3482
COMXUNICATIONS TO
THE
EDITOR
VOl. 80
method (b), it should be noted that t-butyl-di-namylborane (b.p. 47.5” at 0.14 mm., n 2 5 ~ 1.4333, dZ5 0.7585) is also a new mixed trialkylborane stable to disproportionation when distilled in vacuo and was prepared by the alkylation of t butyldichloroborane with n-amylmagnesium bromide in ether. Anal. Calcd. for C I ~ H ~ ~B, B: 5.15. Found: B, 5.26; M R D calcd.,371.93; obsd., 72.06. A number of other mixed trialkylboranes stable to distillation have been prepared in This Laboratory. Details regarding their properties and methods of preparation will be reported at a later date.
mones2 to demonstrate that their respective biological activities do not reside in relatively large proteins but in smaller peptides. We wish to report a simple micromethod of electrodialysis utilizing membranes implanted in starch gels which has allowed us to study this question with regard to pituitary thyrotropin. Based on the passage or non-passage of thyrotropic activity through cellophane membranes whose permeabilities were calibrated with proteins of known molecular weight, we have tentatively assigned a molecular weight in the range of 2(i,OO030,000 to the active principle. The method makes use of the starch gel electroDEPARTMENT OF CHEMISTRY G. F. HEYNION phoresis of Smithies4and is carried out in a trough, UNIVERSITYOF XOTREDAME P. X MCCUSKER 2.3 by 30 cm., 1 cm. deep. The membrane is SOTRE DAME, ISDIANA J. V . MARRA placed in the gel in either of two ways. One way RECEIVED JUSE 2, 1958 is to insert the membrane across half the width of the gel, parallel to and about 1 cm. away from the A MICROMETHOD OF ELECTRODIALYSIS AND ITS filter paper4 containing the sample. The effect of the membrane can thus be seen in direct comparison APPLICATION T O THYROTROPIC HORMONE with the electrophoretic pattern of the sample. Sir: If the membrane, however, is impermeable to the Electrodialysis has been used successfully with protein, the protein tends to travel around it. corticotropin’ and the posterior pituitary hor- This can be prevented by placing the nicmbrane in a semicircle held in place by masking tape and + S a m p l e / JMernbrane + Sample1 l M e m b r o n e - then pouring the hot starch solution in the appara- ._ tus. The latter method was used when bioassay 5 1 UNSTRETCHED MEMBRANE experiments were carried out. 1 q -3 __ Visking 20,‘32 dialysis tubing, which has been LYSOZYME (14,OOOi k o Z y M E t OVALEUM;N used by Craig and co-workers in studying dialysis of proteins by diffusion,j was found to be most suitable for the studies with thyrotropin (30 U.S.P. units per mg.”). With this membrane it was found that neither the biological activity nor any stainI __ able material would pass through a t pH 5.0 or a t pH 9.5. If stretching by hydrostatic pressure is f _ _ _ ~ - carried out,5 the membrane allows both the acCHYMOTRYPSINOGEN CHYMOTRYPSINOGEN f24.000) tivity and the staining components to pass a t pH 5.0. These results afford strong evidence that the I ‘b. I j\: thyrotropic activity does not reside in a small peptl - 3 __I __. PROLACTIN tide bound solely by electrostatic forces to a larger protein and thus separable by electrodialysis. The results together with those on known proteins, IL-upon which the tentative molecular weight is THYROTROPIN PREP THYROTROPIN PREP based, are shown in Fig. 1. The resolving power of the starch gel electrophoresis and the selectivity of the membranes6 are clearly illustrated in the case of the prolactin preparation, which separates into three components. Xone of these will pass the unstretched membrane but one component passes through the stretched membrane a t both PH 9.5 and 5.0. With respect to thyrotropin it should be rememFig. 1.-Diagram of starch gel electrodialysis with a series of proteins of increasing molecular weight (mol. wts. from bered that other methods may still demonstrate ref. 5) in acetate buffer, pH 5.0, r/2 = 0.012: lysozyme that the activity resides in a small molecule. .-I ovalbumin was run a t PH 3 . 5 ; duration of runs 2-3 h r . ; full report of this work together with other at260-300 volts; 13 ma. Proteins were commercial prepara- tempts to dissociate the thyrotropic activity from
r-
i
,
’
2%.
-
+
tions except prolactin.^ Those proteins not passing the membrane were detected as heavily stained areas just behind the membrane. With pH 9..5 glycine buffer, analogous results were obtained with trypsin, prolactin, in-iimuciiict and ovalbumin.
4. (1)
G. P. Hess, J . I. Harris, I?. H. Carpenter and C . H. Li, THIS 73, 5918 (1951).
JOURNAL,
( 2 ) C . H . Haselbach and .4 K . P i g u e t , ,Vel>~,Ciiint. A c l n , 36, 2131 (1952)): R . Acher, J . Chauvet and G O l i v r y , l?iochinz. Biop1iy.s Arlil,
22,401 (1966). (3) J , G . Pierce, L K , U’ynston and h l . 12. Carsten, Riociiiiii. Iliophys. A t l n , 28, 134 (1958). (4) 0. Smithies, B i o c h ~ mJ. , , 61, 629 (193J). ( 3 ) L. C . Craig, T. P. King and A . StrachPr, T H I s J O U R N A I . , 7 9 , 3729 (1957).
