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The chemical synthesis of human and sheep insulin
The Chemical Synthesis of Human and Sheep Insulin PANAYOTIS G . KATSOYANNIS, PH .D .
Upton, New York HE LAST
two decades are probably the most
exciting in the history of protein chemistry . T It was during this period that the development of a variety of degradative technics, together with the refinement of other tools for structural analysis, made possible a better understanding of the refined texture of protein molecules . Equally impressive developments occurred in the synthetic field . Great advances in peptide chemistry, spearheaded by the synthesis of the neurohypopheseal hormones by du Vigneaud and co-workers [1-3] have provided valuable information regarding the relationship between chemical structure and biological activity of several physiologically important polypeptides . Parallel to the developments in the synthetic field there has been a comparable development both of technics which allow the separation of closely related peptides, and of highly sensitive analytical tools for assessing their chemical and stereochemical homogeneity . The combination of advances in synthetic methodology and of refinements in purification and analytical technics has set the stage for the synthesis of polypeptide chains approaching the size of low molecular weight proteins . As an outgrowth of six years of systematic studies, with the collaboration at various times of Tometsko, Fukuda, Suzuki and Tilak, we have accomplished the synthesis of the two poly-
peptide chains that comprise the molecule of sheep insulin [4,5] . Combination of the two chains generated insulin activity . This work represents the chemical synthesis of a protein . The structure of insulin, as determined by Sanger [6], is shown in Figure 1 . In this structure, two polypeptide chains are present : the A chain containing 21 amino acid residues and the B chain containing 30 amino acid residues . In the insulin molecule these two chains are linked together by two disulfide bridges . In addition, there is an intrachain disulfide bridge between the cysteine residues occupying positions 6 and 11 in the A chain . In undertaking the synthesis of insulin we made the basic assumption that if chemically synthesized A chains and B chains were available it might be possible to obtain insulin by air oxidation of a mixture of the sulfhydryl forms of the two chains . This assumption was substantiated, even before the completion of our synthetic work, by Dixon and Wardlaw [7] in Canada and Du et al . [8] in China . These investigators simultaneously reported the cleavage of insulin into its two chains, separation of the chains in the S-sulfonate form, and subsequent regeneration of insulin activity by air oxidation of a mixture of the sulfhydryl forms of the chains . The Chinese group further reported the isolation of crystalline insulin, regenerated in this way,
S, S 5
15 18 2I N1H1 14.1-12 I 2 3 4 1 2 I6 7 8 9 10 III 12 13 14 16 17 19 20 A Chain GIy-lieu -Val-GIu-GIu-Cy-Cy-Ala-Gly-Val-Cy-Ser-Lou -Tyr-Giu-Leu-GIu-Asp-Tyr-Cy-Asp S
S
I
S S 3 4 IH1 N,H2 5 I 2 N 6 17 8 9 10 1I 12 13 14 15 16 17 18 20 21 22 23 24 25 26 27 28 29 30 B Chain Phe -WI-Asp-GIu-His-Leu-Cy-Gly-Ser-His-Leu-Val-GIu-Ala-Leu-Tyr-Leu-Vol-Cy-GIy-GIu-Arq-GIy-Phe-Phe Tyr-Thr-Pro-Lys-Ala
4
FIG . 1 .
Structure of sheep insulin .
* From The Division of Biochemistry, Medical Research Center, Brookhaven National Laboratory, Upton, New York .
652
AMERICAN JOURNAL O F MEDICINE
Chemical Synthesis of Insulin--Katsoyannis
NH2 •G ly
(I)
S (6)Cy s
(21)
---5 .Cys(7) ___(I I) C y s
NH2 (20)
S I S NH2 • Phe
(1)
653
Cys -Asp • OH S i S
C y s (7)
(19)
Cys
(30)
Ala •0 H
INSULIN No 2SO 3 I . -SH 2 . 02, pH 8 .5
Na t S4 0 6 SO3 (I) NH2 . 61y
(6)l
(7) Cys •Cys I S03 S03
NH2 •P he (I)
Cys (7)
_ S03 (II) I -Cys A-CHAIN B-CHAIN
(21) NH2 (20) I Cys •A sp •O H I S03 SO-3 (19) Cys
(30) Ala •O H
Fie . 2 . Cleavage and resynthesis of insulin .
