COMMUNICATIONS TO THE EDITOR
Aug. 5 , 1957
form inosinic acid in these extract^,^ inhibits Compound I formation by about 500j0. These results suggest that Compound I is nicotinic acid mononucleotide and is formed by condensation of P R P P with NA. To characterize these compounds, a large-scale incubation of erythrocyte acetone powder extracts with nicotinic a ~ i d - 7 - C 'was ~ performed. A neutralized perchloric acid filtrate was then passed through a Dowex 1 formate column; 15-ml. aliquots were collected and assayed for radioactivity and absorption at 260 mp. The various fractions were pooled and components identified by paper chromatography. Radioactive fractions were adsorbed on a Norit X column and, after washing with water, were eluted with lOy0aqueous isoamyl alcohol. Ribose and phosphate analyses and absorption spectra suggested that Compounds I and I1 were the nicotinic acid analogs of NMN (desamido-NMN) and DPN (desamido-DPN), respectively. Compound I1 was not active with alcohol dehydrogenase and did not form a fluorescent Further work on the acetone addition p r o d ~ c t . ~ identification of these compounds is in progress. TABLE I SYNTHESIS OF D P N
FROM
4247
enzymes, and a study of their distribution in bacterial and mammalian systems are in progress. DEPARTMENT O F BIOCHEMISTRY JACKPREISS DUKEUNIVERSITY SCHOOL OF MEDICINE PHILIPHANDLER DURHAM,KORTHCAROLINA JUNE 14, 1957 RECEIVED UNSATURATED MACROCYCLIC COMPOUNDS. V.' LARGE RING POLY-ACETYLENES
Sir: We have shown recently that the oxidation of terminal diacetylenes of type I with oxygen in the presence of cuprous chloride and ammonium chloride in aqueous ethanol leads to the cyclic dimers I1 besides linear cornpounds.l.2 We have studied this type of coupling of terminal diacetylenes under a variety of conditions, especially in homogeneous media. The surprising discovery has been made that when diacetylenes of type I in pyridine solution are simply heated with neutral cupric acetate,a not only the cyclic dimers I1 are formed, but also the cyclic trimers 111, tetramers IV, pentamers V, hexamers VI and higher cyclic rc4c-czc7 HCEC-(CH,in-C~CH
(CH
DESAMIDO-DPX
Complete incubation system consisted of 1.7 pmoles of desamido DPK, 20 pmoles of glutamine, 2 pmoles of ATP, 5 pmoles of ATP, 40 pmoles of Pi buffer, p H 7.4, and 4 cc. of dialyzed yeast autolysate. Total volume 1.0 cc. after incubation of 100 minutes 1 cc. of H 2 0 was added and the protein denatured a t 100' for 1.5 minutes; 1-cc. aliquots were taken for alcohol dehydrogenase assay of D P N formed. Omission and/or Additions
Fmoles DPN synthesized
None - Desamido D P N -ATP -lllg++ -Glutamine NH4C12U pm. -Glutamine glutamate 20 pm. -Glutamine "0 time"
0.116 ,028 ,011 ,014 ,026 ,015 ,019 ,007
+ +
Dialyzed yeast autolysates have been found slowly to synthesize DPN upon addition of glutamine, ATP, PRPP, h/lg++, F- and NA but not NAm. With nicotinic a ~ i d - 7 - Cin~ ~ the medium, accumulation of compounds I and I1 was again observed. Compound I1 prepared with erythrocytes or yeast exhibited an Rfin 70% ethanol, 30% 1 M ammonium acetate, $H 5.0 identical to that of an authentic sample of the nicotinic acid analog of DPN kindly supplied to us by Dr. N. 0. Kaplan. This solvent system adequately separates Compound I1 from DPN. Synthesis of Compound I1 from NA by yeast autolysate is dependent on ATP, P R P P and Mg++. R5P is 10% as effective as PRPP. Glutamine, A T P and M g f f are required for synthesis of DPN from desamido-DPN (Table I). NH4+ and glutamate are inactive as amide donors. Further investigations of the mechanisms of these enzymatic syntheses, purification of the (5) N.Lcvitas, J Robinson, F Rosen and W A Perleweig, J Btof. Chrm., 167, 511 (1947).
