8244 20 r
I
I 0
0.1
I .o
I 10
WEIGHT ( n g )
Figure 2. Double antibody immunoassays: ( 0 ) natural bovine C-peptide; (0)synthetic peptide I; and (A) synthetic peptide 11. 1 2 5 1 synthetic tyrosinated bovine connecting peptide was used as a tracer.
almost identically with natural bovine connecting peptide, while I1 failed to displace the tracer completely (Figure 2). Accurate evaluation of the cross-reactivity of I1 was difficult because of nonparallelism in the displacement curves of the synthetic peptide and the natural bovine proinsulin or connecting peptide standard. Formyl protection of lysine residue at position 59 was shown to have no effect on the immunological reactivity of the connecting peptide. Both peptide 111 and IV, which were used as the amino components in the fragment condensations, showed no cross-reaction with bovine proinsulin or the connecting peptide. Detailed immunological evaluation of the synthetic bovine connecting peptide and the related peptides will be published elsewhere. It was clearly shown that full cross-reactivity of the bovine connecting peptide is obtained with the revised amino acid sequence. The distinct difference of immunological reactivity observed which was caused by an inversion of residues 50 and 52 seems to indicate that the 47-60 sequence is indeed involved in the immunological activity tested. The fact that synthetic peptide I11 was immunologically inactive could mean that only part of the antigenic determinant resides in the 47-60 sequence or that the whole 31-60 sequence is necessary for the determinant to acquire the conformation in which it is immunologically active with the particular antisera used. In addition, the present demonstration shows that the structural change in the sequence in which the antigenic determinant is located can be detected by the immunological technique when homogeneous synthetic peptides of definite structure are used. Further synthetic studies on the antigenic determinant of bovine connecting peptide are now under way. The syntheses of I and I1 were accomplished by the Rudinger azide modification. For the synthesis of I, the azide derived from Z-Arg(NO*)-Arg(H+)-Glu-
with 111 [[C~]*~D - 112.0" (c 1.0, 10% acetic acid); R f l 0.02; Rfrl 0.30; amino acid ratios in acid hydrolysate, Lyso dqO, 97Glul.92Pro2,91Glys.1sAlao.9sLeul,0 9 ; peptide content 88 %I, which had been obtained by catalytic hydrogenolysis, followed by gel filtration on Sephadex G- 10, of Z-Gly-Pro-Gly-Ala-Gly-Gly-Leu-Glu-Gly-ProPro-Gln-Lys(F)-Arg(H')-OH acetate pentahydrate [mp 184-192'; [c?!]*'D -44.0" (C 1.0, D M F ) ; Rr' 0.19; R?' 0.59: amino acid ratios in acid hydrolysate, L y s ~glArg0.93Glu1 . .s6Pro3.oYGly;.lsAlal,06Leu0,g8. Anal. Found: C, 49.1; H,7.0; N , 16.81. The resulting crude protected triacontapeptide was hydrogenated to give a crude preparation of I, which was purified by chromatography on CM-Sephadex C-25 using ammonium acetate buffer as an eluent and desalted by gel filtration on Sephadex G-25 [[a]*'D - 100.0" ( e 1.0, 10% acetic acid); RfI 0.08; RrI1 0.54; amino acid ratios in acid hydrolysate, Lyso.9BArg3.09Glu5.70Pro1. ~ G l y. ,d d a 3.ogVal~ .s;Leu*. ~ s N ( 2H. 1 1~) ; peptide content 87 73. The synthesis of I1 was carried out in the same manner as described above. The azide derived from V was coupled with IV [[o!]~~D -117.5' (c 0.3, 10% acetic acid); Ri' 0.05; Rr1I 0.28; amino acid ratios in acid hydrolysate, Lyso,g6Argo.9zGh .9sPr03.oaGlyj.16Ala1.07Leuo,y9;peptide content 91 %I, which was prepared by hydrogenation of Z-Gly-Pro-Gly-Gly-Gly-Ala-LeuGlu-Gly-Pro-Pro-Gln-Lys(F)-Arg(HT)-OH acetate trihydrate [mp 166-168'; [a]?'D -51.8" ( e 1.0, D M F ) ; RrI 0.17; Rirl 0.53: amino acid ratios in acid hydrolysate, L y s ~YArg0. . g2G1u1.d r o 3 .03Glyj.16Alal,oZLeuo.99. Anal. Found: C , 50.5; H, 7.2; N , 16.21. The resulting mixture of crude materials was hydrogenated, and in the manner as described for I pure triacontapeptide 11 was isolated [[a]*'D -104.9' ( e 0.5, 10% acetic acid): RI1 0.09; RiI1 0.55; amino acid ratios in acid hydrolysate, L y s o . 9 ~ ~ r g ~ , g l ~ ~ u 6 . ~ ~ ~ r 0 3 . 