identical with the natural hormone . The overall process of cleavage and regeneration of insulin is illustrated in Figure 2 . Insulin is treated with sodium sulfite (Na 2 SO 3 ) and sodium tetrathionate (Na 2 S 4 O 6) . As a result of the sulfite treatment (sulfitolysis), the disulfide bridges of insulin are broken and the liberated sulfhydryl groups are converted to the S-sulfonates . The chains are separated and purified in the S-sulfonate form and on treatment with a thiol, such as mercaptoethanol or thioglycolic acid, are converted to the sulfhydryl form . On air oxidation of a mixture of the sulfhydryl forms of the two chains, insulin is regenerated . The yield of insulin thus regenerated is, however, an open question . It seems to depend greatly on the method used to cleave the original insulin molecule, the conditions employed in the conversion of the S-sulfonates to the sulfhydryl form, and the conditions employed for the oxidation step and, of course, the manner in which the biological assays are carried out . Consequently, the recombination yields reported by various laboratories vary considerably . The Chinese group reported a yield of 10 per cent for recombination of chains prepared by sulfitolytic cleavage of insulin [8] and, more recently, the same group claims a yield of 40 to 50 per cent [9] . Almost all the other laboratories engaged in a similar type of work have reported recombination yields of sulfitolytically prepared natural chains ranging from 1 to 4 per cent . VOL,
40,
MAY
1966
Chemically synthesized insulin chains would differ, however, in that their functional groups, including the sulfhydryl functions, would be protected . The most convenient way to protect the sulfhydryl groups is by benzylation [10] . Recombination of the insulin chains in the manner described earlier would then require, eventually, debenzylation of the protected chains by the classic method of du Vigneaud, namely, by treatment with sodium in liquid ammonia [11] . The Chinese group using natural insulin chains, which had been fully benzylated and then debenzylated by sodium in liquid ammonia, obtained upon recombination a maximum yield of only 1 to 2 per cent of theory [72] . This implies that the comparative efficiency of insulin regeneration from natural chains prepared by treatment of fully benzylated chains with sodium in liquid ammonia is 10 to 20 per cent of that obtained by recombination of chains prepared by sulfitolytic cleavage of insulin . Similar results were obtained in our own laboratory. I will discuss shortly what we believe to be an explanation of this observation, which is of importance considering the fact that debenzylation by sodium in liquid ammonia is the final step in the chemical synthesis of the insulin chains . It is apparent, therefore, that if the S-sulfonate forms of the A chains and B chains could be synthesized, insulin synthesis should be achieved by a similar series of reactions .
Chemical Synthesis of Insulin-Katsoyannis
654
y-OBut NH2 Bz Bz NZ •G Iy •I leu •Val •G Iu •Glu •Cys •Cys •A Ia •GIy •OH (I) NH2 NH2 Bz NH 2 Br lu •A yr •C ys •Asp ONBz H •Val •Cys •S er •Leu •Tyr-Glu •Leu •G sp •T (U)
r-OBut NH2 Bz Bz
Bz
NH2
NH 2
Bz NH2
NZ •G Iy •Ileu •Val •G Iu •GIu •Cys •Cys •AIa •Gly-VaI •Cys •Ser •Leu •Tyr •Glu •Leu •Glu-Asp Tyr •Cys •Asp-ONBz (III)
1 . CF3 000H 2 . Na/NH 3 3 . Na 2 S0 3 + Na2 S4 06
NH2 NH2 S03 S03 B03 NH 2 NH2 i03 H •G Iy •I leu •Val •G Iu •GIu •Cys •Cys •A Ia •GIy •Val •Cys •Ser •Leu •Tyr •Glu •Leu •Glu •Asp •Tyr •C ys •Asp •OH (IQ) Final step in the synthesis of the S-sulfonate of sheep insulin A chain . NZ : pNitrocarbobenzoxy ; y-OBut : tert-Butyl ester ; Bz : Benzyl ; ONBz : p-Nitrobenzyl ester . FIG . 3 .
