up when a glass electrode is immersed in a solution depends on the activities of the ions in solution and the composition of the glass electrode used to make the measurement. Workers in the field have known for some time that certain formulations of glass can be used to measure certain ions, excluding others. For instance, incorporating aluminum and boron into the glass makes electrodes highly specific or selective for alkali metal cations. This selectivity and its influence on potential is measurable. However, just how the
ion acts on the electrode to produce a potential has been uncertain for some time. Classically, the potential has been thought to be set up either by movement of the ion into the "bulk" phase of the glass—the so-called diffusion mechanism—or by the ion coming to equilibrium on ion exchange sites in the glass, called the phase boundary mechanism. Initial results of attempts to analyze which of these two mechanisms is responsible for causing a potential has convinced Dr. Eisenman and Dr. Karreman that the potential comes from a combination of the two. However, one may dominate the other. Selectivities. Dr. Eisenman and Dr. Karreman first had to find a relationship between the selectivity constant of the electrode (determined by measuring voltage) and the diffusion component and phase boundary component. They did this by adapting standard equilibrium ion exchange equations and flux equations to relate over-all selectivity of a resin to ionic diffusion components and ionic selectivity. This still left both the phase boundary component and the diffusion component unknown, so one had to be measured. Measurements of the rate of uptake of radioactive isotopes provided a measurement of the diffusion component, allowing calculation of the phase boundary component. Isotopic experiments were run using sodium - aluminum - silicate glasses. These glasses were modified to be selective for potassium ion in one case and sodium ion in another. The two scientists also used a potassium-aluminum-silicate glass that's selective for potassium ion, and a lithiumaluminum-silicate glass selective for sodium ion. These experiments show that the surface of all glasses is altered, presumably by hydration, from "dry" glass structure when it contacts liquid. And the extent and depth of alteration depend on the glass composition. Those glasses that are potassium-selective seem to be altered to a greater depth (the change can be considered a swelling with water) than those that are sodium selective. And in these glasses, the diffusion component accounts for almost the entire electrode potential. Glasses selective for sodium seem to be altered to a lesser depth, and potential here comes primarily from the phase boundary component.
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