California Association of Chemistry Teachers
Harold Pa~koff and Choh Hbo Li University of California Berkeley
I
I(
Hormone Structure and Biochemical studies o f three pituitary hormones
Biological studies extending over a quarter of a century established by 1930 the fact that the uituitarv dand secretes manv biolomcallv active subsiances. " Gidence was obtained whrch sGggested that the three lobes of the pituitary (anterior, intermediate, and posterior) produce at least nine different hormones which are responsible for the regulation, maintenance, and function of a host of physiological activities: reproductive function, body growth, mineral metabolism, carbohydrate metabolism, and color adaptation in cold-blooded vertebrates, to name but a fern. It remained, however, for the biochemists, and protein chemists in particular, to establish unequivocally the existence and identity of these hormones, all of which can be described chemically as proteins and polypeptides. Thus, within the last 20 years, investigations have led to the isolation and characterization of most of the oituitarv hormones. I n several cases not onlv has the total phmary structure (amino acid sequence) of the hormone been elucidated, but chemical synthesis has been achieved as well. The investigations of the past decade involving the isolation, physicochemical characterization, immunochemistry, synthesis, and biology of the various pituitary hormones have resulted in the development of a number of important general concepts in what may be called the field of molecular endocrinology. These have been previously summarized by Li ( 1 ) . For the purposes of this discussion some of the more pertinent concepts are paraphrased as questions: (a) Is the same hormone isolated from the glands of various animal species chemically identical? (b) Is the activity of a protein or polypeptide hormone dependent upon the integrity of the whole molecule? (c) Can a biological activity common to two different hormones be explained on the basis of strnctural features? (d) I s the Presented in part a t the California hsociation of Chemistry Teachers Meeting, December 29, 1964, California. State Polytechnic College, San Luis Obispo, California. Harold Papkoff i s a Cnreer Thvelonrnmt, Awnrdee of the United States Public
hormone isolated from one species necessarily active in other species? If not, does this species-specificity with respect to biological action have a structural basis? To answer and illustrate these questions, we propose to consider in greater detail three of the pituitary hormones which have been extensively studied in our laboratory over the past 15 years: growth hormone (GH), adrenocorticotropin (ACTH), and melanocyte stimulating hormone (MSH). Growth Hormone (GH)
The isolation of purified growth hormone from extracts of bovine pituitaries by Li el al. in 1945 (8) conclusively established the existence in the pituitary of this hormone as a discrete entity. Bovine growth hormone was found to be a globular type of protein, consisting entirely of amino acids with a molecular weight of about 45,000. The preparation was very potent in promoting body growth in the rat, the laboratory test animal. When tested clinically in humans, however, the bovine growth hormone preparation was completely inactive (5). The reason for this discrepancy was puzzling and not apparent at the time. I n 1954, Wilhelmi (4) presented evidence which suggested that there were physicochemical differences among preparations of growth hormone obtained from the pituitaries of fish, sheep, horses, and oxen. With respect to biological action, it was shown that the fish pituitary growth hormone preparation, although active in the fish, as expected, was inactive when tested in the rat. To complicate the picture the observation was made that bovine growth hormone was active in the fish as a growth-promoting agent. These and other observations relating to the species-specificity of biological action evidenced by various growth hormones are summarized in Table 1. Thus, a hormone isolated from one species is not necessarily active in another species. It was suggested that the observed species-specificity in biological responses may be related to and perhaps correlated with molecular variations among the various growth hormones. To explore this possibility, we have, in the past 10 years, isolated growth hormone from the Volume 43, Number
I , January 1966
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41
Toble 1. Biological Responses of Different Animals to Growth Hormones from Vorious S ~ e c i e s
Experimental animd Humen Monkey Sheep Goat ox Rat Mouse Guinea PX Dog Cat Tadpole Fish
-----Pituitary growth hormone SheepHumanMonkey Pig Whale Horse
Ox
-
-
+
+
-
Fish
for the determination of the isoelectric points of the growth hormones. All the preparations were electrophoretically homogeneous. A typical pattern is shown in Figure 1. From the mobility data obtained in
?