Experience has shown that the most practical approach for the synthesis of polypeptides of the length and complexity of the insulin chains is by fragment condensation [13] . In this approach, peptide subunits are synthesized stepwise by adding one amino acid at a time onto the amino terminus of the chain and the peptide subunits are then condensed to form larger peptide fragments . This approach was implemented by first undertaking the synthesis of the A chain of sheep insulin [4] . Since insulin chains, obtained b% debenzylation of the respective benzylated natural chains with sodium in liquid ammonia, can be recombined to produce insulin, and since the protecting groups, N-p-nitrocarbobenzoxy and O-pnitrobenzyl, could be cleaved with sodium in liquid ammonia, and the protecting group O-t-butyl could be cleaved with trifluoroacetic acid, we reasoned that if we could synthesize the protected derivative of A chain in (Fig. 3), removal of the blocking groups with trifluoroacetic acid and sodium in liquid ammonia followed by sulfite-tetrathionate treatment should give the S-sulfonate form of the A chain (iv) . Consequently, we have synthesized the partially protected N-terminal nonapeptide I and the C-terminal dodecapeptide ii of the A chain, and by their condensation, using either the azide or the carbonyldiimidazole method, we have obtained the desired product . Removal of the blocking groups, followed by sulfitolysis, led to the syn-
thesis of the S-sulfonate of the A chain of sheep insulin . Two consecutive chromatographic steps on G-25 Sephadex, ® using 1 M pyridine and then 1 M acetic acid as the eluting solvents, sufficed to purify the crude product . On paper chromatography, in two solvent systems, the synthetic material exhibited a single Pauly-positive spot with the same Rf as the natural bovine A chain . On high voltage paper electrophoresis, this material migrated in a fashion similar to the natural chain . (Fig . 4 .) Amino acid analysis of the synthetic chain after acid hydrolysis shows a composition (Table i) in molar ratios which is consistent with the theoretically expected values for the A chain, with only a minor discrepancy : the molar ratio cys :ala in the synthetic chain is 3 .5 :1 .4, whereas in the natural chain obtained by sulfitolysis of insulin it is 4 : 1. This indicates partial desulfuration of the synthetic chain during the deblocking step with sodium in liquid ammonia, which results in a decreased cysteine content with a concomitant increase in the alanine content of that material . We believe that this finding offers one explanation for the lower efficiency in regenerating insulin, of natural A chains and B chains obtained by debenzylation of the fully benzylated chains with sodium in liquid ammonia, as was mentioned earlier . Resynthesis of insulin by combining the synthetic A chain with the natural B chain provided further proof that the synthetic material was AMERICAN JOURNAL O F MEDICINE
tathemlcal synthesis
of
insulin-
-Aatso.Vannis
() 77
'FABLE I AMINO ACID COMPOSITION OF ACID HYDROLYSA'rES OF SYNTHFTIC S-SULFONATES OF SHEEP INSULIN A' AND B CHAINS
Number of Amino Acid Residues per Molecule Amino Acid
Aspartic acid Glutamic acid Glycine Alanine Valine Leucine Isoleucine Serine Threonine Half-cystine Proline Phenylalanine Tyrosine Lysine Histidine Arginine
A Chain
B Chain
Found
Theory
Found
Theory
2 .00 4 .28 2 .11 1 .40 1 .98 1 .78
2 4 2 1 2 2
1 .10 3 .20 3 .40 1 .60 3 .20 4 .00
1 3 3 2 3 4
1 .00
1
0
0
0 .83
1
0
0
3 .50
4
1 .00 0 .76 1 .80
1 1 2
0 0
0 0
1 .00
1
3 .00
2
t 0 .83 2 .00
3 2 1 2
1 .00
1
0 0 0
t
0 0 0
* Purified only by chromatography on Sephadex . t Not determined .
