330 DOROTHYM. WFXNCH Vol. 63 chloroform melted at 214-216'. The pure ester of the not be secured but the crystals obtained were shown to terephthalic acid is recorded" as melting at 225'. Neu- be identical with an authentic sample in having parallel tralization equivalent of the mixture of acids was 84.3; extinction, pleochroism, centered acute bisectrix, and calculated for phthalic acid 83.3. No trace of benzoic acid elongated obtuse bisectrix, and were not in whole agree- could be detected among the oxidation products. Ap- ment with observations on known samples of the like parently the mixture is largely isophthalic acid. The derivatives of acetone and butyraldehyde. A trace of yield calculated on the basis of four propyl chloride mole- propionaldehyde may also be present, for a few crystals pre- cules to one of the butylpropylbenzene mixture varied pared from one of the fractions appeared to have similar from 14 to 38%, an estimation based upon the quantity of optical properties.12 high boiling residue left after distilling the butylbenzene Summary from each experiment. About 6 to 12 g. of a mixture of saturated and un- Organosodium compounds from n-butyl and saturated compounds, boiling in the hexane and nonane n-propyl chlorides have been prepared, the ease range, was obtained from each of these preparations, the of reaction being progressively less, the yields quantity being larger the higher the of re- lower, and the ratio of di- to monocarboxylic acid action. Careful fractionation in an eighteen-plate column greater than observed with the amyl homolog. failed to show any single product. All fractions also showed presence of unsaturated material. Upon ozonoly- n-Butyl- and n-propylsodium are progressively sis of a 63 to 66' cut, decomposition of the ozonides with less reactive with benzene and toluene than is n- zinc and water, and careful fractionation of the resulting amylsodium. aldehydes and ketones, definite evidence in the form of the n-Butylbenzene can be prepared conveniently dinitrophenylhydrazone derivative was obtained for the by adding propyl chloride to sodium in toluene presence of acetaldehyde. The melting point observed was 146-150"; that of an authentic sample was 147.5148'; at 72'. and that of the mixture was 148-150 '. A mixed melting Metalation of butylbenzene takes place largely point with the like derivative of propionaldehyde (m. p. in the meta position. 190") was 141". Owing to the small amount of material (12) We are greatly indebted to Mr. Gibb for assistance on the left after attempts at purification, a satisfactory melting optical studies. point for 2-butanone-2,4-dinitrophenylhydrazonecould CAMBRIDGE,MASS. RECEIVEDOCTOBER 7, 1910

[CONTRIBUTION FROM THE CHEMICAL LABORATORYOF THEJOHNS HOPKINSUNIVERSITY ] The Geometrical Attack on Structure BY DOROTHYM. WIUNCH It is unnecessary at the present time to state discuss, necessarily in a preliminary manner, two the case for the cyclol hypothesis, since authorita- issues (first raised in my publicationslOJ1) to tive accounts have already been given by Lang- which Pauling and Niemann refer, namely (a) cer- muir of the way in which the theory accounts sat- tain short interatomic distances in the original isfactorily for many of the well-known properties cage structures and (b) the of formation of the globular .132 In a recent summary,a of these structures. In particular it is shown that however, Pauling and Niemann repeat a number the claim by Pauling and Niemann that the cyclol of statements purporting to disprove the theory hypothesis can be disposed of by means of Anson already made by other writers. Attention must and Mirsky's heat of denaturation of trypsin therefore be directed to a number of publications must be rejected. in which these criticisms have already been dis- (a) In my fist studies of possible protein struc- cussed, at least so far as their scientific importance tures, very exacting metrical conditions were appeared to ~arrant.~,~-~We then proceed to adopted, mainly in order to demonstrate in a (1) Langmuir, Cold Spring Harbor Symposia Quanl. Bid, 6, 135 simple manner the possibility of handling prob- (1938). lems of by strictly mathemati- (2) Langmuir, Proc. Phys. SOC.(London), 61, 542 (1939). (3) Pauling and Niemann, THISJOURNAL, 61, 1860 (1939). cal methods. These, it appears from crystallo- (4) Wrinch, Cold Spring Harbor Symposia Quant. Biol., 6, 122 graphical data, are unnecessarily onerous. Evi- (1938). (5) Langmuir, Nafurr, 143, 280 (1939). dently a discussion of certain short interatomic (6) Langmuir and Wrinch, ibid., 143, 49 (1939). distances in the original cyclol structures can only (7) Wrinch, ibid., 143, 482 (1939). (8) Wrinch, ibid., 143, 763 (1939); 146, 660, 1018 (1940). (10) Wrinch, Proc. Roy. SOC.