Norbert Isenberg' University of Wisconsin-Parkside Kenosha, Wisconsin 53140 and Marcel Grdinic University of wisconsin-~arathon Wausau, 54401
I1
I
cyclic Disulfids Their functions in health a n d disease
M a n ' s quest for better health is neverending: it includes the story of alchemy, iatrochemistry, and, in our times, biochemistry and chemotherapy. As this story unfolds, more and more compounds enter into our lives, and our knowledge, as well as its applications, continues to grow. While many sulfur compounds have been used in medicine for some time, the isolation of biologically active cyclic disulfides and the elucidation of their structures and functions is a recent advance. Progress in these endeavors during the past few decades typifies the growth of our modern chemistry and its uses. Usually, there is an indication of the presence of a substance which our body needs for normal functioning. Painstaking chemical research leads to its isolation and characterization. A combination of classicaL techniques and up-to-date instrumental methods yield information about strncture and give clues about physiological roles. Within a short time a host of problems are tackled by various research teams leading to syntheses, unraveling of biochemical pathways, and new interpretations. Of particular interest among the cyclic disulfides are a-lipoic acid, a coenzyme found in plants and animals; oxytocin, vasopressin, and related neurosecretory hormones produced by the posterior lobe of the pituitary gland, and insulin, the hormone secreted by the islets of Langerhans in the pancreas. We shall describe them in order of increasing structural complexities.
A naturally occurring "acetate-replacing factor" with growth promoting function for lactic acid bacteria was first described in 1946 (1) and a "pyruvate oxidation factor" was reported in 1947 (92). It was soon shown that the biological activity exhibited by both factors was also found in concentrates of protogen, an essential growth factor for the protozoon tetrahymena gelii (3). These growth factors were then recognized as a-lipoic acid, 30 mg of which was obtained from 40 tons of beef liver in 1951 (4).
'Present address: Department of Chemistry, The University of Wisconsin, Madison, Wis. 53706. 1The synthetic compound, which was optically inactive, was named thioctie acid, while the naturally occuring compound continued to he called e-lipoie acid.
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/ Journal of Chemical Educafion
Although the presence of a disulfide linkage in alipoic acid was indicated (5), early structural assignments were uncertain and the presence of a six-membered ring was suggested in 1952 (6) (x = 2)
The name thioctic acid was proposed for this compound, indicating a sulfur-containing organic acid with eight carbon atoms. Early synthetic work did not resolve the problem of the ring size because of possible rearrangements (7). Structural assignment followed a new synthesis, proceeding without rearrangement, which indicated that synthetic thioctic acid, and also naturally occurring, optically active a-lipoic acidZ have a five-membered disulfide ring (8). Optically active (+)-a-lipoic acid and its optical antipode were synthesized in 1955 (9) and the stereochemical configuration was reported in 1956 (10). Since the biological activity of a-lipoic acid is related to the reactivity of the disulfide bond in the fivemembered dithiolane ring, the chemistry of 1,2-dithiolanes is of interest. The strain in the dithiolane ring is about 4 6 kcal/mole (11, 18). This value is much lower than was originally assumed (IS), and early attempts to correlate reactivity and ring strain were unsatisfactory. More recently the reactivity of disulfide bonds in 1,2-dithiolanes has been described in radical, electrophilic, and nucleophilic reactions (Ida, 15). In contrast to six-membered ring 1,2-dithianes, unsubstituted 1,2-dithiolanes polymerize readily to linear disulfides via a free radical mechanism (13). Whlle 3-methyl-1,2-dithiolane polymerizes easily, substituted 1,2-dithianes are quite stable and the stereochemistry of cis- and trans-3,6dimethyl-l,2-dithianehas been described (16). The dithiyl radical was found by esr spectroscopy following irradiation of a-lipoic acid (17)
This energy absorption and its role in photosynthesis had been suggested earlier (IS). The increase in the rate of electrophilic attack on the disulfide bond going from open-chain disulfides to dithiolane rings has been ascribed to a decrease in activation energy and activation entropy (15). This in-
terpretation is based on the reaction rates which were observed for oxidation of disulfides to thiosulfinates (IS) -... *.*
--
Oxytocin and Vasopressin
.-" &
I n contrast to electrophilic reactions, nucleoph~llc reactions seem to indicate little difference in activation energies for reactions of l,&dithiolanes and of the open-chain dihutyl disulfide with butanethiolate (12) (R = -CaHd
S
I I GLU I
ILEU
/ARG-NH'
FY I
PRO-LEU-GLY-CDNH.
NH..