BUFFER NH 4 HCO 3 , : VOLTAGE : 1,900 V TIME : 1 hour
pH 10
High voltage paper electrophoresis of synthetic (As-SSO3 - ) and natural (A N -SSO3 - ) sheep insulin A chain S-sulfonate . FIG . 4 .
indeed the A chain of sheep insulin and that the structure proposed by Sanger for that chain is correct .* As judged by biological assays using the mouse diaphragm method and immunologic assays, the efficiency of the system of synthetic A chains, natural B chain in generating insulin activity was 41 per cent of that of the system consisting of natural A chains and B chains obtained by sulfitolysis of insulin, and therefore somewhat higher than the efficiency of the system consisting of natural A chains and B chains *The recombination experiments of this early work and the biological assays were carried out by Dr . G . H . Dixon of the University of British Columbia . voL, . 40, MAY 1966
prepared by sodium in liquid ammonia debenzylation of the respective benzylated chains . The synthesis of the B chain of sheep insulin was then undertaken, also using the fragment condensation approach [5,14] . On the basis of considerations similar to those discussed earlier in the synthesis of the A chain, we reasoned that if we could synthesize the protected B chain in (Fig . 5), removal of all the protecting groups in one step, namely, with sodium in liquid ammonia followed by sulfitetetrathionate treatment should give the S-sulfonate of the B chain (iv) . After exploratory synthetic studies, it was decided to attempt to prepare the protected B chain by combining the N-terminal protected nonapeptide azide i with the C-terminal partially protected heneicosapeptide u . This route to the synthesis of the protected B chain was successful and removal of the protecting groups by sodium in liquid ammonia, followed by sulfitolysis, led to synthesis of the S-sulfonate of the B chain . Ion exchange chromatography on Dowex-50 was used to purify the synthetic chain . This material on paper chromatography and paper electrophore-
Chemical Synthesis of Insulin-Katsoyannis
656
NH2 NH2 Bz
Bz
Z •P he •Val -Asp- Glu • His- Leu •C ys •G ly •S er •O N 3 (I)
L
Bz
Bz
u-Tos
E-Tos
H-His-Leu •Val-Glu •A la •Leu-Tyr-Leu •Val •Cys •G lyGlu •A rg • GIyPhe •Phe-Tyr •T hr •Pro •Lys-Ala-OH (II) NH2 NH2Bz
Bz
Bz
1
Bz
w-Tos
Z•Phe •Val-Asp -Glu •H is- Leu •Cys •Gly-Ser •H is •Leu •Val-Glu •Ala •Leu •Tyr •Leu •VaI •Cys •Gly •G lu •A rg (III) Gly E-Tos _ Phe HO •A la •Lys •Pro •Thr •Tyr •Phe
I . Na/NH 3 2 . Na2 S03 + Na2 S4 06 NH2 NH 2
S03
S03
H •P he •VaI •A sp-Glu •H is •Leu •Cys •G ly •S er •H is •Leu •Val • Glu •A la-Leu-Tyr-Leu •VaI •Cys •G ly •G iu •A rg (~1 G)y Phe HO •AIo-Lys •P ro Thr •Tyr•Phe FIG. 5 . Final step in the synthesis of the S-sulfonate of sheep insulin B chain . Z : Carbobenzoxy ; Tos : p-Toluenesulfonyl .
sis was indistinguishable from the natural chain . Amino acid analysis, after acid hydrolysis, shows the average amino acid ratios illustrated in Table i to be in good agreement with those theoretically expected . Generation of insulin activity by combination of the synthetic B chain with natural or synthetic I
I
I
I
I
I
I
I
I
I
I
I
1 .20 1 .10 1 .00
n
E 0 .90
A-SSO; INJECTION POINT
0
1
N 0 .80 N 2
a
B - SSOy
0 .70 0.60
J
r 0
0.50 0.40 0.30 0.20 0.10
0
5
10 15 20 25 30 35 40 45 50 55 60 TUBE NUMBER
Separation of the S-sulfonates of bovine insulin A and B chains by continuous flow electrophoresis . 2,200 volts ; 0.02M NH4HCOa buffer, pH 7 .8 . FIG . 6 .
