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COMMUNICATIONS TO TIIIS ELJIIUK
and 11% cyclohexene, respectively) and cis- atid tmns-3,4-dihydroxytetrahydrofuran (yielding 58 and 47% of dihydrofuran, respectively) bear this out. Mono ethers and esters of 1,2-glycols, chlorohydrins and amino-alcohols might be amenable to a similar "dehydroxylation" reaction. Acknowledgment.--H. W. gratefully acknowledges a Fullbright Research Fellowship a t the University of Leiden, as well as stimulating discussions with Prof. n r . E. c. Kooyinati.
VCI1.
s:j
Water and ether were added to the solution and the two phases were separated. The aqueous layer was reextracted with ether arid then neutralized with Amberlite IRC-50-H ion exchange resin. After removal of the resin by filtration, the filtrate (4,800 optical density units a t 200 nip and PH 6.5, 1 cm. light path) was concentrated and poured onto a column of Dowex-1-OH- (200-400 mesh, 8% crosslinked, 4 X 16 cm.) and fractionated by elution with water. The first fraction (lithium hydroxide and a small, but variable, amount of ultravioletDEPARTMENT OF ORGANIC CHEMISTRY Continued THET-NIVERSITY HANSWYNBERC absorbing material) was discarded. GRONINGEN, HOLLAND A . KRAAK elution with water gave a single ultraviolet-absorbing peak. Evaporation of this solution gave a colorRECEIVED JULY 24, 1961 less gum which was assumed to be a mixture of aand P-2,4-dimethoxy-5-~-ribofuranosylpyrimidine (111, 550 optical density units, Xmax 261.5 mp, Xmin SYNTHESIS OF PSEUDOURIDINE 240, O D m a x / O D m i n 2.3, I?, 0.75 in n-butyl alcoholSir: acetic acid-water (4: 1 :5),6 positive periodate test Pseudouridine, a naturally occurring nucleoside, for vicinal hydroxyl groups).' No further ultrais particularly interesting because of its unique violet-absorbing material wa.s eluted with water. structure (IV). We wish to report the first chemiI11 (750 optical density units) was taken up in cal synthesis of pseudouridine and to confirm the %)yo (v./v.) dichloroacetic acid and hydrolyzed for 5-D-ribofuranosyluracil structure recently assigned 4 hours a t 100' to remove the blocking groups. The to it.2 residue was dissolved in 0.02 111 H3B03 adjusted to p H 10 with NH40H and poured onto a Dowex-lHCOa-- column (200-400 mesh, 8% cross-linked, 1 X 8 cm.). Linear gradient elution, modified from the procedure of Cohn,2bwith 0.02 M H3BOa (adjusted to pH 9 with NH40H) in the mixing flask I1 and 0.05 i l l NH4HC03 in the reservoir, gave five I major peaks. These. were identified by their ultraviolet spectra as unhydrolyzed 2,4-dimethoxy-5-Dribofuranosylpyrimidine followed in order by the pseudouridine isomers Ap, As, I3 and C described by Cohn.8 The fraction containing the C isomer was freed from NH4HCOs with Ihwex-50W-H+ and from borate by repeated addition and evaporation of methanol. The solid then was purified by paper chromatography on Whatmxn 40 paper in nbutyl alcohol-acetic acid--water.fi The major ultraviolet-absorbing band (Rf 0.24) was eluted with water and the solution was evaporated to dryness. The white solid (12 mg.) was recrystallized twice IV v from 95';) ethanol yielding 5.2 mg. (needles) of n-Butyllithium3(l?i nimoles) was added dropwise pseudouridine C. The synthetic material was idento 18 mmoles of 2,4-dimethoxy-5-bromopyriniidirie tical with natural pseudouridiiie (isolated from in 50 ml. of tetrahydrofuran under rigorously an- urineg) by the following criteria: m.p. 223-224' hydrous conditions a t -7.5'. The resulting clear, (uticorr., reported 220--221'2"), mixed melting point, yellow to reddish-brown solution of 2,4-dimethoxy- paper chromatography in four solvent systems,10 pyrimidine-5-lithium4(11)was allowed to stand for 5 paper electrophoresis i n borate buffer and ultraviolet spectra compared a t pH 2, 13 and 14. minutes and then 2,3,5-tri-O-benzoyl-~-ribofuranosyl chloride (I),6 (prepared from 2 mmoles of 1-0- ( 6 ) S . M . Partridge, Biochcm J . . 42, 238 (1948). (7) hl. Viscontini. D . Morh and P. Karrer, Ilelv. Chim. Acta. 88, acetyl-2,3,5-tri-O-benzoyl-P-~-ribose)~ was added slowly (10 minutes) with stirring. The reaction 0.12( 8()19.55). CohnZh has estahlished that naturally occurring pseudouridine mixture was stirred for 1 hour a t -75' and then ( C isomer) is converter! to a n equilibrium mixture of pseudouridine isomers (designated AF, A s , 11 and C ) by heating with acid at 100". allowed to warm to room temperature overnight. (1) See C. A. Dekker in A n n . Rm. niochcrn., 89, 453 (19fiO), f o r a recent review. (2) (a) C. Yu and F. W. Allen, Biorhem. Biophys. A d a . 81, 393 (1959); J. P. Scanaell. A . &I. Crestfield end R . W. Allen, ibid., 406 (19.59); W . Cohn, ibid., 560 (1950); (b) W. Cohn, J . Biol. Ciicm., ass, 1488 (1960). (3) R . G. Jones and H. Gilman. Organic Rcacfions, VI, 339 (1951). The butyllithium solution should be clear before use. (4) B. W. Langley. J . A m . Chrm. Soc., 7 8 , 2136 (1956). (5) H. M. Kissman. C. Pidacks and R. R . Baker, ibid., 77, 18 (1955).