I
Since the rate of the reaction with 1,2-dithiolane is almost 10,000 times faster than that of the open-chain disulfide, a lower entropy of activation may be responsible for the differcnce. The ground state of 1,2-dithiolane is morc similar in structure to the transition state than is the case for open chain disulfides (15). Such an interpretation is also consistent with the observed ultraviolet absorption spectra of disulfides which show a marked shift toward longer wavelengths going from open-chain disulfides (A, = 23&255 mp) to 1,2-dithiolanes (X,., = 330 mp) (IS). The selective desulfurization of elipoic acid on treatment with amino-phosphines has been described recently (14b). There arc several biochemical reactions in which lipoic acid functions as a coenzyme. For example, in the oxidative decarhoxylation of pyruvate, the acetyl group is transfcrrcd to lipoic acid and the dithiolane ring is reductively opened (28-20)
~CH.),COOH HS S-C-CH,
+
CO,
II
0
Thc acctyl group is thcn transferred to coenzyme A and the resulting dihydrolipoic acid is enzymatically oxidized to lipoic acid. The overall acetyl transfera to coenzyme.A. has..been (21,22) . -summarized -. . . - - .. - ... ...
d CII-C-SCoA
+ COI + N A D H + H t
This reaction is required for initiation of the Krebs Cycle. For a detailed description of the mechanisms of these reactions the reader is referred to more comprehensive reviews (15,19,23). There are several rcports of therapeutic applications of lipoic acid in liver diseases as well as heavy-metal poisoning, such as As, Pb, Hg, and Se (24, 25). I n t,he latter cases, lipoic acid may be superior to British Anti-Lewisite (BAL). The ahbrevistions used here are: NAD/NADH, oxidizedl reduced nicotine adenine dinncleotide.
-
PRO-ARG-GLY-CQNH,
The role of extracts of the posterior pituitary gland in producing a flow of milk in lactating animals (26, 27) and in causing uterine contractions (28) was recognized early in this century. There was reason to believe that an oxytocic principle was the milk-ejecting and uterine-contracting hormone (29). Isolation of highly purified oxytocin in the form of a crystalline flaviante (SO) was followed by the structural elucidation of the nonapeptide (31). This advance, in turn, led to the synthesis of physiologically active oxytocin (32), a monumental step which opened the doors to further peptide syntheses. The antidiuretic activity of pituitary extracts had been established and methods of assay were developed (29, 33). Understanding of this hormonal activity was especially important, since lack of the active pressor principle was known to cause the disease diabetes insipidus in which there is a high output of dilute urine. Injections of extracts of the posterior lobe were shown to be helpful in the control of fluid output (34). In 1951 du Vigneaud and cou-orkers reported the isolation of a substance with pressor activity from beef pituitary extracts (35). The structure of this substance, vasopressin, was proposed by du Vigneaud and coworkers in 1953 (36). The synthesis of vasopressin, the first major triumph of synthetic peptide chemistry, was achieved in du Vigneaud's laboratory (36, 371, and the award of the Nobel Prize t,o du Vigneaud was made in recognition of his achievements in disulfide and polypeptide chemist,ry. The highlights of development,^ of our understanding of the structures of oxytocin andvasopressin are: (1) selective extraction and purification leading to isolation of pure substances, (2) complete chemical analyses resulting in assignment of amino acid sequence and showing the presence of disulfide linkages in 20-memVolume 49, Number 6, June 1972
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393
bered rings,' and (3) confirmation of structure proposals by synthetic approaches. Fractional extraction of oxytocin and pressor activity with butanol was first reported in 1919 (88). Change of solvents led to improved fractionation methods during the next thirty years. Isolation of pure oxytocin (SO) and vasopressin (55) was achieved by making use of countercurrent distribution (39). More recent techniques include gradient elution from carboxylic resin (40) and zone electrophoresis (41). The structures of oxytocin and vasopressin were established four years after the isolation of pure oxytocin (SO-32, 85-87). This remarkable feat was achieved by a combination of classical methods of analysis and newer techniques (49, 45). Cleavage of the disulfide bonds with performic acid yielded only one compound with the same amino acid composition as the starting materials, oxytocin and vasopressin. Rupture of the sulfufsulfur link transformed cystine to 2 moles of cysteic acid. Cleavage studies thus revealed that cystine was involved in a ring structure. The sequence of the amino acids was worked out by use of dinitrofluorobenzene (4.9) and carbon disulfide (43). Vasopressin and oxytocin were found to have six amino acids in common: aspartic acid, cystine, glutamic acid, glycine, proline and tyrosine; oxytocin also has isoleucine and Ieucine, while vasopressin has arginine and phenylalanine. Recent advances based on helix-coil transitions (4) have made it possible to calculate the conformations of oxytocin and vasopressin. Synthesis of oxytocin was carried out by stepwise build-up of suitable polypeptide precursors to yield a benzylated oxytocin (Slb). Such a route appeared promising after oxytocin had been reduced by sodium in liquid ammonia (46) and converted to a benzyl derivative from which oxytocin was regenerated by debenzylation with sodium in liquid ammonia (46) followed by air oxidation. The first reduction of a disulfide with sodium in liquid ammonia --. -.-.had been..-.,.***,carried out in 1930 (&)
.~-
-.--..