A chain provided final proof that the synthetic B chain is indeed the B chain of insulin and that the structure proposed by Sanger for that chain also is correct . As judged by biological assays using the mouse-diaphragm method, the efficiency of the system, synthetic B chain, natural A chain, in generating insulin activity was 100 per cent of that of the system of natural A and B chains . The efficiency of the system of synthetic A chain and synthetic B chain in generating insulin activity was 11 per cent of that of the system of the sulfitolytically obtained natural A and B chains, and therefore within the range of the efficiency of the system of the natural chains obtained by debenzylation of the respective benzylated chains with sodium in liquid ammonia . As mentioned earlier, the efficiency of the latter system in generating insulin activity is 10 to 20 per cent of that of the A and B chains prepared by sulfitolysis of insulin . It is apparent from this discussion, however, that in order to isolate and purify the synthetic insulin from the recombination mixture and to give a practical meaning to the chemical synthesis of this protein hormone, reproducible and considerably higher recombination yields must be attained . With this in mind we turned our * See footnote on page
655 .
AMERICAN JOURNAL O F MEDICINE
Chemical Synthesis of Insulin- -Kalsoyai?nis
BUFFER : Pyridine-Acetate-8 .M VOLTAGE : 1,700 V TIME : 2 hours
6j/
Urea, pH 4 .2
FIG . 7 . High voltage paper electrophoresis of the S-sulfonates of the separated bovine insulin A and B chains and of a mixture of these chains at an acidic pH .
BUFFER : Tris, pH 9 VOLTAGE : 1,800 V TIME : 1 hour FIG . 8 . High voltage paper electrophoresis of the S-sulfonates of the separated and of a mixture of bovine insulin A and B chains at an alkaline pH .
efforts, with Tornetsko, during the past several months to the problem of increasing the yield and standardizing the procedure for chain recombination . One of the main problems we faced in this work was the difficulty in obtaining large quanVOL .
40,
MAY 1966
tities of pure natural chains . Procedures for splitting insulin and isolating its individual chains have been reported [7,8] but these could not be used for the production of large amounts of chains ; moreover, the chains prepared by these methods are often contaminated with each other,
6 58
Chemical Synthesis of Insulin-Katsoyannis
FIG . 9 . Crystalline insulin isolated from the recombination mixture of natural bovine insulin A and B chains .
with uncleaved insulin and probably with other side products, particularly in the methods in which urea is used to solubilize insulin . We have now developed in our laboratory a method for the cleavage of insulin and isolation of its individual chains in highly purified form and on a preparative scale . Briefly, in our procedure a solution of insulin in 8 M guanidine is treated at pH 9 with excess of sodium sulfite and sodium tetrathionate for 24 hours . After dialysis and lyophilization, the split product is placed in a continuous flow electrophoresis machine under conditions illustrated in Figure 6 and the S-sulfonates of the A chains and B chains are readily separated . The contents of the tubes containing each peak are pooled and lyophilized, and the S-sulfonates of the A and B chains are obtained as white fluffy powders . Based on the amount of insulin used, the individual chains are obtained in yields of approximately 75 per cent . The chains isolated in this manner are homogeneous by all criteria available to us . In high voltage paper electrophoresis at acidic (Fig . 7) and alkaline (Fig . 8) pH's, they exhibit sharp single Pauly-positive spots . Amino acid analysis of acid hydrolysates by a Beckman-Spinco analyzer, to which a computing system has been attached, did not show any measurable contamination of each chain with the other . Finally, injection of each chain in mice at doses in excess of 300 µg . per mouse did not cause any convulsion . With a good supply of pure natural A chains