+ + ++ ++ + + + + + + + + +
Figure 1. Free boundary electroohoretic ootlemr of whale .row* " hormone in an acetate buffer of dl 4.0 ond imic strength 0.03; pictures token after 10,680 seconds of electroohoreris fnotentiol aradient = 5.6 "~ v/cml; u m e r oattern i s the ascend-
I
~~
~
A
+
- represents no response; a definitive response; ? response doubtftil or not yet established. Takes from (11).
pituitaries of the following mammalian species in addition to the ox (2): the sheep (5),the pig (6), the humpback whale (r), the Rhesus monkey (a), and the human (8). The availability of these purified growth hormone preparations has allowed us to perform a comparative study of a number of physical and chemical properties. The growth hormones from all the species were purified by similar fractionation procedures. I n general, the hormone is extracted from ground pituitaries with either a dilute saline solution or a Ca(OH)2solution adjusted to pH 10. Addition of ammonium sulfate t o the extract (1.9 M ) effects precipitation of the hormone. Further purification is achieved by chromatography on Amherlite IRC-50 cation exchange resin. All the species of growth hormone studied were found to be adsorbed onto the resin at pH 5.1 in the presence of 0.45 M (NH4),SOn. Elution of the hormone is effected with a buffer of pH 6 except in the case of human growth hormone which could be eluted with water. Final purification of the hormones was accomplished with isoelectric precipitation and alcohol fractionation. Countercurrent distribution was employed in the case of porcine growth hormone (6) and gel filtration was used for both porcine (6) and human growth hormone (9). The purified growth hormones when bioassayed by the tibia test (10, 11) were shown to he potent and of comparable activity. The basis of this test is that the epiphyseal cartilage plate of the tibia bone of hypophysectomized rats increases in width when stimulated by growth hormone. A summary of bioassay data comparing the activities of the various growth hormones is shown in Table 2. The technique of free boundary electrophoresis was employed not only for examination of purity, but also Toble 2.
buffers of various pH, the isoelectric points (pH of zero mobility) were determined. These are summarized in Table 3. It can be seen that the isoelectric points vary from about 6.8 for bovine and ovine growth hormone to 4.9 for human growth hormone. Since the isoelectric point is a function of the charged groups in the molecule, it can be predicted that these various growth hormones have different amounts of acidic and basic amino acids which give rise to the observed differences in isoelectric points. The preparations were also examined by techniques of zone electrophoresis. I n these experiments it was also shown that the preparations were homogeneous, but in addition it was possible to demonstrate that the hornlone activity was associated with the protein peak.
-
Table 3. lsoelectric Points and Molecular Weiahts of Vorious Species of Growth Hormone
Species
The molecular weights of the various grovth hormones were determined from data obtained by scdimentation-velocity experiments in the ultracentrifuge, from diiusion data, and from partial specific volumes calculated from amino acid analysis. The results are shown in Table 3. Examination of these results indicates a wide range in n~olecularweights among the species studied. Thus, the primate hormones, (human and monkey growth hormone) appear to be the smallest, around 25,000; pig and whale are intermediate at around 40,000; and ox and sheep growth hormone are
Assay of Various Purified Growth Hormones b y the Tibio Test
-
20 rrga
Reaponse 60 a"
120 pg'
Ox Sheep Pig Whale Monkey Human
2'32 zt 6(4) 209 + 2(4) 226 =t 4(7) zao 4(5) 210 + 4(4) 213 + 2(8)
250 i 2(4) 231 =t 3(5) 243 + 4(5)b 250 i 2(5) 242 =t5(6) 23.5 =t 2(8)
288 + G(4) 249 =t 4(6) 279 zt 4(6)' 268 + 4(5) 261 2z 3(5) 2.56 J; 2(6)
Slope 69.6 52.6 60.8 62.6 65.7 32.9
Taken from (11) In terms of mean tihial width =t standard error. Number of rats in parentheses. "Total dose in 4 days. 40 wg ttotal dose. 100 pg ttotal dose.
42
/
Journal of Chernicol Education
Molecular weight
ox Sheep Pig Whale Monkey Human
Growth hormone
*
PI
Index of precision ( A ) 0 152 0.157 0.168
o.no 0.147 0.12Y
the largest at around 45,000. These results firmly establish the fact that there are large differences between the molecules of the various growth hormones. Chemical studies have shown that the growth hormones are all simple proteins; i.e., they consist entirely of amino acids and contain no carbohydrate, nucleic acid, lipid, or other prosthetic groups. The ultravioletadsorbing characteristics of the growth hormones are such that the spectra ohtained can be interpreted solely on the basis of the constituent residues of tyrosine, tryptophan, and phenylalanine. Quantitative amino acid analyses revealed that all the common amino acids are present, but there is a large variation in the number of amino acid residues present in each species-a reflection, of course, of the differences in n~olecularsize. In Table 4 are listed a few of the amino acids which are present in small numbers. Table 4.