ICH,),
j
i';=c-cE: -J
c
C
Ill C
C
m
N
C ;\ C \ C
\
a,n = 5 b,n = 4 c,n=3 d,n = 2
I CH21,Cz
C-CE
C- (CH2 ) n
VI
(1) Part IV, F . Sondheimer, Y.Amiel and R. Wolovsky, THIS JOURNAL, in
press.
(2) (a) F. Sondheimer and Y.Amiel, ibid., 78, 4178 (1956); i b i d . , in press; (b) Y.Amiel. F. Sondheimer and R . Wolovsky, Proc. Ckcm. SOC.,22 (1957). (3) This sort of oxidation has been shown to result in the smooth
-
coupling of simple acetylenes and to give the cyclic monomer, and 10 (and of a diacetplenic diester) when cardimer in the case of I, n ried out under conditions of high dilution (G.Eglinton and A. R. Galbraith, Chem. and I n d . , 737 (1956)).
4248
Vol. 79
COMMUNICATIONS TO THE EDITOR
polyacetylenes. Except in the case of Id, only exploded on attempted combustion and gave low small amounts of the linear coupling products carbon values. I n all the four series studied, cyclic result. polyacetylenes of higher mol. wt. than those deFor example 1 part of nona-l&diyne (Ia) in 100 scribed were also obtained; their structures are now parts of pyridine was heated and stirred with 15 under investigation. parts of neutral cupric acetate a t 55' for 3 hours. The present method makes available in one step The product on chromatography on 200 parts of highly unsaturated large ring alicyclic hydrocarbons alumina into ca. 300 fractions gave successively : (a) (and by hydrogenation the corresponding saturated the Cis-tetrayne IIa (loyo), m.p. 210-212' (identi- ones) over a wide range of size, including considercal with that made previously1-2b); (b) the CZi- ably larger ones than the thirty-four membered hexayne IIIa (130/0), m.p. 125-126' (hydrogenated cycle which is the biggest alicyclic ring prepared to m.p. 47-48', mol. wt., 3745; calcd. 378) ; previously. (c) the Cas-octayne IVa (11%)) m.p. 135-136' DANIELSrEFF RESEAKCH FKANZ SOXDHEIMER INSTITUTE (hydrogenated t o C ~ B H ,m.p. ~ , ~ 70-71', mol. wt., INSTITUTE OF SCIENCE YAACOV AYIEI, 517; calcd., 504); (d) the C%-decayne Va (4y0), WEIZMANN ISRAEL REUVEN WOLOVSKY m.p. 144-145' (hydrogenated to C46H0~,4 m.p. 78.5- REHOVOTH, RECEIVED JUNE 17, 1957 79') mol. wt., 618; calcd., 630); (e) probably the C ~ d o d e c a y n eVIa (4%), m.p. 144-145' (hydrogenated to m.p. 90-91', mol. wt. not THE CHANGE OF OPTICAL ACTIVITY OF POLY-)BENZYL-L-GLUTAMATE I N AN ELECTRIC FIELD determined due t o insolubility). A similar coupling of octa-1,7-diyne (Ibj gave: Sir: (a) the Cia-tetrayne I I b (9%), m.p. 162-163' The optical activity of a molecule is different (identical with that made previously2"); (b) the along different directions. Therefore we expect C ~ h e x a y n eI I I b (14%)) m.p. 173-174' (hydro- the optical activity of a solution of these molecules genated to C24H@,m.p. 46.547', mol. wt., 330; to change if the molecules are oriented. However, reported: m.p. 46-17') mol. wt., 336); (c) the this effect has not been described. We wish to reC ~ o c t a y n eIVb (8%), m.p. 154-155' (hydrogen- port the change of optical activity of a solution of ated to C32H64, m.p. 58-59', mol. wt., 429; re- poly-7-benzyl-L-glutamate (PBLG) caused by oriported: map. 59-60') mol. wt., 448); (d) the GO-entation of the molecules in an electric field. decayne Vb (9%), m.p. 155-157' (hydrogenated to An equation relating the change in optical acC40H80,4 m.p., 74-75', mol. wt., 568; calcd. 