Oct. 5 , 1957
COMMUNICATIONS TO THE EDITOR
complete destruction of the metal-porphyrin complexes, the durability of the porphyrin components of the crude oils and asphalts was unexpected. The data (footnote b ) indicate that simple shielding by the petroleum is an insignificant factor as expected from the energy range of gamma radiation from spent fission products.‘ Crude petroleum may afford some protection against radiationinduced oxidation. However, the data (footnote c) indicate that the metal-porphyrin complexes are extensively decomposed even under anaerobic conditions. The metal-porphyrin complexes were separated from the petroleum by mild physical methods.2 The possibility remained that the complexes in the petroleum were chemically different from those isolated. However, the enriched petroleum samples suffered essentially the same degree of decomposition as did the natural samples. These data are interpreted as indicating that crude petroleum acts as a “protective” solvent for the metal-porphyrin complexes. Such action has been the subject of considerable research3 but is only incompletely u n d e r ~ t o o d . ~However, it appears that petroleum may be a natural protective agent against gamma irradiation by either of the commonly accepted mechanism^^-^ in which i t scavenges free radicals or provides an effective medium for internal energy transfer. The protective action of petroleum may have considerable practical importance in the development of nuclear engineering processes. The author gratefully acknowledges the assistance of Mr. J. W. Moore in making the porphyrin analyses listed above.
532 1
activity. The amorphous acid which was isolated was indistinguishable from amorphous I by ultraviolet measurements, elemental analyses and specific rotation. The amorphous nature of the material impaired infrared comparison, but no interpretable differences were detected. This ap-
0 OH
HO
c=o I
..2:”.‘ “I
RT.
?
c=o “I
“co+cH3
0
4
‘
OH
CH,
parently homogeneous material was partially resolved, however, by countercurrent distribution into 2 main components after 2000 transfers using a solvent system comprising water, acetone, methyl ethyl ketone, and Skellysolve B (3:9 :2 :6 by volume). Analysis of the distribution data2showed the mixture contained ca. 67% of I and ca. 33% of a new material. This inactive component, very similar in physical properties t o I, was named isonovobiocin. When pure I1 was subjected to the original isomerization conditions, i t was converted back t o I in 55-60% yields. An equilibrium was thus indicated. Since no spectral changes were detected, i t seemed most likely that the sugar moiety was involved. The material used for the structure determina(1) J. Moteff, Nucleonrcs, 13, No 5 , 28 (1955). (2) H. N. Dunning and N. A. Rabon, I n d . Eng. Chem., 48, 951 tion of I1 was obtained as follows: With the (1956). knowledge gained from the initial countercurrent (3) B. M. Tolbert and R. M. Lemmon, Radiation Res., 3 , 52 (1955), distribution experiment a fractional crystallization including 58 references. procedure was developed to remove most of I from (4) M. Burton, S. Lipsky and J. L Magee, presented at Division of Physical and Inorganic Chemistry, Am. Chem. SOC.Meeting, Miami, the equilibrium mixture. The remaining mixture Fla. (Apr. 7, 1957); Chem. E n g . News, 36, No. 15, 27 (1957). was separated by countercurrent distribution (2000 (5) J. L. Magee, J. Phys. Ckcm., 56, 555 (1952). transfers) and found to contain about 65% IT, (6) J P. Manion and M. Burton, i b i d . , 56, 560 (1952). 