and B chains on hand, we then turned our efforts to the problem of chain recombination . As a result of these efforts, we were able to recombine the natural A and B chains in yields up to 16 per cent .* The insulin activity in these experiments was determined by the mouse convulsion method . In the recombination process, which is a slightly modified version of the method used by the Chinese team [9], a mixture of the S-sulfonates and A and B chains is treated with mercaptoethanol at 100 degrees and at pH 5 for a few minutes under nitrogen atmosphere . The reduced chains are precipitated at pH 3 .8 and then air oxidized in dilute aqueous solution at pH 10 .6 for 24 hours at 0 degrees. From the crude recombination mixture, crystalline insulin is isolated (Fig . 9) with a specific activity of 24 I . U . The experiences gained in connection with the purification and the recombination work of the natural insulin chains enabled us to purify further our synthetic sheep chains to a point that the synthetic A chain, upon combination with natural bovine B chain, generates insulin activity in a yield of 2 to 4 per cent, and the synthetic B chain, upon combination with natural bovine A chain, generates insulin activity in a yield of approximately 8 per cent of theory . From the recombination mixtures of synthetic sheep B * We have recently developed in our laboratory a procedure by which natural bovine A chains and B chains can be recombined to produce insulin in yields ranging from 45 to 55 per cent. AMERICAN JOURNAL O F MEDICINE
Chemical Synthesis of Insulin---Katsoyannis
65`)
Fre . 10 . Top, crystalline hybrid insulin isolated from the recombination mixture of synthetic sheep insulin B chain and natural bovine insulin A chain . Bottom, crystalline hybrid insulin isolated from the recombination mixture of synthetic sheep insulin A chain and natural bovine insulin B chain .
chain and natural bovine A chain, and synthetic sheep A chain and natural bovine B chain the respective hybrid insulins were isolated in crystalline form .* (Fig . 10 .) It might be pointed out that the recombination yields mentioned throughout this discussion are based on the amounts of the S-sulfonates of the A and B chain used and involve three distinctive steps : (1) conversion of the S-sulfonates to the sulfhydryl form, (2) isolation of the chains * In this work we had also the collaboration of Drs . A. Trakatellis, G . Schwartz and S . Johnson of our laboratory .
VOL .
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1966
in the reduced form, and (3) the oxidation step . Encouraged by the successful outcome of the work on sheep insulin, we have undertaken recently, with Tometsko, Zalut, Ginos and Tilak, the synthesis of human insulin . The structure of this insulin [ 15], which differs from sheep insulin in the amino acid sequence at the intrachain portion of the A chain and in the C-terminal amino acid residue of the B chain, is shown in Figure 11 . We first attempted the synthesis of the B chain of human insulin . On the basis of assumptions similar to those considered in the preparation of
66 0
Chemical
Synthesis
of
S
Insulin-Katsoyannis S
5 1
2
3
15
Ni,
4
6 7
8
10
9
11 12
13
14
NI
18 z
16
17
21
NIHZ
19
20 NI z
A Chain . H •G LY •I LEU •VAL . GLU •G LU •CY •C Y •THR •S ER •I LEU •C Y •SER •LEU •T YR •G LU •L EU •G LU •ASP •T YR •C Y •ASP •OH
I S
S
I
S 3 1
NHZ
2
S
4 N
HZ 5 6 7 8 9 10 11 12 13 14 15 16 17 18 /19 B Chain- H •P HE •V AL •A SP- GLU •HIS •LEU •C Y •G LY •S ER-HIS •L EU •VAL •G LU •A LA- LEU • TYR-LEU • VAL •CY •G LY
20
GLU 21 OH •T HR •L YS •PRO •T HR •T YR •PHE •PHE •G LY •A RG 30
FIG . 11 . NH, NH, Bz
29
28
27
26
25
24
23
22
Structure of human insulin.