8 9 Ala3,1aVal~.03Leu~.91NH3~2.16): peptide content 90%]. Acknowledgment. We wish to express our sincere appreciation to Drs. D. F. Steiner, A. H. Rubenstein, and M. B. Block, Department of Biochemistry and Medicine, University of Chicago, School of Medicine, for the immunoassays using lz5I tyrosinated bovine connecting peptide and to Drs. R . E. Chance and M. A. Root, the Lilly Research Laboratories, for the ethanol precipitation immunoassays using [12jI]bovine proinsulin. Noboru Yanaihara,* Naoki Sakura, Chizuko Yanaihara, Tadashi Hashimoto Laboratory of Bioorgaiiic Chemistry, Sliizuoka College of Pharmacy Shizuoka. Japaii Rrceiced August I O , I972
An Electron Spin Resonance Study of Some Group IVb Organometallic Peroxy Radicals
Val-Glu-Gly-Pro-Gln-Val-Gly-Ala-Leu-Glu(0Bu')Sir: Leu-Ala-Gly-NzHz-Boc acetate trihydrate (V) [mp 228-230'; [a]25D -35.1' (C 1.1 DMSO); RfI 0.45; Ri'I 0.75; amino acid ratios in acid hydrolysate, Arg 4- Orn1.89Glu~.o~Proo, 96G1y2,g1Ala2olValz.ogLeul.98. Anal. Found: C 50.4; H, 7.2; N, 17.01 was coupled
Electron spin resonance spectroscopy ( e x ) has proved to be a useful technique for the study of the chain carrying peroxy radicals (ROO . ) involved in the autoxidation of organic compounds. For example, absolute
( 1 5 ) J. Honzl and J. Rudinger, Collect. Czech. Chem. Commun., 26, 2333 (1961).
(1) J. A. Howard, Adcan. Free-Radical Chem., 4, 49 (1972). a n d references cited therein.
Journal of the American Chemical Society / 94.23 / "ember
15, 1972
8245
values of the rate constants, 2kt, for the chain termination step
Table I. Esr Parameters of ( C H 3 ) 3 M O 0 .in the Solid Phase and in Solutiona
2kt
ROO.
+ ROO. +molecular products
(1)
and changes in the enthalpy and entropy for the equilibrium process ROO.
+ ROO. e ROOOOR
t-BuO.
hv --f
2t-BuO.
+ (CHa)aMH +t-BuOH + (CH3)aM.
or by reaction of the trimethylorganometallic halide with sodium in a rotating ~ r y o s t a t . ~ ~When * these reactions were carried out in the presence of oxygen, the appropriate peroxy radicals were produced by the reaction (CH3)aM.
Si
CsDs Cyclopropane
Solution
----Solid phase-gzz
guu
gzz
2.0027 2.0086 2.0395 2.0022 2,0079 2.0587
(2)
have been determined by this technique. These studies have, however, been almost entirely confined to hydrocarbon peroxy radicals although FOO . , HOO ., and some pho~phoranylperoxy4~ and a r ~ a n y l p e r o x yradi~~ cals have received some attention. In this communication we report an esr investigation of some group IVb organometallic peroxy radicals of the structure ( C H 3 ) 3 M O 0 . ,where M = Si, Ge, Sn, or Pb. The identification of these radicals and a knowledge of their stabilities are of fundamental importance to the development of a better understanding of the autoxidation of organosilicon, -germanium, -tin, and -lead compounds. The organometallic radicals (CH3)3M* have been prepared and identified by esr either by liquid-phase photolyses of di-tert-butyl peroxide in the presence of the hydride5sG t-BuOOBu-t
M
c
Solvent or matrix
+ +(CHs)aMOO. 0 2
The principal g factors for these radicals are given in Table I together with those for tert-butylperoxy radicals. With the exception of silicon, the average g factors for the radicals (CH3)3MO0.in the solid phase and the isotropic values in solution increase with increasing atomic number of M. This trend is similar to that found for the group IVb metal centered radicals (CH3),M. in which the unpaired electron is located mainly in a valence shell p orbital: gi,,(M = C) = 2.0029,9 g(Si) = 2.0031,; g(Ge) = 2.0104,6 g(Sn) = 2.0163,6sJg(Pb) = 2.0389.* The origin of the deviation of the principal g factors in a-electron radicals from the free spin value (2.0023) is reasonably well understood.1° The g shifts are asso(2) (a) P. H . Kasai and A. D. Kirshenbaum, J . Amer. Chem. SOC., 87,3069 (1965); (b) R. W. Fessenden and R. H. Schuler, J . Chem. Phjs., 44,434 (1966); (c) F. J. Adrian, ibid., 46,1543 (1967). (3) (a) G. Czapski, Annu. R e c . Phjs. Chem., 22, 171 (1971); (b) 3. E. Bennett, B. Mile and A. Thomas, Eleventh International Symposium on Combustion, Pittsburgh, Pa., 1967, p 853. (4) (a) G. B. Watts and I