22% I, and 13% sodium chloride and unidentified U. S. DEPARTMENT OF THE INTERIOR BUREAUOF MINES H. N. DUNNING materials. The purified isonovobiocin was heated under BARTLESVILLE, OKLAHOMA reflux in methanol containing a slight excess of one RECEIVED AUGUST23, 1957 equivalent of acetyl chloride, a cleaving the molecule selectively t o crystalline novobiocic acid (IV) in NOVOBIOCIN.’ V. CARBAMYL MIGRATION AND 98% yield and the methyl glycoside (111). The ISONOVOBIOCIN novobiocic acid was identical with that obtained Sir: from I by infrared, ultraviolet and melting point The studies reported here show that novobiocin comparisons. Compound 111, crystallized from acetone and (I) isomerizes to isonovobiocin (IT) in dilute alkali and establish the structure of I1 as 4-hydroxy-3- Skellysolve B, was found to be isomeric with methyl .~ for C10H19N06: [4-hydroxy-3-(3-methyl-2 - butenyl) - benzamido] - 8- 3-U-carbamyl n o ~ i o s i d e l ~(calcd. methyl-2-0~0-2H-l-benzopyran-7-yl 2-0-carbamyl- C, 48.19; H , 7.68; N, 5.62. Found: C, 48.46; H, 7.59; N, 5.54). I t was different on the basis of 4-0-methyl-5,5-dimethyl-~-lyxoside. When a solution of novobiocin was held a t pH infrared absorption. Barium hydroxide a t 25’ 10 for 2 hours a t 25’, statistically significant bio- converted TI1 t o methyl novioside (V)lcv3in 96% assays indicated a loss of 30-35y0 of its antibiotic yield, showing that I11 differed from methyl 3-Ocarbamylnovioside only in the position of the (1) (a) J. W. Hinman, H. Hoeksema, E. L. Caron and W. G. Jackcarbamyl group. Acid hydrolysis of I11 yielded an son, THISJOURNAL, 7 8 , 1072 (1956); (b) C. H. Shunk, C. H. Stammer, E. A. Kaczka, E. Walton, C. F. Spencer, A. N. Wilson, J. W. Richter, F. W. Holly and K. Folkers, ibid., 78, 1770 (1956): (c) H. Hoeksema, E. L. Caron and J. W. Hinman, ibid., 7 8 , 2019 (1956); (d) E. Walton, J. 0. Rodin, C. H. Stammer, F. W. Holly and K. Folkers. ibid., 7 8 , 6 4 5 4 (1956).
(2) L. C. Craig and D. Craig, Chapt. I V in “Technique of Organic Chemistry,” Vol. 111, A. Weissberger, Editor, Interscience Publishers. Inc., New York, N. Y.,1950. (3) J. W. Hinman, E. L. Caron and H. Hoeksema, THISJOURNAL, 79, 3789 (1957).
COMMUNICATIONS TO THE EDITOR
6322
Vol. 79
aldose which failed to react with periodate. These findings along with published datal permit the assignment of the structure of methyl 2-0-carbamyl-4-0-methyl-5,5-dimethyl-~-lyxoside to 111 and show that I1 is the 2-carbamyl analog of 1.
tyrosine hydrogens, as suggested. l I n aqueous solutions these shifted - 20 to - 30 C.P.S. relative to the aromatic proton of toluene as 0. I t probably also includes the 8 CH hydrogens on the imidazole of histidine, found a t somewhat lower fields. The total number of non-exchangeable hydrogens in the CH3 protein being 697,4this will account for S.970of the CH3COCI Me0 k 0 total area under the absorption curve, in agree:I -A MeOH A’: ment with the 9 =t1% measured. IVhile in water the guanidino group of arginine gives rise to an HO OCONHz additional peak in the same region, this disappears after equilibration with D,O, so that it would not likely contribute to peak I of ribonuclease in this Me0 0 instance. H ,OMe MH eFGH,OH Y“ A second peak, due to the 123 wCH protons, of ‘ I 1 1 HO O C O N H z HO OH all amino acids the 30 @-protons of serine and 10 @-protonsof threonine should be observed at +90 YI v to +I10 C.P.S. relative to toluene if no shifts due to peptide bond formation were to be expected. I n addition, the 6 glycine protons and 8 protons of the CH, group in arginine are observable at slightly OH HO higher fields of +I17 to +I20 C.P.S. in a region CH3 intermediate between the second and third peaks. 