560). tivity to molecular parameters a t infinite dilution Coupling of hepta-1,6-diyne (IC)did not give the has been derived.' For a helical molecule the (214-tetrayne ITc (if formed, it probably decom2 ____ but the C21-hexayne IIIc (3%), m.p. = -- ( [ a n ) - ia,tl)[P*2 (P3J - 4n)I E2 43 174-17.5' (hydrogenated to C21H42,4 m.p. 63-64', mol. wt., 289; calcd. 294) and the Cz8-octayne IVc change in the specific optical activity in an electric (4%), m.p. 213-214' (hydrogenated t o CrsH56, field2 E is proportional to the difference in optical activity for light incident parallel to the helical m.p. 47-48', mol. wt., 389; reported: m.p. 47-45', axis [a331 and perpendicular to the helical axis mol. wt., 398). Coupling of hexa-1,5-diyne (Id) and chromatog- [a!~].The proportionality factor is the electrical raphy into 450 fractions gave: (a) the linear dimer orientation term which involves the permanent (9%), m.p. 98-99' (identical with that made pre- and induced dipole moments of the molecule. viously1,2b); (b) the &-hexayne I I I d (6%), de- The specific optical activity in the absence of a composes on heating (hydrogenated t o C18H36, field [a10is just (2[a11] [(~33]),/3. I n order to measure the change of optical acm.p. 72-73', identical to that obtained from IIa) ; (c) the Cz4-octayneIVd (67,), decomposes on heat- tivity in an electric field, the light must be incident ing (hydrogenated to C24H4.3, n1.p. 46-17', identi- parallel to the field. Only along this direction is cal t o that obtained froin IIIb); (d) the C30- the refractive index and optical absorption the decayne Vd (6%)) decomposes on heating (hydro- same for all directions of the plane of p ~ l a r i z a t i o n . ~ A one-em. cell with transparent conducting glass6 genated to C30Hso,m.p. 57-58"; mol. wt., 416; reported m.p. 57-58O, mol. mt., 420); (e) probably electrodes was used. The optical rotation with the cyclic Cd*-tetradecayne, decomposes on heat- the electric field off, then on, was measured from ing (hydrogenated to C4*HBl14 m.p. 75-78', mol. X = 330 to 550 mp in a Rudolph automatic spectropolarimeter6 for two concentrations of AVw wt., 598; calcd., 588). All the above cyclic polyacetylenes were highly = 64,000 PBLG' in ethylene dichloride. The crystalline. They were shown to differ from each (1) I. Tinoco, Jr., and W.G. Harnrnerle, J . Phys. Chcm., 60, 1619 other since they gave mutual depressions in m.p. (1956). (2) The units of E are e.s.u./cm. (1 e.s.u. 300 volts). Their cyclic nature mas shown b y the absence of (3) The change of refractive index in an electric field is electrical acetylenic hydrogen (no band a t ca. 3300 cm.-' in birefringence The change of optical absorption is or Kerr effect.' the infrared, no precipitate with silver nitrate) and electrical dichroism; it has not been reported. (4) C. G. LeFevre and R. J. W. LeFCvre, Revs. P u r e A p p l . Chcm., by the absence of terminal methyl groups (no band 261 (1955). at ca. 1380 cm.-l) in the corresponding saturated 6, (5) wlsh to thank Dr. E. M. Grcist of Corning Glass Worka for compounds. The latter gave satisfactory an- klndly We furnishing this glass (E-C 67720). alytical results, but most of the polyacetylenes (6) We wish to thank Professors J. B. Nielanda and H.K. Schachman, and Mr. B. Burnham for t h e use of thls Instrument. (4) Thfa Is a previously unknown cpcloalkane.
+
+
-
(5) All molecular weights were determlncd by the Rast method in camphene.
(7) We wlsh to thank Dr. E.R.Blout for klndly supplylug a sample of PBLG ({ES-508) for these measurements.
