this note is to report the use of organic inclusion compounds for the ... of free radicals on exposure to X-irradiation.10 Using urea compounds as examples,.
Proceedings of the NATIONAL ACADEMY OF SCIENCES Volume 48 * Number 11
* November 15, 1962
ELECTRON SPIN RESONANCE OF X-IRRADIATED ORGANIC INCLUSION COMPOUNDS* BY 0. HAYES GRIFFITHt
AND
HARDEN M. MCCONNELLT
GATES AND CRELLIN LABORATORIES OF CHEMISTRY,§ CALIFORNIA INSTITUTE OF TECHNOLOGY
Communicated by Norman Davidson, September 24, 1962
In recent years, there have been many electron-spin-resonance (ESR) investigations of X- and -y-irradiated single crystals of organic compounds.' Studies of the anisotropic hyperfine spectra of the trapped radicals have been used both to identify the radicals formed and to determine their electronic structure. However, there are two troublesome limitations to this technique: (1) Many organic compounds of theoretical importance are liquids at room temperature, and (2) the crystal structures of many compounds of interest have not been determined. The purpose of this note is to report the use of organic inclusion compounds for the investigation of long-lived free radicals produced by X-irradiation. Inclusion compounds are crystalline compounds in which two or more components are associated without ordinary chemical bonds and in which one of the components fits into cavities formed by the other. The cavity formed by the host may be tube-shaped or cage-shaped, or it may consist of open layers.2 Perhaps the most useful for X-ray damage studies are the urea and thiourea crystalline inclusion compounds. In these compounds, the cavity formed by the host (e.g., urea) is tube-shaped. Crystallographic investigations of the urea and thiourea compounds have been reported,3-6 and a large variety of organic molecules have been found to include with urea and thiourea7'9 (in general linear molecules include with urea and highly branched or cyclic compounds include with thiourea). In addition, urea and thiourea have been reported to yield a negligible concentration of free radicals on exposure to X-irradiation.10 Using urea compounds as examples, three applications of organic inclusion compounds are reported below. These are: (1) The identification of stable free radicals not observed in X-ray-damaged single crystals of the molecule itself, (2) the study of radiation damage in a molecule with unknown crystal structure, and (3) the investigation of X-ray damage of a compound which is a liquid at room temperature. Single crystals of the adipic acid (HO2C(CH2)4C02H)-urea inclusion compound were X-irradiated at room temperature. The ESR spectra indicate the presence of two stable free radicals, I and II, present in different concentrations. 1877
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CHEMISTRY: GRIFFITH AND McCONNELL
PROC. N. A. S.
The radical present in the greater concentration is H H H
HO2C-C-C-C-C-CO2H
I
II H H H This radical has been thoroughly studied in a y-irradiated single crystal of adipic acid.II The ESR spectra of radical I in the urea inclusion compound unambiguously identified the orientation of I, and hence the direction of the tubular cavity, with respect to the external crystal morphology. The crystals grown from an aqueous solution elongate along the tube direction, making the adipic acid chain direction easily identifiable. From the ESR spectra, it is evident that all molecules of I are magnetically equivalent when the laboratory magnetic field is in a direction perpendicular to the tubular cavity. Therefore, there exists either a unique conformation of I in the cavity or two conformations related by a twofold rotation. As expected, all molecules of I are magnetically equivalent when the magnetic field is parallel to the tubular cavity. In addition to the 6- to 8-line spectrum of I, a single line was observed and is attributed to the presence of radical II. Spectroscopic splitting factor data and limited C13 coupling constant data are reminiscent of the C02- radical ion previously detected in ay-irradiated single crystals of sodium formate.'2 Radical II is apparently associated with the pink color acquired by the adipic acid-urea inclusion crystal during X-irradiation. Upon heat treatment, both the pink color and the ESR spectrum of radical II disappear, while the spectrum of I remains. A similar pink color persists for a few seconds after X-irradiation in single crystals of adipic acid, possibly indicating the presence of II as a transient radical in the adipic acid crystal. Sebacic acid analogues of I and II have also been observed in single
crystals of the sebacic acid-urea inclusion compound. The ESR spectrum of X-irradiated single crystals of the fumaric acid'3 (trans HO2CCH=CHCO2H)-urea inclusion compound is dominated by the six-line spectrum of radical III: H
HO2CC-C
CCO2H
III
HH This radical has previously been produced in X- and a-irradiated single crystals of succinic acid14 I' and in X-irradiated single crystals of dl-aspartic acid.16 The formation of III from fumaric acid is an example of the "hydrogen aufbau process"17; that is, the formation of a long-lived oriented free radical by the addition of a hydrogen atom to the undamaged molecule. 18 As confirmed by the ESR spectra, fumaric acid-urea crystals, like those of adipic acid-urea, elongate along the tubular direction. There exist, however, multiple orientations of radical III in the tubular cavity. The liquid compound investigated was ethyl heptanoate. The ESR spectrum
VOL. 48, 1962
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of X-irradiated single crystals of the ethyl heptanoate-urea compound consists of six lines and is attributed to the radical H H H H
I H3C-C-C-C-C-C- COSCH2CH3
IV
H H H H H Again, the crystals (grown from methanol) were elongated along the tubular direction, simplifying the analysis of the spectra. At 300'K and with the magnetic field perpendicular to the tubular cavity, the spectrum of IV is isotropic. As the temperature is lowered, this spectrum broadens until at 770K the spectrum consists of eight broad lines. This behavior suggests that at room temperature radical IV is undergoing a large degree of molecular motion, if not actually rotating in the tubular cavity. These observations are consistent with both dielectric absorption19 and nuclear magnetic resonances investigations of urea compounds with long-chain hydrocarbons and their derivatives. In addition to the above compounds, ESR data have been taken of X-irradiated powders of nitrile, amide, carbonyl, alcohol, thiol, ether, unsaturated (R2C=CR2 and RC CR), and other derivatives of the straight chain hydrocarbons. Useful ESR data have also been recorded for organic inclusion compounds of thiourea and for the clathrates of hydroquinone. In particular, single crystals of cycloheptatriene-thiourea, cyclooctatetraene-thiourea, and formic acid-hydroquinone yield high concentrations of free radicals on exposure to X-irradiation. In all the above-mentioned urea, thiourea, and hydroquinone inclusion compounds, the free radical concentration produced from the host was negligible compared to that produced from the included molecules. A detailed investigation of some of these organic inclusion compounds is in progress. We are greatly indebted to A. L. Kwiram for helpful discussions.