1Y Together with the protons of the second peak they It appears, therefore, that the antibiotic activity would account for 23.47,of the total area, as com2%. of novobiocin is highly dependent on the presence pared t o the experimental estimate of 26 As peptide bond formation causes a displacement of and location of the carbamate group. We gratefully acknowledge the contributions of a-CH peaks 15-20 cycles toward lower fields, the Drs. W. G. Jackson, W. T. Sokolski, J. L. Johnson, maximum of the second peak of ribonuclease, should appear at about +SO to +90 c.p s. where it Mrs. G. S. Fonken, and Mr. W. A. Struck. is actually found. RESEARCH LABORATORIES J. W. HINMAN A third peak with a maximum between 13.5 to THEUPJOHNCOMPANY E. L. CARON KALAMAZOO, MICHIGAS H. HOEKSEMA +145 C.P.S. is accounted for by CH2 groups as follom-s: 16 from cysteine-cystine, 8 from methRECEIVED AVGUST8, 1957 ionine, 32 from aspartic acid, 20 from lysine ( E -CH2), 10 from proline, 8 from histidine, 6 from phenylalanine and 12 from tyrosine. This amounts AN INTERPRETATION OF THE PROTON MAGNETIC t o 15.27, of the total area, the experimental estiRESONANCE SPECTRUM OF RIBONUCLEASE mate for the peak being 18 + 3c/,. The 64 CH2 Sir: groupsof glutamicacid fallat +156c.p.s., accounting Saunders, Wishnia and Kirkwoodl recently have for an additional !3,2YGof the area, which could be reported the first proton magnetic spectrum due to a attributed either t o the third or the fourth peak. protein, pancreatic ribonuclease, in D 2 0 . The It is probable that peak I V is actually a fusion of spectrum consists of four broad peaks, falling two broad peaks, one with a maximum a t +180 to roughly into the range between the aromatic and the +190 c.P.s., the other a t $210 to $220. The aliphatic peaks of toluene. The authors have first of these is due to aliphatic CH groups of identified the first peak tentatively, corresponding leucine (2 hydrogens) isoleucine (3) and valine t o lower field strength, as due to the aromatic (9), CH2 groups of leucine (4 hydrogens), isohydrogens and the fourth peak, a t highest field leucine (G), proline (20)) lysine (BO’, and arginine strength as due to “aliphatic carbon atoms attached (16) and the CH3 group of methionine (12 hydroonly t o other aliphatic carbons,” stating that a gens), accounting for 19.17, of the area. The complete interpretation is not possible a t present. second, due to CH3 groups of alanine (36 hydroHowever, our studies on the proton magnetic gens), valine (M),leucine (12), isoleucine (18),and resonance spectra of amino acids and peptidesz3 threonine (30) would respresent 21.6%. The together with the amino acid composition of combined area under peak IV. including glutamic ribonuclease determined by Hirs, Stein and Moore, acid is 49.9$,, closely in agreement with the experipermit the prediction of a complete n m . r . spec- mental value of 47 =k 37,. It would be of interest trum for the protein, which is in excellent agree- if it were possible to sufficiently average the field ment with experiment. seen by the aliphatic protons-perhaps a t higher There is little reason to doubt that peak 1 is temperature-to actually resolve the fourth peak. due essentially to the 18 phenylalanine and 36 It might also be noted, that due t o peptide bond formation small and somewhat variable shifts (1) XI Saunders A Wishnia a n d J G Kirkwood THISJ O U R V A L 79, 3289 (1957) toward lower fields can be expected for hydrogens (2) M Takeda end 0 Jardetzky, J Chem Phvs 26, 1346 (1957) contributing to peaks XI1 and I T T making :>nevact (3) 0 Jardetzky end C D Jardetzky, t o be publlshed prediction of their maximum dificult. However, (4) C H W Him, w k , Fltdbl and 9 M o o r e J BIdl Ckrm 911, 841 (1964) even the foregoing illustrates that I\TM R spectra
VHpMe
u+ ’,
fs
m J H c o y J + , I
*
+