COMMUNICATIONS TO THE EDITOR
July 5 , 1955
3483
the material of 26,000-30,000 molecular weight will be published.‘j
been supposed3 that such a hybridization change decreases the covalent radius of carbon by 0.04 A,, just accounting for the difference between the C-H (6) Supported by a grant No. C-2290 from the National Cancer Institute, National Institutes of Health. The prolactin used in this distances in ethane and acetylene. We suggest work was a gift from the Endocrinology Study Section, National that C-C distances are much more sensitive to Institutes of Health. hybridization changes than C-H distances, so that DEPARTMEST OF PHYSIOLOGICAL CHEMISTRY sp carbon has a smaller covalent radius toward JOHN G. PIERCE carbon than toward hydrogen. The radius toward UNIVERSITY OF CALIFORSIA MEDICAL CENTER Los ANGELES, CALIF. MARYE. CARSTEN halogens is apparently still smaller, as evidenced by the even larger differences between C-X disRECEIVED MAY29, 1958 tances in the methyl halides and the haloacetylenes or cyanogen, halide^.^ The variability of the coABSENCE OF HYPERCONJUGATIVE EFFECTS ON T H E valent radius may be due to the fact that the hySTRUCTURE OF MALONONITRILE’ bridization change must alter the repulsive as well Sir: as the attractive forces associated with the bond. Microwave studies of derivatives of acetonitrile The repulsive forces should be particularly sensitive and methylacetylene suggest2 that the length of a to the size and electron distribution in the atom carbon-carbon single bond adjacent to a triple bond bonded to the sp carbon. never deviates significantly from an average value We feel that these results call for a searching rejust over 1.46 A. Several different hypotheses examination of the common assumption that varihave been used to explain, for each compound sep- ations in C-C bond lengths are primarily deterarately, the shortening of the bond relative to the mined by bond-order changes, and that they may standard value, 1.54 8. In acetonitrile, a major be used as unequivocal evidence for bond-order part of the bond shortening has been attributed to changes due to resonance. n-bonding arising through hyperconjugation. To (3) C . A. Coulson, “Valence,” Oxford University Press, London, test this assumption, we have studied the structure 1952, pp, 206, 310. (4) W. Gordy, W. V. Smith and R. F. Trambarulo, “Microwave of malononitrile, CHZ(CN)Z. Consideration of the Spectroscopy,” John Wiley and Sons, hTew York, N. Y., 1953, p. 371. possible valence-bond structures shows that each DEPARTMENT OF CHEMISTRY C-C bond here should have appreciably less doublePURDUE UNIVERSITY NORBERT MULLER bond character than in acetonitrile, and hence LAFAYETTE, INDIANA DONALD E. PRITCHARD should be appreciably longer. RECEIVEDMAY23, 1958 We have measured about two hundred frequencies in the microwave rotational spectrum of gaseous malononitrile, and assigned fifteen of SIMULTANEOUS SYNTHESIS OF AROMATIC ACID CHLORIDES AND METAL CHLORIDES these to low-J transitions. The rotational conSir: stants are A new simultaneous synthesis of aromatic acid a = 20,882.35 mc. chlorides and metal chlorides from trichloromethylb = 2,942.15 mc. ated aromatic compounds and metal oxides has c = 2,616.75 rnc. been discovered. The method of preparation is The structural parameters which we feel best fit simple and consists of heating the reactants in the proportions as shown in equations 1, 2 and 3. the data are r(C-H) r(C=X) r(C-C) LH-C---H LC-C--C
= 1 . 0 9 A. (assumed) = 1.158 A. (assumed) = 1.460 A.
7ClS
= 113’39’
An unambiguous structure determination will be possible when we complete work on malononitriledz. It seems certain that the correct C-C distance is again very nearly 1.46 A. Taken with the data on related molecules, this makes i t appear very unlikely that n-bonding arising through hyperconjugation is a major factor in fixing bond lengths. According to the principle of parsimony, one should seek a common explanation for the essentially constant single-bond distances in all these molecules. We suggest that the bond contraction is principally attributable to the change from spa to sp hybridization a t one end of the bond. It has (1) Supported by the Purdue Research Foundation acting under contract AT-(ll-1)-164 with the Atomic Energy Commission, (2) W. Zeil and 1. P. Pfrommer, Z . EIPkliochem., 61, 938 (1957), and references given there.
-
+ TiOz
(1) &CCI. /
= 105’39’
yoc1
6 C O C I
Cocl
CCl3 (2) 312 &GI,
/
YCla
+ TiCI4
+ vzos
-+
312 &COCI
+ 2voc13
YOCl
Metal oxides such as Ti02 and V Z Ogive ~ the best yields of both the aromatic acid chloride and the metal chloride. Other oxides such as those of arsenic, antimony and zirconium are operable but give lower yields of the aromatic acid chloride. Aromatic compounds containing mono- or bis(trichloromethyl) groups, which are obtained readily by the side-chain chlorination of the corresponding hydrocarbon, work equally well. With the mono-(trichloromethyl) compounds, monoacid