f
The periodate titration data he reports leave little doubt that the A isomers are 5-a- and 5-8-n-ribopyrnnosyluracil ( V ) and B and C are the 5-ribofirmnosyl anomers (IV). (9) We are indebted to Dr. Waldo Cohn for this sample (estimated h y him as 75% pure). We purified it further by paper chromatography (ref. loa) and two recrystallizations from 06'% ethanol. (10) (a) n-Butyl alcohol-acetic acid-water,a R f 0.26; (b) isopropyl alcohol-ammonia-water ( 7 . 1 :2), R . Markham and J. D. Smith, Riochcm. J.,SI, 552 (1952). Rf 0.40; (c) n-butyl alcohol-saturated with water, R. D. Hotchkiss. J . Biol. Chem., l T S , 315 (1948), R f 0.11; (d) isopropyl alcohol-lYo ammonium sulfate (2:1), N. Anand, V. M. Clarke, R. H. Hall and A. R. Todd, J. Chem. Sot., 3665 (1950). Rf 0.54.
1 3 0 0 ~REVIEWS
Calcd. for C9HI2N2Oa (244.20) : C, 44.26; H, 4.9,5; N, 11.47. Found: C, 44.54; H I 5.20; N, 11.71. X max 262 mp, Cmax 7,900; Xmin 232.5, Emin 2,090, OD2”0,/ODZM 0.06 a t PH 2. Xmax 287, Cmax 7,960; Xmin 244.5, emin 1,070; OD290/OD260 2.13 a t p H 12.12 The yield of pseudouridine C based on the sugar derivative (I) was 20/,. The total yield of all four pseudouridine isomers was 3.5%. This synthesis conclusively establishes the 5 - ~ ribosyluracil structure of natural pseudouridine, but the important question concerning the configuration of the anomeric carbon still remains unanswered. Hydrolysis experiments have indicated that the A isomers are produced, as expected, during acidic hydrolysis of the methoxyl groups. Since the benzoyl groups are removed from the sugar by a side reaction,13 the /?-directing influence of the 2(11) Schwarzkopf
Microanalytical
Laboratory, Woodside,
New
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benzoyl group“ may be lost. From a consideration of the spectral data and by analogy with other nucleosides i t seems likely that pseudouridine C is 5-/?-D-ribofuranosyluracil and the B isomer is 5-CYD-ribofuranosyluracil. Attempts to settle this structural point and to improve the yield of pseudouridine by utilizing the less reactive cadmium reagent are in progress. Acknowledgments.-We are indebted to Mr. - Viktor Kurkov for technical assistance and to hlr. Peter Lengyel for preparing l-O-acety1-2,3,5-tri-Obenzoyl-P-D-ribose. This work was supported by grants from the National Science Foundation (No. 1602 C4) arid the United States Public Health Service (No. RG-7262 C l ) . DEPARTMENT OF BIOCHEMISTRY ROBERT SHAPIRO NEW YORKUNIVERSITY SCHOOL OF MEDICINE NEWYORK16, NEWYORK ROBERTWARNERCHAMBERS RECEIVEDJUNE 30, 1961
York. (12) These em=
values are in fair agreement with those reported
by Yu and Allen.% but they are about 10% lower than those reported b y Cohn.*b The sample provided by Dr. Cohn and purified as described in ref. 9 gave values corresponding t o those given above.