(London), A160,59 (1937); A161,505 (9) Langmuir and Wrinch, Proc. Phys. SOC.(London), 61, 613 (1937). (1939). (11) Wrinch, Nafure,138, 241 (1936). Feb., 1941 GEOMETRICALATTACK ON PROTEINSTRUCTURE 33 1 be profitable if accompanied by a detailed study weight.) Denaturation, on the cyclol therory, of the propriety and the feasibility of relaxing corresponds to any breakdown of the intact struc- the metrical conditions. Further, it must be re- ture produced by the opening of any subset of membered that cases are known (e. g., in the ter- cyclol bonds.14 The theory thus readily offers an penes, the and phenanthrene derivatives) interpretation of the well-known fact that very in which the accumulated evidence of organic different denatured products can be obtained from chemistry makes it necessary to place certain non- one and the same protein (cf. the film studies of bonded nearer together than the minimum which have been shaken for different distances allowed by Pauling and Niemann. times, heated for different times at 65’) etc.’), These distances in any case refer to interatomic regarding each type of denatured product as due distances in different molecules. Thus the state- to the opening of some definite subset of cyclol ment that methyl groups in the case of hexa- bonds and possibly the re-formation of some of methylbenzene are 4.0 to 4.1 8. apart refers to them. A denatured product (particularly when, intermolecular distances. Within a single mole- as in the case under discussion, the denaturation cule of hexamethylbenzene, methyl groups ap- is reversible) may have only a small or even neg- proach as near as 2.93 8. Further, these dis- ligible decrease in its complement of cyclol bonds. tances have been derived from a study of simple In the case of a CZcyclol cage, such as Pauling molecules which bear no resemblance to the very and Niemann are assuming for trypsin, a net de- complex highly organized structures which, on crease of one in n cyclol bonds, where n is 12 or any plausible theory of protein structure, must even 24, is sufficient to allow a gross change in characterize the globular proteins. Nothing is structure and so may be sufficient to account for known as to the nearest approach of groups denaturation. A degradation product of trypsin within a single molecule in such cases. (b) It is may therefore possess or even 23/24 of its full known that, under certain closely defined condi- complement of cyclol bonds. Thus if it were the tions, the heat of denaturation of trypsin amounts case that a cyclol cage structure is about 28 kcal./ to less than 1 kcal./mole of residues.12 mole of amino acid residues less stable than the Pauling and Niemann use this figure in an attempt corresponding linear polypeptides, it still would to disprove the cyclol hypothesis as follows: (1) not follow that the heat of denaturation should they claim that a cyclol cage structure is about 28 be of this order. With reversible denaturation kcal./mole of amino acid residues less stable than the figure should in this event be 28/12, or say 2.3 the corresponding linear polypeptide containing or 1.17 if n were 12 or 24, respectively. NHCO groups: (2) they claim that Anson and (1) But there is, in any case, no reason to sup- Mirsky’s denatured trypsin consists of linear poly- pose that the figure 28 kcal./mole of residues rep- . They then deduce that the trypsin mole- resents the situation even approximately. cule cannot be a cyclol cage structure, since if it We consider first the attempt of Pauling and were the heat of denaturation obtained by Anson Niemann to estimate the difference between the and Mirsky would have been about -28 kcal./mole heats of formation of a glycyl linear residue and a of amino acid residues. It is my opinion that both glycyl cyclol residue by the explicit use of bond claims (1) and (2) were unfounded, and that the . Repeating without acknowledgment suggested deduction therefore falls to the ground. the calculation (yielding the value 27.3) which I (2) Little or nothing is known as to the structure published in 1936 to ventilate the question,16 of any denatured protein, nothing as to the struc- Pauling and Niemann write for the heats of for- ture of the denatured trypsin under discussion. mation in the gaseous state It is assumed by Pauling and Niemann that de- linear glycyl residue - cyclol glycyl residue naturation on the cyclol theory means the open- = C=O + N-H + ( in CO-NH bond) - ing of all cyclol bonds. This is not the case. (C-0 + 0-H + C-N) = 152 83.3 21 - (110.2 70 48.6) = 27.5 (In particular the difference between trypsin and + + + + (1) denatured trypsin in this case cannot consist in or say 28, since the standard energy of NH is opening of all cyclol bonds, since, as Mirsky and usually taken to be 83.7.16 It is my opinion that Pauling state,I3 there is no change in molecular (14) Wrinch, Phil. Mag., 26, 706 (1938). (15) Wrinch, Nafztre, 158, 241 (1936). This calculation was (12) Anson and Mirsky, J. Gen. Physiol., 17, 393 (1934). brought to the notice of Professor Pauling in January, 1938. (13) Mirsky and Pauling, Proc. Naf. Acad. Sci. U. S., 22 439 (16) Pauling, “Nature of the ,” Cornell University (1936) Press, Ithaca. N.Y., p. 53. 