,.
'...
e ~ ~ * . ~ . . * ~ a > . ~ . a . .a' a~ -
....
v.x
..-
......-.*.~h.'
Removal of a benzyl group from N-carbobenzoxypalanine with sodium in liquid ammonia was first achieved in 1935 (46). Almost quantitative yields of P-alanine obtained ... . a.' .,, ,, ,. -,'"were * -.",, , ',*.*,. A -
A
Du Vigneaud and coworkers first synthesized hog vasopressin in which lysine replaced arginine (86, 87). Several new preparations of both oxytocin (47, 48) and vasopressin (47, 49, 50) have been reported in the literature after Menifield's method of solid-phase peptide synthesis (61) had provided researchers with a versatile new synthetic approach. Analogs of oxytocin, such as deaminooxytocin (52) as well as its 28-membered ring homolog, (1-[w-mercaptoundecanoic acid)]-oxytocin (531, have also been synthesized. Merrifield's technique of "solid phase peptide (protein) synthesis," SPPS, uses conventional protein chemistry (51). However, the first amino acid in the synthetic sequence is attached by a chemical bond to a polymer. All the successive amino acids thus hecome part of the solid polymer. This makes the p e p tides "insoluble," and the soluble reactants can be added in large excesses, easily removed after the reaction by washing, etc. At the end, the peptide or protein is chemically removed from the polymer. SPPS synthesis can be carried out in completely automatic apparatus. Oxytocin and vasopressin are neurohormones which are released by separate neurosecretory cells in the posterior lobe of the pituitary gland. The process of secretion, especially the biosynthetic mechanisms have been under investigation for some time and a precursor model has been proposed for vasopressin (64). The functions of the neurosecretory hormones are distinct: oxytocin is a milk-ejecting and uterine-contracting hormone, while vasopressin has antidiuretic properties. Oxytocin plays an important role at the termination of pregnancy: it causes contracting of smooth muscle, particularly that of the uterus. It is used in medicine to induce labor. Furthermore, its milk-ejecting property plays an important role in the maternal reflex process of milk-ejection (29). The release of the antidiuretic vasopressin appears to be regulated by the osmotic pressure of the blood. High osmotic pressure causes release of the pituitary hormone and results in the retention of water. Vasopressin-release control is exerted by osmoreceptors which respond to high concentrations of sodium chloride in blood (55). I t has been suggested that a related hormone, called arginine vasotocine (8-Arginine oxytocin) fouhd in primitive vertebrates is ancestral to the other neurosecretory hormones (66). Two peptide series may have evolved as a result of doubling of the vasotocin controlling gene (57). Vasopressin may have been formed when phenylalanine was substituted for isoleucine in position 3. Such a mutation presumably resulted from a change from adenine to uracil in the genetic coding material, changing the triplet codings for isolencine to those for phenylalanine: AUC or AUU to UUC or UUU. Insulin
These two reactions which make use of reduction by sodium in liquid ammonia havc had many important. applications in disulfide and polypeptide chemistry, especially in connection with synthet,ic work of disulfide-containing polypeptides and their analogs. 394
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Journal o f Cherniml Education
The formula of human insulin, a cyclic disulfide which regulates the blood sugar level, reveals its complex protein nature. A deficiency of this hormone is the
'
As we shall see later, insulin d s a contains a 20-membered disulfide ring.