Bz
I
I
Z •P HE •V AL •A SP •G LU •H IS •L EII •C YS •G LY •S ER •O N,
(I)
L
Bz
Bz
w-Tos
I
I
I
•- Tog
H •HIS •LEU •VAL •G LU •A LA •LEU •TYR •LEU •VAL •C YS •G LY •G LU •A RG •G LY •PHE •PHE •T YR •THR •PRO •LYS •T HR •O rl
NH,z
NHz
Bz
Bz
Bz
I
Bz
I
m-Tos
I
Z •PHE •VAL •ASP •G LU •HIS- LEU •CYS •G LY •S ER • HIS- LEU •V AL •G LU •ALA •LEU •TYR •LEU •VAL •C YS •G LY •G LU •A
(III)
G
GLY 6-Tog
PHE
HO •THR •LYS •PRO •THR •T YR •P HE 1 . Na /NHS 2 . Na z SO3 + Na Z S6 06 NH,z
s
SO,
NH, Z
H • PHE •VAL • ASP • GLU • HIS . LEU • CYS •G LY • SER • HIS- LEU • VAL• GLU •ALA • LEU • TYR • LEU • VAL . CYS • GLY • GLU •ARG GLY
(iv)
r
PHE
HO • THR •LYS •PRO •THR •TYR •PHE
FIG . 12 .
Final step in the synthesis of the S-sulfonate of human insulin B chain .
NH, I
Bz Bz I
I
Z •G LY •I LEU •VAL-GLU •G LU •C YS •CYS •T HE SER •O NI
(I)
Bz
NHz
I
I
NH,
Bz
NH,
I
I
I
r H •I LEU •C YS •S ER •L EU •T YR •G LU •LEU •G LU •ASP-TYR .CYS •A SP- ONBz
(II)
NHz
Bz iz
Bz
NIZ
NIHz
Hz
NIHZ
Z •G LY •I LEU •VAL •G LU •G LU •C YS •CYS •T HR •S ER•I LEU •C YS •S ER •L EU •T YR •G LU •LEU •G LU •ASP •TYR •C YS •A SP-ONBz 1 . Na /NHS 2 . Na Z SO l + Na Z S4 NI2
S033
S03j
Si ~
06
NiZ
NH,
SO,
NH Z
H •G LY •ILEU •V AL •G LU •G LU •C YS •C YS •T I1R •SER •I LEU •C YS •S ER •L EU •T YR •G LU •LEU •G LU •ASP •T YR •C YS •ASP •O H
FIG . 13 .
(IV) Final step in the synthesis of the S-sulfonate of human insulin A chain . AMERICAN
JOURNAL
O
F
MEDICINE
Chemical Synthesis of Insulin
the chains of sheep insulin, we approached the synthesis of the human B chain through preparation of the appropriately protected triacontapeptide derivative in shown in Figure 12 . Synthesis of this derivative was accomplished by coupling the N-terminal nonapeptide azide i with the C-terminal partially protected heneicosapeptide it . Removal of the blocking groups with sodium in liquid ammonia and treatment of the ensuing product with sodium sulfitesodium tetrathionate yielded the S-sulfonate of the B chain of human insulin iv, which was purified by continuous flow electrophoresis . The purified material on high voltage paper electrophoresis is homogeneous and has a mobility similar to that of the natural bovine B chain . Amino acid analysis of an acid hydrolysate shows average amino acid ratios in good agreement with those theoretically expected for the human B chain . Combination of this synthetic human B chain with the natural A chain of bovine insulin generated considerable insulin activity . As judged by the mouse convulsion assay method, the yield of the hybrid insulin generated was 4 to 8 per cent of theory . The synthesis of the A chain of human insulin was then undertaken by a route similar to that followed in the synthesis of the A chain of sheep insulin, namely, through the preparation of the respective protected heneicosapeptide iii . (Fig . 13 .) We have, therefore, prepared the C-terininal partially protected dodecapeptide it and the N-terminal nonapeptide derivative i . Condensation of these two polypeptide fragments by the azide method gave the desired heneicosapeptide derivative in, which was subsequently deblocked by sodium in liquid ammonia and treated with sodium sulfite and sodium tetrathionate . The S-sulfonate form of the human A chain thus obtained was purified by chromatography on G-25 Sephadex . 'The purified material on 'high voltage paper electrophoresis exhibits a single Pauly-positive spot and has a mobility similar to that of the natural bovine A chain . Amino acid analysis of an acid hydrolysate of the synthetic chain showed a composition in molar ratios in good agreement with the theoretically expected values for the human A chain . Resynthesis of insulin by combining the synthetic human A chain with the natural bovine B chain or the synthetic human B chain provided the final proof that the polypeptide chains synV CAL . 40, MAY 1966
-K'atsoyannis
6 6 1.