Some Amino Acids Present in Various Species of Growth Hormone'
Ox S h e e ~ Pie Tyrosine 11 12 11 Tryptophan 3 3 3 Histidine 7 6 5 Cystine 4 5 3 Methianine 7 9 6 * Residues per mole of hormone.
Whale Human 12 3 5 3 7
Monkev
10 1 4 2 4
7 1 5 4 6
The terminal amino acids, both amino and carboxyl, were determined for the various growth hormones. The fluorodinitrobenzene technique of Sanger (12) and Edman's phenylisothiocarbonyl method (IS) were used for the evaluation of the N-terminal residues. The use of carboxypeptidase (14) revealed the carboxyl terminal amino acids. These results are found in Table 5. Four points of interest were derived from these studies. First, it is apparent that there are two fundamentally different types of growth hormone. One type, of which bovine and ovine growth hormone are examples, possess two polypeptide chains as indicated by the presence of the N-terminal phenylalanine and alanine residues. The others are a11 of a single polypeptide chain type, possessing only a single N-terminal residue, phenylalanine. Second, it is of interest that all the growth hormones, regardless of species and molecular size, possess phenylalanine as an N-terminal residue. Third, the results ohtained with oarboxypeptidase showed that all the growth hormones have the ubiquitous phenylalanine as the carboxyl terminal residue. Finally, biological experiments demonstrated that complete removal of the C-terminal residue with carboxypeptidase did not alter the growth-promoting activity of the preparations, i.e., the entire molecule may not be needed for biological activity to be manifested. Table 5. NHz-terminal and COOH-terminal Amino Acid Sequences of Various Species of Growth Hormone S~eoiea
Ox Sheeo
NHeTermind sequence
Phe.Ala.Thr.. . . Ala.Phe.Ala... . Phe.. . .
COOH-Terminal sequence
. . . .Leu.Ala.Phe.Phe .. . .Ale.Leu.Phe
Table 6.
Action of Chymotrypsin on Porcine Growth Hormone
Time (min)
Percentage hydrolysis"
0 30 60 90 120 180 240
0 8.3 15.0 21.2 26.2 37.5 53.2
Activit? (w) 243 i 4(12)' 245 =t 4(6) 236 i 6(5) 216 + 3(5) 178 + 4(4)
Taken from ( 6 ) . " As indicated by solubility in 5 percent triehloroaeetie acid. "4 total dose of 40 rcg in 4 days. (Mean =t standard error; number of animal6 shown in parenthem
I n order to explore more fully the possibility that the entire growth hormone molecule may not be necessary for biological activity, but that all growth hormones may possess a common "core" or active center, studies were undertaken employing proteolytic enzymes to digest the growth hormones and to correlate degree of digestion with hormonal activity. I n this respect, the action of chymotrypsin on bovine growth hormone has been the most extensively studied (15, 16). The other species have been studied as well, however. I n a typical experiment with porcine growth hormone, the results shown in Table 6 were obtained. Thus, when porcine growth hormone is digested with chymotrypsin, hydrolysis to the extent of 260jo NPN (NPN, material soluble in 5% trichloroacetic acid) does not result in a significant change in biological activity. Further digestion, however, ultimately results in complete loss of hormone activity. I n the case of bovine growth hormone (15, 16) analysis of digests retaining full biological activity (26T0 NPN) by various physical and chemical techniques indicated that there was little if any unrnodified native hormone present. It was concluded, therefore, that it was indeed possible to degrade the hormone to a smaller molecular species which would still retain biological activity. A summary of results obtained with all six purified growth hormones is shown in Table
-
Table
7. Action of Chymotrypsin on Various Species of Growth Hormone*
Soecies
Retention of activity (% . . .NPN)
Time (min) . .
Loss of activity ('% . . NPN)
Time (min)
Pig Whde Beef Sheep Monkey Human
26 26 25 25 19 10
120 80 105 120 120 160
32 38 30 39 34 21
180 150 135 240 300 420
a pH 9.5 borate buffer; enzyme:substrate = 1 :300; time is that required to achieve indicated yo NPN.