332 DOROTHYM. WRINCH Vol. 63 no weight is to be attached to this formal evalua- of cyclol residues, we obtain 1302.8 = 1293.0. tion, since little or nothing is known as to the Again the equation values of several of the bond energies in these CsHizNi + l2C2He = 4N(CzHs)s + 6CH4 particular cases. Particularly I question the say D + 12 E = 4F + 6C, using again the values validity of (1) assuming that the C-CHz bonds used for evaluating the glycyl cyclol residue, we have the same energy in both structures, (2) as- obtain the result 5446.7 = 5405.6. Plainly such suming the C-0 bonds in cyclols have an energy equations are untrue in the case of these com- as small as in the primary alcohols. These uncer- pounds. As little reliance can be placed upon tainties appear to me to make the calculation them for the evaluation of cyclol residues. Evi- valueless, except in so far as it calls attention dently the only way of interpreting such an equa- (as was my reason for publishing my calculation tion, P = Q + R, is to write it in the form in 1936) to the types of data which are needed, Heat of combustion of P, K(P,) = Heat of cornbusdon namely, bond distances (and derivatively bond + of QO + Heat of combustion of R, + K(Qo) + K(R,) energies) in long linear peptides-particularly those containing residues other than glycyls-and where K(Xc) is the heat of sublimation of X plus the amount by which the heat of formation of in ring compounds in which a carbon bearing an OH group is linked to two nitrogens in the same X,,, exceeds the sum of bond energies, when the ring, and to another carbon bearing a variety standard values (e. g., N - C = 48.6) are used. of substituents. When written in this form, all such equations of course yield the result = In other words, However, as Pauling and Niemann rest the 0 0. greater part of their case against the cyclol hy- such an equation as (2) states simply that, if pothesis upon this calculation and upon two equal energies be given to the corresponding bonds, then others, it is necessary (A) to point out that these two other calculations are strictly equivalent to 4C-C + 8C-H + 4C4+ 40-H = 4(C-C 5C-H C-0 0-H) - 12C-H the first and (B) to consider how far present data + + + offer any guide as to the bond energies involved After the same manner, the equations for the in the first calculations. (A) Pauling and Nie- cyclol residue constructed by Pauling and Nie- mann state that they have obtained without the mann, which may be written use of bond energies other values (in fact 32 and linear glycyl residue - cyclol glycyl residue 24) agreeing closely with and so further supporting =I/* diketopiperazine - (F + B - 3E) = diketopiperazine - (A + D - 3C)/4 the value 28 found with the use of bond energies. First, we have to point out that, assuming that give simply my original calculation. In view of the heat of combustion of crystalline diketopi- the claim that such equations give estimates with- perazine is 474.6, it is erroneous to conclude that out the use of bond energies, it must be empha- the heat of formation of crystalline diketopipera- sized that they are simply bond energy equations. zine from elements in their standard states is 128. They make no use of the heats of combustion The correct value is cited and add nothing to my original calcula- tion,15to which in fact they are strictly equivalent. -474.6 + 4 X 94.45 + 3 X 68.37 = 108.3 (B) The fact that a C-C bond length as small as These two calculations, as they stand, are cer- 1.47 + 0.03 A. has been found in the only cyclic tainly remarkable in that they give values which so far investigated, l7 whereas in glycineIR differ from the fist by +4 and -4, respectively, the distance is 1.52 * 0.02 A. shows that (1) is or using the correct value for the heat of forma- not the most plausible assumption. Rather it tion of crystalline diketopiperazine, by -6 and would seem natural to assume that this bond is - 14, respectively. The reason for these discrep- shorter and therefore stronger in the cyclol struc- ancies is easily seen. For if we write for the heats ture. Further, it seems difficult to reconcile the of combustion of crystalline compounds for which assumption of Pauling and Niemann that the ter- experimental data are available a typical equation tiary cyclol OH has its C-0 bond as low in energy such as as the standard value for primary alcohols, with C(CH20H)r = 4CsH5OH - 3CH4 (2) Pauling’s statementlQthat this bond in secondary say A = 4B 3C, with the values used by Paul- (17) Corey, THISJOURNAL, 60, 1598 (1938). - (18) Albrecht and Corey, ibid., 61, 1087 (1939). ing and Niemann to assess the heat of formation (19) Pauling, ibid., 64, 3570 (1932). Feb., 1941 GEOMETRICALATTACK ON PROTEINSTRUCTURE 333 and tertiary alcohols “seems to be 0.3 to 0.5 v. e. the stability of a native protein is impaired when more stable than corresponds to this C-0 value.’’ the PH goes outside some central “stability” Rather it would seem more reasonable to follow range suggests that these terms make some con- Pauling’s 1932 lead in attributing say 0.4 v. e. siderably greater contribution to the heat of for- or 9.2 kcal. more energy to this bond, as is in fact mation of the cage cyclol than to that of the linear suggested by the observed heat of combustion of peptide. In egg albumin, for example, where trimethylcarbinol. These corrections can easily there are say 27 basic and 27 acidic R groups, an add 12 to 18 to the heat of formation of the glycyl arrangement of these groups in isolated pairs at cyclol residue, reducing the balance in favor of the distances apart of 4 A. would contribute to the linear peptide in the gaseous state to (say) 13. So heat of formation of the cyclol cage an amount of far everything said has had reference only to glycyl the order of 2 or more per residue. This changes residues and only to the gaseous state. Now pro- the balance in favor of the cyclol to 1 or more. teins in general have at least one CHZ group per TO Sum Up.-The estimate of a balance in residue in their R groups. If any serious estimate favor of the linear peptide of 28 kcal./mole of of the relative stability of the cyclol cage and the residues rests upon many assumptions which lack extended peptide is to be attempted, this fact must plausibility. With assumptions which are less be taken into account. In view of the solubilities unplausible, the estimate can be changed to a of the native proteins it may be presumed that the balance in favor of cyclol cages of at least 1. interarrangement in space of this large number of However the lack of information makes any such CH2 groups is such as to contribute considerably estimates, ranging from 28 to -1 or less, of little more, say 2, to the heat of formation of the former usefulness. Since in any case, a reversible dena- than to that of the latter, and to facilitate a higher turation probably destroys only a small part of the degree of hydration in the former than in the fine structure, the heat of denaturation should latter. This higher degree of hydration, if it tally not with the balance in favor of cyclols per amounts to two more bonded water residue but with some small fraction of this molecules per residue in the cyclol than in the amount. peptide, would add a further 10 or more to the It must be concluded that no case has been balance in favor of cyclol cages. These two made out for deducing that the cyclol theory is terms thus may further reduce the balance in false on the basis of Anson and Mirsky’s figure for favor of linear peptides to 13 - 2 - 10 = 1. Finally, the heat of denaturation of trypsin; further, that the association of oppositely ionized R groups has no measurements of the heat of denaturation of to be taken into account. The dipole moments proteins can provide a test of the cyclol hypothesis of the native proteins which have been measured unless the structure of the denatured products in are all low (180 X for egg albumin, a mole- question is known and the heat of formation of such cule of diameter 30 8.or more and 500 X 10-l8 structures and of cyclol cages can be estimated. for hemoglobin, a molecule of about twice the Summary weight).21 While it must again be emphasized that the data necessary for an estimate are lack- Arguments against the cyclol hypothesis, which ing (since nothing is known as to the arrangement have been collected by Pauling and Niemann in a of the ionizable R groups on any native protein) recent article, are examined. It is found that it appears to be illegitimate and certainly un- they do not disprove it. In particular their state- plausible to assume that the coulomb energy terms ments purporting to prove that a protein with the would be unchanged when the cyclol cage struc- cyclol structure would be less stable than the ture opens into linear peptides. The fact that polypeptide chain structure by a very large amount is examined and found to be unproven. (20) Kharasch, Bur. Sfandards J. Research, 4, 359 (1929). (21) Coho, Chem. Rm., 34, 210 (1939). BALTIMORE,MD. RECEIVEDAPRIL 30, 1940