Supported by the National Science Foundation. t National Science Foundation Predoctoral Fellow. $ Alfred P. Sloan Fellow. § Contribution No. 2889. 1Weissman, S. I., Ann. Rev. Phys. Chem., 12, 155 (1961). 2Schlenk, W., Jr., Fortsch. Chem. Forsch., 2, 92 (1951). 3 Schlenk, W., Jr., Ann. Chem., Liebigs, 565, 204 (1949); ibid., 573, 142 (1951). 4 Smith, A. E., J. Chem. Phys., 18, 150 (1950). 6 Smith, A. E., Acta Cryst., S. 224 (1952). 6 Lenne, H.-U., Acta Cryst., 7, 1 (1954). *
7 Angla, B., Ann. Chim., 4, 639 (1949). 8 Redlich, O., C. M. Gable, A. K. Dunlop,
and R. W. Millar, J. Am. Chem. Soc., 72, 4153 (1950); ibid., 72, 4161 (1950). 9 Kobe, K. A., and W. G. Domask, Petrol. Refiner, 31, 151 (1952). '0Jaseja, T. S., and R. S. Anderson, J. Chem. Phys., 35, 2192 (1961). Morton, J. R., and A. Horsfield, Mol. Phys., 4, 219 (1961). 12 Ovenall, D. W., and D. H. Whiffen, Mol. Phys., 4, 135 (1961). 13 A detailed crystallographic investigation of fumaric acid has not, to the authors' knowledge, appeared in the literature.
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PROC. N. A. S.
14 Heller, C., and H. M. McConnell, J. Chem. Phys., 32, 1535 (1960). 15
Pooley, D., and D. H. Whiffen, Mol. Phys., 4, 81 (1961). 16 Jaseja, T. S., and R. S. Anderson, J. Chem. Phys., 36, 2727 (1962). 17 Kwiram, A. L., and H. M. McConnell, these PROCEEDINGS, 48, 499 (1962). 18 Preliminary work on X-irradiated single crystals of maleic acid (cis HO2CCH=CHCO2H) indicates that radical III is also formed in this system. 19 Meakins, R. J., Trans. Faraday Soc., 51, 953 (1955). 20 Gilson, D. F. R., and C. A. McDowell, Mel. Phys., 4, 125 (1961).
THE FUSION OF TWO PEPTIDE CHAINS IN HEMOGLOBIN LEPORE AND ITS INTERPRETATION AS A GENETIC DELETION CORRADO BAGLIONI* BIOLOGY, MASSACHUSETTS INSTITUTE OF TECHNOLOGY Communicated by S. E. Luria, August 22, 1962
DEPARTMENT OF
Gerald and Diamond' have described an abnormal hemoglobin, called hemoglobin Lepore (Hb-LeporeB.8t~n). The present communication reports the results of a chemical investigation of Hb-LeporeBo8ton and an interpretation of the genetic events leading to the formation of a single peptide chain from the fusion of two different chains. Abnormal hemoglobins electrophoretically identical to Hb-LeporeB.8t~n have have been reported among Greeks,2 Italians,3 and Papuans4 and designated HbPylos, Hb-G, and Hb-LeporeHollandia respectively. These hemoglobins are always found in concentrations of about 10-15 per cent of the total hemoglobin in the heterozygotes. One individual homozygous for the Hb-Pylos gene and two homozygous for the Hb-LeporeHollandia gene have been reported.2 4 They were characterized by the absence of normal adult hemoglobin (Hb-A) and of the minor component hemoglobin A2(Hb-A2).2 4 Neeb et al.4 have reported, however, the presence of traces of a hemoglobin component migrating like Hb-A2 in HbLeporeHollan(lia homozygotes; the identification of this trace component remains to be established. The three normal human hemoglobins Hb-A, Hb-F (fetal hemoglobin), and Hb-A2 consist of two a peptide chains which are under the control of a single structural gene and of two other peptide chains which differ in different hemoglobins: 1 chains in Hb-A, -y chains in Hb-F, and 5 chains in Hb-A2.5 These are under the control of different structural genes.5 The 13 gene is linked to the 6 gene.5 The 1 and 8 chains have extremely similar amino acid compositions and sequences.6-8 This similarity has suggested that the 8 gene may have originated through a duplication of the 13 gene followed by independent evolution.9 The Hb-Pylos and Rb-LeporeHollandia mutations seem to affect both the two linked genes 13 and 8, suppressing the synthesis of both Hb-A and Hb-A2 in homozygotes.2 4y 4 The a and peptide chains are synthesized normally.2' 6 Gerald et al.'0 have suggested that the Lepore abnormality is a "mutation possibly involving two cistrons." These authors fingerprinted Hb-LeporeBo8ton and HbPylos and analyzed some of their tryptic peptides. The fingerprints were found to be indistinguishable from those of Hb-A2. The composition of the tryptic peptides