The action of other enzymes has been studied as well. Trypsin has been found to give results similar to those obtained with chymotrypsin in the case of bovine growth hormone (17). Studies with pepsin showed that it very rapidly inactivates bovine, porcine, and ovine growth hormones. However, human growth hormone can he digested to the extent of 40% NPN without loss of activity (18). The foregoing has served to demonstrate that various Volume 43, Number 1, k n u a r y 1966
/
43
species of growth hormone display individual differences with respect to their physical and chemical properties. Only six species of growth hormone have been extensively studied, and it is reasonable to expect other differences to become apparent as this type of study is extended. Although the results do not resolve the problem of the species-specificity observed with respect to biological response, it is likely that the explanation can be found, in part, in the chemical differences hetween the various growth hormones. The enzyme experiments suggest that the integrity of the entire molecule is not required for biological activity. The concept is supported of a common active core which may ultimately be amenable to chemical synthesis.
evidence was found in the observation that when porcine (2s) or ovine (24) ACTH is treated with the enzyme pepsin, the COOH-terminal undecapeptide (i.e., from position 29-39) is released with no loss of hormonal activity. I n contrast to the above observations, modification of the amino acid residues within the NH,-terminal portion of the molecule results in loss of biological activity. Thus, White (25) showed that aminopeptidase rapidly inactivated ACTH when more than one half of both the first two amino acids (serine and tyrosine) are removed. Oxidation of the N-terminal serine residue with periodate (26, 27, 28) causes almost complete loss of ACTH activity. Furthermore, selective acetylation of the terminal amino group of ACTH (29) results in a derivative possessing less than 10% of the activity of the untreated hormone. Thus, it is evident that the ACTH activity must reside in the N-terminal amino acid portion of the 39amino acid polypeptide chain. Synthetic studies amply confirmed this view. I n Table 8 are listed a number of synthetic polypeptides corresponding to various portions of the N-terminal sequence of the ACTH molecule. It can be seen that the heptadecapeptide, a'-IT-ACTH (first 17 amino acids of ACTH)
Adrenocorlicotropin (ACTH) and Melanocyte Stimulating Hormone (MSH)
Of the anterior pituitary hormones, ACTH has been the most intensively studied from the chemical point of view (1,19). It has been isolated in pure form from the pituitaries of three different species: sheep (20), pig (21), and ox (22). I n each, the complete amino acid sequence of its polypeptide chain is known. I n addition, large portions of the ACTH molecule and related molecules have been chemically synthesized (1). I n part, the extensive progress made with this hormone is due to its relatively low molecular size, 39 amino acids (molecular weight = 4560). I n Figure 2 the structure of bovine ACTH is shown and compared with the structures of sheep and porcine ACTH. There are a numher of interesting features to this molecule. All the basic amino acid residues (arginine and lysine) are to be found in the first 21 residues. I n addition, there is a concentration of basic amino acids from positions 15-18. The acidic amino acid residues (aspartic and glutamic acids) with the exception of the glutamyl residue a t position 5 are all located in the carboxyl portion of the molecule from residue 25 on. The differences observed among the ACTH's from the three species studied are minor and are all located between residues 2533. Thus, between the porcine and ovine hormones, the only differencein composition is the additional residue of leucine in the porcine, and one more serine in the ovine hormone. There are, however, juxtapositions of several of the amino acid residues which they both have in common. From these structural considerations it was inferred that the COOH-terminal portion of the ACTH molecule was not necessary for the maintenance of biological activity. Supporting
Spocics
Table 8.
Steroidogenic Activity of ACTH and Structurally Related Peptides
- Aslay
Partial Amino Acid S e ~ u e n o e
Peptide
25
26
nh'-'LACTH m'-"NYrACTH o'-LLACTH
al-'"NArACTH .T-"~~+ACTH
.z-IT-ACTH
SerTry.. . .Lya-Lya-Arg-Arg-Pro... . P h e 1 2 15 16 17 18 19 39 Ser-Try... .Lys-Lys-Arg-Arg-Pro... .Giy 1 2 15 16 17 18 19 26 Ser-Try... .Lye-Lys-Arz-Arg-Pro-NH? 1 2 15 16 17 18 19 Ser-Try... .Lys-Lys-Arg-Arg-Pro-OH 1 2 15 16 17 18 19 Ser-Try... .Lya-Ly-ArgB.4rg-NH~ 1 2 15 16 17 18 S~P-TW.. . .Lye-Lye-Arg-NHH 1 2 15 16 17 Ser-Try.. . .L,yLys-hrg-OH 1 9
.
."
"
%G
.,
21
28
29
30
y.