cause of diabetes mellitus, a dreaded and fatal disease that had been plaguing mankind for many millenia of recorded history (58). Progress in the conquest of this disease had been slow. In 1650's Thomas Willis, an English court physician, observed that urine of diabetic patients had a sweet taste. More than a century later, Cowly (59) suggested a relationship between defective pancreas and diabetes mellitus. Probably the biggest step forward was made when Minkowski and Mering (60) found in 1889 that surgical removal of the pancreas produced all symptoms of diabetes mellitus. This discovery, combined with recognition of the high level of sugar in the blood of diabetic patients, opened an intensive search for a sugar-regulating pancreatic secretion. Although the initial efforts in that direction were unsuccessful, it was found that the Langerhans islet @-cells of the pancreas were responsible for the secretion of the elusive substance @I), later named "insulin" (68) (after the Latin insula, an island). It was not until 1921, by which time almost all the efforts to isolate "insulin" were abandoned, that Bane ing and Best attacked the problem by using a simple but bold hypothesis: the pancreas, in addition to the internal secretion product-insulin, generates the enzyme trypsinogen, an insulin "antagonist," delivered into the duodenum via the pancreatic ducts. While insulin is produced by the Langerhans islets, trypsinogen is secreted by the acinous tissue of the pancreas. A surgical ligation of the ducts should degenerate the acinous tissue and prevent formation of the insulin "antagonist," and thus allow the "survival" and isolation of insulin. The hypothesis was correct; several months after the initiation of the work, Banting and Best obtained insulin preparations capable of eliminating dramatically all the symptoms of diabetes mellitus in experimental dogs (65). This discovery, a milestone in the history of medicine, has not only paved the way for remedy of a fatal disease, but also laid the cornerstone of chemical research which led to the preparation of crystalline insulin (64, and culminated in the elucidation of the structure of insulin by Sanger (65) in 1955. Both Banting and Sanger received the Nobel Prize for their work on insulin. Since these momentous events, there has been a steady progress, particularly in the areas of ext,raction techniques and preparation of insulin with con-
trollable duration of action. The latter can be achieved, for example, by combining insulin with small amounts of Zn2+or some other ions (66) and certain proteins, such as protamines. In the initial stages of the study of insulin structure, du Vigneaud recognized its protein nature (Bra). A qualitative and quantitative analysis of insulin hydrolysates showed the presence of 16 different amino acids (67b). The presence of the disulfide rings was established by Sanger in 1945 (48). These results laid the groundwork for Sanger's primary structure elucidation (65, 68). In order to determine the number of peptide chains in insulin, Sanger developed a mcthod for determination of terminal amino groups, known as dinitrobensene (DNB) method (42). In this method 1-fluoro-2,4-dinitrobenzene (FDNB) reacts with the free (terminal) amino group of a protein to give the corresponding DNB derivative
In subsequent acid hydrolysis of DNB-protein all peptide bonds are broken to give free amino acids and one DNB-amino acid per each protein chain
DNB-amino acids are brightly yellow and, in contrast to free amino acids, soluble in ether. DNB-amino acid separation and analysis is then carried out. DNB-method revealed the presence of two peptide chains in insulin. In the next step, a separation of the two chains was achieved by an oxidative cleavage of the disulfide bonds by performic acid (69) Volume 49, Number 6, June 7 972
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395
I I
CH,
I
CH.-SO?
The cysteic acid chains were separated by fractional precipitation. The shorter (21 amino acid, glycine ~ - ~ ~A ,.h~ain ~andi the~ longer ~ l (30) amino acid, phenylalanine ~ - t ~ ~B chain i ~ were ~ l then ) to a number of different acid catalyzed and proteolytic enzyme catalyzed degradations into a large number of simpler peptides. By an extensive use of paper chromatography (yo), and a painstaking jigsaw puzzle appreach, it was possible to determine the amino acid sequence of different peptides and eventually a total primary structure of both chains, as well as the sites of disulfide bonds (65), ~h~ stmctnre of insulin reveals presence of two disulfide rings: the 20.membered intrachain ring, analogous to those found in vasopresdouble sin and oxytocin and a larger, disulfide ring. ~i~~ sizes and positions of the disulfide bonds in all insulins are identical, hi^ is of interest, particularly in view of the fact that the amino acid sequence and composition in insulins from different species varies. Though the amino acid sequence and composition of both A and B chains of insulins isolated from the pancreas of various representatives of the animal king. dam differ, t,hey a~~exhibit the same sugar-regulating activit,y (71). ~~~t