(13) Presumably by reaction of the lithium derivative with the ester groups t o form bis-(2.4-dimethoxy-5-pyrimidyl)-phenylmethanol. (14) B . R . Baker, J. B . Jo’ieph, R . E. Schaub and J. H. Williams, J . Org. Chem., 19, 1786 (1954).
BOOK REVIEWS interest to biochemists will be the compilation, into a very useful table, of all known enzyme reactions that require either coenzyme A or one of its derivatives. Botanically Chemistry, University of Minnesota, Minneapolis, Min- inclined readers will be interested t o know that royal nesota, HENRY LARDY,Institute for Enzyme Research, jelly is a plant (Table, 2 p. 14). On the whole, this is a University of Wisconsin, Madison, Wisconsin, and KARL masterly review and one which more than adequately sumMYRBACK,Institute for Organic Chemistry and Bio- marizes Lynen’s investigations and thoughts on coenzyme chemistry, University of Stockholm, Sweden. Academic A . The chapter was originally written in German, was Press Inc. 111 Fifth Avenue, New York 3, N.Y., 1960. admirably translated by Dr. Helmut Beinert, but somehow a discrepancy between the numbered equations, figures xiv 497 pp. 15.5 X 23 cm. Price, $16.00. and tables and their corresponding numbers in the text did The first edition of “The Enzymes” has proved to be a appear in the final printed version. useful well of information; undoubtedly the second edition Most aspects of the chemistry and enzymology of the will turn out likewise. The information in these volumes pyridine coenzymes are discussed by N. 0. Kaplan. This is presented in a streamlined fashion which should provide discussion summarizes, and usefully so, recent investigapainless reading with maxinial absorption for the reader. tions on the chemical and physical properties of DPN and The biologist of today is presented with numerous volumes TPN. Possible reaction mechanisms of DPN and TPN, which are published for the dual purposes of keeping him as studied with these coenzymes or with one of their derivaabreast of diverse fields of interest, not necessarily his own, tives, are also discussed. The chapter points out again the and to present an appraisal of the current status of these complexities introduced by the discovery of different fields. In reading Volume 3 (Part B) of “The Enzymes,” isomeric forms of the pyridine coenzymes. This reviewer this reviewer had the impression that he was reading a found the discussion of the enzymatic destruction of DPN super volume of an annual review work. One wonders if most helpful, but was disappointed that plants weren’t we really need or can afford such comprehensive treatises included in the section on the distribution of pyridine which in so many ways duplicate each other, duplicate the nucleotides. efforts of the varied annual summarizing publications arid E. M. Kosowcr presents a series of interesting considerado away so completely with the personal aspects of scientific tions on what is known about charge transfer complexing. writing. I t would seem that more effort could be devoted Even though little is known about such reactions in biologitoward publishing memoirs or monographs, contributed by cal systems, Kosower does consider the evidence and theories individuals who wish to sum up a point of view or provide for such reactions in pyridinium systems and possible applia record of their investigations. Such publications need cations of these theories to reactions of pyridine nucleotides. not be subject to the rigid time relations that are needed for This chapter is a solid discussion of things that are either multi-volume treatise publication and could benefit thereby. partially known or need to be investigated. Volume 3 (Part B) of “The Enzymes” contains eleven L. J . Reed contributes a chapter on lipoic acid. The contributions of varying length; all contributions suffer chapter comprises a comprehensive review of the chemistry from irritating editorial troubles. In this volume there is and biological role, in terms of reaction mechanisms, of considerable unevenness among the eleven contributions, lipoic acid. an unevenness that does not reflect the spirit of a revised “Metal and Enzyme Interactions: correlation of comedition of an advanced treatise on enzymology, the contriposition, function, and structure” is the title of a very butions to which should be authoritative treattnents of each ambitious chapter contributed by B. L. Vallee. The main topic and remain so for some years. emphasis of this chapter is on the non-heme metal-coenzyrnes Jaenicke and Lynen provide a lengthy and thorough dis- and consequently centers on Vallee’s own contributions. cussion on almost all aspects (if coenzyme A . Of particular After a brief introductory discussion of metal containing
The
Enzymes.
Second
Edition,
Completely Revised.
Volume 3. Prosthetic Groups and Cofactors (Part B). Edited by PAULD. BOYER,Department of Physiological
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