31
32
Asp-Gly-Ala-Glu-Asp-C~u-Lcu-AI~-Gl~
Sheep
Univerlily ol Cslilornis
AIP-Gly-Ch-i\sp-Asp-G1u-AI1-44-GIu
Beel
University ol Calilornis
Asp-Gly-Gh-Aia-Clu-Asp-Ser-*l.-Gh
33
7".
/
Slructuroi differences omong odrenocorti~otro~lnr iroloted from
Journal of Chemical Education
278
345
122
110
82
299
74
69
88
11
11
3,
7%
44
300
is the shortest peptide possessing significant ACTH activity. As the polypeptide chain is lengthened, there is a corresponding increase in potency. Thus, the hexacosapeptide (a1-26-ACTH) has very nearly full activity, and peptides shorter than the heptadecapeptide are virtually inactive. The relationship of the MSH molecule to ACTH
h e r i e a n Cyanamid Company
Figure 2.
*mole ,'mole 481 817
Taken from (30).
Pig
Token from ( 1 ) .
in vifro
"",
cs-ACT13
Amino acid residue Ln position
laborlory
in vwo
pig, sheep and beef pituitary glands.
forms another interesting aspect of the chemistry of ACTH [see reference (51) for a review]. I t was known for many years that crude ACTH preparations stimulate the melanophores of cold-blooded invertebrates, causing them to disperse and thus darken the skin of the animal. It was thought that this activity in the crude preparation represented contamination with MSH which is found in the intermediate lobe of the pituitary. I t was, therefore, surprising to find a persistent MSH activity of considerable magnitude in the purest ACTH sample which could be obtained. Research into the purification and structure of MSH led to the resolution of this problem. Figure 3 shows the structure of various MSH molecules compared to the partial structure of ACTH. Several points are worthy of mention. First, it became apparent that there are two different types of MSH molecule. One, designated o~-i\lSH, is common to all species studied and consists of a structure which corresponds to the first 13 amino acids of the structure of ACTH except that the amino terminus is acetylated and the carboxyl group of the terminal valine is amidated. The other variety of MSH, 8MSH, has an 18 amino acid structure, except in the case of the human, and several minor structural variations are apparent when the P-MSH of different species
are compared. The most interesting aspect to these structures is that all, including ACTH, have in common a heptapeptide sequence . . . Met-Glu-His-PheArg-Try-Gly. . . . It seemed likely that this sequence was responsible for the MSH activity found in ACTH preparations, and indeed, synthesis of this peptide fragment shows it to be active as an MSH. A large number of peptides containing various portions of the a- and p X S H structures have been synthesized and their activities measured. These are summarized in Table 9. In brief, the peptide H-His-Phe-Arg-TryGly-OH is the smallest polypeptide which will show observable 34SH activity. Extension on either side of this sequence (Table 9) results in polypeptides with increasing potencies, approaching eventually the activity of the native hormone. Summary
We have tried in this necessarily brief discussion t o demonstrate a chemical approach to the study of polypeptides with hormonal activity. The growth hormones represent molecules of a size such that synthetic and extensive structural studies have been thus far precluded. However, chemical techniques, where applicable, extended our knowledge of these hormones and point to the course of future studies.
Table 9. Melanocyte-Stimulating Activity of Synthetic Peptides
MSH Activitv
Peptides
nil
H-~er-~et-bln- is- he-~rg-~ry-~ly-~~ (54) 3
6
4
7
8
9 1 0
Tos
H-His-Phe-Arg-Try-Gly-~ys-Pro-VaL-NRZ (54) 6
7
9
8
10 11
12 13
NHs I
nil
~-~et-(3rlt1-~is-~he-~rg-0h (54) 4 6 7 8 H-His-Ph-Arg-Try-Gly-OH (56, 56) 6 7 8 9 1 0 H - G I L I - H I ~ - P ~ ~ - A ~ ~ - T ~ (36) ~-GI~-OH 5 6 7 8 9 1 0
1.5-3 X 10' 2 . 2 X lo5
H-Me6Gli1-His-Phe-Arg-Try-Gly-OH (57) 4
5
6
7
8
9
1
1 . 4 X loG
0
"Literaturereferences indicated in parentheses following peptide sequence. Taken in part from (31). Volume 43, Number I, January 1966
/ 45
The studies on ACTH and RlSH have been truly in the realm of molecular endocrinology. I t is to be expected that future chemical studies on ACTH will take us closer yet to a fuller understanding of how these hornlones act from the physiological point of view. Also, it may now be possible to tailor-make ACTH's of special properties (i.e., long-acting, potentiated, etc.) which will have ilnportant therapeutic value.
en.