often, the amino acid composition variation is found in positions 8, 9, and of l the A chain, and the position 30 of the chain, ~ less frequent variations are found in the positions of A chain and 1, 2, 3, 2,, and 29 of the B chain, ~h~ relatively small number of variations suggests that all insulins have been developed by a small number of mutations from a common.precursor. While the amino acid variation in insulin may indicate a low conformational specificity, this is not so: both interchain and intrachain disulfide rings appear to be essential for insulin activity; also, elimination or exchange of the A 21 amino acid results in a total loss of the hormonal activity (66b), ~h~~~facts suggest that not only the primary, hut also the secondary and higher of insulin, i.e., insulin conformation, are essential for it,s art,ivit,v. The conformation of insulin is medeter~< mined to a high degree by its disulfide brihges. A -65% or-helical secondary stmcture of insulin has been suggested on the basis of deuterium exchange experimerits (7% Tertiary and quaternary structures may he assumed on the basis of a strong aggregation tendency of insulin molecules (66b). Three-dimenof has produced only insional X-ray conclusive conformational results. Ever since the primary structure of was elucidated, attempts have been made to synthesize the hormone. Three independent groups, in the USA, Germany and mainland China, succeeded in that effort (73). With minor variations, all three groups achieved ~~~
~
396
~~~
/
Journal of Chemical Education
the synthesis in a similar manner: a separate build-up of the A and B chains by conventional methods, followed by the coupling of the chains by disulfide bonds, using mild oxidation conditions. Prior to coupling, the protected S H groups of cysteine were freed by the use of sodium in liquid ammonia (46). In a more recent alternative of the original process, the S H group is converted into the S-sulfonate, which can easily be purified, and then reduced by use of thioglycolic acid or similar mercapto compound (73). In each case the over-all yields were quite low, particularly due to the low yields of chain-coupling oxidation, which gives a number of other disulfide compounds. The procedures were also lahor and time consuming, requiring several years to produce milligram quantities insulin. By using SPPS, Merrifield and Marglin synthesized insulin in good yield (74). The synthesis can be carried out by one person and completed in several days. This approach certainly opens the possibility of a commercial 'ynthetic insulin. In a healthy organism insulin regulates the metabolism not only of carbohydrates but also of fats and proteins. There are numerous theories of insulin mode of action; probably the most widely accepted is that of Levine and Goldstein (75). In accordance to their theory, the primary function of insulin is that of a promoter and regulator of glucose diffusion into the cell. However, recent studies indicate that the theory should be modified to accommodate new findings. There is as yet very little understanding and information concerning the active site of insulin. Insulin is at this time still the only substance capable of controlling diabetes mellitus caused by a complete failure of the Langerhans islet function. Despite extensive efforts, some of which are very recent (7% no effectiveinsulin analog has been synthesized as yet. number of diabetic conditions, however, ~A significant ~ , are caused by inadequate insulin production rather than total pancreatic failure. In such cases the disease hypOglycemics~ Can he treated by a number chemically unrelated to insulin. Their function is to production. In addition to the cyclic disulfides surveyed here, there are others that play vital roles in the body &ernistry. The enzymes ribonuclease with 124 amino acid residues (77a), and lysozyme with 129 residues (77b), as as shake (77c) may be cited as additional important examples of cyclic disulfides. Cyclic disulfides are also found among immunoglobulins where they mag be involved in stabilizing the site c7'). Literature Cited (1) GVIRARD. B. M..SNELL. E. E., A N D WILLIAMS, R. J.. A W ~ ~. i ~ ~ h ~ ~ . 9,381 (1946). (,, o t K A D. ~ =J.,. G U N S A L U ~ .J. c.,.T. B G C ~ . .s4. 20 (1947). (3) STORSTID. E. L. R.. HOIPMANN, C. E.. REDAN, M. A.. FURDA*Y,'D.. A N D JUKEB.T . H.. Amh. Biochem. Biophvsies. 20.75 (19491. (4) REED. L. J.. DBBUSH.B. G.. G U N B A ~ . I. U ~c., , A N D HORNBEROER JR., C. 6.. Science. 114.93 (19511. (5) REED. L. I.. AND DEBUSK.B. G., J . Amer. Chcm. Soe.. 73,5920 (1951). (6) ~ n o c x n a ~J. x . A,, STOX~TAD,E . L.. PATTERSON, E. L., P ~ E R CJ. E .v., MACCLI, M . A , , A N D D A Y , F . , J . A m w . Chom. Soc., 74,1868 (1952). (7) BULLOCK.M.., BRoCIXAN, J. A,, PATTBABON, E. L., PIEROE, J. V.. AND ROXBTAD, E . L.. J . A ~ S ,chern.soc..74,3455 . (18521. (8) SOPEE.Q. F.. Bmmo. W. E.,Cocnnm Jn., J. E..A N D PoaLmo. A,. 3 . Amer. Chem. Soc., 76,4109(1954). (9) WAITON, E.. WAONER.A. F . . BACXELOR, F . w.. PETERSON. I,. A.. HOLLY,F. W.,A N D FOLKERS,K., J . A m w . Chem. Soe., 77, 5144 (1965).
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