thesized are indeed the chains of human insulin and that the structure proposed for that insulin is correct . As judged by the mouse convulsion assay method, the yield of the hybrid insulin produced by combining the synthetic human A chain with the natural bovine B chain is 8 per cent of theory, and the over-all yield of the allsynthetic human insulin produced is 2 per cent of theory . This last result represents the first chemical synthesis of a human protein . REFERENCES 1 . DU VIGNEAUD, V ., RESSLER, C ., SWAN, J . M ., ROBERTS, C . W ., KATSOYANNIS, P . G . and GORDON, S . The synthesis of an octapeptide amide with the hormonal activity of oxytocin . J. Am . Chem . Soc ., 75 : 4879, 1953 . 2. Du VIGNEAUD, V ., RESSLER, C ., SWAN, .1 . M., ROBERTS, C . W . and KATSOYANNIS, P . G . The synthesis of oxytocin . J. Am . Chem . Soc., 76 : 3115, 1954 . 3 . DU VIGNEAUD, V ., GISH, D . T., KATSOYANNIS, P . G . and HESS, G. P . Synthesis of the pressor-antidiuretic hormone, arginine-vasopressin . J . Am . Chem . Soc ., 80 : 3355, 1958 . 4 . KATSOYANNIS, P. G ., TOMETSKO, A . and FUKUDA, K . The synthesis of the A-chain of insulin and its combination with natural B-chain to generate insulin activity . J. Am . Chem . Soc ., 85 : 2863, 1963 . 5 . KATSOYANNIS, P. G ., FUKUDA, K ., TOMETSKO, A ., SuzuKi, K . and TILAK, M . The synthesis of the B-chain of insulin and its combination with natural or synthetic A-chain to generate insulin activity . J. Am . Chem . Soc ., 86 : 930, 1964. 6 . SANGER, F . Chemistry of insulin . (Nobel lecture .) Science, 129 : 1340, 1959 . 7 . DIXON, G . H . and WARDLAW, A . C . Regeneration of insulin activity from the separated and inactive A and B chains . Nature, 188 : 721, 1960 . 8 . Du, Y .-C ., ZHANG, Y .-S ., Lu, Z .-X . and Tsou, C .-L . Resynthesis of insulin from its glycyl and phenylalanyl chains. Sc . Sinica, 10 : 84, 1961 . 9 . Du, Y .-C ., JIANG, R.-Q . and Tsov, C.-L . Conditions for successful resynthesis of insulin from its glycyl and phenylalanyl chains . Sc . Sinica, 14 : 229, 1965 . 10 . WOOD, J. L . and Du VIGNEAUD, V . Racemization of benzyl-L-cysteine, with a new method of preparing D-cystine . J. Biol, Chem ., 130 : 109, 1939 . 11 . SIFFERD, R . H . and DU VIGNEAUD, V. A new synthesis of carnosine, with some observations on the splitting of the benzyl group from carhohenzoxy derivatives and from benzylthio ethers . J. Biol . Chem ., 108 : 753, 1935 . 12. Tsou, C .-L ., Do, Y .-C . and XC, G .-J . The reduction of insulin and its benzyl derivatives by sodium in liquid ammonia and the regeneration of activity from the reduced chains . Sc . Sinica, 10 : 332, 1961 . 13 . KATSOYANNIS, P . G . Peptide synthesis and protein structure. J. Polymer Sc ., 49 : 51, 1961 . 14 . KATSOYANNIS, P . G . Synthetic studies on the A- and B-chains of insulin . Vox Sang ., 9 : 238, 1964 . 15 . NICGL, D. S. H . W . and SMITH, L . F . Amino acid sequence of human insulin . Nature, 187 : 483, 1960 .