Literature Cited (1) LI, C. H., Recent Prog. Hormone Res., 1 8 , l (1962). (2) LI, C. H., EVINS, H. M., A N D SIXPSON,M. E., J. B i d . Chem., 159, 353 (1945). L. L., ET AL., J. Clin. Endocrinol., 10,492 (1950). (3) BENNETT, (4) WILHELMI,A. E., in "Hypophyseal Growth Hormone, 0. H., Nature aod Actions," SMITH,R. W., GAEBLER, AND LONG,C. N. H. editors, XcGraw-Hill Book Co., New York, 1955, p. 59. H., A N D LI, C. H.,Biochim. Biophys. Acla, 29, (5) PAPKOFB, 145 (1958). . (6) PAPKOFF,H., LI, C. H., A N D LIU, W-K, A T C ~Biochem. Biophys., 96, 216 (1962). H.. AND LI, C. H., J . Biol. Chem., 231,367 (1958). (7) PAPKOFF, H., Science, 124,1293 (1956). (8) LI, C. H., AND PAPKOFF, (9) LI, C. H., Lm, W-K., AND DIXON,J. S., Arch. Biochem. Biophys. Supple., 1, 237 (1962). (10) GREENSPIN,F. S., ET AL., Endocrinology, 45, 544 (1949). H., A N D LI, C. H., in "Methods in Hormone Re(11) PAPKOFF, R. I., editor, Academic Press, search," Yol. 2, DORFMAN, New York, 1962, p. 671. (12) SINGER,F..Riochem. J.,39,507 (1945). P., Actn Chem. Seand., 4,283 (1950). (13) EDMAN,
46
1 Journal
of Chemical Education
LI, C. H., PARCELLS, A. J., AND PAPKOFF, H., J . Bid. Chem., 233, 1143 (1958). LI, C. H., ET AL., J . Biol. Chem., 218,41(1956). LI, C. H., PMKOPF,H., AND HAYASHIDA, T.,A ~ e h .Bio&em. Biophys., 85, 97 (1959). LI, C. H., ET AL., in "Hypophyseal Growth Hormone, Nature and Actions," SMITH,R. W., GAEBLER,0. H., AND LONG.C. N. H.. editors. McGraw-Hill Book Co. New York: 1955. D. 70, LI, C. H., Fhwiol., 45, 169 (1962). LI, C. H., Sn'. Am., 209, 46 (1963). LI, C. H., ET AL.,Nature, 176, 687 (1955). HOWARD, K. S., ET AL.,J. Am. Ckem. Soe., 77,341 (1955). LI, C. H., DIXON,J. S., AND CHUNG, D., J . Am. Chem. Soc., 80, 2587 (1958). BELL,P. H., ET AL.,J. Am. Chem. Soc., 78,5059 (1956). COLE,R. D., ET AL., J . Bwl. chem., 219,903 (1956). WHITE.W. F.. J. Am. Ckem. Soc.. 77. 4691 (1955). ' DIXON; H. B.F., Biochem. J., 6 2 , ' 2 5 ~(1956). GESCHWIND, I. I., AND LI, C. H., Biochim. Biophys. Acla, 15, 442 (1954). GESCHWIND, I. I., AND LI, C. H., Endocrinology, 63, 449 (1959). WALLER,J. P., AND DIXON,H. B. F., Bwchem. J., 66, 320 (1960). RAMACHANDRAN, J., C a u ~ o ,D., AND LI, C. H., J. Am. Chem. Sue.. 87.2696 (1965). LI, C. H., viamins H o m a e s , 19,313 (1961). LI, C. H., ET AL.,J. Am. Ckem. Soe., 82,5760 (1960). GUTTMANN. S., AND BOISSONNAS, R. A,, Helv. Chtm. Ada, 42,1257 (1959). HOPMANN, K., ET AL., J. Am. Chem. Soc., 82, 3721 (1960). HOFMANN, K., ET AL.,J. Am. Chem. Soe., 80,1486 (1958). SCHWYZER, R., AND LI, C. H., Nature, 182, 1669 (1958) LI, C. H., ET AL..Nature, 189,143 (1961).
i26j (27) (28) (29) (30)
.
.
